Society of General Physiologists Series • Volume 46, Part 2

The Sodium Pump: Recent Developments

Society of General Physiologists • 44th Annual Symposium

Edited by
Jack H. Kaplan
and
Paul De Weer
University of Pennsylvania

Marine Biological Laboratory

Woods Hole, Massachusetts

5–9 September 1990

© The Rockefeller University Press

New York

Copyright © 1991 by The Rockefeller University Press

All rights reserved

Library of Congress Catalog Card Number 91-052922

ISBN 0-87470-049-3

eISBN 978-0-87470-093-0

Printed in the United States of America

Contents

Preface

Molecular Biology and Expression

Cloning and Analysis of the 5'-Flanking Region of Rat Na,K-ATPase αl-Subunit Gene
Yuriko Yagawa, Kiyoshi Kawakami, and Kei Nagano

Characterization and Sequence Analysis of the Multiple Genes Encoding the Human Na,K-ATPase Catalytic Subunit
E. D. Sverdlov, G. S. Monastyrskaya, N. E. Broude, K. E. Petrukhin, I. V. Malyshev, D. A. Bessarab, S. V. Volik, V. E. Sverdlov, M. Y. Orlova, A. V. Grishin, and N. N. Modyanov

Evolutionary Conservation of the (Na+ + K+)-ATPase Genes
Kunio Takeyasu, Atsushi Mizushima, Andrew M. Barnstein, Maura Hamrick, and Douglas M. Fambrough

Na,K-ATPase Gene Expression during Cytodifferentiation of a Toad Bladder Cell Line
F. Jaisser, C. Canessa, H. P. Gaeggeler, L. Racine, J.-P. Kraehenbuhl, and B. C. Rossier

Molecular Analysis of Na,K-ATPase Subunit Assembly
Karen Jane Renaud and Douglas M. Fambrough

Molecular Cloning of Na,K-ATPase α-Subunit cDNA from Hydra vulgaris
Victor A. Canfield, Kai-Yuan Xu, Thomas D'Aquila, Andrew W. Shyjan, and Robert Levenson

Molecular Cloning and Immunological Characterization of the γ-Subunit of the Na,K-ATPase
Robert W. Mercer, Daniel Biemesderfer, David P. Bliss, Jr., John H. Collins, and Bliss Forbush III

Expression of DNA for α, β, and γ Polypeptides of Na,K-ATPase in Yeast: Does γ Have a Function in Na,K-ATPase Activity?
Georgios Scheiner-Bobis, Kurt A. Eakle, Kyu S. Kim, and Robert A. Farley

Expression of Domains of the Na,K-ATPase in a Run-away Plasmid Replication System in Bacteria
Douglas M. Fambrough and Ru Chih C. Huang

Expression of Exogenous β1-Subunits of Na,K-Pump in Xenopus laevis Oocytes Raises Pump Activity
Günther Schmalzing, Halit Omay, Silke Kröner, Heribert Appelhans, and Wolfgang Schwarz

Expression of the Large Cytoplasmic Domain of Na,K-ATPase in Escherichia coli
Kena Wang, Edward E. Huston, and Robert A. Farley

Localization of Na,K-ATPase α- and β-Subunits Expressed in Xenopus Oocytes
Kazuo Takeda, Shunsuke Noguchi, and Masaru Kawamura

Functional Expression of the Na,K ATPase in Spodoptera frugiperda Cells
Anthony W. De Tomaso, Peter Zdankiewicz, and Robert W. Mercer

Cellular Mechanisms in the Induction of Na,K-ATPase Subunit mRNAs by Serum and Growth Factors in Clone 9 Cells
Alok Bhutada and Faramarz Ismail-Beigi

Serum-independent Induction of Na,K-ATPase and c-fos by Low K+ in a Rat Liver Cell Line
E. Cayanis, J. J. Russo, Y.-S. Wu, and I. S. Edelman

Low K+ Stimulates Differential Regulation of Na,K-ATPase Subunits in Kidney Cells
Leslie Lescale-Matys, Ming-Jer Tang, and Alicia A. McDonough

Inhibition of the Na+/K+ Pump in Xenopus Oocytes by Diacylglycerol Analogues
Larisa A. Vasilets, Kristin Mädefessel, Günther Schmalzing, and Wolfgang Schwarz

Regulation of Expression of Na,K-ATPase in MDCK Cells
Christopher P. Cutler and Gordon Cramb

Enzyme Isoforms

Identification of Cis Elements Regulating the β2-Subunit Gene of the Na+, K+-ATPase
Kiyoshi Kawakami, Yuriko Yagawa, Hiroshi Okamoto, and Kei Nagano

Alternately Spliced cDNAs Encode the Chicken Sarco/Endoplasmic Reticulum Slow Ca2+ ATPase
A. Malcolm Campbell, Paul D. Kessler, and D. M. Fambrough

Deletions in the 3' Untranslated Region of the βl-Subunit mRNA Affect mRNA Stability
Joseph P. Taormino and Douglas M. Fambrough

Analysis of Isoform-specific Regions within the α- and β-Subunits of the Avian Na,K-ATPase
Victor Lemas, Jayson Rome, Joe Taormino, Kunio Takeyasu, and Douglas M. Fambrough

Expression and Assembly of Different α- and β-Subunit Isoforms of Na+, K+ ATPase in Yeast
Kurt A. Eakle, Burton Horowitz, Kyu Seong Kim, Robert Levenson, and Robert A. Farley

Functional Properties of the Na,K-ATPase α2-Subunit Isoform Expressed in Mammalian Cell Lines
Andrew W. Shyjan, Victor A. Canfield, and Robert Levenson

Distribution of Isoforms of the α-Subunit of the Na,K-ATPase in Adult Rat
Paul M. Rowe, W. Timothy Link, Charlene P. Osborn, and R. Wayne Albers

αl and α3 Isoforms of (Na,K)-ATPase Are Both Produced in Neurons and Show Unique Distributions in the Rat CNS and PNS
George J. Siegel, Marina Mata, Virginia Hieber, and David J. Fink

Expression of α- and β-Subunit Isoforms of the Na,K-ATPase in the Nervous System
William R. Anderson, Peter E. Filuk, Auday A. Maki, and William L. Stahl

Diversity of Na,K-ATPase Isoform Distribution in the Nervous System
Kevin M. McGrail, Joseph M. Phillips, and Kathleen J. Sweadner

Cellular Distribution and Differential Expression of the Na,K-ATPase α Isoform (α1, α2, α3), βl, and β2/AMOG Genes in the Ocular Ciliary Epithelium
Miguel Coca-Prados, Pablo Martin-Vasallo, Nati Hernando, and Sikha Ghosh

Na,K-ATPase α1, α2 Isoform and β-Subunit Are Differentially Expressed in Different Regions of the Colonic Circular Muscle Layer
Burton Horowitz and Edmund Burke

Regulation of Na,K-ATPase αl and α2 Isoforms in Rat Myocardial Cells by Thyroid Hormone
Charles B. Hensley, Ming-Jer Tang, Kay K. Azuma, and Alicia A. McDonough

Differential Regulation of Na,K-ATPase αl, α2, and β in Heart, Skeletal Muscle, and Brain of Hypokalemic Rats
Kay K. Azuma, Charles B. Hensley, Daun S. Putnam, and Alicia A. McDonough

A Highly Ouabain-sensitive Isoform of Na,K-ATPase in Nervous Cells: Functional Evidence for a Multigenic Expression of Its Catalytic Subunit
Gustavo Blanco and Luis Beaugé

Differential Expression of Three Na,K-ATPase Catalytic Subunit Isoforms in Human Tissues, Organs, and Cell Lines
N. S. Akopyanz, N. E. Broude, N. G. Vinogradova, Yu. A. Balabanov, G. S. Monastyrskaya, and E. D. Sverdlov

Transfection of Na,K-ATPase α1- and βl-Subunit cDNAs into Clone 9 Cells: Analysis of Na,K-ATPase mRNA Abundance and Enzyme Activity
Mary Ann Hatala, Isidore S. Edelman, and Faramarz Ismail-Beigi

Structure

Esterification of a Carboxyl Residue in the Na Pump α-Subunit Abolishes K Sensitivity
Jose M. Argüello and Jack H. Kaplan

Lysine 480 Is an Essential Residue in the Putative ATP Binding Site of Lamb Kidney (Na,K)-ATPase
Terence L. Kirley and Hellmuth R. Hinz

Disulfide Bonds in the (Na,K)-ATPase: Are They Important?
Terence L. Kirley

Three Proline Residues in the Fifth Transmembrane Segment of Na+,K+-ATPase α-Subunit Inhibit Its Membrane Insertion
Haruo Homareda, Kyoshi Kawakami, Kei Nagano, and Hideo Matsui

Determination of the Four Disulfide Bond Locations Contained in Na,K-ATPase Inhibitor (SPAI)
KengoAraki, Manabu Kuwada, Osamu Ito, Jun Kuroki, and Shinro Tachibana

Site-directed Mutagenesis of Cys in the β-Subunit of the Torpedo californica Na,K-ATPase
Shunsuke Noguchi, Yoshinobu Mutoh, and Masaru Kawamura

NMR Studies on Intermediate States of SR Ca2+ -ATPase Using Co(III), Cr(III), and Rh(III) Nucleotides
Theresa A. Kuntzweiler and Charles M. Grisham

Expression of Functional Sodium and Calcium Pump Chimeric Molecules
Douglas B. Luckie, Kimberly L. Boyd, Atsushi Mizushima, Zhifeng Shao, Andrew P. Somlyo, and Kunio Takeyasu

Three-dimensional Structure of CO(NH_3)4ATP-induced Membrane Crystals of Na,K-ATPase
Elisabeth Skriver, Hans Hebert, Urban Kavéus, and Arvid B. Maunsbach

Structural Analysis of Na,K-ATPase Isoforms in Mammalian Tissues
Elena A. Arystarkhova, Natalia M. Vladimirova, Natalia A. Potapenko, and Nikolai N. Modyanov

Function and Orientation of the COOH-terminal Domain of the α-Subunit
T. Krämer, R. Antolovic, H.-J. Brüller, U. Richter, G. Scheiner-Bobis, and W. Schoner

Location of Amino Acid Residues Critical for Ca2+ Transport by Sarcoplasmic Reticulum Ca2+-ATPase in Light of Membrane Protein Folding Proposed for Na+,K+-ATPase
Jesper V. Møller, Marc le Maire, and Philippe Champeil

Structure-Function Studies of Na/K-ATPase Isozymes
Bruce C. Kone, Kunio Takeyasu, and Douglas M. Fambrough

Posttranslational Processing and Basic Properties of a Putative β3-Subunit of Na,K-ATPase
Philippe Jaunin, Peter Good, Irène Corthesy, and Käthi Geering

Immunodetection and Characterization of the β-Subunit of Na+,K+-ATPase in Mammalian Liver Tissues
Ying Sun and William J. Ball, Jr.

Association and Dissociation of Solubilized Na+/K+-ATPase by Phosphatidylserine and ATP
Yutaro Hayashi, Kunihiro Mimura, Hideo Matsui, and Toshio Takagi

Identification of a 97-kD Protein from Rat Kidney Related to the Gastric H,K-ATPase
Mark D. Okusa and Michael J. Caplan

An Analysis of Digitalis Sugar Binding Site Characteristics
Dwight S. Fullerton, Arthur H. L. From, and Khalil Ahmed

Studies of Digitalis Binding Site(s) in Na,K-ATPase by Covalent Labeling with a Photoactive Probe Located in the Genin Moiety
Reginald A. McParland, Dwight S. Fullerton, Robert R. Becker, Arthur H. L. From, and Khalil Ahmed

Characterization of Carbohydrate in Na+,K+-ATPase β-Subunit from Electrophorus
W. Timothy Link, Paul M. Rowe, and R. Wayne Albers

The Na-Pump α-subunit Has N-linked Glycans Facing the Cytosol
Carlos H. Pedemonte and Jack H. Kaplan

Reaction Kinetics and Transport Modes

Steady-State Phosphorylation and Dephosphorylation of Na,K-ATPase in the Presence of Na+ and Na+ + K+
Jens Chr. Skou

Effect of Oligomycin on the Rate of Phosphorylation of Na,K-ATPase in the Presence of Na+
Jens Chr. Skou

Anion Effects on the Steady-State Ratio of the Phosphoenzymes of Na,K-ATPase as Measured by Dephosphorylation and Oligomycin Inhibition
Irena Klodos and Liselotte Plesner

Transient Kinetics of Dephosphorylation of Na,K-ATPase after Dilution of NaCl
Irena Klodos and Bliss Forbush III

Effect of Lyotropic Anions on the Dephosphorylation of Na,K-ATPase Phosphointermediates
Irena Klodos

Oligomycin Activation of Na,K-ATPase Hydrolysis
Liselotte Plesner and Igor W. Plesner

Order-of-Magnitude Estimation of Rate Constants for the Interconversion of the Phosphoenzymes of Na,K-ATPase, Based on Oligomycin Inhibition Experiments
Igor W. Plesner

A Procedure for the Simulation of Unidirectional (Exchange) Rates in Enzyme Catalyzed Reactions in the Presence of Both Substrates and Products
Igor W. Plesner

Chemical Relaxation Studies of Fluorescein 5'-Isothiocyanate Modified Sodium Pump
Larry D. Faller, Georgios Scheiner-Bobis, and Robert A. Farley

Fluorescence Energy Transfer between N-[P-(2-benzimidazolyl)-phenyl]maleimide, N-(1-anilinonaphthyl-4)maleimide, Fluorescein 5'-isothiocyanate, and Anthroylouabain in Na+, K+-ATPase
Eiji Shinoguchi, Eiichi Ito, Akinobu Kudo, Shinji Nakamura, and Kazuya Taniguchi

Effect of Na+ and Amino Compounds on the E_2 → E_1 Transition, Studied by Eosin Fluorescence
T. J. F. Van Uem, F. M. A. H. Schuurmans Stekhoven, G. F. Borm, A. G. M. Theeuwes, H. G. P. Swarts, and J. J. H. H. M. De Pont

Slow and Rapid Components of Dephosphorylation Kinetics of Na,K-ATPase
Kuniaki Suzuki and Robert L. Post

High Concentrations of ATP Induce a Transient Extra Phosphorylation of the Na,K-ATPase
Ruben D. Peluffo, Patricio J. Garrahan, and Alcides F. Rega

High Affinity Inhibitory Effect of Cytoplasmic K+
Flemming Cornelius and Jens Chr. Skou

Sidedness of Na and K Effects on Phosphorylation and Conformations of Pig Kidney Na,K-ATPase Interacting with Inorganic Phosphate or Acetyl Phosphate
G. Berberián and L. Beaugé

Phosphatase Activity and Potassium Transport
G. Berberián and L. Beaugé

The Order of ADP and P_1 Release in a Single Cycle of Na,K-ATPase
Tim J. H. Walton and José D. Cavieres

High and Low Affinity ATP Binding Sites as Well as Na+ and K+ Forms Coexist in Na+,K+-ATPase
E. Buxbaum, E. H. Serpersu, R. Antolovic, E. Hamer, M. Willeke, and W. Schoner

Kinetics of Magnesium Interaction with Na,K-ATPase
Yolanda G.-M. Covarrubias and Paul De Weer

The Change in Na+ Binding States before and after Accumulation of ADP-sensitive Phosphoenzyme in Na+,K+-ATPase
Toshio Sasaki, Eiji Shinoguchi, Yoshiko Kamo, Eiichi Ito, and Kazuya Taniguchi

Occlusion of the K+-Congener T1+ through the Phosphointermediates of Na,K-ATPase
Rolando C. Rossi and Jens G. Nørby

Competitive Relationship beteen K+ and (Z)-5-Methyl-2-[2-(1-naphthyl)ethenyl]-4-piperidinopyridine for Access to the K+-translocating Channel of (Na,K)-ATPase
Yoshihiro Fukushima, Junji Takada, Hideaki Nagamune, and Mitsuo Yano

Characterization of Lanthanides as Competitors of Na+ and K+ in Occlusion Sites of Renal (Na+,K+)-ATPase
Peer David and Steven J. D. Karlish

Kinetics of Na+,K+-ATPase Conformational Change with Alternate Substrates
P. R. Pratap, J. D. Robinson, and M. I. Steinberg

Photo-cleavable Cryptands as a New Tool to Perform Na+ and K+ Concentration Jumps for Binding and Transport Studies
R. Warmuth, E. Grell, and J. M. Lehn

Precision Titrations to Determine Affinity and Stoichiometry of Alkali, Alkaline Earth, and Buffer Cation Binding to Na,K-ATPase
E. Grell, R. Warmuth, E. Lewitzki, and H. Ruf

Ca-dependent Inhibition of the Na,K-ATPase: Estimation of the Molecular Weight of Calnaktin
Jing Ye-Hu, Virginia Barrett, Guangsheng Wang, and Douglas R. Yingst

Na/K-Pump Activity and Cell Volume Changes during Maturation of Rat Reticulocytes
Heimo Mairbäurl and Joseph F. Hoffman

Is the Pump of Intact Red Blood Cells Saturated for ATP at Physiological Concentrations?
Marina Marjanovic and John S. Willis

Characterization of Purified Na,K-ATPase Isolated from Mink (Mustela vison) Kidney
Otto Hansen, Tove N. Clausen, and Søren Wamberg

Immunocytochemical Evidence for Protein Kinase C-Induced Internalization of Sodium Pumps in Xenopus laevis Oocytes
Günther Schmalzing, Kristin Mädefessel, Winfried Haase, and Kathi Geering

Effect of Phosphatidylcholine on the Na,K-ATPase Activity of Basolateral Plasma Membranes from Proximal Tubular Cells of Rat Kidney
Reinaldo Marín, Teresa Proverbio, and Antonio J. Rodriguez

Cellular Regulation of the [Na+,K+]-ATPase Activity
Haywood Blum and Robert G. Johnson, Jr.

Defective Na Transport of Murine Ouabain-resistant Alpha-1 Na,K-ATPase
B. M. Anner, E. Imesch, and M. Moosmayer

Regulatory Role of ATP in the Na,K-ATPase Operation
A. A. Boldyrev, N. U. Fedosova, O. D. Lopina, and E. V. Pindel

A Ouabain-sensitive Na,K-ATPase in the Sea Anemone Stoichactis helianthus: Preliminary Characterization
Roberto Lopez Rosado and Susan Corey

Spectral Tagging of Reaction Substates in a Sodium Pump Analogue: The Plasma Membrane H+-ATPase of Neurospora
Georg Nagel, Esther Bashi, and Clifford L. Slayman

Interaction of NaK-ATPase Proteoliposomes with Palytoxin at Picomolar Concentrations
Atsunobu Yoda, Marsha Rich Rosner, Peter Morrison, and Shizuko Yoda

Palytoxin Forms Ion Channels through Na,K-ATPase
Soung Y. Kim, Chau H. Wu, and Lászlo Béress

Properties of the Ion Channel Formed in Bilayer Lipid Membranes by α-Subunits of (Na+ + K+)-ATPase
Galina D. Mironova and Nina I. Zolotarjova

Lack of K+ Stimulation of the Na-ATPase Activity of Red Cell Ghosts from the LK Sheep Ovis aries
Fulgencio Proverbio, José A. Duque, and Reinaldo Marín

Ouabain, Amiloride, and Gossypol Inhibit the Channels Formed by α-Subunit of Na+,K+-ATPase in Bilayer Lipid Membranes
Nina I. Zolotarjova, Larisa V. Ivkova, Galina D. Mironova, Nailya M. Mirsalikhova, and Svetlana F. Sokolova

Electrogenic Aspects

Correlation of Charge Translocation with the Reaction Cycle of the NaK-ATPase
K. Fendler, J. Froehlich, S. Jaruschewski, A. Hobbs, W. Albers, E. Bamberg, and E. Grell

Charge Translocation by the Sodium Pump: Ion Binding and Release Studied by Time-resolved Fluorescence Measurements
W. Stürmer, R. Bühler, H.-J. Apell, and P. Läuger

Comparison of the Na/K and the Na-only Mode of the Sodium Pump
Hans-Jürgen Apell and Milena Roudna

Na/K Pump Current in Giant Membrane Patches Excised from Ventricular Myocytes
Donald W. Hilgemann, Georg A. Nagel, and David C. Gadsby

Sodium Pump Current in Cardiac Purkinje Cells Dialyzed with ATP-free Solutions
Helfried G. Glitsch and André Tappe

Study of the Voltage Dependence of the Na,K Pump in Cardiac Cells Using Simulation
D. Renald Lemieux and J. Mailen Kootsey

Effects of Intra- and Extracellular pH on Na+/K+ Pump Current in Single Rabbit Cardiac Purkinje Cells
Frieda V. Bielen and Fons Verdonck

Current–Voltage Relation of the Na-K Pump–generated Current in A6 Cells
Jean-Daniel Horisberger and Marie-Christine Broillet

Transport Properties of an Electrogenic Proton Pump from Enterococcus hirae
H.-J. Apell, C. Hölscher, and M. Solioz

Regulation and Adaptation

Regulation of Expression and Polar Distribution of Na,K-ATPase in Renal Epithelium during Recovery from Ischemic Injury
M. Kashgarian, S. K. Van Why, F. Hildebrand, A. S. Mann, K. M. Gaudio, and N. J. Siegel

Regulation of the Myocardial Na+,K+-ATPase and Ca2+ during Ischemia and Reperfusion
TaiAkera and Takashi Koyama

Quantification of Rat Cerebral Cortex Na+,K+-ATPase with High Recovery: Evaluation of Age and K+-Depletion
Jim S. Larsen, Thomas A. Schmidt, and Keld Kjeldsen

Adaptation of Brain Na,K-ATPase to Changes in Neural Activity
Alan C. Swann

Changes in Responses of Na Pump Isoforms to Glutamate Stimulation of Cerebral Neurons During Maturation in Culture
Nobuo Inoue and Hideo Matsui

Induction of α2 by Growth-promoting Concentrations of Insulin in 3T3-L1 Preadipocytes
James J. Russo, K. J. Sweadner, and I. S. Edelman

Chronic Diabetes Increases Na,K-ATPase Activity, α1 Isoform Protein, and mRNA Abundance in Rat Small Intestinal Mucosa
Kassem Barada, Charles Okolo, Michael Field, and Nadim Cortas

Glucagon Changes the Kinetics of Ouabain Inhibition of Na,K-ATPase in Caco2 Cells: An Effect Reversed by Insulin
S. L. Lucak, R. W. Infantino, C. Okolo, M. Field, and N. Cortas

Clinical Aspects

Clinical Implications of Digitalis Glycoside Receptor Studies in Human Heart and Skeletal Muscle
Keld Kjeldsen, Thomas A. Schmidt, and Jim S. Larsen

Preferential Occupancy of High-affinity Cardiac Na,K-ATPase Isoenzymes (α2 or α3) by LND-796 during Positive Inotropy in the Dog
Jean-Michel Maixent, Richard M. Kawamoto, James R. Skuster, Bruce M. Halpryn, Antoine Baggioni, and Simone Fenard

An Outward K Current in Cat Ventricular Myocytes Shows Sensitivity to Dihydroouabain
Ruth L. Martin and Robert E. Ten Eick

Na Pump in Vascular Smooth Muscle: Regulation of Cell Proliferation
J. C. Allen, C. L. Seidel,, S. S. Navran, T. A. Pressley, and R. M. Medford

Isoforms of Na,K-ATPase in Myocardial Tissues of the Milan Hypertensive Rat
Christopher P. Cutler and Gordon Cramb

Quantification of the Total Na,K-ATPase Concentration in Mammalian Heart: Regional Distribution and Regulatory Aspects
Thomas A. Schmidt, Jim S. Larsen, and Keld Kjeldsen

Dissociation Kinetics of [3H] Ouabain Na,K-ATPase Isoform Complexes
Alain De Pover and Gudrun Feige

Ouabain-induced Changes in Intracellular Free Calcium Concentration Examined in Different Cell Types of Chick Heart Monolayer Cultures
Barbara Ahlemeyer, Horst Weintraut, Georg Seibold, and Wilhelm Schoner

Presence of an Inward Background Current Sensitive to Sodium in the Isolated Rabbit Sinoatrial Node Cells
N. Hagiwara, H. Irisawa, H. Kasanuki, and S. Hosoda

Author Index

Preface

Every few years, researchers from many nations gather to present and discuss the latest data and ideas on the workings and structure of the sodium pump. Successive meetings have been held under the auspices of the New York Academy of Sciences (1973), Aarhus University in Denmark (1978), Yale University (1981), Cambridge University in England (1984), and Aarhus University again (1987). The 6th International Sodium Pump Meeting, organized by the editors of this volume, was held in September 1990 at the Marine Biological Laboratory in Woods Hole, Massachusetts, as the 44th Annual Meeting of the Society of General Physiologists. There were nearly 400 participants.

This volume contains condensed papers presented in poster form at the Woods Hole symposium, as well as several that are based on workshop contributions. A companion volume, entitled The Sodium Pump: Structure, Mechanism, and Regulation, contains the plenary presentations made during the five-day gathering; it is distributed as a supplement to the Journal of General Physiology, and can also be purchased separately. Both volumes are divided into thematic sections that follow the organizational format of the meeting itself. Because of its large number of contributors, this volume contains, in addition, an author index. We trust that these printed proceedings convey the sense of excitement and expectation that we as participants enjoyed.

We thank all the participants in the meeting, the director and staff of the Marine Biological Laboratory, Woods Hole, Massachusetts, and the officers of the Society of General Physiologists. The Conference would not have been possible without financial support from the Lucille P. Markey Charitable Trust, The National Institutes of Health, the National Science Foundation, and the U. S. Army Medical Research and Development Command. We were particularly pleased to be able to offer two dozen accommodation awards to young investigators from around the world. We are grateful to Jane Leighton and Sue Lahr for their tireless efforts to ensure the smooth running of the meeting, and to Jeanne Hess and her colleagues at the Rockefeller University Press for their patient efficiency in enabling us to bring these volumes to timely publication.

Paul De Weer

Jack H. Kaplan

Molecular Biology and Expression

Cloning and Analysis of the 5'-Flanking Region of Rat Na,K-ATPase α1-Subunit Gene

Yuriko Yagawa, Kiyoshi Kawakami, and Kei Nagano

Department of Biology, Jichi Medical School, Tochigi, 329-04, Japan

Introduction

To analyze the molecular mechanism of the differential expression and the stoichiometric mutual regulation between the α- and β-subunits of Na,K-ATPase, we have initiated a systematic study of the genomic DNA of α and β isoform genes, especially including their 5'-flanking regions where important regulatory functions must reside. We have cloned a 13.3-kb fragment of the rat Na,K-ATPase αl-subunit gene (ATP1A1) with its 5'-flanking region and identified the transcription initiation site, which was located 262 bp upstream from the translation initiation codon and which was identical among six tissues of adult rat (kidney, brain, heart, thyroid, liver, and lung) (Yagawa et al., 1990). This suggests that the level of expression of the ATP1A1 is modulated by the same promoter, at least in these tissues. Maximum homology between rat, horse (Kano et al., 1989), and human (Shull et al., 1990) ATP1A1 was observed in the region from 98 bp upstream from the transcription initiation site to the 3' end of the first exon, where the TATA-like box and Sp1-binding and ATF consensus sites were located.

For the purpose of identifying the cis-acting elements that regulate the expression of the α1-subunit gene, we have constructed 5' and 3' sequential deletion mutants fused to luciferase reporter gene. The promoter activities were assayed after transfection with MDCK (epithelial cell line from female cocker spaniel kidney) and B103 (rat neuroblastoma cell line) cells, which have demonstrated the ATP1A1 expression.

Methods

Plasmid Construction

The Sal I-Nco I fragment of ATP1A1 spans from the 5'-flanking region (–1,537; the nucleotides are numbered in reference to the major transcription initiation site designated +1) to just before the translation initiation site in the first exon (+261). The cohesive end of the Nco I site was deleted with Mung bean nuclease to prevent reconstruction of the translation initiation codon (ATG at +263) and that of the Sal I site was filled in with Klenow fragment. The blunt-ended DNA fragments were ligated with Hind III linker (dCAAGCTTG). After digestion and fractionation, these fragments were ligated to Hind III-digested pSVOA/LΔ5' (Wet et al., 1987), a vector containing luciferase gene (pA1LF-1). pSV2A/LΔ5' was used as positive control. pSV2CAT was used as internal control of the luciferase gene expression. All plasmids were isolated by a modified method of lysozyme–triton extraction and purified by CsCl gradient centrifugation.

DNA Transfection and Enzyme Assay of Luciferase and CAT

The cells were maintained in Dulbecco's modified Eagle medium (DMEM; Gibco Laboratories, Grand Island, NY) supplemented with 10% (for MDCK cells) or 15% (for B103 cells) fetal calf serum (FCS; Gibco Laboratories) in a CO_2 incubator (5% CO_2) at 37°C. Each 16 μg of the plasmid was cotransfected with 4 μg of pSV2CAT into MDCK or B103 cells (3–4 × 10^5 cells in 9-cm dishes) by the calcium phosphate coprecipitation method. At 48 h after transfection the cells were harvested and lysed. Luciferase activity was measured as described (Wet et al., 1987) using a luminometer (model 1251; LKB Instruments) and normalized with CAT activity in the same cell lysate. CAT activity was assayed as described (Gorman et al., 1982). The acetylated chloramphenicol was separated by thin layer chromatography. The radioactivity was assayed using a radioanalytic imaging system (AMBIS System, Inc., San Diego, CA).

Figure 1. Reporter gene assays of 5' sequential deletion mutants. The following restriction sites were used for the construction of the deletion mutants: Bgl II (–1110), Hpa I (–689), Xho I (–375), Rsa I (–201), Nhe I (–155), Pvu II (–102), Mlu I (–61), BspM I (–49), and PmaC I (+53). The terminal nucleotide of the deletions is indicated. The bottom line shows the location of common sequence element between α1- and βl- or β2-subunit gene (B1 and B2) and the consensus sequence of Sp1, ATF, and TATA-like box.

Results and Discussion

To delineate the sequences essential for transcription of the rat ATP1A1 gene, we fused nine and six portions of the 5' and 3' sequential deletion fragments to the luciferase gene as a heterologous reporter gene (Figs. 1 and 2). Transient transfections of the longest (wild type) construct (pA1LF-1) into MDCK and B103 cells produced 20–40% of the luciferase activity of that of the pSV2A/LΔ5'. Shortening the upstream sequences from positions –1,537 to –375, which contained the common sequence elements of the β2-subunit gene, did not produce major changes in luciferase activity in either cell (Fig. 1). Cells transfected with the next two shorter constructs, pA1U-201LF and pA1U-155LF, gave about twofold lower activities in MDCK cells, similar to those in B103 cells. This result indicates the existence of a positive regulatory region that is responsible for the promoter activity only in MDCK cells (PRRM in Fig. 3). Deletion to position –102 (pA1U-102LF) decreased the luciferase activity twofold more than that of pA1U-155LF, and deletion to position –61 (pA1U-61LF) reduced the luciferase activity fivefold more than that of pA1U-102LF in both cells. Further deletion to position –49 (pA1U-49LF) reduced the promoter activity to the background level in both cells. These results indicate that major elements responsible for transcriptional activation are confined to the region from –155 to –49 in both MDCK and B103 cells (PRR1 and PRR2 in Fig. 3). Shortening the downstream sequence from positions +260 to +92, where it contains a common sequence with the β1-subunit gene, brought about no significant changes in luciferase activity in both cells (Fig. 2). Deletion to position +66 (pA1D+66LF) reduced the luciferase activity twofold more than the wild type in both cells, suggesting the existence of positive regulatory element(s) between +92 and +66 (PRR3 in Fig. 3).

Figure 2. Reporter gene assays of 3' sequential deletion mutants. The 3' deletion mutants were constructed with use of Bal31 nuclease and ligated onto pSVOA/LΔ5' (luciferase gene vector). Each terminal nucleotide of the deletions is indicated. The bottom line shows the location of the common sequence element between α1- and β1-subunit gene (B1) and TATA-like box.

Figure 3. Regulatory regions of rat ATP1A1 expression in MDCK and B103 cells.

From the results shown in Figs. 1 and 2, at least four positive regulatory regions responsible for transcriptional activation were identified (Fig. 3). The PRR1 contains an Sp1 binding site, whereas the PRR2 contains ATF consensus site and Sp1 binding site. A 3.5-fold increase of the luciferase activity was observed when pA1LF-1 was cotransfected with E1A expression plasmid, whose product is known to act through the ATF element. The most important region of the ATP1A1 expression is considered to be the PRR2 because of the highest ability to activate the transcription. The sequence of this region is highly conserved between rat, horse, and human. An interesting feature of the gene is the presence of PRRM, which is active only in MDCK, not in B103 cells. Even though the α1 gene is transcribed in every cell type, the reporter of positive regulatory regions may be different among various cells types.

Conclusion

The presence of three positive regulatory regions of rat ATP1A1 (PRR1: –155 to –61; PRR2: –61 to –49; and PRR3: +66 to +92 in Fig. 3) which were active both in MDCK and B103 cells, and a positive regulatory region between –375 to –155 (PRRM in Fig. 3) which was active only in MDCK cells, were suggested from the results of reporter gene assays of 5' and 3' deletion mutants. The PRR1 contains Sp1 binding site, whereas the PRR2 contains an ATF consensus site and an Sp1 binding site.

Acknowledgments

This work was supported in part by grants from the Ministry of Education, Science and Culture of Japan (01617001, 02780233, and 02258219).

References

Gorman, C. M., L. F. Moffat, and B. H. Howard. 1982. Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Molecular and Cellular Biology. 2:1044–1051.

Kano, I., F. Nagai, K. Satoh, K. Ushiyama, T. Nakao, and K. Kano. 1989. Structure of the α1 subunit of horse Na,K-ATPase gene. FEBS Letters. 250:91–98.

Shull, M. M., D. G. Pugh, and J. B. Lingrel. 1990. The human Na,K-ATPase α1 gene: characterization of the 5'-flanking region and identification of a restriction fragment length polymorphism. Genomics. 6:451–460.

Wet, J. R., K. V. Wood, M. DeLuca, D. R. Helinski, and S. Subramani. 1987. Firefly luciferase gene: structure and expression in mammalian cells. Molecular and Cellular Biology. 7:725–737.

Yagawa, Y., K. Kawakami, and K. Nagano. 1990. Cloning and analysis of the 5'-flanking region of rat Na,K-ATPase α1 subunit gene. Biochimica et Biophysica Acta. 1049:286–292.

Characterization and Sequence Analysis of the Multiple Genes Encoding the Human Na,K-ATPase Catalytic Subunit

E. D. Sverdlov, G. S. Monastyrskaya, N. E. Broude, K. E. Petrukhin, I. V. Malyshev, D. A. Bessarab, S. V. Volik, V. E. Sverdlov, M. Y. Orlova, A. V. Grishin, and N. N. Modyanov

Shemyakin Institute of Bioorganic Chemistry, USSR Academy of Sciences, 117871, Moscow, USSR

Introduction

In man there is a family of related genes encoding the multiple forms of the Na,K-ATPase catalytic subunit. Shull and Lingrel (1987) and Sverdlov et al. (1987) have shown that the family consists of five α-subunit genes and one related gene encoding the H,K-ATPase. Only three of the α-subunit genes that encode the α1, α2, and α3 isoforms were demonstrated to be transcriptionally competent. In this paper we present the results on the cloning and restriction enzyme mapping of all genes making up the multigene family of the Na,K-ATPase catalytic subunit. We have also demonstrated that the fourth gene of this family (ATP1AL1) is functionally active and expressed in human brain and kidney. In addition, we shall consider some distinctive features of the promoter regions of two genes coding for the α2 and α3 isoforms. The nucleotide sequences of these regions have been published elsewhere by us and others (Shull et al., 1989; Sverdlov et al., 1989; Malyshev et al., 1990).

Results and Discussion

Characterization of the Genomic Clones for the Multiple Na,K-ATPase Genes

The object of this study was to clone and map the known α-subunit genes, including their 5' regulatory regions, and to identify possibly existent new members of the Na,K-ATPase gene family. To isolate the DNA clones that contain overlapping inserts which encompass the human Na,K-ATPase genes, phage λ and cosmid libraries were constructed from human brain, placenta, and sperm genomic DNA. The libraries were screened with probes that represented the most conservative regions of the pig kidney α1 cDNA. Mapping and hybridization studies, as well as partial sequencing of the isolated clones that mainly represented the central parts of the known α-subunit genes, allowed us to conclude that the Na,K-ATPase gene family to date consists of five genes for the catalytic subunit. To approach the 5' and 3' termini of the genes we used the method of chromosome walking, but the isolation of the genomic fragments containing the 5' regulatory region of the α3 gene was found to be a somewhat difficult objection because of the long intron (10 kb) containing Alu repeats, which is located between the first and second exons. In an attempt to surmount this problem, the cDNA clone containing the 140 bp of the α3 mRNA 5'-untranslating region was isolated from the human brain cDNA library. This cDNA fragment was used as a hybridization probe to find the λ phage clones that contain the promoter region of the ATP1A3. The physical maps of the α-subunit genes are depicted in Fig. 1 and have minor variations in the size and array of the Eco RI fragments in comparison with published data (Shull and Lingrel, 1987).

Figure 1. Restriction enzyme maps of the overlapping genomic clones representing ATP1A1 (A), ATP1A2 and ATP1AL2 (B), ATP1A3 (C), and ATP1AL1 (D) genes for the catalytic subunit of human Na,K-ATPase.

ATP1AL1 Is a New Transcribed Gene of the Na,K-ATPase Gene Family

The ATP1AL1 gene, which was previously described under the names αD (Shull and Lingrel, 1987) and SW3.2 (Sverdlov et al., 1987), was cloned as a set of five overlapping phage λ clones (Fig. 1 D). Portions of the λ phage inserts that hybridized with pig kidney α1 cDNA were recloned and sequenced. The nucleotide sequences of exons 5, 6, 7, 9, 15, and 20 were determined. The structure of these exons as well as the structure of the exon/intron boundaries gave no indication that the ATP1AL1 represents a pseudogene, but its functional status remained unknown. To determine whether this gene is transcriptionally competent we decided to use the method of polymerase chain reaction (PCR) for the mRNA detection after the conversion of an RNA preparation to the complementary DNA. The specificity of PCR amplification is based on the specificity of two primers that flank the DNA sequence. In the case of ATP1AL1 mRNA detection we chose two oligonucleotides from exons 6 and 7 (coordinates 756–775 and 888–913, respectively; numbering and sequence are as Shull and Lingrel [1987] gave for the αD genomic sequence). The primers were shown to be very specific for the ATP1AL1 and gave no amplification product when phage λ DNAs containing the corresponding regions of the other α-subunit genes were used as templates in PCR reaction (Fig. 2). Searching for the ATP1AL1 transcript in the poly (A+) fraction of human brain, kidney, liver, and renal carcinoma RNA included the cDNA synthesis and 25–30 cycles of PCR followed by electrophoresis and hybridization analysis of the amplified product. The PCR analysis revealed the presence of the ATP1AL1 transcript in human brain and kidney (Fig. 3) and this result clearly demonstrated that the ATP1AL1 encodes a functional isoform of the Na,K-ATPase or a closely related cation-transporting ATPase.

Figure 2. Specificity tests for the oligonucleotides which were used to search the ATP1AL1 gene transcript. 1 ng of the phage λ and cosmid DNAs from the clones containing exons 6 and 7 of five α-subunit genes were subjected to 25–30 cycles of PCR followed by gel electrophoresis in 2% agarose. Lanes 1–5, PCR from the DNA of λSW21 (gene ATP1AL1), λRS9-1 (gene ATP1A1), cosRC16-10 (gene ATP1A2), λRS8.1 (gene ATP1A3), and λSAKS35 (gene ATP1AL2), respectively. Lane 6, PCR from 10 ng of the pGC 35 DNA (full-length pig αl cDNA). Lane 7, size markers (Pst I digest of phage λ DNA).

Figure 3. PCR-based search for the presence of the ATP1AL1 mRNA in the poly(A+) fraction of different human tissues. The poly(A+) fraction of total cellular RNA was converted to the randomly primed complementary DNA and subjected to 25–30 cycles of PCR with ATP1AL1-specific oligonucleotides from exons 6 and 7. The PCR products were electrophoretically analyzed on the 8% polyacrylamide gel (A) followed by Southern blotting and low stringency hybridization to the 32P-labeled α1 pig cDNA (B). Lanes 3–6, PCR from the poly(A+) RNA of human brain, kidney, liver, and renal carcinoma, respectively. Lane 2, PCR from the DNA of genomic clone λSW21 (contains intron 6 that increases the size of the fragment). Lanes 1 and 7, size markers (Hinc II digest of ΦX 174 DNA and Hae III digest of pBR 322 DNA, respectively).

Putative Regulatory Regions of the ATP1A2 and ATP1A3

The nucleotide sequences of the 1.5-kb 5'-flanking region of the ATP1A3, as well as 2.0-kb promoter-containing portion of the α2 gene were determined and have been published elsewhere (Malyshev et al., 1990). Transcription start sites of the α3 gene have been mapped in positions –152 and –155 relative to the translation initiation codon by primer extension and S1 nuclease protection analysis of the poly(A+) RNA from human brain. The absence of a distinctly pronounced TATA box and high G + C content are the main features of the ATP1A3 promoter region, and this is in common with characteristics of the 5'-flanking sequences of many housekeeping genes. Another notable property of the ATP1A3 promoter is the presence of the CAAT box in the noncoding strand.

The structure of the 5' regulatory region of the ATP1A2 which encodes the α2-isoform of the catalytic subunit was discussed in detail by Shull et al. (1989) and Sverdlov et al. (1989). The only interesting feature that should be noted here is thepresence of additional promoter elements (TATA and CAAT boxes) found on the opposite DNA strand in the reverse orientation at a distance of ~ 0.5 kb from the α2 TATA box. These putative promoter elements flank the long open reading frame (ORF), which can represent the beginning of a new gene orientated head-to-head relative to the ATP1A2. The structure of the ORF and putative polypeptide which is 120 amino acids in length is published in Malyshev et al. (1990) and is seen to have no obvious homology with protein and DNA sequences from PIR and GENBANK computer databases.

References

Malyshev, I. V., D. A. Bessarab, M. Y. Orlova, K. E. Petrukhin, S. V. Volik, N. E. Broude, G. S. Monastyrskaya, N. N. Modyanov, and E. D. Sverdlov. 1990. Analysis of the putative regulatory regions of human genes coding for α-subunit of Na,K-ATPase. Biomedical Science. In press.

Shull, M. M., and J. B. Lingrel. 1987. Multiple genes encode the human Na+,K+-ATPase catalytic subunit. Proceedings of the National Academy of Sciences, USA. 84:4039–4043.

Shull, M. M., D. G. Pugh, and J. B. Lingrel. 1989. Characterization of the human Na+,K+-ATPase α2 gene and identification of intragenic restriction fragment length polymorphism. The Journal of Biological Chemistry. 264:17532–17543.

Sverdlov, E. D., D. A. Bessarab, I. V. Malyshev, K. E. Petrukhin, Y. V. Smirnov, Y. A. Ushkarev, G. S. Monastyrskaya, N. E. Broude, and N. N. Modyanov. 1989. Family of human Na,K-ATPase gene: structure of the putative regulatory region of the α+ -gene. FEBS Letters. 244:481–483.

Sverdlov, E. D., G. S. Monastyrskaya, N. E. Broude, Y. A. Ushkarev, R. L. Allikmets, A. M. Melkov, Y. V. Smirnov, I. V. Malyshev, I. E. Dulubova, K. E. Petrukhin et al. 1987. The family of human Na+,K+-ATPase genes: no less than five genes and/or pseudogenes related to the α-subunit. FEBS Letters. 217:275–278.

Evolutionary Conservation of the (Na+ + K+)-ATPase Genes

Kunio Takeyasu, Atsushi Mizushima, Andrew M. Barnstein, Maura Hamrick, and Douglas M. Fambrough

Department of Physiology, the University of Virginia, Charlottesville, Virginia 22908; and Department of Biology, the Johns Hopkins University, Baltimore, Maryland 21218

Introduction

The primary structures of the sodium pump subunits deduced from the cDNA sequences of a variety of species have indicated a great deal of sequence conservation through evolution (reviewed by Lingrel et al., 1990). We previously demonstrated that three α-subunit isoforms (α1, α2, and α3) of the chicken (Na+ + K+)-ATPase correspond one-to-one to three isoforms of the rat (Takeyasu et al., 1990) on the basis of (a) cDNA sequences and the deduced amino acid sequences, and (b) distinct tissue distribution of isoform-specific mRNA. This suggests that the gene multiplication and subsequent isoform evolution occurred before the separation of avian and mammalian lineages. In this report we describe evolutionary conservation of exon–intron structures between these isoform genes as well as between β-subunit genes.

Avian α1-Subunit Gene Structure and Evolutionary Conservation

We have constructed chicken genomic DNA libraries in λ-phage, EMBL_4. The screening of approximately five million recombinant phage with a 32P-labeled chicken α1-cDNA (Takeyasu et al., 1988) yielded three overlapping genomic DNA fragments containing the protein coding regions as well as ~10 kb 5'-flanking and ~10 kb 3'-flanking sequences. DNA sequence analysis revealed that (a) the chicken α1 gene consists of 23 exons, (b) the position of exon–intron junctions in the encoded protein are identical to those of the horse α1 gene, (c) these positions are also conserved in the human α2 and α3 genes, and (d) the chicken α1 gene structure is virtually identical to that of human (H+ + K+)-ATPase gene (except that exons 6 and 7 are combined in the (H+ + K+)-ATPase gene). Fig. 1 shows the combined overall structure of the avian α1-subunit gene as well as the structures of mammalian α-isoform and (H+ + K+)-ATPase genes. It should be noted that the sizes of introns in species- and isoform-specific genes are different, while the sizes of exons are identical.

Fig. 2 shows degrees of homology between corresponding exons of α-subunit isoform genes as well as between the chicken sodium pump and the human (H+ + K+)-ATPase genes. Comparison of percent conservation between exons of different isoform genes revealed that the first three exons (1–3) are highly variable, suggesting that these domains may have isoform-specific structure and function relationships. Comparison between different ion pumps shows that exons 9 and 16, which encode the phosphorylation site, and the 5'-(p-fluorosulfonyl)-benzoyladenosine (FSBA) binding domain, respectively, are highly conserved, suggesting that these domains are important for general ion pump structure/function. The last five exons (19–23) are the least conserved between different ion pumps, although they remain highly homologous between sodium pump isoforms. This suggests that these regions might be involved in ion-specific functions. One such domain responsible for Ca2+-stimulated ATPase activity was identified by exchanging the domain encoded by the exons (19–23) between the sodium pump α1-subunit and the sarcoplasmic reticulum calcium pump (Luckie et al., 1991).

Figure 1. Exon-intron structures of ATPase isoform genes. The structures of the chicken (Na+ + K+)-ATPase α1-subunit gene (this work), horse α1 gene (Kano et al., 1989), human α2-subunit gene (Shull et al., 1989), and human (H+ + K+)-ATPase α-subunit gene (Maeda et al., 1990) were compared. It has been pointed out that human (H+ + K+)-ATPase α-subunit gene has the same exon–intron structure as mammalian (Na+ + K+)-ATPase α3 gene (Ovchinnikov et al., 1987), except that intron 6 in the (Na+ + K+)-ATPase gene is missing in the (H+ + K+)-ATPase gene.

Figure 2. Comparison of degrees of conservation between corresponding exons of different ion pump and isoform genes. For calculation, a common structure of the sodium pump gene consisting of 23 exons was applied to the rat α-subunit isoform sequences (Shull et al., 1986), and exon 6 of the (H+ + K+)-ATPase gene was divided into two on the basis of the corresponding exon–intron sequence of the (Na+ + K+)-ATPase gene. C1, chicken (Na+ + K+)-ATPase α1-subunit gene; R1, R2, and R3, rat α1, α2, and α3, respectively; HK, human (H+ + K+)-ATPase α-subunit gene.

In a previous report (Takeyasu et al., 1990) we demonstrated that three α-subunit isoforms (α1, α2, and α3) of the chicken (Na+ + K+)-ATPase correspond one-to-one to three isoforms of the rat. This suggests that the gene multiplication and subsequent isoform evolution occurred before the separation of avian and mammalian lineages. In this report we isolated and analyzed the chicken α1-subunit gene. A comparison of the exon–intron structures shows 100% conservation between avian and mammalian isoform genes. These data are consistent with the idea that the exon–intron structures of the (Na+ + K+)-ATPase α-subunit genes were established before the isoform gene multiplication.

Figure 3. Exon–intron structures of the (Na+ + K+)-ATPase β1-subunit genes and comparison of degrees of conservation between corresponding exons. The structures of the chicken β1 gene (this work) and the human β1 gene (Lane et al., 1989) were compared.

Avian β1-Subunit Gene Structure and Evolutionary Conservation

We have isolated five overlapping genomic DNA fragments encoding the chicken β1-subunit using 32P-labeled chicken β1-subunit cDNA (Takeyasu et al., 1987). One of these clones (cGβ1) contains the longest 5'-upstream region and was used for further analysis. The gene is ~ 15 kb long, and consists of six exons as reported in the human β1 gene (Lane et al., 1989). DNA sequence analysis revealed that the positions of exon–intron junction in the encoded proteins are identical between chicken and human β1 genes. As is the case for the structure of α-subunit isoform genes, while the sizes of the exons are identical, the sizes of the corresponding introns of both β1 genes are different, suggesting that the mutation rates of introns are much higher than those of exons.

It is generally believed that highly conserved regions of proteins carry structural and/or functional importance. A comparison of conservation between exons (Fig. 3) indicates that exons 1 and 2, which encode a cytoplasmic NH_2-terminal region and a unique membrane spanning domain, respectively, are most conserved. Surprisingly, the recent experiments involving expression of β-subunit deletion mutants have demonstrated that neither the cytoplasmic region nor the membrane spanning domain are necessary for assembly with the α-subunit. Instead, the ectodomains of both subunits are involved in subunit assembly (Renaud and Fambrough, 1991). In this sense, it is interesting to notice that the ectodomain of the β1-subunit is not highly conserved through evolution (Fig. 3). It might be possible that several different ectodomains of the β-subunit form a distinct tertiary structure that may play a critical role in assembly with the α-subunit. What roles the well-conserved transmembrane and endodomain of the β-subunit play in sodium pump function remains unknown.

Toward Better Understanding the Regulatory Mechanism of Sodium Pump Gene Expression

It has been well documented that sodium pump gene expression can be regulated by a variety of signals (for review, see Rossier et al., 1987). A line of evidence supporting the idea that the expression of the β-subunit is the limiting step in sodium pump biosynthesis has been provided in developing Xenopus oocytes (Geering, 1990) and cultured skeletal muscle cells (Wolitzky and Fambrough, 1986; Taormino and Fambrough, 1990). To better understand the regulatory mechanisms of sodium pump gene expression and biosynthesis, we have introduced the chicken β1 gene into mouse cells, and the expression of the chicken β1-subunit was monitored using a chicken specific monoclonal antibody (mAb), IgG 24 (Fambrough and Bayne, 1983). Fig. 4 shows fluorescence micrographs of cloned mouse L cells and C_2H_2 cells transfected with cGβ1 DNA, illustrating distribution of the chicken β1-subunit expressed on mouse cell surface. Metabolic labeling of cells with [35S]methionine, immunoprecipitation with mAb 24–coupled Sepharose 4B beads, and SDS-PAGE/fluorography analysis demonstrated that the expressed chicken β1-subunit is assembled with the mouse α-subunit, as shown previously by using a cDNA expression system (Takeyasu et al., 1987). Treatment of the transfected L cells and C_2H_2 cell with low K+ medium and 10 μM veratridine, respectively, resulted in upregulation of the chicken β-subunit expression, suggesting that this cloned β1-subunit gene contains responsive element(s) for these treatments. By using the heterologous gene expression system presented in this study, we hope to identify such regulatory sequences involved in response to demand for ion transport.

Figure 4. Transfected mouse cells expressing the chicken β1-subunit gene. (Top) Mouse Ltk–cells were transfected with 0.1 μg of pHSVtk5' 0.2 DNA and ~10 μg of isolated cGβ1 DNA/100-mm-diam tissue culture plate. The transfected cells were grown for 48 h in Dulbecco's modified Eagle's minimal essential medium (DMEM) containing 10% fetal calf serum, and then transferred to HAT medium for cloning. (Bottom) Mouse C_2H_2 cells were grown in the same way except that the medium contained 20 fetal calf serum, 0.1 μg of pSVNeo was used for transfection instead of pHSVtk5' 0.2 plasmid, and selection was done in medium containing G418 (400 μg/ml). Immunofluorescence microscopy was used to detect expression of the avian (Na+ + K+)-ATPase β-subunit in live mouse cells. Transfected mouse cells grown on glass coverslips were incubated for 45 min in HEPES-buffered MEM containing monoclonal antibody 24 (5 μg/ml), and incubated for 45 min in HEPES-buffered MEM containing tetramethylrhodamine (TRITC)–labeled goat anti–mouse IgG (5 μg/ml) After extensive washing with PBS, labeled cells were examined with an inverted fluorescence microscope (Carl Zeiss, Inc., Thornwood, NY).

Acknowledgments

This work was supported by NIH grants R01-NS23241 (to D. M. Fambrough) and RO1-GM-44373 (to K. Takeyasu) and a Grant-in-Aid from the American Heart Association National Center (#901107 to K. Takeyasu).

References

Fambrough, D. M., and E. K. Bayne. 1983. Multiple forms of (Na+ + K+)-ATPase in the chicken: selective detection of the major nerve, skeletal muscle, and kidney form by a monoclonal antibody. Journal of Biological Chemistry. 258:3926–3935.

Geering, K. 1990. Subunit assembly and functional maturation of Na,K-ATPase. Journal of Membrane Biology. 115:109–121.

Kano, I., F. Nagai, K. Satoh, K. Ushiyama, T. Nakao, and K. Kano. 1989. Structure of the α1 subunit of horse Na,K-ATPase gene. FEBS Letters. 250:91–98.

Lane, L. K., M. M. Shull, K. R. Whitmer, and J. B. Lingrel. 1989. Characterization of two genes for the human Na,K-ATPase β subunit. Genomics. 5:445–453.

Lingrel, J. B., J. Orlowski, M. M. Shull, and E. M. Price. 1990. Molecular genetics of Na,K-ATPase. Progress in Nucleic Acid Research and Molecular Biology. 38:37–89.

Luckie, D. B., K. L. Boyd, A. Mizuchima, Z. Shao, A. P. Somlyo, and K. Takeyasu. 1991. Expression of functional sodium and calcium pump chimeric molecules. In The Sodium Pump: Recent Developments. J. H. Kaplan and P. De Weer, editors. The Rockefeller University Press, New York. In press.

Maeda, M., K. Oshiman, S. Tamura, and M. Futai. 1990. Human gastric (H+ + K+)-ATPase gene: similarity to (Na+ + K+)-ATPase genes in exon/intron organization but difference in control region. Journal of Biological Chemistry. 265:9027–9032.

Ovchinnikov, Yu. A., G. S. Monastyrskaya, N. E. Broude, R. L. Allikmets, Yu. A. Ushkaryov, A. M. Melkov, Yu. V. Smirnov, I. V. Malyshev, I. E. Dulubova, K. E. Petrukhin et al. 1987. The family of human Na+,K+-ATPase genes: a partial nucleotide sequence related to the α-subunit. FEBS Letters. 213:73–80.

Renaud, K. J., and D. M. Fambrough. 1991. Molecular analysis of Na,K-ATPase subunit assembly. In The Sodium Pump: Recent Developments. J. H. Kaplan and P. De Weer, editors. The Rockefeller University Press, New York. 25–29.

Rossier, B. C., K. Geering, and J.-P. Kraehenbuhl. 1987. Trends in Biochemical Sciences. 12:483–487.

Shull, G. E., J. Greeb, and J. B. Lingrel. 1986. Molecular cloning of three distinct forms of the Na+,K+-ATPase α-subunit from rat brain. Biochemistry. 25:8125–8132.

Shull, M., D. G. Pugh, and J. B. Lingrel. 1989. Characterization of the human Na,K-ATPase α2 gene and identification of intragenic restriction fragment length polymorphisms. Journal of Biological Chemistry. 264:17532–17543.

Takeyasu, K., V. Lemas, and D. M. Fambrough. 1990. Stability of the (Na+ + K+)-ATPase α-subunit isoforms in evolution. American Journal of Physiology (Cell Physiology). 28:C619–C630.

Takeyasu, K., M. M. Tamkun, K. J. Renaud, and D. M. Fambrough. 1988. Ouabain-sensitive (Na+ + K+)-ATPase activity expressed in mouse L cells by transfection with DNA encoding the α-subunit of an avian sodium pump. Journal of Biological Chemistry. 263:4347–4354.

Takeyasu, K., M. M. Tamkun, N. R. Siegel, and D. M. Fambrough. 1987. Expression of hybrid (Na+ + K+)-ATPase molecules after transfection of mouse Ltk- cells with DNA encoding the β-subunit of an avian brain sodium pump. Journal of Biological Chemistry. 262:10733–10740.

Taormino, J. P., and D. M. Fambrough. 1990. Pre-translational regulation of the (Na+ + K+)-ATPase in response to demand for ion transport in cultured chicken skeletal muscle. Journal of Biological Chemistry. 265:4116–4123.

Wolitzky, B. A., and D. M. Fambrough. 1986. Regulation of the (Na+ + K+)-ATPase in cultured chick skeletal muscle: modulation of expression by demand for ion transport. Journal of Biological Chemistry. 261:9990–9999.

Na,K-ATPase Gene Expression during Cytodifferentiation of a Toad Bladder Cell Line

F. Jaisser, C. Canessa, H. P. Gaeggeler, L. Racine, J.-P. Kraehenbuhl, and B. C. Rossier

Institut de Pharmacologie et de Toxicologie de l'Université de Lausanne, CH-1005 Lausanne, Switzerland; Institut de Biochimie de l'Université de Lausanne, Chemin des Boveresses, CH-1066 Epalinges, Switzerland; and Institut de Recherche Expérimentale Sur le Cancer (ISREC), Chemin des Boveresses, CH-1066 Epalinges, Switzerland

Introduction

The TBM cell line was established in 1979 by J. Handler from the urinary bladder of the toad Bufo marinus (Handler et al., 1979). Like the tissue of origin, the derived cell line expresses high resistance to ouabain and responsiveness to aldosterone. The clonal cell line (TBM 18–23) was derived in our laboratory from the original TBM cells. Like the parental cells, the TBM 18–23 clone undergoes a high degree of morphological and functional differentiation in culture. The epithelial cytodifferentiation can be conveniently monitored in culture by functional, morphological, and molecular techniques. For instance, transepithelial Na transport, a well-defined epithelial function, can be followed during the process of cytodifferentiation. In this study we report the molecular cloning of Na,K-ATPase α- and β-subunits in the TBM clonal cell line and their expression as markers of sodium transport.

Results and Discussion

Functional and Morphological Differentiation of TBM 18–23 Cells

When TBM 18–23 cells are seeded at high density (10^6 cells/cm^2) on polycarbonate filters, transepithelial Na transport is established within 3 or 4 d. The epithelium displays a high transepithelial resistance (> 4,000Ω. cm^2) and vectorial Na reabsorption can be monitored by the short circuit current method (SCC ~ 10–20 μA/cm^2). Upon addition of aldosterone, Na transport increases three- to five-fold above control values, while transepithelial electrical resistance decreases, as observed in the urinary bladder.

The time course of cytodifferentiation can be followed when the cells are seeded on filter cups at low density (10^4 cells/cm^2). Within 24 h after seeding, cells form a monolayer with tight junctions between some of the cells, indicating that morphological polarity is already established. However, no transepithelial electrical resistance can be recorded. During the next 4–6 d of culture the cells proliferate and form a multilayer. Only the cells that face the apical medium keep their epithelial morphology, but again no functional polarity is observed. Between day 6 and day 10 picnosis is observed within the intermediate layers of cells and a bilayer is progressively formed with a concomitant appearance of Na transport and high transepithelial resistance. When fully differentiated (day 12 of culture), the upper layer consists of typical granular cells with conspicuous apical microvilli, tight junction apparatus, and basolateral membrane infolding. The lower layer is formed by cells with morphological characteristics of microfilament-rich cells as observed in the urinary bladder (Kraehenbuhl et al., 1979).

TBM 18–23 Cells Express One α-Subunit and Two β-Subunit Isoforms of Na,K-ATPase

Using a 2.4-kb α1 cDNA probe from Xenopus laevis, an Okayama-Berg cDNA library constructed with size-fractionated poly A+ RNA from TBM cells was screened and several positive clones were isolated. A 3.4-kb cDNA clone was sequenced, which showed 85% (nucleic acid) and 95% (protein) identities with the α1-subunit of Xenopus laevis (Verrey et al., 1989). The sequence of the H1-H2 ectodomain was of special interest since it has been clearly involved in the ouabain-resistant phenotype of the α1-subunit (Lingrel et al., 1990). Table I shows the amino acid composition of the 1 H1-H2 region of ouabain-resistant and ouabain-sensitive species. As observed in the rat sequence, a positively charged amino acid (arginine) was observed at the NH_2 terminal border of our sequence. But contrary to the rat sequence, the al TBM sequence is not characterized by a negatively charged amino acid (aspartate at the COOH-terminal border). By contrast, we do observe a second positively charged amino acid at the NH_2-terminal border, as recently observed in the brine shrimp, another species which is characterized by its ouabain resistance. It will be interesting to test directly whether the observed charged amino acid distribution in Bufo marinus is necessary and sufficient to confer the ouabain resistance that is observed in this cell line (Ki ~ 30 μM).

TABLE I
Amino Acid Composition of the α1 H1-H2 Region of Different Species

Alignment of amino acid sequence of α1 H1-H2 ectodomain from ouabain-resistant vs. ouabain-sensitive species. Relevant and specific references reviewed in Horisberger et al. (1991) and Lingrel et al. (1990).

The screening of the same cDNA library with a Xenopus heterologous β1 cDNA probe (Verrey et al., 1989) was unsuccessful and an alternative strategy using polymerase chain reaction (PCR) methodology was used. Degenerate oligonucleotides coding for conserved domains of the β1-subunit (amino acids 32–39 and 242–248) were designed and used to amplify a 640-bp cDNA fragment from a single-strand cDNA made from TBM cell poly A+ RNA. the 640-bp cDNA was sequenced and found to be 75% identical to the Xenopus β1-subunit. The TBM homologous β1 probe was then used to screen a double-strand cDNA library constructed from the single-strand cDNA used for the PCR reaction. A 2.3-kb β1 cDNA was cloned and showed 75 and 70% identity with the Xenopus β1-subunit in the coding region (906 bp) and the 3' untranslated region (1,109 bp), respectively. Using the same methodology, we screened our cDNA library for the presence of α2, α3, β2, and β3 isoforms. The search for both α isoforms was negative but we were able to amplify a 490-bp cDNA fragment coding for a β isoform, sharing 78% identity with a recently described Xenopus β3 isoform (Good et al., 1990). The physiological role of two β isoforms with one α isoform in a clonal cell line is not known. This finding opens the possibility of a differential regulation and/or function of α1β1 and/or α1β3 heterodimeric proteins in these cells. Alternatively, each β isoform might segregate in different cell types within the bilayer described above. Interestingly, preliminary data show that the expressions of β1 and β3 isoforms are differentially regulated by aldosterone. As shown in Fig. 1, aldosterone increases β1 mRNA abundance by a factor of 3 to 5 after a 24-h stimulation (300 nM), while the abundance of β3 isoform is apparently not increased and might even be repressed.

Figure 1. Northern blot analysis of 1 μg poly A+ RNA from TBM 18–23 cells grown on Petri dishes according to the method described previously (Verrey et al., 1987). Cells were treated on day 8 with 300 nM aldosterone for 24 h. The blot was successively hybridized with TBM β1 and TBM β3 probes.

Figure 2. Northern blot analysis of 6 μg total RNA from TBM 18–23 cells grown on nucleopore filters and collected at different times of cytodifferentiation. The blot was hybridized successively with TBM α1 (1,700 bp) and TBM β1 (640 bp) probes.

Expression of α1, β1, and β3 Isoforms During Cytodifferentiation

The expression of the three subunits during cytodifferentiation was studied in parallel with the establishment of Na transport. TBM 18–23 cells were seeded at low density on polycarbonate filters and Na transport was measured by the SCC method. As shown in Fig. 2, the abundance of α1 and β1 mRNA did not change during the first 8 d of culture despite large changes in Na transport and electrical transepithelial resistance (Table II). The abundance of β3 mRNA was low and did not change to a great extent up to 12 d of culture. Our data suggest that no major change in Na,K-ATPase mRNA abundance occurs during the establishment of functional polarity. We are presently testing the possibility that the electrophysiological changes are accompanied by an increase in the expression of Na,K-ATPase at the protein level.

TABLE II
Development of Sodium Transport during Cytodifferentiation

Na transport measured by the short circuit current method (Verrey et al., 1987) in TBM cells 18–23 during 10 d of culture. n, Number of dishes; PD, potential difference; SCC, short circuit current; R, transepithelial resistance.

The experimental model described here gives us a unique opportunity to study terminal epithelial differentiation in culture, following the expression of Na transport markers such as Na,K-ATPase or Na channels. The possible existence of different Na,K-ATPase pools and their cell-specific expression and cell-specific regulation during cytodifferentiation are open to direct experimental examination.

Acknowledgments

We thank Irène Corthésy-Theulaz, Käthi Geering, and Anne Seiler for their suggestions, and Chantal Verdumo for technical assistance.

This work was supported by grant 31–26497.89 to B. C. Rossier and J.-P. Kraehenbuhl.

References

Good, P. J., K. Richter, and I. B. Dawid. 1990. A nervous system-specific isotype of the β subunit of Na+-K+-ATPase expressed during early development of Xenopus laevis. Proceedings of the National Academy of Sciences, USA. 87:9088–9092.

Handler, J. S., R. E. Steele, M. K. Sahib, J. B. Wade, A. S. Preston, N. L. Lawson, and J. P. Johnson. 1979. Toad urinary bladder epithelial cells in culture: maintenance of epithelial structure, sodium transport, and response to hormones. Proceedings of the National Academy of Sciences, USA. 76:4151–4155.

Horisberger, J.-D., V. Lemas, J.-P. Kraehenbuhl, and B. C. Rossier. 1991. Structure-function relationship of Na,K-ATPase. Annual Review of Physiology. 53:565–584.

Kraehenbuhl, J. P., J. Pfeiffer, M. Rossier, and B. C. Rossier. 1979. Microfilament-rich cells in the toad bladder epithelium. Journal of Membrane Biology. 48:167–180.

Lingrel, J. B., J. Orlowski, M. M. Shull, and E. M. Price. 1990. Molecular genetics of Na,K-ATPase. Progress in Nucleic Acid Research and Molecular Biology. 38:37–89.

Verrey, F., P. Kairouz, E. Schaerer, P. Fuentes, K. Geering, B. C. Rossier, and J.-P. Kraehenbuhl. 1989. Primary sequence of Xenopus laevis Na+-K+-ATPase and its localization in A6 kidney cells. American Journal of Physiology. 256:F1034–F1043.

Verrey, F., E. Schaerer, P. Zoerkler, M. P. Paccolat, K. Geering, J.-P. Kraehenbuhl, and B. C. Rossier. 1987. Regulation of Na+-K+-ATPase mRNAs, protein synthesis, and sodium transport in cultured kidney cells. Journal of Cell Biology. 104:1231–1237.

Molecular Analysis of Na,K-ATPase Subunit Assembly

Karen Jane Renaud and Douglas M. Fambrough

Department of Biology, The Johns Hopkins University, Baltimore, Maryland 21218

Introduction

We have demonstrated that when a complementary DNA (cDNA) encoding the chicken β-subunit is transfected into mouse L cells, the avian protein will assemble with the endogenous murine α-subunit (Takeyasu et al., 1987). We chose this system to examine the nature of the interaction between the α- and β-subunits. cDNAs encoding β-subunit molecules with truncated amino or carboxyl termini, or with deletions in the membrane spanning domain, were engineered. These were transfected into L cells, and the subcellular localization of the mutant proteins and their ability to assemble with mouse α-subunit were assessed with monoclonal antibodies against avian β-subunit by immunofluorescence microscopy and immunopurification.

Methods

Construction of cDNAs Encoding Deleted β-Subunits

Portions of avian β_1, cDNA (Takeyasu et al., 1987) were removed by oligonucleotide-directed mutagenesis to produce cDNAs encoding β-subunit molecules with truncated amino or carboxyl termini. Human/chicken β-subunit chimeras were constructed as follows: Oligonucleotide-directed mutagenesis was used to introduce a unique Nde I restriction site into the coding region of our avian β-subunit cDNA at a position corresponding to that of the existing site in a human Na,K-ATPase β-subunit cDNA (Kawakami et al., 1986). A large portion of the avian β-cDNA encoding the extracellular domain of the β-subunit was joined to the cytosolic and transmembrane domain-encoding portions of the human β-cDNAs via this restriction site. The human β-subunit cDNA clone and derivatives of the clone were generously provided by Dr. Kiyoshi Kawakami (Jichi Medical School, Minamikawa-chi, Tochigi, Japan).

Transfection of L Cells

cDNAs encoding full-length or mutant avian β-subunits were cloned into the mammalian expression vector pSVDF (Takeyasu et al., 1987). These were transfected into mouse L cells using DEAE dextran followed by DMSO shock by the method of Seiden (Ausubel et al., 1988). Before immunofluorescence localization or metabolic labeling for immunoprecipitations, transfected cells were grown for 48 h in the presence of 10 mM butyrate to increase expression of the transfected gene products (Gorman et al., 1983).

Immunofluorescence Localization

Cells were fixed and permeablized in 100% methanol to expose both internal and surface epitopes or were fixed with 1% formaldehyde to permit binding of antibody only to the cell surface. Fixed cells were incubated with a monoclonal antibody specific for avian β-subunit, followed by rhodamine-conjugated goat anti–mouse IgG as described previously (Lippincott-Schwartz and Fambrough, 1986).

Immunoprecipitations

Cells were maintained in methionine- and serum-free DMEM supplemented with 50 μCi of [35S]methionine and [35S]cysteine per ml for 3 h. The cells were then lysed in buffer containing 1% Triton X-100. Extracts from single 60- or 100-mm culture dishes were incubated with Sepharose beads conjugated with anti–chicken β-subunit monoclonal antibody as described (Tamkun and Fambrough, 1986). Antigen was eluted in SDS-PAGE loading buffer, electrophoresed in a 5–15% gradient polyacrylamide gel, and fluorographed.

Results

Deletions were made in each of three “domains” of the β-subunit to study the role(s) of these domains in α-β subunit assembly. Fig. 1 depicts in cartoon form some examples of the mutant β-subunit proteins encoded by β-subunit cDNAs containing deletions in the cytoplasmic tail, transmembrane, and extracellular domains. Since the epitopes recognized by our monoclonal antibodies are located in the extracellular domain of β-subunit, the entire cytoplasmic tail domain could be removed without interfering with antibody binding. In addition, nearly one-third of the carboxyl-terminal domain could be deleted and still allow antibody binding. To investigate the role of the membrane spanning domain of β-subunit in assembly, we used a series of human β-subunit cDNAs containing deletions in this domain (Kawakami and Nagano, 1988) to construct human/avian chimeric β-subunit molecules that could be detected with our avian monoclonal antibodies. cDNAs encoding these proteins were transfected into mouse L cells.

Fig. 2 shows the results of immunoprecipitations from extract preps of transfected cells for representative mutant β-subunit proteins. Mouse α-subunit and an amino-terminally truncated avian β-subunit coprecipitate with immunobeads specific for avian β-subunit (Fig. 2, lane 2). This example and the results from other amino-terminal deletions demonstrate that removal of the amino-terminal (cytoplasmic) tail domain of the β-subunit does not prevent assembly with the α-subunit. Human/chicken chimeric β-subunits, containing deletions in the membrane spanning domain ranging in size from 3 to 14 amino acids, are also capable of assembly with the α-subunit. Immunoprecipitations from cells expressing two of these chimeras are shown in Fig. 2, lanes 3 and 4. Endogenous mouse α-subunit coprecipitates with each of these internally deleted β-subunits. Due to the high amino acid sequence similarity between species in the amino-terminal and transmembrane domains of the β-subunit, it has been postulated that these domains may be involved in α-β subunit assembly. The findings here would argue otherwise. Interestingly, truncations of the extracellular domain of the β-subunit prevent assembly with the α-subunit. The example shown in Fig. 2, lane 5 is a β-subunit with only 11 amino acids removed from the carboxyl terminus. No mouse α-subunit is seen to coprecipitate with this mutant β-subunit. Larger truncations give the same result. It appears, then, that α-β subunit assembly involves the extracellular domains of the two subunits.

Figure 1. Schematic representations of proteins encoded by full-length avian β-subunit cDNA (A), and β-subunit cDNAs containing deletions in the amino-terminal (B) and the carboxylterminal (C) portions of the coding region. (D) Human/chicken chimeric β-subunits were constructed in which the amino-terminal one-third of the molecules are encoded by human cDNAs containing deletions in the transmembrane region with the remaining carboxylterminal two-thirds encoded by avian β-cDNA. The membrane spanning domain is symbolized by a thickened line; disulfide bridges are shown; and the positions of the three N-glycosylation sites are marked by vertical bars. The avian β-subunit monoclonal antibodies used in this study bind to epitopes located in the carboxyl-terminal two-thirds of the molecule.

It has been proposed that one role the β-subunit may have is in the targeting and/or transport of the Na,K-ATPase to the plasma membrane (McDonough et al., 1990). The intracellular location of the truncated and deleted avian β-subunits was determined by immunofluorescence microscopy. Fluorescence micrographs of fixed, nonpermeablized cells show an amino-terminal deleted β-subunit at the cell surface (Fig. 3 A). It appears, then, that the cytoplasmic tail domain of the β-subunit is not required for correct transport to the plasma membrane. Because we have also shown that the amino-terminal truncated β-subunit assembles with the α-subunit, it is presumed that assembled avian mutant β-subunit/mouse α-subunit complexes are being expressed at the cell surface. A deletion of three amino acids from the transmembrane domain still allows expression at the cell surface (Fig. 3 B), but deletion of five residues from this domain results in the intracellular accumulation of this mutant (Fig. 3 C) even though each of these mutants is capable of assembly with the α-subunit. Deletions of the extracellular domain of the β-subunit also prevent expression at the cell surface (Fig. 3 D). This result is consistent with findings that misfolded proteins or unassembled subunits are often retained in the ER (Rose and Doms, 1988).

Figure 2. Immunoprecipitations from L cells expressing avian β-subunit or mutated β-subunits. Lane 1, Full-length avian β-subunit. Lane 2, β-subunit with deletion in the aminoterminal (cytoplasmic) domain. Human/chicken chimeric β-subunits with transmembrane deletions of three amino acids (lane 3) and five amino acids (lane 4). Lane 5, β-subunit with deletion in the carboxylterminal (extracellular) domain. α, Mouse α-subunit; β, mature avian β-subunit; β', high-mannose form of β-subunit.

Figure 3. Fluorescence micrographs of cells expressing amino-terminally deleted β-subunit (A), human/chicken chimera with three amino acid transmembrane region deletion (B), human/chicken chimera with five amino acid transmembrane deletion (C), and carboxyl-terminally deleted β-subunit (D). Cells in A and B were fixed with 1% formaldehyde to show surface staining only. Cells in C and D were fixed and permeablized in 100% methanol. All cells were labeled with avian β-subunit-specific monoclonal antibodies followed by rhodamine-conjugated goat-anti–mouse IgG.

References

Ausubel, F. M., R. Brent, R. E. Kingston, D. D. Moore, J.G. Seidman, J. A. Smith, and K. Struhl. 1988. Current Protocols in Molecular Biology. Vol. 1. John Wiley & Sons, Inc., New York. 9.2.1–9.2.6.

Gorman, C. M., B. H. Howard, and R. Reeves. 1983. Expression of recombinant plasmids in mammalian cells is enhanced by sodium butyrate. Nucleic Acids Research. 11:7631–7648.

Kawakami, K., N. Hiroshi, T. Ohta, and K. Nagano. 1986. Molecular cloning and sequence analysis of human Na,K-ATPase β-subunit. Nucleic Acids Research. 14:2833–2844.

Kawakami, K., and K. Nagano. 1988. The transmembrane segment of the human Na,K-ATPase β-subunit acts as the membrane incorporation signal. Journal of Biochemistry. 103:54–60.

Lippincott-Schwartz, J., and D. M. Fambrough. 1986. Lysosomal membrane dynamics. Structure and interorganellar movement of a major lysosomal membrane glycoprotein. Journal of Cell Biology. 102:1593–1605.

McDonough, A. A., K. Geering, and R. A. Farley. 1990. The sodium pump needs its β subunit. The FASEB Journal. 4:1598–1605.

Rose, J. K., and R. W. Doms. 1988. Regulation of protein export from the endoplasmic reticulum. Annual Review of Cell Biology. 4:257–288.

Takeyasu, K. T., M. M. Tamkun, N. R. Siegel, and D. M. Fambrough. 1987. Expression of hybrid (Na++K+)-ATPase molecules after transfection of mouse Ltk- cells with DNA encoding the β-subunit of an avian brain sodium pump. Journal of Biological Chemistry. 262:10733–10740.

Tamkun, M. M., and D. M. Fambrough. 1986. The (Na+ + K+)-ATPase of chick sensory neurons: studies on biosynthesis and intracellular transport. Journal of Biological Chemistry. 261:1009–1019.

Molecular Cloning of Na,K-ATPase α-Subunit cDNA from Hydra vulgaris

Victor A. Canfield, Kai-Yuan Xu, Thomas D'Aquila, Andrew W. Shyjan, and Robert Levenson

Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06510

Introduction

One approach to understanding the relationship between Na,K-ATPase structure and function is to compare the Na,K-ATPases of evolutionarily diverse organisms. To date, the only Na,K-ATPase genes from invertebrates that have been examined are those from the arthropods Drosophila (Lebovitz et al., 1989) and Artemia (Baxter-Lowe et al., 1989). We have chosen to use the simple freshwater coelenterate Hydra vulgaris to study the evolution of Na,K-ATPase isoforms. Hydra appears to be especially useful for analyzing Na,K-ATPase structure and function because coelenterates are highly divergent from all other metazoans, both by traditional phylogenies and by molecular criteria. In addition, Hydra possesses a simple body architecture consisting of two epithelial layers, with the outer layer in contact with the environment. Since Hydra does not survive in salt concentrations > 10 mM, we reasoned that this animal may express a Na,K-ATPase with unique structural and transport properties.

Results

The feasibility of a molecular approach to the Hydra Na,K-ATPase was initially demonstrated by probing Hydra genomic DNA blots with rat Na,K-ATPase α-subunit cDNAs. As shown in Fig. 1, a single Hydra DNA fragment hybridizes with rat α1-, α2-, and α3-subunit cDNA probes. These results suggest that Hydra contain a single α-subunit gene which is detectable with rat cDNA probes.

To characterize the Hydra Na,K-ATPase α-subunit, we screened a Hydra vulgaris (formerly H. attentuata) λgt11 cDNA library with full-length rat α1-subunit cDNA. Seven clones were isolated and characterized. DNA sequencing indicates that all clones represent a single class of cDNA. These observations are consistent with the view that Hydra possesses only one α-subunit gene. Northern blot analysis of Hydra poly A+ RNA (Fig. 2) indicates that the Hydra α-subunit gene encodes a 3.7-kb mRNA transcript.

The Hydra α-subunit cDNA contains a continuous open reading frame encoding a polypeptide of 1,031 amino acids with a calculated molecular weight of 114,160. The deduced amino acid sequence of the Hydra α-subunit shows 71, 71, and 73% identity to the rat α1-, α2-, and α3-subunits, respectively (Shull et al., 1986), and 70% identity to the Drosophila α-subunit. Thus, the Hydra Na,K-ATPase is slightly more divergent from vertebrate and insect ATPases than the latter are from each other (73–75% identity). These results suggest that the evolutionary ancestor of all metazoans possessed a Na,K-ATPase that was highly conserved with respect to its vertebrate counterparts.

Figure 1. Southern blot analysis of Hydra genomic DNA. Hydra DNA was digested with Eco RI, electrophoresed through a 0.8% agarose gel, transferred to Zetabind, and hybridized with radiolabeled rat α1-, α2-, and α3-subunit cDNAs. Eco RI–digested rat liver DNA was run in control lanes. 25 μg DNA was run in each lane. Positions of DNA size standards are indicated at the left.

Figure 2. Northern blot analysis of Hydra Na,K-ATPase α-subunit gene expression. Hydra vulgaris poly A+ RNA (0.5 μg) was electrophoresed through a 1% agarose-formaldehyde gel, transferred to Zetabind, and hybridized with radiolabeled Hydra α-subunit cDNA. Positions of 18S and 28S markers are indicated at the left.

A comparison of portions of the Hydra Na,K-ATPase α-subunit with Drosophila and vertebrate α-subunits is shown in Fig. 3. The amino terminus of the Hydra α-subunit (residues 1–19) is similar to that of Drosophila, but differs considerably from the vertebrate α-subunits. The region containing the FITC site (residues 492–524) is highly conserved between Hydra and the other organisms, except for a short nonconserved region preceding the invariant sequence Lys-Gly-Ala-Pro-Glu. The carboxyl terminus (residues 985–1,031) is highly conserved in all species, suggesting an important functional role for this domain. The region containing the nonpolar segment H4 and the phosphorylation site (residues 321–414, not shown) is 89% identical to the rat α1-subunit (residues 310–403). A region encompassing residues 693–795 (not shown) is 99% identical to the rat α1-subunit (residues 680–782). Interestingly, the hydropathy plot of the Hydra Na,K-ATPase α-subunit is virtually identical to those of Drosophila and rat (data not shown). These observations suggest that the transmembrane organization and secondary structure of the Hydra α-subunit are virtually identical to those of vertebrate and Drosophila α-subunits.

Figure 3. Amino acid sequences of selected regions of the Hydra, vertebrate, and Drosophila Na,K-ATPase α-subunits. The Hydra α-subunit sequence is numbered above. Gaps are inserted in the sequences (hyphens) to allow for optimal alignment. Residues 1–44: Amino termini. Residues 118–141: H1-H2 region. Arrows indicate residues at the termini of the H1-H2 domain. Residues 492–524: FITC region. Arrow indicates the FITC-reactive lysine. Residues 985–1031: Carboxyl termini.

Figure 4. Northern blot analysis of Na,K-ATPase β-subunit gene expression. Hydra poly A+ RNA (3 μg), total rat kidney (25 μg), and brain (25 μg) RNA were electrophoresed through a 1% agarose-formaldehyde gel, transferred to Zetabind, and hybridized with radiolabeled rat β1- and β2-subunit cDNAs. Positions of 18S and 28S markers are indicated at the left.

A comparison of the H1-H2 region from Hydra (residues 118–141) and other species is shown in Fig. 3. Charged residues (Arg and Asp) at the termini of the region between the H1 and H2 transmembrane segments appear to be responsible for the natural ouabain resistance of the rat αl isoform (Price and Lingrel, 1988; Canfield et al., 1990). Ouabain-sensitive isoforms (human α1, rat α2, rat α3) contain neutral residues at these positions. The presence of charged residues (Arg and Glu) at the termini of the H1-H2 region of Hydra (arrows in Fig. 3) suggests that the Hydra Na,K-ATPase may be resistant to ouabain. The ability of Hydra to grow in concentrations of ouabain ≥ 10-4 M, and the ability of the Hydra α-subunit cDNA to confer ouabain resistance to ouabain-sensitive CV-1 cells are consistent with this hypothesis.

To address the question of whether the Hydra Na,K-ATPase contains a β-subunit, we probed Northern blots of Hydra poly A+ RNA with rat β1 and β2 cDNA probes. As shown in Fig. 4, no β-subunit mRNA was detectable by hybridization. Further, β-subunits were not detected when antibodies specific for the rat β1- and β2-subunits (Shyjan and Levenson, 1989; Shyjan et al., 1990) were used to probe Western blots of Hydra membranes. It is possible that the Hydra Na,K-ATPase contains a β-subunit that is significantly divergent from the vertebrate β-subunit isoforms. Alternatively, our results raise the interesting possibility that the Hydra Na,K-ATPase may not contain a β-subunit.

Conclusions

Hydra vulgaris contains a Na,K-ATPase α-subunit with extensive primary and secondary structure similarity to vertebrate α-subunit isoforms. Transfection experiments suggest that the Hydra α-subunit gene may encode a ouabain-resistant Na,K-ATPase. The availability of cell lines expressing the Hydra α-subunit may provide insight into whether the Hydra Na,K-ATPase can function without a β-subunit and the enzymatic properties of a Na,K-ATPase which normally functions in a freshwater environment.

Acknowledgments

We thank Douglas Fisher (University of California, Irvine) for generously providing Hydra cDNA libraries and poly A+ RNA.

This work was supported by PHS grant CA-38992 and NIH grant HL-39263 to R. Levenson, who is an Established Investigator of the American Heart Association.

References

Baxter-Lowe, L. A., J. Z. Guo, E. E. Bergstrom, and L. E. Hokin. 1989. Molecular cloning of the Na,K-ATPase α-subunit in developing brine shrimp and sequence comparison with higher organisms. FEBS Letters. 257:181–187.

Canfield, V., J. R. Emanuel, N. Spickofsky, R. Levenson, and R. F. Margolskee. 1990. Ouabain-resistant mutants of the rat Na,K-ATPase α2 isoform identified by using an episomal expression vector. Molecular and Cellular Biology. 10:1367–1372.

Lebovitz, R. M., K. Takeyasu, and D. M. Fambrough. 1989. Molecular characterization and expression of the (Na+ + K+)-ATPase alpha-subunit in Drosophila melanogaster. EMBO Journal. 8:193–202.

Price, E. M., and J. B. Lingrel. 1988. Structure-function relationships in the Na,K-ATPase α subunit: site-directed mutagenesis of glutamine-111 to arginine and asparagine-122 to aspartic acid generates a ouabain-resistant enzyme. Biochemistry. 27:8400–8408.

Shull, G. E., J. Greeb, and J. B. Lingrel. 1986. Molecular cloning of three distinct forms of the Na+,K+-ATPase α-subunit from rat brain. Biochemistry. 25:8125–8132.

Shyjan, A. W., C. Gottardi, and R. Levenson. 1990. The Na,K-ATPase β2 subunit is expressed in rat brain and copurifies with Na,K-ATPase activity. Journal of Biological Chemistry. 265:5166–5169.

Shyjan, A. W., and R. Levenson. 1989. Antisera specific for the α1, α2, α3, and β subunits of the Na,K-ATPase: differential expression of α and β subunits in rat tissue membranes. Biochemistry. 28:4531–4535.

Molecular Cloning and Immunological Characterization of the γ-Subunit of the Na,K-ATPase

Robert W. Mercer, Daniel Biemesderfer, David P. Bliss, Jr., John H. Collins, and Bliss Forbush III

Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110; Departments of Cellular and Molecular Physiology and Internal Medicine, Yale University School of Medicine, New Haven, Connecticut 06510; and Department of Biological Chemistry, University of Maryland School of Medicine, Baltimore, Maryland 21201

Introduction

The Na,K-ATPase is generally described as being composed of a catalytic α-subunit of ~ 100,000 mol wt, and a glycoprotein β-subunit of ~ 55,000 mol wt. However, studies of the cardiac glycoside binding site on the Na,K-ATPase (Forbush et al., 1978; Rogers and Lazdunski, 1979; Lowndes et al., 1984) have shown that in addition to the α- and β-subunits, the binding site also consists of a small peptide of ~ 10,000 mol wt, termed the γ-subunit (previously “proteolipid component”). Evidence obtained in these studies indicates that the α- and γ-subunits are labeled with photoaffinity compounds with approximately equal probability when the reactive groups are 23–24 Å from the lactone ring of the cardiotonic steroid (Forbush, 1983). The significance of the γ-subunit has been unclear. The functional role of the small peptide is unknown, and its necessity for Na,K-ATPase activity has been experimentally questioned (Hardwicke and Freytag, 1981). Evidence has been presented both for (Harris and Stahl, 1988) and against (Forbush et al., 1978) the hypothesis that the γ peptide is a proteolytic fragment of the α- and/or β-subunits. However, partial peptide sequencing of the HPLC-purified γ-subunit has clearly established that the polypeptide is unique and different from the α- and β-subunits (Collins and Leszyk, 1987).

Here we summarize recent molecular, biological, and immunological studies of the γ-subunit. We have obtained cDNA clones for the rat and sheep γ, and deduced the full-length amino acid sequence. γ-Specific antibodies demonstrate that: (a) the sequenced protein is the same as that labeled by [3H]nitroazidobenzoyl-ouabain (NAB-ouabain); and (b) this protein colocalizes with the α-subunit of the Na,K-ATPase to the basolateral membrane of renal tubular epithelial cells.

Results and Discussion

The strategy for the isolation of the cDNA for the γ-subunit is based on the procedure of Frohman et al. (1988). mRNA from sheep kidney was isolated and reverse-transcribed using a 35-base oligonucleotide with 17-dT residues and an 18-base adaptor sequence containing three endonuclease sites. This reaction creates a single-stranded antisense DNA strand. Next, a 38-base oligonucleotide (5'-GAGAA(T,C)GAGGA(C,T)CCITTCTA(C,T)TA(C,T)GA(C,T)TA(C,T)GAGACIGT, derived from the 13 amino acid NH_2-terminal γ peptide sequence was annealed to the cDNA strand and used as a primer to generate the complementary second strand. Repetitive PCR cycles using the γ-subunit primer and the 3' adaptor primer resulted in the specific amplification of the double-stranded γ-subunit cDNA. Using this technique a cDNA clone corresponding to a portion of the sheep kidney γ-subunit was isolated. This clone was subsequently used to identify and isolate a cDNA containing the complete coding region for the rat kidney γ-subunit.

The nucleotide and deduced amino acid sequences from the rat and sheep γ-subunits are shown in Fig. 1. The amino acid sequence for the sheep kidney γ-subunit as determined by Collins and Leszyk (1987) is shown underlined, above the deduced amino acid sequences for the rat and sheep γ-subunit. The portion of the sheep cDNA nucleotide sequence corresponding to the γ sequence primer is underlined. The first 13 bases of the primer were apparently lost during amplification or cloning. The deduced amino acid sequence includes the initial eight amino acids determined by the γ sequence primer, followed by the complete carboxy-terminal sequence of the sheep kidney γ-subunit. The deduced amino acid sequence matches perfectly the sequence determined by peptide analysis.

The rat cDNA consists of 645 nucleotides with an open reading frame beginning from the first ATG triplet downstream of an in-frame termination codon (TGA) beginning at position –12. The initiation codon is found within the consensus sequence thought to be necessary for efficient translation of eucaryotic mRNAs (Kozak, 1989). An open reading frame extends from this ATG triplet to position 177 and codes for a polypeptide of 58 amino acids with a molecular mass of 6,497 D. The rat γ-subunit exhibits 92% amino acid similarity (78% identity, 14% favored substitutions) with the sheep subunit. There are no sites for N-linked glycosylation. Hydropathy analysis (Kyte and Doolittle, 1982) of the deduced amino acid sequence of the rat kidney γ predicts a single hydrophobic transmembrane domain consisting of 19 amino acids (amino acids 21–39) and highly charged intra- and extracellular domains. Until the site that interacts with the cardiac glycosides is determined, it is not known which portion of the subunit is cytoplasmic or extracellular.

Rat kidney γ-subunit cDNA was used to analyze the RNA products of γ-subunit gene expression in several rat tissues by hybridization to total cellular RNA (not shown). The γ-subunit gene encoded two RNA species ~ 1.5 and 0.8 kb in length. The smaller transcript is most abundant in the kidney, with lower levels in the spleen, lung, and heart. In contrast, the larger transcript is most abundant in the spleen and lung. Thus, the expression of γ-subunit mRNA appears to be regulated in a tissue-specific fashion. Moreover, in contrast to α-subunit mRNA expression, total γ mRNA levels are higher in the spleen and lower in the brain. However, in the kidney it appears that the levels of α1, β, and γ mRNA expressed are similar, suggesting that the subunits may be coordinately regulated.

The γ polypeptide was characterized using two polyclonal antibodies to the γ peptide. The first of these (Ab-gam) was raised by immunization with dog kidney γ peptide obtained by elution from SDS gels of purified Na,K-ATPase. The second (Ab-G17) was raised by immunization with hemocyanin-conjugated synthetic peptide prepared according to the first 17 amino acids of the sequence obtained by Collins and Leszyk (1987). For some purposes the antibodies were purified by adsorption to nitrocellulose-immobilized γ peptide from dog kidney.

Figure 1. The amino acid sequence for the sheep kidney γ-subunit determined from peptide analysis is shown underlined, above the deduced amino acid sequences for the rat and sheep γ-subunits. The portion of the sheep cDNA nucleotide sequence corresponding to the γ primer is underlined.

To characterize the specificity of the γ-reactive antisera, renal membranes were analyzed by SDS-polyacrylamide gel electrophoresis (PAGE). Ab-gam and Ab-G17 were found to react with a doublet occurring at 8.8 and 9.9 kD (reported in recent abstracts as 6.7 and 7.6 kD because of an error in commercial molecular weight standards; Kratzin et al., 1989). The antisera do not recognize either the α- or β-subunits. The reason γ runs as a doublet is not clear, but importantly the same protein doublet (and only this protein) is seen when synthetic γ-subunit RNA is translated in vitro using rabbit reticulocyte lysate (data not shown).

To determine if the γ antisera recognize polypeptides that have been specifically labeled with [3H]NAB-ouabain, labeled proteins were separated by SDS-PAGE, transferred to nitrocellulose, and analyzed for reactivity to the Ab-gam antiserum. Only the polypeptides that correspond to the labeled γ-subunit of the Na,K-ATPase are immunoreactive. In addition, when compared with preimmune sera, both antisera immunoprecipitate solubilized [3H]NAB-ouabain-labeled γ-subunit. This immunoprecipitation is partially (Ab-gam) or completely blocked (Ab-G17) by the presence of the G-17 peptide.

Figure 2. Indirect immunofluorescence demonstrating the localization of γ in the rat kidney. 0.5-μm-thick cryosections of rat renal cortex were labeled with affinity-purified rabbit anti–γ antibody and detected with FITC goat anti–rabbit IgG. Note that the fluorescence is restricted to the basolateral membrane of the tubular epithelium. Magnification, 800×.

To determine the localization of the γ-subunit within the renal tubular epithelium, indirect immunofluorescence experiments were carried out on 0.5-μm cryosections of PLP fixed rat kidney cortex (Biemesderfer et al., 1991). Fig. 2 demonstrates that γ is restricted to the basolateral membrane of epithelial cells, which is consistant with that described for the α-subunit of the Na,K-ATPase (Kashgarian et al., 1985).

These results demonstrate that the γ-subunit is a unique polypeptide separate from the other subunits, and that the identified and sequenced peptide is specifically associated with the Na,K-ATPase.

References

Biemesderfer, D., G. Dekan, P. S. Aronson, and M. G. Farquhar. 1991. Assembly of the renal brush border: biogenesis of distinctive coated pit and microvillar plasmalemmal microdomains. American Journal of Physiology. In press.

Collins, J. H., and J. Leszyk. 1987. The “γ subunit” of the Na,K-ATPase: a small, amphiphilic protein with a unique amino acid sequence. Biochemistry. 26:8665–8668.

Forbush, B., III. 1983. Cardiac steroid binding to Na,K-ATPase. Current Topics in Membranes and Transport. 19:167–201.

Forbush, B., III, J. H. Kaplan, and J. F. Hoffman. 1978. Characterization of a new photoaffinity derivative of ouabain: labeling of the large polypeptide and of a proteolipid component of the Na,K-ATPase. Biochemistry. 17:3667–3676.

Frohman, M. A., M. K. Dush, and G. R. Martin. 1988. Rapid production of full-length cDNAs from rare transcripts: amplification using single gene-specific oligonucleotide primer. Proceedings of the National Academy of Sciences, USA. 85:8998–9002.

Hardwicke, P. M. D., and J. W. Freytag. 1981. A proteolipid associated with Na,K-ATPase is not essential for ATPase activity. Biochemical and Biophysical Research Communications. 102:250–257.

Harris, W. E., and W. L. Stahl. 1988. Origin of the γ polypeptide of the Na+/K+-ATPase. Biochimica et Biophysica Acta. 942:236–244.

Kashgarian, M., D. Biemesderfer, M. Caplan, and B. Forbush III. 1985. Monoclonal antibody to Na,K-ATPase: immunocytochemical localization along nephron segments. Kidney International. 28:899–913.

Kozak, M. 1989. The scanning model for translation. An update. Journal of Cell Biology. 108:229–241.

Kratzin, H. D., J. Wiltfang, M. Karas, V. Neuhoff, and N. Hilschmann. 1989. Gas-phase sequencing after electroblotting on polyvinylidene difluoride membranes assigns correct molecular weights to myoglobin molecular weight markers. Analytical Biochemistry. 183:1–8.

Kyte, J., and R. F. Doolittle. 1982. A simple method for displaying the hydrophobic character of protein. Journal of Molecular Biology. 157:105–132.

Lowndes, J. M., M. Hokin-Neaverson, and A. E. Ruoho. 1984. Photoaffinity labeling of (Na+K+)-ATPase with [125I]iodoazidocymarin. Journal of Biological Chemistry. 259:10533–10538.

Rogers, T. B., and M. Lazdunski. 1979. Photoaffinity labeling of a small protein component of a purified (Na+ + K+)-ATPase. FEBS Letters. 98:373–376.

Expression of DNA for α, β, and γ Polypeptides of Na,K-ATPase in Yeast: Does γ Have a Function in Na,K-ATPase Activity?

Georgios Scheiner-Bobis, Kurt A. Eakle, Kyu S. Kim, and Robert A. Farley

Department of Physiology and Biophysics, School of Medicine, University of Southern California, Los Angeles, California 90033

Introduction

Na,K-ATPase is an oligomeric membrane protein. Its α-subunit, a polypeptide of Mr 112,000 is directly involved in the coupling of ATP hydrolysis to the transport of the ions. The role of the β-subunit, a glycoprotein of Mr 56,000, is not very well understood. However, recent work shows that this second subunit is probably involved in the stabilization and transport of the α-subunit to the plasma membrane.

Labeling experiments with radioactive derivatives of cardioactive steroids show that in addition to the incorporation of the label into the α-subunit, a protectable amount of radioactivity is also incorporated into a smaller peptide which runs on polyacrylamide gels with Mr 11,000–12,000 (Forbush et al., 1978; Collins et al., 1982). This observation, and earlier studies with ATP analogues, have been interpreted to indicate the existence and participation of a third γ-subunit in the Na,K-ATPase.

Separation of the γ-subunit from the α/β-subunit complex under nondenaturing conditions, however, shows that the ouabain-sensitive ATPase activity is independent of the smaller γ-proteolipid (Hardwicke and Freytag, 1981). Since the peptide seems to be present in various preparations of Na,K-ATPase from different tissues and has a highly conserved primary structure (Forbush et al., 1978; Hardwicke and Freytag, 1981; Collins et al., 1982), it might participate in some other aspect of ion transport. One possibility would be its participation in the formation of the ionophoric part of the Na,K-ATPase (Hardwicke and Freytag, 1981). Hydropathy plots of the deduced γ-subunit sequence show a long hydrophobic part, sufficient for the insertion of the peptide in the cell membrane.

The possibility that a γ-subunit is involved in ion transport cannot be verified with Na,K-ATPase isolated from animal cell membranes after treatment with detergent. The possible role of the putative γ-subunit in the Na,K-ATPase activity can be investigated, however, in a heterologous expression system lacking its own Na,K-ATPase. We have used the recently described yeast expression system for Na,K-ATPase (Horowitz et al., 1990) to test this hypothesis after transformation of yeast cells with vectors containing cDNA encoding for α-, β-, and γ-subunits. The ouabain-sensitive ATPase, phosphatase, and ion transport characteristics of the transformed cells were studied. Nontransformed yeast cells or cells transformed with either the γ-subunit alone or the α- and β-subunits simultaneously served as controls.

Figure 1. Yeast cells, grown overnight at 30°C with agitation, were centrifuged at 5,000g for 10 min, washed with water, and centrifuged again under the same conditions. The pelleted cells were resuspended in 2% glucose and stored on ice. 500 μl of the cell suspension was incubated for 30 min at 37°C with or without 0.1 mM ouabain in 10 mM Tris/HCl, pH 7.5, 5 mM PO_4(Tris), pH 7.0, and 5 mM MgCl_2. Thereafter, 2.5 mM 86RbCl was added to each sample. The time-dependent accumulation of 86Rb+ into the cells was followed at 22°C. The reaction was interrupted by centrifugation for 3 min in an Eppendorf centrifuge. Nonspecifically entrapped 86RbCl was removed by resuspending the cells in cold water, followed by an additional centrifugation. Cells were dissolved in TS-1 tissue solubilizer before counting for radioactivity. (A) 86Rb uptake by nontransformed yeast cells. (B) 86Rb uptake by yeast cells expressing α- and β-subunits. (C) 86Rb uptake by yeast cells expressing α-, β-, and γ-subunits.

The initial results of our investigation which are reported here do not support the direct involvement of the putative γ-subunit in the function of the Na,K-ATPase.

Results and Discussion

The goal of the investigation presented here is to identify the function of the putative γ-subunit of the Na,K-ATPase. Although this hydrophobic proteolipid is found in Na,K-ATPase preparations from different tissues, its role in the function of the pump has not been determined. Earlier experiments showed that the γ-subunit is not required for ATP hydrolysis by the Na,K-ATPase (Hardwicke and Freytag, 1981), but did not exclude the possibility that this oligopeptide might be involved in the transport of ions (Hardwicke and Freytag, 1981).

We used a recently described heterologous expression system for the Na,K-ATPase in yeast (Horowitz et al., 1990) to investigate this possibility. cDNA, coding for the rat γ-subunit (gift of Dr. R. W. Mercer, Washington University, St. Louis, MO) was ligated into the expression vector pAAH5 (gift of Dr. J. B. Lingrel, University of Cincinnati, Cincinnati, OH). The resulting new vector pA-RG1 was used together with the previously described plasmid pCGY1406αβ, coding for both α- and β-subunits (Horowitz et al., 1990), for double transformations of yeast cells. Control cells contained either one of the above plasmids, or neither of them.

Successful transcription of the cDNA molecules for each subunit of the Na,K-ATPase in the yeast cells was verified by Northern blot analysis (not shown). Although we have not yet measured the presence of the γ-subunit in yeast due to the absence of appropriate antibodies, it is likely that the yeast cell will translate the mRNA for the γ-subunit into protein since the same organism is capable of translating the mRNA for α- and β-subunits (Horowitz et al., 1990), and since the mRNA for the γ-subunit is produced in the full length (not shown).

Cells transformed with cDNA encoding the α- and β-subunits, or the α-, β-, and γ-subunits of the sodium pump, were used to study the ouabain-sensitive uptake of 86Rb+ ions. Nontransformed cells were used as a control. Fig. 1 shows the results of this experiment. While a ouabain-sensitive 86Rb+ transport cannot be detected in the nontransformed cells (Fig. 1 A), 86Rb+ uptake in cells transformed with the α- and β-subunits, or the α-, β-, and γ-subunits of Na,K-ATPase, is inhibited ~ 30% by ouabain (Fig. 1, B and C). This fraction of the transported ions into the cells can be attributed to the action of the Na,K-ATPase. Transformation of cells with cDNA encoding all three subunits does not lead to a difference in ouabain-sensitive transport activity when compared with the cells expressing only α- and β-subunits. This result does not support the involvement of the γ-subunit in the transport of the ions.

We have also investigated the ATPase and phosphatase activities in membranes isolated after SDS treatment from the different cells. The results are presented in Table I. It is shown that the ouabain-sensitive ATPase or phosphatase activity is comparable in both cases. Enzymatic activities from nontransformed cells and from cells transformed with only the γ-subunit are not affected by the cardioactive steroid. These data indicate that the ouabain-sensitive ATPase and phosphatase activities are due to the presence of the α- and β-subunits of Na,K-ATPase, and that the γ-subunit appears not to be essential or involved in these activities. The ratio of ATPase to phosphatase activities is in the same range as for the kidney Na, K-ATPase (Horowitz et al., 1990).

The data presented here suggest that the γ-subunit is not essential for either ATPase or phosphatase activities of Na,K-ATPase. This conclusion is in agreement with previous investigations (Hardwicke and Freytag, 1981). Additionally, the participation of this oligopeptide in the formation of the ionophoric part of the sodium pump is very unlikely, since no differences in the rate of 86Rb+ uptake could be detected between cells expressing α-, β-, and γ-subunits and cells expressing only α- and β-subunits (Fig. 1, A-C). This conclusion, however, is based on the assumption that the γ-subunit is present within the cells, and efforts to demonstrate this are in progress.

TABLE I
Ouabain-sensitive ATPase and Phosphatase Activity in Yeast Membranes Treated with SDS

The preparation of the membranes and the conditions for the measurement of the ouabain-sensitive ATPase or phosphatase activity are published (Horowitz et al., 1990).

The results of this investigation do not allow us to exclude the possibility that the γ-polypeptide is important in a different way for the sodium pump; e.g., in the asymmetric distribution of the Na,K-ATPase observed in epithelial tissues. Such a possibility, however, cannot be verified with the yeast expression system used for the experiments described here.

Acknowledgments

This work was supported by NIH grants GM-28673 and HL-39295, NSF grant DMB-8919336, and American Heart Association, Greater Los Angeles Affiliate, award 809 IG5.

References

Collins, J. H., B. Forbush, L. K. Lane, E. Ling, A. Schwartz, and A. Zot. 1982. Purification and characterization of an (Na+ + K+)-ATPase proteolipid labeled with a photoaffinity derivative of ouabain. Biochimica et Biophysica Acta. 686:7–12.

Forbush, B., J. H. Kaplan, and J. F. Hoffman. 1978. Characterization of a new photoaffinity derivative of ouabain: labeling of the large polypeptide and of a proteolipid component of the Na,K-ATPase. Biochemistry. 17:3667–3676.

Hardwicke, P. M. D., and J. W. Freytag. 1981. A proteolipid associated with Na,K-ATPase is not essential for ATPase activity. Biochemical and Biophysical Research Communications. 102:250–257.

Horowitz, B., K. A. Eakle, G. Scheiner-Bobis, G. R. Randolf, C. Y. Chen, R. R. A. Hitzeman, and R. A. Farley. 1990. Synthesis and assembly of functional mammalian Na,K-ATPase in yeast. Journal of Biological Chemistry. 265:4189–4192.

Expression of Domains of the Na,K-ATPase in a Run-away Plasmid Replication System in Bacteria

Douglas M. Fambrough and Ru Chih C. Huang

Department of Biology, The Johns Hopkins University, Baltimore, Maryland 21218

A definitive understanding of ion transport by the Na,K-ATPase requires knowledge of the three-dimensional structure of the enzyme in all relevant conformations. Only with such information can central questions about the transport process be answered. These questions include: Is the selectivity for potassium ions in the E2 conformation mediated by the same coordinating groups that mediate sodium selectivity in E1? How are potassium ion binding sites and cardiac glycoside binding sites related? and Are the low and high affinity ATP sites different conformations of the same surface? Given the extraordinary difficulty of obtaining ordered arrays of membrane protein molecules of sufficient size and alignment for high-resolution structural studies, it seems worthwhile to explore other approaches to structural analysis. One such approach is to subdivide the problem into parts and first determine the structures of fragments of the Na,K-ATPase. This approach is predicated upon the hypothesis that within large, complex proteins there exist smaller, quasi-independent domains that mediate different aspects of the overall function of the protein. This idea is implicit in hypothetical models of E1E2 cation transporters in which parts of the structure bear labels such as “nucleotide binding domain” and “phosphorylation domain.” This way of thinking about protein structure has been validated, for example, in the recent structural analysis of the ATP binding domain of one of the heat-shock cognate proteins. In this case, proteolytic cleavage of the molecule released a crystallizable fragment, for which the structure is now determined (Flaherty et al., 1990). Molecular biological techniques now make possible production of large quantities of protein fragments of predetermined length and amino acid sequence. Thus, the limitations of enzyme purification, proteolytic fragmentation, and purification of fragments can, in part, be bypassed.

In the case of the Na,K-ATPase, the problems of structural analysis are severe. Not only are there many membrane-spanning segments, but these have not yet been identified with certainty. There remains a very real possibility that some regions of the molecule intimately involved in ion translocation across the membrane are not obviously identified as candidates for this role through analysis of hydrophobic or amphipathic character. The one region of the molecule that is generally thought to exist as one or two hydrophilic, more-or-less globular domains is the region of the α-subunit between amino acid residues about 340 and 780. Secondary structure predictions for this region suggest an alternating series of α-helices and β-pleated sheets. Weak sequence similarities to other nucleotide binding proteins suggest that a portion of this region may have a folding pattern similar to that of hexokinase (Taylor and Green, 1989). Thus, there is reason to hope that this region, representing > 40% of the catalytic subunit, might be amenable to structural analysis as an isolated unit. Further, this unit might be expected to display some characteristics of the enzyme, such as ATP binding, p-nitrophenylphosphatase activity, sensitivity to FITC, and, conceivably, even ATPase activity. Finally, this unit might embody some isoform-specific aspects, such as unique epitopes for monoclonal antibodies or even isoform-specific functional characteristics. With these optimistic thoughts, we set out to isolate the central cytoplasmic domain of the Na,K-ATPase α-subunit by expressing its encoding DNA in a high-expression system in bacteria.

Figure 1. Schematic representation of cloning and helper plasmids for the run-away plasmid replication system of Giza and Huang (1989).

The run-away plasmid replication system developed by Giza and Huang (1989) was used to express the central cytoplasmic domain of an avian Na,K-ATPase α-subunit in bacteria (see Fig. 1). In this system, plasmid copy number in the bacterium is regulated by the gene Rop (repressor of primer). The Rop gene product is a small protein (6,000 mol wt) that interacts with primer RNA at the site of DNA replication, blocking DNA synthesis. The Rop gene is interrupted in the cloning plasmid by insertion of a cDNA encoding part of the Na,K-ATPase. Regulation of cloning plasmid replication requires a functional Rop gene on another plasmid in the same bacterium. This helper plasmid is kept in the bacterium by selecting for tetracycline resistance (TetR), while the cloning plasmid is required for resistance to ampicillin (AmpR). When the bacteria are inoculated into medium at very low concentration and grown under Amp selection but not Tet selection, the helper plasmid is lost at high frequency, resulting in uncontrolled replication of the cloning plasmid in those bacteria. Transcription through the Rop gene is controlled by the tryptophan promoter, a very strong promoter. This results in mRNA molecules that encode a fusion protein: the first 51 amino acids of the Rop protein, followed by the amino acids encoded in the cloned DNA.

Figure 2. Design of PCR oligonucleotides for generating the DNA encoding the central cytoplasmic domain of the Na,K-ATPase α-subunit. Coding and noncoding strand oligonucleotides are shown with the encoded amino acids below and above, respectively. As shown below, when cloned into the BglII site of the pPG vector, a fusion protein is encoded by the plasmid, consisting of 51 amino acids of the Rop gene product followed by an arginine and then the DNA encoding residues 342–750 of the Na,K-ATPase α-subunit.

We chose initially to express almost the entire central domain of the α-subunit: amino acids 342 to 750. The cDNA encoding this region of the α3-isoform (Takeyasu et al., 1990) was prepared by polymerase chain reaction (PCR) methods, using oligonucleotides designed with BamH1 restriction enzyme sites flanking base sequences encoding the ends of the cDNA fragment (Fig. 2). In addition, a stop translation codon was inserted in the 3'-end oligonucleotide so that the expressed protein would terminate properly at this position. PCR product was cut with BamH1 to generate sticky ends and the DNA was ligated into BglII-cut pPG cloning vector. (The BglII site in the cloning vector was generated by inserting a commercial BglII linker in the PvuII cloning site used by Giza and Huang [1989].) The resulting plasmid, pPGα3m, was used to transform bacteria carrying the helper Rop plasmid, and colonies were selected for Amp resistance.

Figure 3. Expression of the α-subunit central cytoplasmic domain in the run-away plasmid replication system. (Top) SDS-PAGE (Coomassie stained). (Bottom) Protein blot of an identical SDS-PAGE run, probed with monoclonal antibody (α5) to the α-subunit central domain followed by 125I-labeled goat anti–mouse IgG. Lane 1, molecular weight standards; lane 2, total bacterial protein, α-subunit expressed; lane 3, inclusion body fraction; lane 4, soluble proteins; lane 5, total bacterial protein, cDNA reversed in plasmid; lane 6, total bacterial protein, plasmid without DNA insert.

When the bacteria harboring the pPGα3m plasmid were grown under expression conditions, a polypeptide of the expected size was expressed at high levels. Bacteria were lysed by treatment with lysozyme and sonication, and the bacterial proteins were fractionated into those soluble in dilute Triton X-100-containing solution, and those that sedimented at 100,000g within 1 h (Kaplan and Greenberg, 1987). The total bacterial protein and the soluble and “inclusion body” fractions were analyzed by SDS-PAGE, and protein blots of the electrophorograms were assayed for binding of a monoclonal antibody (α5) (Lebovitz et al., 1989) specific for the central domain of the Na,K-ATPase. Fig. 3 shows that bacteria carrying the pPGα3m plasmid with the cDNA in the correct orientation express a polypeptide of ~ 50 kD that is the major component of the inclusion body fraction. This component specifically binds the α5 monoclonal antibody, and thus is the expected Na,K-ATPase fragment.

The occurrence of the α-subunit fragment in inclusion bodies is a mixed blessing. It affords rapid partial purification but so far has required denaturing conditions for solubilization. As a first approach to isolating properly folded Na,K-ATPase fragment, we have solubilized the inclusion body fraction in 10 M urea, 0.5% deoxycholate and then dialyzed away the urea. The clear solution was then applied to a DEAE ion exchange column and the fragment was eluted in 600 mM NaCl/80 mM Na borate buffer, pH 8.0. In preliminary tests done in collaboration with Dr. Jack Kaplan (University of Pennsylvania), this fragment showed partial protection by ATP from reaction with FITC. Heat-denatured samples of this fragment did not show such ATP-preventable FITC binding.

The run-away plasmid replication system has also been used to express the NH_2-terminal 350 amino acid fragment of the Na,K-ATPase α-subunit. In this case the resultant peptide is expressed at a lower level but is recovered principally in the soluble fraction from the bacteria. This fragment should possess four membrane-spanning segments of the α-subunit. It will be interesting to know if this fragment is incorporated into the bacterial membrane in the topology it is believed to assume in eukaryotes, and whether this fragment displays any affinity for cardiac glycosides.

References

Flaherty, K. M., C. DeLuca-Flaherty, and D. B. McCay. 1990. Three-dimensional structure of the ATPase fragment of a 70K heat-shock cognate protein. Nature. 346:623–628.

Giza, P., and R. C. C. Huang. 1989. A self-inducing runaway-replication plasmid expression system utilizing the Rop protein. Gene. 78:73–84.

Kaplan, H. B., and E. P. Greenberg. 1987. Overproduction and purification of the luxR gene product: transcriptional activator of the Vibrio fischeri luminescence system. Proceedings of the National Academy of Sciences, USA. 84:6639–6643.

Lebovitz, R. M., K. Takeyasu, and D. M. Fambrough. 1989. Molecular characterization and expression of the (Na+ + K+)-ATPase α-subunit in Drosophila melanogaster. EMBO Journal. 8:193–202.

Takeyasu, K., M. V. Lemas, and D. M. Fambrough. 1990. Stability of the (Na+ + K+)-ATPase α-subunit isoforms in evolution. American Journal of Physiology. 259 (Cell Physiology 28):C619-C630.

Taylor, W. R., and N. M. Green. 1989. The predicted secondary structures of the nucleotide-binding sites of six cation-transporting ATPases lead to a probable tertiary fold. European Journal of Biochemistry. 179:241–248.

Expression of Exogenous β1-Subunits of Na,K-Pump in Xenopus laevis Oocytes Raises Pump Activity

Günther Schmalzing, Halit Omay, Silke Kröner, Heribert Appelhans, and Wolfgang Schwarz

Max-Planck-Institut für Biophysik, Frankfurt/Main, Germany

Introduction

Oocytes of Xenopus laevis synthesize a large excess of α1-subunits over β-subunits (Geering et al., 1989; Geering, 1991). The excess α-subunits that cannot complex with a β-subunit reside in the endoplasmic reticulum and retain high sensitivity for trypsin (Geering, 1991) characteristic of nonfunctional sodium pumps (Geering et al., 1985). By expression of exogenous β1-subunits of the sodium pump of Xenopus kidney cells in the oocytes, the catalytically immature α-subunits can be induced to assemble as functional holoenzymes in the plasma membrane (Geering et al., 1989). In this study we examined the question of whether or not expression of the β1 isoforms of species other than Xenopus can raise sodium pump activity in Xenopus oocytes. The three β1-subunits of the sodium pump investigated were previously cloned from electric organ of Torpedo californica (Noguchi et al., 1986), rat brain (Mercer et al., 1986), and chicken brain (Takeyasu et al., 1987). According to derived amino acid sequences, these β1-subunits have 61–67% identity and 73–79% similarity with the sequence of the β1-subunit of Xenopus published recently by Verrey et al. (1989).

Results

We have previously demonstrated that intracellular sodium pumps can be rendered accessible for ouabain binding in Xenopus oocytes by permeabilizing the plasma membrane with digitonin and intracellular membranes with the nonselective detergent SDS (Schmalzing et al., 1989). Taking advantage of this technique, we quantified the number of sodium pumps in oocytes injected with cRNA specific for sodium pump subunits. As illustrated in Fig. 1 a, injection of cRNA for the α-subunit of the sodium pump of Torpedo californica did not significantly change ouabain binding to permeabilized oocytes. In contrast, injection of cRNA for the β-subunit of the Torpedo sodium pump induced a 40% increase of ouabain binding in both the plasma membrane and intracellular membranes (Fig. 1 b, P < 0.001 according to paired one-tailed t test). In accordance with the previous results of Noguchi et al. (1987), coinjection of cRNAs for both subunits caused a several-fold increase in the number of ouabain binding sites.

The Scatchard plot given in Fig. 2 demonstrates that the effect of expression of a heterologous β1-subunit is not due to a change in the affinity for ouabain, but to an increase of the ouabain binding capacity to both the plasma membrane and intracellular membranes by 54 and 60%, respectively. The binding capacity of intracellular membranes is calculated as the difference in the abscissa intercepts of the straight lines fitted to data obtained by incubation of the cells with and without SDS. Similar increases in ouabain binding could also be elicited by injection of cRNA encoding the β1-subunits of chicken brain and mouse brain, as will be described in detail elsewhere (Schmalzing, G., S. Gloor, H. Omay, S. Kröner, H. Appelhans, and W. Schwarz, manuscript submitted for publication).

Figure 1. Rise of ouabain binding by sole expression of heterologous β1-subunits, but not of α-subunits. Capped cRNA was synthesized from cDNA clones using templates linearized in the 3'-untranslated region and SP6 RNA polymerase. Oocytes defolliculated by collagenase treatment were injected with 50 ng of cRNA for either the α1-subunit (a) or the β1-subunit (b) of the Torpedo sodium pump. After 2 d at 17°C, the cells were permeabilized with digitonin and assayed for ouabain binding in the absence and presence of SDS as described (Schmalzing et al., 1989). Results are means ± SEM from a typical experiment (a) or from 14 different experiments (b) with 8–12 separate determinations in individual oocytes. Open bars, noninjected controls; filled bars, cRNA-injected oocytes.

Figure 2. Scatchard plot for ouabain binding to oocytes injected with cRNA for a heterologous β1-subunit. Capped cRNA derived by SP6 RNA polymerase-directed in vitro transcription was injected into oocytes (50 ng/cell). After 2 d at 17°C, cells were permeabilized with digitonin and ouabain binding was determined in the absence (○,●) or presence (,■) of 0.02% SDS at 1–50 nM [3H]ouabain. Results in this and the following experiments are means ± SEM of 8–12 separate determinations in individual oocytes. Noninjected oocytes were assayed in parallel and are indicated by the open symbols. Bound/free is given in fmol × cell-1 × nM-1.

Figure 3. ATP and K+ sensitivity of sodium pumps induced by expression of a heterologous β1-subunit. Oocytes injected with cRNA for the β1-subunit of the Torpedo sodium pump were kept for 2 d at 17°C before being permeabilized with digitonin. Ouabain binding (filled bars) was determined with or without 1 mM ATP and 50 mM KCl as indicated at a final concentration of 120 nM [3H]ouabain. Noninjected oocytes were assayed in parallel (open bars).

To examine whether or not sodium pumps induced by injection of cRNAs for heterologous sodium pumps are functional, the effect of ATP and K+ on ouabain binding was studied. As illustrated in Fig. 3, the sodium pumps of oocytes injected with cRNA for the Torpedo β-subunit have a similar sensitivity toward omission of ATP or inclusion of K+ as noninjected control oocytes. This indicates that the additional sodium pumps induced by expression of heterologous β-subunits must be able to undergo at least some of the conformational changes expected for a functional enzyme.

Figure 4. Sodium pumps induced by expression of a heterologous β1-subunit are functional. Oocytes were injected with 5–50 ng of cRNA for the β1-subunit of the rat sodium pump. After 2 d at 17°C, the cells were loaded with Na+ by incubation in a NaCl-based medium nominally free of divalent cations and assayed either for ouabain binding or ouabain-sensitive 86Rb+ uptake.

In another series of experiments, oocytes injected with various amounts of cRNA for the β1-subunit of rat brain were assayed for ouabain-sensitive 86Rb+ uptake in addition to surface binding of ouabain. Fig. 4 suggests that the increase in the number of ouabain binding sites was associated with a proportional increase in active 86Rb+ uptake.

Discussion

The present findings demonstrate that the capacity of exogenous β1-subunits of the sodium pump of Xenopus laevis kidney cells to raise sodium pump activity in Xenopus oocytes (Geering et al., 1989) is shared by heterologous β1-subunits. The additional sodium pumps probably result from a complexation of exogenous β1-subunits with endogenous α-subunits that reside in a nonfunctional form in the endoplasmic reticulum because they lack endogenous β-subunits (Geering, 1991). Association with any of the β1-subunit isoforms as with the endogenous β3 isoform (Geering, 1991) possibly induces a critical refolding of immature α-subunits required for ouabain binding (Figs. 1–3) and ouabain-sensitive transport of K+ (or Rb+, Fig. 4) and Na+. The proportional increase of ouabain binding capacity and ouabain-sensitive Rb+ uptake (Fig. 4) suggests that interspecies hybrid sodium pumps have a turnover similar to the endogenous enzyme which consists of α1 and β3 subtype isoforms (Geering, 1991). Expression of active sodium pumps from cDNA specific for either the α- or β-subunit alone has been shown to occur in other cells as well, where it has also been attributed to the formation of hybrid sodium pumps between exogenous α-subunits and endogenous β-subunits (Takeyasu et al., 1988; Lebovitz et al., 1989) and vice versa (Takeyasu et al., 1987). Changes in the transcriptional or translational activity for one of the β isoforms alone might be a mechanism for sodium pump regulation in cells with a pool of immature α-subunits such as Xenopus oocytes.

Acknowledgments

We thank Drs. E. J. Benz, D. Fambrough, P. Graham, and M. Kawamura for the generous gifts of the cDNAs. We also thank Dr. Hermann Passow for helpful comments on the manuscript.

The work was financially supported by the Deutsche Forschungsgemeinschaft (Sonderforschungsbereich 169).

References

Ackermann, U., and K. Geering. 1990. Mutual dependence of Na,K-ATPase α- and β-subunits for correct posttranslational processing and intracellular transport. FEBS Letters. 269:105–108.

Geering, K. 1991. Posttranslational modifications and intracellular transport of sodium pumps: importance of subunit assembly. In The Sodium Pump: Structure, Mechanism, and Regulation. J. H. Kaplan and P. De Weer, editors. The Rockefeller University Press, New York. 31–43.

Geering, K., D. I. Meyer, M.-P. Paccolat, J.-P. Kraehenbühl, and B. C. Rossier. 1985. Membrane insertion of α- and β-subunits of Na+,K+-ATPase. Journal of Biological Chemistry. 260:5154–5160.

Geering, K., I. Theulaz, F. Verrey, M. T. Häuptle, and B. C. Rossier. 1989. A role for the β subunit in the expression of functional Na+-K+-ATPase in Xenopus oocytes. American Journal of Physiology. 257:C851-C858.

Lebovitz, R. M., K. Takeyasu, and D. M. Fambrough. 1989. Molecular characterization and expression of the (Na+ + K+)-ATPase alpha-subunit in Drosophila melanogaster. EMBO Journal. 8:193–202.

Mercer, R. W., J. W. Schneider, A. Savitz, J. Emanuel, E. J. Benz, Jr. and R. Levenson. 1986. Rat-brain Na,K-ATPase β-chain gene: primary structure, tissue-specific expression, and amplification in ouabain-resistant HeLa C+ cells. Molecular and Cellular Biology. 6:3884–3890.

Noguchi, S., M. Mishina, M. Kawamura, and S. Numa. 1987. Expression of functional (Na+ + K+)-ATPase from cloned cDNAs. FEBS Letters. 225:27–32.

Noguchi, S., M. Noda, H. Takahashi, K. Kawakami, T. Ohta, K. Nagano, T. Hirose, S. Inayama, M. Kawamura, and S. Numa. 1986. Primary structure of the β-subunit of Torpedo californica (Na+ + K+)-ATPase deduced from the cDNA sequence. FEBS Letters. 196:315–320.

Schmalzing, G., S. Kröner, and H. Passow. 1989. Evidence for intracellular sodium pumps in permeabilized Xenopus laevis oocytes. Biochemical Journal. 260:395–399.

Takeyasu, K., M. M. Tamukun, K. J. Renaud, and D. M. Fambrough. 1988. Ouabain-sensitive (Na+ + K+)-ATPase activity expressed in mouse L cells by transfection with DNA encoding the α-subunit of an avian sodium pump. Journal of Biological Chemistry. 263:4347–4354.

Takeyasu, K., M. M. Tamkun, N. R. Siegel, and D. M. Fambrough. 1987. Expression of hybrid (Na+ + K+)-ATPase molecules after transfection of mouse Ltk- cells with DNA encoding the β-subunit of an avian brain sodium pump. Journal of Biological Chemistry. 262:10733–10740.

Verrey, F., P. Kairouz, E. Schaerer, P. Fuentes, K. Geering, B. C. Rossier, and J.-P. Kraehenbuhl. 1989. Primary sequence of Xenopus laevis Na+/K+-ATPase and its localization in A6 kidney cells. American Journal of Physiology. 256:F1034–F1043.

Expression of the Large Cytoplasmic Domain of Na,K-ATPase in Escherichia coli

Kena Wang, Edward E. Huston, and Robert A. Farley

Department of Physiology and Biophysics, University of Southern California School of Medicine, Los Angeles, California 90033

Introduction

Na,K-ATPase, like other membrane-embedded, cation-transporting ATPases, utilizes the energy of ATP hydrolysis to drive ion transport across the membrane. The identification of amino acids within the ATP binding site of the enzyme has been made using affinity labeling techniques with specific ATP binding site probes. Fluorescein-5'-isothiocyanate (FITC) is generally believed to react at the adenine ring binding site of the enzyme. 5'-(p-fluorosulfonyl)benzoyladenosine (FSBA), 8-azido-ATP (8-N_3ATP), and r-[4-(N-2-chlorethyl-N-methylamino)]benzylamide ATP (CIRATP) are ATP analogues that have protein-reactive groups at different positions of the ATP molecule. All the amino acid residues that have been labeled by the probes (Farley et al., 1984; Ohta et al., 1986; Ovchinnikov et al., 1987; Tran et al., 1988) are located in the largest cytoplasmic domain of the α-subunit (amino acid residues 342–779 from sheep kidney [Shull et al., 1985]). The primary sequences surrounding these labeled residues are highly conserved among different cation-transporting ATPases. Predictive secondary and super-secondary structure analyses suggest that in this region a parallel β-stranded sheet structure may exist, and that the folded pattern may be similar to the nucleotide binding domain of adenylate kinase (Taylor and Green, 1989). All the available evidence suggests that the ATP binding domain of Na,K-ATPase is located in this large cytoplasmic domain of the α-subunit.

The question remains, however, whether this large cytoplasmic loop alone contains sufficient information to form the complete ATP binding site of the enzyme. To address this question, we have expressed the large cytoplasmic domain (DP47) of Na,K-ATPase in Escherichia coli.

Methods and Results

Construction of the Expression Plasmid pK1.3

pK1.3 was derived from E. coli expression plasmid pKK233–2 (Clontech Laboratories Inc., Palo Alto, CA). The cDNA fragment coding for the amino acids Met^344 to Glu^779 was isolated from sheep kidney Na,K-ATPase α-subunit cDNA (Shull et al., 1985). Oligonucleotide-directed mutagenesis was used to introduce a translational stop codon into the sequence immediately after the Glu^779 codon. This modified cDNA fragment of ~ 1.3 kb was inserted into the plasmid pKK233–2 between Nco I and Hind III sites. In this plasmid (pK1.3) the expression of DP47 is under the control of the Ptrc promoter and the codon for Met^344 of the Na,K-ATPase α-subunit is used as the translational start codon.

Expression of DP47 in E. coli

Both pKK233–2 and pK1.3 were transformed into E. coli strain JM105. Cells were incubated overnight at 37°C in M9 medium containing thiamine and ampicillin. The overnight culture was diluted 100-fold in LB broth containing ampicillin. At OD_600 ~ 0.5–0.6, isopropyl-β-D-thiogalactopyranoside (IPTG) was added to the culture to induce the expression from the promoter Ptrc. Immunoblot with monoclonal antibody #5 (gift of D. Fambrough, The Johns Hopkins University, Baltimore, MD) directed against the Na,K-ATPase α-subunit was used to detect the expression of DP47 in E. coli. It was found that an immunopositive protein of ~ 47 kD was only present in the cells carrying pK1.3, and not in the cells carrying pKK233–2. The expression level of DP47 was increased when the temperature of the growth medium was changed from 37 to 30°C after IPTG addition.

Cell Lysis and Fractionation

Cell pellets were suspended in ice-cold buffer containing 50 mM Tris-HCl, pH 8.0, 1 mM EDTA, 100 mM NaCl, 0.5 mM DTT, and 150–200 μM PMSF. The cells were then lysed with lysozyme as described by Marston (1987). The lysate was centrifuged in a Ty65 rotor at 52,000 rpm for 1 h at 4°C. The supernatant was separated from the pellet and both fractions were analyzed for the presence of DP47. The presence of DP47 was detected only in the lysate supernatant. In most cases, the amount of DP47 is ~ 1% of the total lysate supernatant protein.

Purification of DP47

DP47 was purified using ion exchange chromatography on QAE-Sepharose and immunoaffinity chromatography.

Ion exchange chromatography. The E. coli lysate supernatant was applied to a QAE-Sepharose column equilibrated with buffer A (50 mM Tris-HCl, pH 8.0, 1 mM EDTA, 0.5 mM DTT, and 100 mM NaCl). After washing extensively with buffer A, proteins were eluted with a linear gradient from buffer A to buffer B (50 mM Tris-HCl, pH 8.0, 1 mM EDTA, 0.5 mM DTT, and 400 mM NaCl). DP47 was eluted from the column at ~ 0.24 mM NaCl. The fractions containing DP47 were pooled and concentrated. It was estimated that the DP47 had been enriched about sevenfold in this step.

Immunoaffinity chromatography. The anti–Na,K-ATPase monoclonal antibody #5 was purified on protein G-Sepharose and conjugated to Affi-Gel Hz (Cat.#153-6047, Bio-Rad Laboratories, Richmond, CA). The fractions containing DP47 after QAE-Sepharose purification were dialyzed against the application buffer containing 50 mM Tris-HCl, pH 7.5, 200 mM NaCl, and 0.05% Tween 20. After applying the sample, the immunoaffinity column was washed extensively with the application buffer. DP47 was eluted with 100 mM glycine-HCl, pH 2.7. It was directly collected into tubes containing the neutralizing buffer (1 M Tris-HCl, pH 9.0, and 0.05% NaN_3).

Fig. 1 shows the results of the expression and purification procedures for DP47. It can be seen that DP47 has been substantially purified from the E. coli lysate supernatant in an abundance suitable for functional analysis.

Functional Studies of DP47

The possible ATP binding function of the purified DP47 has been investigated with various assays. One of the assays was designed to test whether ATP could prevent the labeling of DP47 by FITC. DP47 was incubated with 10 μM FITC at pH ~ 9.2 in the presence or absence of 1 mM ATP in the dark at room temperature for 30 min. The reaction mixtures were run on an SDS-polyacrylamide gel and viewed under UV light. In some preparations of DP47, partial protection (~ 50%) by ATP against FITC labeling was observed. In other DP47 preparations, however, no difference in the FITC labeling between –ATP and +ATP reactions was detected.

Inconsistent results were also obtained with DP47 in an eosin fluorescence assay. Eosin is a fluorescent ATP binding site probe for Na,K-ATPase (Skou and Esmann, 1981). When incubating 1 μM eosin with Na,K-ATPase in the presence of Na, the fluorescent signal was enhanced, and after the subsequent addition of micromolar concentrations of ATP it fell to the level of eosin without the enzyme. A similar but smaller response was observed in some DP47 preparations. Eosin fluorescence was increased after addition of DP47 and was reduced after ATP addition. In other DP47 preparations, however, no change of fluorescence was observed when ATP was added to the eosin solution containing DP47. DP47 preparations that initially showed a positive response to ATP in both the FITC labeling experiment and the eosin fluorescence assay eventually lost the response after several cycles of freezing and thawing.

Figure 1. Purification of DP47 from E. coli. E. coli cells were transformed with plasmid pK1.3, grown in nutrient broth, and lysed as described in the text. Aliquots of the lysate supernatant (lanes 1), the immunopositive pool after QAE-Sepharose chromatography (lanes 2), and the immunopositive pool after immunoaffinity chromatography (lanes 3) were fractionated by SDS-PAGE. The Coomassie blue-stained gel is shown on the left (150 μg protein in lanes 1 and 2; 20 μg in lane 3) and the Western blot is shown on the right (15 μg protein in lanes 1 and 2; 2 μg in lane 3). Molecular weight markers in the Coomassie-stained gel are phosphorylase (94 kD), bovine serum albumin (68 kD), ovalbumin (45 kD), carbonic anhydrase (29 kD), soybean trypsin inhibitor (21.5 kD), and lysozyme (14 kD). For the immunoblot, Na,K-ATPase was digested with trypsin in the presence of KCl to generate fragments (E/Try) of 94, 58, and 41 kD that were used as molecular weight markers.

Conclusions

DP47 is expressed in E. coli JM105 carrying pK1.3 after IPTG induction. The protein is soluble and is located in the E. coli lysate supernatant. The expression level of DP47 was improved when the temperature of the growth medium was changed from 37 to 30°C after IPTG addition. DP47 has been purified to near homogeneity in two steps using ion exchange chromatography on QAE-Sepharose and immunoaffinity chromatography. Preliminary experiments indicate that DP47 may not be folded into a stable conformation capable of binding ATP with high affinity. Additional assays of both ATP binding and of DP47 conformation are in progress to test this hypothesis.

Acknowledgments

This work was supported by NIH grants GM-28673 and HL-39295, and American Heart Association, Greater Los Angeles Affiliate, award 885 F1–2.

References

Farley, R. A., C. M. Tran, C. T. Carilli, D. Hawke, and J. E. Shively. 1984. The amino acid sequence of a fluorescein-labeled peptide from the active site of (Na,K)-ATPase. Journal of Biological Chemistry. 259:9532–9535.

Marston, F. A. O. 1987. The purification of eukaryotic polypeptides expressed in Escherichia coli. In DNA Cloning. Vol. III. D. M. Glover, editor. IRL Press, Oxford, UK. 59–88.

Ohta, T., K. Nagano, and M. Yoshida. 1986. The active site structure of Na+/K+-transporting ATPase: location of the 5'-(p-fluorosulfonyl)benzoyladenosine binding site and soluble peptides released by trypsin. Proceedings of the National Academy of Sciences, USA. 83:2071–2075.

Ovchinnikov, Y. A., K. N. Dzhandzugazyan, S. V. Lutsenko, A. A. Mustayev, and N. N. Modyanov. 1987. Affinity modification of Ei form of Na+,K+-ATPase revealed Asp-710 in the catalytic site. FEBS Letters. 217:111–116.

Shull, G. E., A. Schwartz, and J. B. Lingrel. 1985. Amino-acid sequence of the catalytic subunit of the (Na+,K+)ATPase deduced from a complementary DNA. Nature. 316:691–695.

Skou, J. C., and M. Esmann. 1981. Eosin, a fluorescent probe of ATP binding to the (Na+ + K+)-ATPase. Biochimica et Biophysica Acta. 647:232–240.

Taylor, W. R., and N. M. Green. 1989. The predicted secondary structures of the nucleotide-binding sites of six cation-transporting ATPases lead to a probable tertiary fold. European Journal of Biochemistry. 179:241–248.

Tran, C. M., G. Scheiner-Bobis, W. Schoner, and R. A. Farley. 1988. The amino acid sequence of a peptide from the ATP-binding site of Na,K-ATPase labeled by 8-N3-ATP. Biophysical Journal. 53:343a. (Abstr.)

Localization of Na,K-ATPase α- and β-Subunits Expressed in Xenopus Oocytes

Kazuo Takeda, Shunsuke Noguchi, and Masaru Kawamura

Department of Biology, University of Occupational and Environmental Health, Kitakyushu, Japan

Introduction

It is proposed that β-subunit of Na,K-ATPase (NKA) possesses a receptor-like function for the insertion of α-subunit into the endoplasmic reticulum (ER) membrane (Zamofing et al., 1988; Noguchi et al., 1990b). Another possible role of β-subunit is to act as a carrier targeting NKA from the ER membrane to the cell surface. Here we employed an immunohistochemical approach to studying the function of β-subunit by using the expression system of Xenopus oocytes.

Materials and Methods

Expression of Subunits in Xenopus Oocytes

mRNAs were synthesized in vitro by using SP6 RNA polymerase from cDNAs of NKA α- and β-subunits of Torpedo californica. mRNAa or mRNAβ (30–40 ng/oocyte) or mRNAα plus mRNAβ (10–13 ng/oocyte) were injected into Xenopus laevis oocytes (stages IV or V), respectively. Oocytes were incubated at 19°C for 3 d.

Affinity Purification of Antiserum

Rabbit anti–α- and anti–β-subunit sera were purified by a blot-affinity purification method (Wedlich, 1989). Purified α- or β-subunits were blotted on the nitrocellulose membrane. The membrane was incubated with periodate-lysine-paraformaldehyde fixative (containing 0.01 M NaIO_4, 0.075 M lysine, and 2% paraformaldehyde in 0.0375 M phosphate buffer) for 10 min, followed by immersion with diluted anti–α- or anti–β-subunit sera at 25°C for 18 h. The bound antibodies were eluted in glycine (0.1 M, pH 2.8) for 5 min. The eluate was immediately neutralized with Tris base.

Immunohistochemistry

Oocytes that had been injected with mRNAs were fixed with periodate-lysine-paraformaldehyde fixative at 4°C for 6–8 h, frozen, and cryosectioned longitudinally parallel to the cell axis. Sections with ~ 10 μm were stained with affinity purified antibodies at 4°C for 16 h, followed by staining with the biotin-conjugated secondary antibody at room temperature for 2 h. The subunits were visualized as brown precipitation of diaminobenzidine by using avidin-biotin-peroxidase complex.

Figures 1–3. Oocytes injected with mRNAα alone (1, a-c), mRNAβ alone (2, a-c), or mRNAα plus mRNAβ (3, a-c) were cryosectioned longitudinally and stained with affinity purified anti–α-subunit antibody (1 a, 2 a, 3 a) or with anti–β-subunit antibody (1 b, 2 b, 3 b), or without primary antibody (1 c, 2 c, 3 c). The large number of yolk platelets are seen in these photographs. The particles near the cell surface are pigment granules. The precipitations of diaminobenzidine are densely seen in these photographs. Magnification, ×260.

Results

Affinity purified antibodies against α- and β-subunit of Torpedo NKA were applied to the sections of oocytes into which mRNA had not been injected. Positive staining was hardly observed in these sections (data not shown), indicating that these antibodies did not crossreact with the intrinsic subunits of NKA of Xenopus oocytes.

When mRNAα was injected alone, α-subunit was detected in the cytoplasm but not on the cell surface (Fig. 1 a). On the other hand, for the oocyte injected with mRNAβ alone, β-subunit was detected not only in the cytoplasm but also on the cell surface (Fig. 2 b).

When mRNAs for α- and β-subunits were injected simultaneously, both subunits were detected on the cell surface as well as in the cytoplasm (Fig. 3, a and b). The alternate staining of the serial sections with anti–α- or anti–β-antibodies revealed that both subunits were similarly distributed in these oocytes.

These results suggest that β-subunit of NKA acts as a carrier for α-subunit from the ER membrane to the cell surface to form αβ complex.

Discussion

Geering et al. (1989) have claimed that the fully maturated Xenopus oocytes synthesized α-subunit in excess over β-subunit and that the excess α-subunit can assemble with β-subunit to form active αβ complex when only mRNAβ is injected into oocytes. It seems likely that this active αβ complex can be transported to the cell surface. We could not, however, detect any positive staining with antibody to α-subunit on the cell surface when Torpedo mRNAβ was injected. This is probably because of non-crossreactivity of our antibody to Torpedo α-subunit with α-subunit of Xenopus. The hybrid molecules between Xenopus α-subunits and Torpedo β-subunits should be synthesized in this, because ATPase activity was increased slightly when Torpedo mRNAβ alone was injected into Xenopus oocytes (Noguchi et al., 1990a).

It has been reported that the exogenous Torpedo α-subunit is inserted into Xenopus membrane in an aberrant fashion in the absence of β-subunit (Noguchi et al., 1990c), and it cannot assemble with β-subunit synthesized later in the same oocyte (Noguchi et al., 1990b). This may explain why Torpedo α-subunit remains in the cytoplasm when expressed alone (Fig. 1 a). The excess Xenopus α-subunit mentioned above could be synthesized in a different way than the exogenous α-subunit.

According to Geering (1990), β-subunit remains in the cytoplasm so far as it does not assemble with α-subunit. Although the β-subunits on the cell surface of oocytes injected with mRNAβ alone derived partly from the hybrid molecule between Xenopus α-subunit and Torpedo β-subunit, the following observations support our conclusion that β-subunit can be largely transported to the cell surface without forming the complex with α-subunit: (a) Iodination of oocytes from their outer cell surface have revealed that the amounts of the fully glycosylated β-subunit on the cell surface were not significantly different from oocytes injected with mRNAβ alone and with mRNAα plus mRNAβ. Nevertheless, ouabain binding activity of the oocyte injected with mRNAα plus mRNAβ is much higher than that injected with mRNAβ alone, suggesting that the oocytes injected with mRNAβ alone contain β-subunit free from α-subunit on their cell surface (Takeda, 1988). (b) The deletion mutant of β-subunit, which lacks 72 amino acid residues from its COOH-terminus can no longer assemble with α-subunit, but the mutant is still partly transported to the cell surface (Noguchi, S., and M. Kawamura, manuscript in preparation).

References

Geering, K. 1990. Subunit assembly and functional maturation of Na,K-ATPase. Journal of Membrane Biology. 115:109–121.

Geering, K., I. Theulaz, F. Verrey, M. T. Häuptle, and B. C. Rossier. 1989. A role for the β-subunit in the expression of functional Na+-K+-ATPase in Xenopus oocytes. American Journal of Physiology. 257:C851–C858.

Noguchi, S., K. Higashi, and M. Kawamura. 1990a. A possible role of the β subunit of (Na,K)ATPase on the biogenesis of the enzyme. Journal of University of Occupational and Environmental Health. 12:67–75.

Noguchi, S., K. Higashi, and M. Kawamura. 1990b. Assembly of the α-subunit of Torpedo californica Na+/K+-ATPase with its pre-existing β-subunit in Xenopus oocytes. Biochimica et Biophysica Acta. 1023:247–253.

Noguchi, S., K. Higashi, and M. Kawamura. 1990c. A possible role of the β-subunit of (Na,K)ATPase in facilitating correct assembly of the α-subunit into the membrane. Journal of Biological Chemistry. 265:15991–15995.

Takeda, K., S. Noguchi, A. Sugino, and M. Kawamura. 1988. Functional activity of oligosaccharide-deficient (Na,K)ATPase expressed in Xenopus oocytes. FEBS Letters. 238:201–204.

Wedlich, D. 1989. Method for epitope selection of antisera for immunohistology. The Journal of Histochemistry and Cytochemistry. 37:273–276.

Zamofing, D., B. C. Rossier, and K. Geering. 1988. Role of the Na,K-ATPase β-subunit in the cellular accumulation and maturation of the enzymes as assessed by glycosylation inhibitors. Journal of Membrane Biology. 104:69–79.

Functional Expression of the Na,K ATPase in Spodoptera frugiperda Cells

Anthony W. De Tomaso, Peter Zdankiewicz, and Robert W. Mercer

Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110

Introduction

Expression systems for the Na,K ATPase have been greatly hindered by the fact that the most commonly used cell lines have high endogenous levels of the Na pump. In contrast, it has been shown that some insect cells have little or no Na,K ATPase activity (Harvey et al., 1983). A recently designed expression system uses the baculovirus Autographica californica to express foreign genes in insect cells. In this system, a foreign gene is inserted in place of the natural polyhedrin gene. The polyhedrin gene product is expressed at extremely high levels late in infection (> 48 h) and is nonessential for viral replication and infection. When recombinant gene expression is directed by the strong polyhedrin promoter, recombinant proteins have been produced at levels up to 500 mg/liter infected insect cells. Using this system, several foreign, biologically active proteins have been characterized. Moreover, it has been shown that the insect cells carry out most of the posttranslational modifications that normally occur in mammalian cells. The system has been used to successfully express multisubunit proteins, and to properly segregate mammalian proteins to their correct cellular domains (Luckow, 1991).

We have made two separate recombinant baculoviruses containing the rodent Na,KATPase α1 and β cDNAs and used them to infect a cell line (Sf-9) derived from the ovary of the fall armyworm, Spodoptera frugiperda.

Methods

Recombinant Baculovirus

cDNAs containing the complete coding regions of the rodent α1 and β Na,K ATPase subunits were introduced into the baculovirus expression vector and used to infect Sf-9 cells according to Summers and Smith (1987). In all experiments cells were assayed 3 d after infection unless stated otherwise.

Expression of Recombinant Protein

RNA blot hybridization, SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and immunoblotting were done as previously described (Mercer et al., 1986).

Transport Activity

Sf-9 cells growing in suspension were gently centrifuged (1,000 g) and resuspended at a density of 1 × 10^7 cells/ml in preincubation media (130 mM NaCl, 50 mM HEPES, and 20 mM sucrose, pH 7.4) in the presence or absence of 1 mM ouabain and/or 100 μM bumetanide. After a 10-min incubation at room temperature, an equal volume of flux medium (130 mM NaCl, 5 mM KCl, 5 mM MgCl_2, 20 mM sucrose, 50 mM HEPES, and 0.1 mM EDTA, pH 7.4, final concentrations) with 86Rb (1 μCi/ml) as tracer and the same concentrations of inhibitors was added. At prescribed intervals, 300-μl aliquots (1.5 × 10^6 cells) were removed and layered on 0.5 ml silicone oil (Versilube F-50; General Electric, Waterford, NY) in a 1.5-ml microcentrifuge tube and centrifuged for 1 min. Cell separation occurred in < 5 s. The aqueous and oil phases were removed by aspiration and discarded. The cell pellets were excised and counted by liquid scintillation. Each time point is the average of three replicates, error bars representing one standard deviation.

Immunofluorescence

Uninfected log phase Sf-9 cells were allowed to attach on ethanol-washed glass coverslips (No. 1) for 1.5 h. 10 μl of viral stock (multiplicity of infection = 10) was added and the cells were incubated for 1 h at room temperature. After 3 d of growth in fresh medium, cells were fixed in 4% paraformaldehyde in PIPES buffer (25 mM PIPES, 50 mM KCl, 2 mM MgSO_4, 1 mM EGTA, and 0.2 mM DTT, pH 7.0) for 1 h at 4°C. Cells were washed three times in PBS, and excess fixative was quenched by incubation in PBS containing 100 mM glycine for 15 min. Cells were permeabilized with 0.1% Triton X-100 in PBS for 15 min, washed three times with PBS, and incubated with 2% goat serum in PBS for 20 min. After three washes with PBS, coverslips were incubated overnight with 1:250 antiserum in 0.2% goat serum at 4°C. Cells were washed for 15 min with three changes of PBS and then incubated for 50 min with 5 μg/ml fluorescein isothiocyanate (FITC)–conjugated goat anti–rabbit secondary antibody (Cappel Laboratories, West Chester, PA). The cells were then washed for 15 min with PBS and mounted in 50% glycerol containing 1% n-propyl-gallate.

Coverslips were examined with a Zeiss Axioplan microscope equipped with a Bio-Rad MRC-500 laser confocal imaging system. Images were averaged over eight scans. Images were photographed directly from a NEC multisync II monitor.

Results and Discussion

While Sf-9 cells are the cells of choice for expression using baculovirus (Luckow, 1991), they have never been well characterized in terms of ion flux. Fig. 1 shows that potassium uptake is linear for 8 min, and that the majority (> 70%) of uptake is bumetanide sensitive. This bumetanide-sensitive uptake was found to be highly dependent on external Na and Cl ions (data not shown). This strongly suggests that the majority of potassium influx in uninfected Sf-9 cells is mediated by the diuretic-sensitive Na,K,2Cl cotransporter.

A small component of the total potassium uptake appears to be ouabain sensitive. This ouabain sensitivity was not apparent unless cells were preincubated in 1 mM ouabain for 10 min in a potassium-depleted medium. We conclude that there is a small amount of endogenous Na,K ATPase in uninfected Sf-9 cells.

Sf-9 cells were infected with α1,β, or coinfected with α1 and β recombinant baculovirus. Total RNA was isolated 3 d after infection and probed with rodent α1 and β cDNAs. Infected Sf-9 cells express α1 and β mRNAs when infected with the corresponding virus. Coinfected cells express both messages (data not shown). No endogenous Na,K ATPase mRNA was detected in uninfected controls. Fig. 2 shows that these messages are translated to immunoreactive polypeptides. Immunoblotting of cell plasma membrane fractions using a Na,K ATPase antiserum identifies α1 polypeptides. When comparing equivalent amounts of plasma membrane protein, the quantity of Na,K ATPase polypeptides expressed in baculovirus-infected cells is comparable to levels in the rat kidney.

Figure 1. Potassium uptake in uninfected Sf-9 cells. Potassium uptake was measured using 86Rb as a tracer. The majority of potassium uptake (> 70%) is bumetanide sensitive.

Next we analyzed the distribution of the α1 polypeptide in α1-infected and α1- and β-coinfected cells using confocal microscopy. Uninfected cells show little reactivity to the Na,K ATPase antisera. However, infected cells show a majority of the immunoreactive protein at the plasma membrane in both α1-infected and α1-and β-coinfected cells (data not shown). Thus, it appears that the rodent α1-subunit is targeted to the plasma membrane in the absence of rodent β-subunit.

Figure 2. Immunoreactivity of Sf-9 cells to Na,K ATPase antiserum. 20 μg of plasma membrane protein was separated by SDS-PAGE, electroblotted to nylon, and probed with Na,K-ATPase antiserum.

Finally, we investigated whether infected Sf-9 cells produced functionally active Na,K ATPase polypeptides. Fig. 3 shows the results from potassium uptake experiments that demonstrate an increase in ouabain-sensitive potassium uptake in Sf-9 cells coinfected with α1 and β recombinant virus 3 d after infection. No such increase was observed when cells were infected with each subunit alone. This suggests that both α1- and β-subunits are required for an increase in ouabain-sensitive potassium influx. The fact that infection with α1 and β virus alone did not increase ouabain-sensitive uptake indicates that the small amount of endogenous Na,K ATPase polypeptides did not contribute to activity.

Figure 3. Functional expression of Na,K ATPase. Ouabain-sensitive potassium uptake in infected Sf-9 cells was measured. Results were normalized to ouabain-sensitive flux in uninfected cells. There is an almost fourfold stimulation in ouabain-sensitive flux 3 d after infection in α1, β-coinfected cells. This is not seen 2 d after infection, nor is it seen with either subunit infected alone.

Preliminary experiments have demonstrated that recombinant baculovirus-infected Sf-9 cells produce functionally active Na,K ATPase polypeptides. They also have demonstrated that the rodent α1 polypeptide is targeted to the plasma membrane independent of the rodent β-subunit. However, functional activity (potassium uptake) appears to require both the α1- and β-subunits.

References

Harvey, W. R., M. Cioffi, and M. G. Wolfersberger. 1983. Chemiosmotic potassium pump of insect epithelia. American Journal of Physiology. 244:R163–R175.

Luckow, V. A. 1991. Cloning and expression of heterologous genes in insect cells with baculovirus vectors. In Recombinant DNA Technology and Applications. C. S. Ho, A. Prokop, and R. K. Bajpai, editors. McGraw-Hill, New York. 97–152.

Mercer, R. W., J. W. Schneider, A. Savitz, J. Emanuel, E. J. Benz, Jr., and R. Levenson. 1986. Rat brain Na,K ATPase β-subunit gene: primary structure, tissue-specific expression and amplification in ouabain-resistant HeLa cells. Molecular and Cellular Biology. 6:3884–3890.

Summers, M. D., and G. E. Smith. 1987. A manual of methods for baculovirus vectors and insect cell culture procedures. Texas Agricultural Experimental Station Bulletin. 55:1–56.

Cellular Mechanisms in the Induction of Na,K-ATPase Subunit mRNAs by Serum and Growth Factors in Clone 9 Cells

Alok Bhutada and Faramarz Ismail-Beigi

Department of Medicine, College of Physicians and Surgeons, Columbia University, New York 10032

Introduction

In a recent study on a “nontransformed” rat-liver cell line, Clone 9, we found that addition of serum to quiescent cells increased the abundance of Na,K-ATPase mRNAα1 and mRNAβ1 at 3 h by 2- and 2.7-fold, respectively, and that the effect was followed by increases in Na,K-ATPase activity, and in protein, RNA, and DNA content (Bhutada et al., 1990). In this study we examine the potential role of activation of protein kinase C (PKC) as a mediating mechanism in the induction of Na,K-ATPase mRNAα1 and mRNAβ1 by serum in Clone 9 cells. In addition, the effects of epidermal growth factor (EGF), platelet-derived growth factor (PDGF), basic fibroblast growth factor (FGF), insulin, dexamethasone, aldosterone, and dibutyryl cyclic AMP on the abundance of the subunit mRNAs are examined in control cells and in cells “depleted” of PKC activity.

Methods

Cell Culture

Clone 9 cells were maintained in 100-mm plastic culture plates in Dulbecco's modified Eagle's medium (DME medium) containing 10% calf serum as described previously (Bhutada et al., 1990). Confluent cells were preincubated in DME medium devoid of serum for 24 h before all experiments.

Measurement of Protein, RNA, and DNA

Previously described methods were used without modification (Bhutada et al., 1990).

Phorbol Dibutyrate Binding

Minor modifications were made to previously described methods (Kaibuchi et al., 1989). Confluent cells preincubated in DME medium for 24 h in the presence of 160 nM TPA or 10 μl of dimethylsulfoxide (Me_2SO, diluent) were rinsed three times with 5 ml of phosphate-buffered saline (PBS) at 37°C over a 10-min period. Cells were then incubated for 1 h in 1 ml of DME medium in the presence or absence of 10 μM phorbol dibutyrate and various concentrations of [3H]phorbol dibutyrate in triplicate ranging from 3.75 to 300 nM (specific activity 5 Ci/mmol). The data were fitted to either a one- or two-site ligand-binding computer model using nonlinear regression and “explicit weighting” of the standard deviations at each of the experimental points to derive the best fit (Enzfitter, Biosoft, Milltown, NJ).

Isolation and Analysis of Cytoplasmic RNA

Cytoplasmic RNA was isolated by methods utilizing an NP-40-containing lysis buffer (Maniatis et al., 1982; Bhutada et al., 1990). Analysis of the abundances of RNAα1 and mRNAβ1 was performed by Northern blotting. Northern blots were hybridized with ~30 × 10^6 cpm of full-length rat Na,K-ATPase cDNAα1 or cDNAβ1 inserts 32P-labeled by nick-translation to ~ 5 × 10^8 cpm/μg DNA (Shull et al., 1985,1986).

Statistical Analysis

All values are expressed as means ± SE. To compare two groups, P values were calculated by Student's unpaired two-tailed t test (Alder and Roessler, 1968). Analysis of variance using the least significant difference was used in cases involving more than two groups.

Results and Discussion

Addition of fresh serum to confluent Clone 9 cells results in an increase in Na,K-ATPase activity that is preceded by an increase in the content of both subunit mRNAs encoding the enzyme, mRNAα1 and mRNAβ1 (Bhutada et al., 1990). Because many actions of serum and associated growth factors are mediated by activation of PKC, we explored the possibility that the induction of Na,K-ATPase subunit mRNAs by serum is likewise mediated by PKC activation (Nishizuka, 1986). We therefore determined whether the increments in subunit mRNA abundances occurred in cells that had been “depleted” of PKC activity by prolonged preincubation in the presence of high concentrations of the phorbol ester, 12-O-tetradecanoyl phorbol 13-acetate (TPA) (Jaken et al., 1981; Rodriguez-Pena and Rozengurt, 1984; Nishizuka, 1986; Blackshear et al., 1988). The efficacy of this technique for “depleting” PKC was examined by measuring the number and affinity of [3H]phorbol dibutyrate binding sites in Clone 9 cells preincubated in the presence or absence of 160 nM TPA for 24 h. The Scatchard plot for control cells was curvilinear and could be resolved into a low-capacity, high-affinity site (Bmax of 1.2 pmol/mg protein; Kd of 5 nM) and a high-capacity site with markedly lower affinity. In contrast, cells pre-exposed to TPA for 24 h expressed only the low-affinity site. Examination of the effect of serum on the induction of subunit mRNAs in control and PKC-depleted cells showed that in control cells, the abundance of both subunit mRNAs was significantly increased at 6 h after addition of either serum or TPA. In contrast, addition of TPA to PKC-depleted cells showed no increase in mRNAα1 and mRNAβ1 abundances, suggesting that the acute stimulatory effect of TPA on the subunit mRNA contents had subsided after the 24-h preincubation period and that the cells were functionally depleted of PKC activity. Serum, however, still increased the abundance of both mRNAα1 and mRNAβ1 in these PKC-depleted cells to values similar to those observed in control cells. These results suggest that the induction by serum is predominantly mediated by pathways other than PKC activation and raise the possibility that the effects of TPA and serum might be additive. To explore this issue, we added serum, TPA, or both to cells preincubated in DME alone. The stimulatory effect of serum and TPA on mRNAα1 content was nearly additive (2.7-fold increment observed versus calculated increment of 2.9-fold for additive effects). In contrast, the combined addition of serum and TPA increased mRNAβ1 content 2.9-fold, a value that was intermediate between the 3.9- and 1.6-fold increments observed in serumand TPA-treated cells, respectively.

We next examined the effects of EGF, PDGF, FGF, insulin, dexamethasone, aldosterone, and dibutyryl cyclic AMP on the subunit mRNA abundances. Additionally, the response to hypothyroid serum was determined because T_3 induces Na,K-ATPase and the subunit mRNAs in these cells (Gick and Ismail-Beigi, 1990). Thyroid hormone–deficient calf serum was nearly as effective as calf serum in the induction of the mRNAs, and EGF, PDGF, and FGF all increased the subunit mRNA contents. Insulin had a stimulatory effect on both mRNA abundances, whereas dibutyryl cyclic AMP had no effect. Dexamethasone (10-8 M) increased mRNAα1 and mRNAβ1 abundances by 1.6- and > 30-fold, respectively, whereas a similar concentration of aldosterone had no effect on the subunit mRNA abundances. Probing the Northern blots with cDNAβ2 failed to show any expression of mRNAβ2 under any of the experimental conditions examined.

Since the stimulatory effects of the above agents on the subunit mRNA abundances might be mediated by activation of protein kinase C, we additionally examined the effect of the above factors and agents in PKC-depleted cells. As noted above, serum was fully effective in inducing the subunit mRNAs in cells pretreated with TPA. EGF, FGF, insulin, and dexamethasone significantly increased both mRNAα1 and mRNAβ1 contents in PKC-depleted cells to values comparable to those obtained in control cells, whereas the stimulatory effect of PDGF, especially on mRNAα1, was blunted. Finally, insulin augmented subunit mRNA abundances both in control cells and in cells depleted of PKC activity, a finding consistent with a PKC-independent mode of action of this hormone (Simpson and Cushman, 1985).

References

Alder, H. L., and E. B. Roessler. 1968. The analysis of variance, one criterion of classification. In Introduction to Probability and Statistics. W. H. Freeman and Company, San Francisco. 260–288.

Bhutada, A., C. Perez, D. Y. Chon, and F. Ismail-Beigi. 1990. Induction of Na+-K+-ATPase and its subunit mRNAs by serum in a rat liver cell line. American Journal of Physiology. 258 (Cell Physiology 27):C1044–C1050.

Blackshear, P. J., A. C. Nairn, and J. F. Kuo. 1988. Protein kinases: A current perspective. FASEB Journal. 2:2957–2969.

Gick, G. G., and F. Ismail-Beigi. 1990. Thyroid hormone induction of Na+-K+-ATPase and its mRNAs in a rat liver cell line. American Journal of Physiology. 258 (Cell Physiology 27): C544–C551.

Jaken, S., A. H. Tashjian, and P. M. Blumberg. 1981. Characterization of phorbol ester receptors and their down-modulation in GH_4C_1 rat pituitary cells. Cancer Research. 41:2175–2181.

Kaibuchi, K., Y. Fukumoto, N. Oku, Y. Hori, T. Yamamoto, K. Toyoshima, and Y. Takai. 1989. Activation of the serum response element and 12-O-tetra-decanoylphorbol-13 acetate response element by the activated c-raf-1 protein in a manner independent of protein kinase C. Journal of Biological Chemistry. 264:20855–20858.

Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Enzymes used in molecular cloning. In Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. 97–148.

Nishizuka, Y. 1986. Studies and perspectives of protein kinase C. Science. 233:305–312.

Rodriguez-Pena, A., and E. Rozengurt. 1984. Disappearance of Ca2+-sensitive, phospholipid-dependent protein kinase activity in phorbol ester-treated 3T3 cells. Biochemical and Biophysical Research Communications. 120:1053–1059.

Shull, G. E., L. K. Lane, and J. B. Lingrel. 1986. Amino-acid sequence of the β-subunit of the (Na+ +K+)ATPase deduced from a cDNA. Nature. 321:429–431.

Shull, G. E., A. Schwartz, and J. B. Lingrel. 1985. Amino-acid sequence of the catalytic subunit of the (Na+ +K+)ATPase deduced from a complementary DNA. Nature. 316:691–695.

Simpson, I. A., and S. W. Cushman. 1985. Regulation of glucose transporter and hormone receptor cycling by insulin in the rat adipose cell. Current Topics in Membranes and Transport. 24:459–503.

Serum-independent Induction of Na,K-ATPase and c-fos by Low K+ in a Rat Liver Cell Line

E. Cayanis, J. J. Russo, Y.-S. Wu, and I. S. Edelman

Department of Biochemistry and Molecular Biophysics, Columbia University College of Physicians & Surgeons, New York 10032

Introduction

Incubation of confluent adult rat liver cells with low extracellular K+ (< 1 mM) increased the Na,K-ATPase specific activity and mRNA (α and β) content (Pressley et al., 1986, 1988; Bowen and McDonough, 1987; Ismail-Beigi et al., 1988). The mRNAs encoding the α- and β-subunits of Na,K-ATPase increased at 6 h, preceding the increase in Na,K-ATPase abundance at 12–24 h (Pressley et al., 1986, 1988). This implies that the Na,K-ATPase response to low K+ is regulated at the genetic level and that some of the early events in the responsiveness of these cells to low K+ could be mediated via a cascade of earlier nuclear protooncogene products.

This study explored the dependence of the low K+ response on serum factors. Furthermore, as serum and serum growth factors induce the nuclear protooncogenes, c-ros and c-myc (Curran et al., 1985; Grinstein and Rothstein, 1986), the effects of low K+ on these were also investigated.

Methods

ARL_15 cells (passage 25–27) were grown to confluency in Williams medium E supplemented with 10% calf serum in 100-mm Corning culture plates at 37°C in a 5% CO_2 atmosphere. The plates were washed twice with 10 ml Williams “K+ free”medium before replacing the medium with either Williams E solution or Williams E medium containing 5% calf serum, 1% BSA, and K+ concentrations of either 0.68 mM (low K) or 4 mM (control) for the required time interval. Crude homogenates were prepared by washing cell monolayers twice with 10 ml ice-cold homogenization buffer (HB; 50 mM Tris-Cl, pH 7.5, 1.25 mM EGTA, and 250 mM sucrose), scraping into 2 ml HB with a rubber policeman, centrifuging for 6 min at 2,000 rpm, and homogenizing with an Elvehjem homogenizer. The Na,K-ATPase assays were performed by the method of Pressley et al. (1986). The amount of inorganic phosphate released was measured by the colorimetric method of Baginski et al. (1967).

The steady-state levels of specific mRNAs were analyzed by Northern blotting using established methods (Chirgwin et al., 1979; Pressley et al., 1988). The specific cDNA probes were purified by digestion with appropriate restriction endonucleases, electrophoresis in 1% low melting point agarose gels, and electroelution.

Results and Discussion

10% calf serum, 1% bovine serum albumin, 1% ovalbumin, and 1% human serum albumin had no effect on the Na,K-ATPase activity (Vmax) of confluent ARL_15 cells. It is of interest that clone 9 cells, also of rat liver origin, did exhibit increases in active transport of K+ mediated by the Na/K pump in response to serum (Bhutada et al., 1990).

When confluent ARL_15 cells were incubated in Williams E medium containing a low concentration of K+ (0.68 mM) for 24 h, the Na,K-ATPase activity increased by 57%. Depletion of serum in the growth medium before the low K+ challenge did not blunt the increase. These findings show that serum and serum factors do not mediate the induction of Na,K-ATPase by low extracellular K+ concentration in confluent ARL_15 cells.

To delineate some of the early events in the response to low K+, the mRNA levels of several nuclear protooncogenes at various times during incubation in serum-free, normal K+, and low K+ media were estimated. C-myc and c-jun mRNAs were relatively unresponsive during a 15-min to 2-h time course, and c-ski showed only slight increases at 2 h. The most prominent response was a marked increase in c-fos mRNA 15–30 min after low K+ exposure. This increase in c-fos in the absence of serum was also elicited by serum alone and by 93.7 ng/ml of phorbol-1,2-myristate-13-acetate (PMA).

The induction of c-fos mRNA by low K+, serum, and PMA at an earlier time point (30 min) than the visible increase in the Na,K-ATPase α- and β-subunit mRNAs at 6 h (Pressley et al., 1988) makes it a candidate mediator in the signal-transducing pathway from low K+ challenge to enhanced Na,K-ATPase activity. During low K+ treatment there is a net loss of intracellular K+ and a net gain of intracellar Na+ (Graves and Wheeler, 1982; Pressley et al., 1986). Hence the present findings are consistent with the report that Na+ deprivation inhibits induction of c-fos expression by epidermal growth factor in cultured hepatocytes (Krüijer et al., 1986).

References

Baginski, E. S., P. P. Foa, and B. Zak. 1967. Microdetermination of inorganic phosphate, phospholipids and total phosphate in biologic material. Clinical Chemistry. 13:326–333.

Bhutada, A., C. Perez, D. Y. Chon, and F. Ismail-Beigi. 1990. Induction of Na+,K+-ATPase and its subunit mRNAs by serum in a rat liver cell line. American Journal of Physiology. 258:C1044–C1050.

Bowen, J. W., and A. McDonough. 1987. Pretranslational regulation of Na+,K+-ATPase in cultured canine kidney cells by low K+. American Journal of Physiology. 252:C179–189.

Chirgwin, J. M., A. E. Przybyla, R. J. MacDonald, and W. J. Rutter. 1979. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry. 18:5294–5299.

Curran, T., R. Bravo, and R. Müller. 1985. Transient induction of c-fos and c-myc is an immediate consequence of growth factor stimulation. Cancer Surveys. 4:655–681.

Graves, J. S., and D. D. Wheeler. 1982. Increase in K+ and α-AIB active transport in CHO cells after low [K+] treatment. American Journal of Physiology. 243:C124–C132.

Grinstein, S., and A. Rothstein. 1986. Mechanisms of regulation of the Na+/H+ exchanger. Journal of Membrane Biology. 90:1–12.

Ismail-Beigi, F., T. A. Pressley, R. S. Haber, G. G. Gick, J. N. Loeb, and I. S. Edelman. 1988. Kinetic analysis of Na,K-activated adenosine triphosphatase induced by low external K+ in a rat liver cell line. Journal of Biological Chemistry. 263:8162–8167.

Krüijer, W., H. Skelley, F. Botteri, H. van der Putten, J. R. Barber, I. M. Verma, and H. L. Leffert. 1986. Protooncogene expression in regenerating liver is stimulated in cultures of primary adult rat hepatocytes. Journal of Biological Chemistry. 261:7929–7933.

Pressley, T. A., R. S. Haber, J. N. Loeb, I. S. Edelman, and F. Ismail-Beigi. 1986. Stimulation of Na,K-activated adenosine triphophatase and active transport by low external K+ in a rat liver cell line. Journal of General Physiology. 87:591–606.

Pressley, T. A., F. Ismail-Beigi, G. G. Gick, and I. S. Edelman. 1988. Increased abundance of Na+,K+-ATPase mRNAs in response to low external K+. Cell Physiology. 24:C252–260.

Low K+ Stimulates Differential Regulation of Na,K-ATPase Subunits in Kidney Cells

Leslie Lescale-Matys, Ming-Jer Tang, and Alicia A. McDonough

Department of Physiology and. Biophysics, University of Southern California School of Medicine, Los Angeles, California 90033

Introduction

When cells are placed in an environment in which the K+ concentration is below the Na,K-ATPase K0.5 for K+, the sodium pump rate will decrease to the point where, if K+ is low enough, there will be a failure of ionic homeostasis in the cell: intracellular sodium will rise and potassium will fall, and the processes that depend on these gradients will be inhibited. To restore ionic homeostasis, cells can compensate by increasing the number of pumps in the plasma membrane, which will increase Na+ and K+ pumping in spite of the fact that the individual pump turnover rate is inhibited. In this report we will focus on the mechanisms used by cultured kidney cells in low K+ to increase sodium pump abundance. In particular, we will illustrate how the application of compartmental analysis to the problem of increasing abundance of Na,K-ATPase revealed key levels of ionic regulation.

Our working model of Na,K-ATPase subunit biosynthesis and assembly is illustrated in Fig. 1. Starting at the bottom, the abundance of Na,K-ATPase αβ heterodimers depends on the pool sizes of α and β protein subunits, the rates of assembly of the two subunits, and the degradation rate of mature αβ heterodimers. The pool sizes of the nascent α and β protein subunits are determined by their respective subunit mRNA pool sizes, synthesis (translation) rates, and degradation rates of nascent subunits. The α and β mRNA pool sizes are, likewise, determined by their synthesis (transcription and processing) rates and their degradation rates. The 1:1 stoichiometry of the αβ heterodimer is determined at the assembly step. Since the α and β genes are distinct, there must be independent, and not necessarily coordinate, regulation of the synthesis of the two subunits. An excess synthesis and accumulation of one subunit over the other could theoretically be handled by the degradation of that unassembled subunit. The arrows in Fig. 1, solid for synthesis and dashed for degradation, define the potential steps for regulation of biosynthesis.

Methods

We chose LLC-PK_1 cells as a model system for the study of ionic regulation of Na,K-ATPase abundance. This renal porcine cell line grows as an epithelium with characteristics typical of proximal tubule cells (Rabito, 1986). When LLC-PK_1 cells are placed in medium K+ of 0.25 mM, intracellular Na+ increases and K+ falls, indicative of a decreased transport rate of the resident sodium pumps. All methods have been previously described in detail (Lescale-Matys et al., 1990). In brief, changes in pool sizes of α and β mRNAs were measured by Northern analysis of constant amounts of total RNA using canine α and β cDNA probes. Changes in pool sizes of α and β proteins were measured by immunoblot analysis of a constant amount of homogenate protein with rabbit anti–canine Na,K-ATPase holoenzyme antiserum. Nascent protein subunit synthesis rates were measured by pulse labeling cells for 30 min with [35S]methionine followed by immunoprecipitation. Nascent protein subunit degradation rates were determined by pulse labeling cells for 10 min and chasing for up to 2 h in unlabeled medium before immunoprecipitation. Degradation rates of mature α- and β-subunits were determined by labeling for 30 min, chasing for 90 min to allow time for the αβ heterodimers to reach the plasma membrane, and then immunoprecipitating at subsequent chase intervals. Na,K-ATPase activity is measured as ouabain-sensitive inorganic phosphate liberated per milligram protein in homogenates. All studies were conducted in low K+ treated cells paired to control cells and results were expressed as pool sizes or rates in low K+ relative to control. All studies were designed as time courses in order to determine new steady-state pool sizes after ionic induction.

Figure 1. Biosynthesis of Na,K-ATPase subunits and assembly into functional heterodimer. ER, endoplasmic reticulum; PM, plasma membrane. Solid arrows represent synthesis rates and dashed arrows represent degradation rates.

Results and Discussion

As summarized in Table I and previously reported (Lescale-Matys et al., 1990), when LLC-PK_1 cells are incubated in low K+, there is a coordinate increase in both α and β protein subunits that plateaus at greater than twofold over control after 24 h in low K+. This increase in subunits is accompanied by an increase in Na,K-ATPase activity that also plateaus after 24 h in low K+ at 1.44-fold over control. We don't understand why the ratio of sodium pump activity to abundance is decreased, but hypothesize that in low K+ there is an accumulation of inactive pumps in the process of degradation that remain immunoreactive. In contrast, when primary cultures of rat renal proximal tubule cells are incubated in low K+, there is a coordinate increase in α- and β-subunits and Na,K-ATPase activity to twofold over control that plateaus at 12 h of low K+ incubation (Tang and McDonough, 1990).

Subunit mRNA levels were measured to determine whether the increased pool sizes of α and β protein subunits were due to pretranslational increases in their respective mRNA pool sizes in low K+. As shown in Table I, α mRNA pool size did not change during low K+ incubation, while β mRNA levels were increased nearly twofold by 6 h of low K+ incubation and remained at this level through the 24-h period when measurements were taken. Thus, the increased β mRNA pool size can nearly account for the increased β protein pool. We did not determine whether the increase in β mRNA was a result of increased transcription or decreased degradation of β mRNA. We hypothesized from these data that the increased α pool size could be accounted for if α mRNA and nascent α protein subunits were normally in excess, and if increased availability of β drives increased αβ heterodimer formation. Inherent in this hypothesis was the testable prediction that unassembled nascent α-subunits are degraded and that the degradation rate is decreased as a consequence of increased αβ heterodimer formation.

Nascent subunit degradation rates were measured in LLC-PK_1 cells incubated in control and low K+ medium to test the hypothesis discussed above. We were surprised to find that nascent α-subunits were not degraded during 2 h after labeling in control and low K+ conditions, while nascent β subunits were rapidly degraded, with an average half-life of 110 min. The β degradation rate was not altered by low K+ incubation. This finding disproved our hypothesis that α protein accumulated in low K+ as a consequence of increased αβ formation and decreased degradation of unassembled nascent subunits.

TABLE I
Steady-State Na,K-ATPase Biosynthetic Pool Sizes in Low K+/Control

Pool

Pool size
(low K+/control)

Time in low K+
to reach new steady state

α protein

2.32

24 h

β protein

2.53

24 h

Na,K-ATPase activity

1.44

24 h

α mRNA

1.00

No change in pool size

β mRNA

1.88

6h

To establish whether the α and β pool sizes were increased as a result of increased synthesis, subunit synthesis rates were measured in low K+ and normalized to control rates. As indicated in Table II, α- and β-subunit synthesis rates in low K+ were 1.45- and 2.60-fold over control, respectively, and at steady state by 16 h in low K+. The changes in synthesis rates for α and β were greater than the changes in mRNA levels, indicating that the “translatability” of the RNA was affected by low K+ incubation. As calculated in Table II, the translatability of both α and β mRNAs is increased ~ 40%. This increased translatability provides the mechanism to explain the increase in the pool size of α protein subunits in low K+. Cation concentrations are known to affect translation efficiency for different mRNAs (Sonenshein and Brawerman, 1977; Tse and Taylor, 1977), so perhaps the elevated intracellular K+ and/or depressed Na+ are responsible for increasing α and β translatability.

The mRNA results predicted, and the synthesis experiments established that low K+ increased the pool of nascent β-subunits more than the pool of nascent α-subunits. The pulse-chase experiments showed that since nascent β is subject to rapid degradation, after a 2-h chase the ratio of α to β increased to 1:1 (based on the intensity of the autoradiographic signal normalized to the number of methionines in α and β). If the degradation rate represents the disappearance of unassembled β, then the degradation rate depends on αβ complex formation and these findings suggest that the ceiling for αβ accumulation in low K+ appears to be α-subunit availability. However, in Table I we show that α and β proteins accumulate 2.4-fold over control in low K+, even though α synthesis increases only 1.45-fold. This discrepancy suggests an additional level of regulation.

The pool size of the αβ heterodimers is determined not only by the αβ synthesis and assembly rates but also by the degradation rate of the mature complexes. When we measured the degradation rate of mature pump subunits in low K+, normalized to control, we found no difference before 16 h and a decrease in α- but not β-subunit degradation rates after 16 h in low K+. The discoordinate nature of the effect suggests that it is unlikely to be a mechanism for increasing the abundance of functional pumps but may represent an effect of the disrupted ionic environment on the enzymes responsible for degradation of α and β (something we know nothing about), resulting in the accumulation of internalized subunits that are still immunoreactive in our assays of abundance. Such a mechanism would explain the decreased ratio of activity to abundance measured during low K+ induction. Nonetheless, this decreased degradation of mature α-subunits in low K+ provides a mechanism to explain the discrepancy between the increased α synthesis rate and increased abundance, discussed above.

TABLE II
Na,K-ATPase Nascent Subunit Biosynthetic Rates in Low K+/Control

All rates at steady state after 16 h in low K+.

In conclusion, we have applied a compartmental analysis approach to the study of ionic regulation of Na,K-ATPase abundance in cultured kidney cells. The determination of initial and new steady-state values for pool sizes and rates is important to this type of analysis. Our studies have determined that there are a number of steps in Na,K-ATPase biosynthesis that are altered in low K+: (a) pretranslational increase in β but not α mRNA pool size, (b) increased translatability of both α and β mRNAs into proteins, and (c) decreased degradation of mature α-subunits after 16 h in low K+. These mechanisms can account for the increase in abundance of the α and β protein subunit pool sizes. The compartmental analysis also revealed that newly synthesized β but not α is subject to rapid degradation in control and low K+ conditions. By increasing β mRNA and not α, β is synthesized in excess and the ceiling for αβ accumulation is limited by α availability.

Acknowledgments

This work was supported by NIH grant DK-34316.

References

Lescale-Matys, L., C. B. Hensley, R. Crnkovic-Markovic, D. S. Putnam, and A. A. McDonough. 1990. Low K+ increases Na,K-ATPase abundance in LLC-PK_1 cells by differentially increasing β and not α subunit mRNA. Journal of Biological Chemistry. 265:17935–17940.

Rabito, C. A. 1986. Occluding junctions in a renal cell line (LLC-PK_1) with characteristics of proximal tubular cells. American Journal of Physiology. 250:F734–743.

Sonenshein, G. E., and G. Brawerman. 1977. Differential translation of rat liver albumin messenger RNA in a wheat germ cell free system. Biochemistry. 16:5445–5448.

Tang, M. J., and A. A. McDonough. 1990. Na,K-ATPase beta subunit is preferentially stimulated by low K+ in cultured rat proximal tubule cells. FASEB Journal. 4:A296.

Tse, T. P. H., and J. M. Taylor. 1977. Translation of albumin messenger RNA in a cell free protein synthesizing system derived from wheat germ. Journal of Biological Chemistry. 252:1272–1278.

Inhibition of the Na+/K+ Pump in Xenopus Oocytes by Diacylglycerol Analogues

Larisa A. Vasilets, Kristin Mädefessel, Günther Schmalzing, and Wolfgang Schwarz

Max-Planck Institut für Biophysik, W-6000 Frankfurt/M 71, Germany

Introduction

Phosphorylation of membrane proteins by stimulation of protein kinase C (PKC) (Nishizuka, 1984, 1986) plays a key role in regulation of membrane permeabilities (Kaczmarek, 1987). In addition, phorbol esters, known to stimulate PKC, can induce exocytotic as well as endocytotic activity (Swanson et al., 1985; Knight et al., 1988). Previously we have reported that the phorbol ester, phorbol 12-myristate 13-acetate (PMA), reduces membrane permeabilities by an unspecific endocytotic removal of membrane proteins, including the Na+/K+ pump, from the cell surface (Vasilets et al., 1990). Here we report that the diacylglycerol analogues 1,2-dioctanoyl-sn-glycerol (diC_8) and 1-oleyl-2-acetyl-sn-glycerol (OAG) also lead to downregulation of the Na+/K+ pump in oocytes of Xenopus laevis by endocytosis. The parameters that were monitored were pump activity by measurements of ouabain- or K+-sensitive membrane current and ouabain-sensitive 86Rb+ uptake, number of pump molecules by ouabain binding studies, surface area by determination of membrane capacitance, and endocytotic activity by measurements of inulin uptake.

Results and Discussion

Fig. 1 illustrates by two voltage-clamp experiments the effects of 50 μM diC_8 or 10 μM OAG. The left panels show records of holding current during the experiment. Before and during application of the diacylglycerol analogues the K+ concentration in the bath solution was repeatedly changed between 0 and 3 mM [K+]. The changes in holding current on switching to 3 mM [K+] represent current generated by the Na+/K+ pump. Pump current can be determined as the K+-sensitive current since all non-pump related K+-sensitive currents were blocked by 5 mM Ba2+ and 20 mM tetraethylammonium (TEA+) in the external medium (see Rakowski et al., 1991; Schwarz and Vasilets, 1991). The right panels show the gradual changes in current–voltage (I-V) relationships of the Na+/K+ pump with time. In addition to the gradual inhibition of pump current, the diacylglycerol analogues also seem to modulate passive conductances (see decline in holding current in the absence of K+).

Similar observations have been made previously during application of PMA (Vasilets et al., 1990). Fig. 2 shows the decline of pump current with time induced by 50 nM PMA in comparison with the decline in the presence of 50 μM diC_8. Steady state is reached after ~ 1 h. The degree of inhibition amounts to ~ 80% with PMA, 70% with diC_8, and 45% with 10 μM OAG (not shown). The additional inhibition by PMA compared with that by diC_8 can be suppressed by inhibition of proteases. This supports the suspicion that, in contrast to diC_8, PMA not only activates PKC but also stimulates proteolytic cleavage of PKC, leading to a Ca2+-independent kinase (Kishimoto et al., 1983). This protein kinase could possibly produce additional suppression of membrane transport.

Figure 1. Effects of 50 μM diC_8 (A) or 10 μM OAG (B) added to the bath solution on membrane current. The applied concentrations yield maximal effects. (Left panels) Traces from pen recorder of membrane current necessary to hold the membrane potential at –60 mV in solution containing either 0 or 3 mM K+ as indicated. The brief deflections in holding current indicated current-voltage measurements (see Lafaire and Schwarz, 1986). (Right panel) Voltage dependence of current generated by the Na+/K+ pump, determined as K+-sensitive current (Rakowski et al., 1991). I-V curves in the absence of K+ determined before and after application of the 3 mM [K+] were used for linear drift corrections.

Figure 2. Time course of reduction of pump current at +20 mV after application of diacylglycerol analogues. (Filled circles) 50 nM PMA. (Open circles) 50 μM diC_8. Open squares refer to data obtained in the presence of 50 nM PMA, but activity of proteases was suppressed by pretreatment of the oocytes with 0.1 mM phenylmethylsulfonyl fluoride and microinjection of 25 nl each of 5 mM antipain and 5 mM leupeptin. Data represent average values (±SEM) from 5–10 oocytes.

Figure 3. Correlation between number of ouabain binding sites and pump-mediated Rb+ uptake (at 1 mM extracellular [Rb+]). The number of binding sites was determined with 3H-labeled ouabain (see Schmalzing et al., 1991). Pump-mediated Rb+ uptake was determined as ouabain-sensitive uptake using 86Rb+ as tracer (see Richter et al., 1984). Data were obtained from different batches of oocytes, but corresponding flux and binding measurements were performed with oocytes obtained from same batch. Squares represent data from oocytes treated with diC_8; diamonds are data from oocytes treated with OAG. Concentrations of 1, 3, 10, and 50 μM were used. Data represent average values (±SEM) from 10 oocytes.

In parallel to the reduction of pump current, both the number of ouabain binding sites and the ouabain-sensitive Rb+ uptake are reduced. Fig. 3 demonstrates a monotonic relationship between the number of binding sites and the rate of Rb+ uptake for different degrees of pump inhibition by various concentrations of diC_8 and OAG.

Figure 4. Scatchard plots of ouabain binding to cells permeabilized by 10 μM digitonin (open symbols) and cells that have been treated in addition with 0.02% SDS (filled symbols) (Schmalzing et al., 1989). SDS permeabilization allows detection of additional pump molecules in membranes of intracellular compartments. Measurements of bound [3H]ouabain were performed with untreated oocytes (circles) and oocytes that were treated for 1 h with 30 μM diC_8 (squares). All data points represent average values (±SEM) from 10 oocytes.

The reduction of pump current, pump-mediated Rb+ uptake, and ouabain binding is accompanied by a reduction of membrane surface area as determined by measurements of membrane capacitance. For PMA it has been demonstrated that the decrease in membrane surface area results from endocytosis exceeding exocytosis (Vasilets et al., 1990; see also Schmalzing et al., 1991) and can at least partially account for the downregulation of the Na+/K+ pump. Elevated endocytotic uptake of [3H]inulin and reduced membrane capacitance can also be induced by application of diC_8 and OAG (not shown). Fig. 4 demonstrates that the ouabain binding sites that disappear from the cell surface during diC_8 treatment (see open symbols) can be revealed in intracellular compartments. This conclusion can be inferred from the observation that the total number of pump molecules (in the cell surface and in intracellular compartments) is not altered after application of diC_8 (filled symbols).

At least for diC_8, the reduction in membrane capacitance (i.e., surface area) can only partially account for the reduction of pump activity and number of ouabain binding sites. In some oocytes, pump inhibition exceeds the degree expected on the basis of the reduction of membrane capacitance. This suggests that pump molecules are selectively withdrawn from the surface membrane, or that additional modifications of pump molecules are caused by activation of PKC.

Acknowledgments

We thank Dr. Hermann Passow for his comments on the manuscript.

This work was supported by the Deutsche Forschungsgemeinschaft (SFB 169).

References

Kaczmarek, L. K. 1987. The role of protein kinase C in the regulation of ion channels and neurotransmitter release. Trends in Neuroscience. 10:30–34.

Kishimoto, A., N. Kajikawa, M. Shiota, and Y. Nishizuka. 1983. Proteolytic activation of calcium-activated, phospholipid-dependent protein kinase by calcium-dependent neutral protease. Journal of Biological Chemistry. 258:1156–1164.

Knight, D. E., D. Sugden, and P. F. Baker. 1988. Evidence implicating protein kinase C in exocytosis from electropermeabilized bovine chromaffin cells. Journal of Membrane Biology. 104:21–34.

Lafaire, A. V., and W. Schwarz. 1986. The voltage dependence of the rheogenic Na+/K+ ATPase in the membrane of oocytes of Xenopus laevis. Journal of Membrane Biology. 91:43–51.

Nishizuka, Y. 1984. The role of protein kinase C in cell surface signal transduction and tumor promotion. Nature. 308:693–698.

Nishizuka, Y. 1986. Studies and perspectives of protein kinase C. Science. 233:305–312.

Rakowski, R. F., L. A. Vasilets, and W. Schwarz. 1991. A negative slope in the current-voltage relationship in Xenopus oocytes produced by reduction of external [K+]. Journal of Membrane Biology. 121:177–187.

Richter, H.-P., D. Jung, and H. Passow. 1984. Regulatory changes of membrane transport and ouabain binding during progesterone-induced maturation of Xenopus oocytes. Journal of Membrane Biology. 79:203–210.

Schmalzing, G., K. Mädefessel, W. Haase, and K. Geering. 1991. Immunocytochemical evidence for protein kinase C–induced internalization of sodium pumps in Xenopus laevis oocytes. In The Sodium Pump: Recent Developments. J. H. Kaplan and P. De Weer, editors. The Rockefeller University Press, New York. 465–470.

Schmalzing, G., S. Kröner, and H. Passow. 1989. Evidence for intracellular sodium pumps in permeabilized Xenopus laevis oocytes. Biochemical Journal. 260:395–399.

Schwarz, W., and L. A. Vasilets. 1991. Variations in voltage-dependent stimulation of the Na+/K+ pump in Xenopus oocytes by external potassium. In The Sodium Pump: Structure, Mechanism, and Regulation. J. H. Kaplan and P. De Weer, editors. The Rockefeller University Press, New York. 327–338.

Swanson, J. A., B. D. Yirinec, and S. C. Silverstein. 1985. Phorbol esters and horseradish peroxidase stimulate pinocytosis and redirect the flow of pinocytosed fluid in macrophages. Journal of Cell Biology. 100:851–859.

Vasilets, L. A., G. Schmalzing, K. Mädefessel, W. Haase, and W. Schwarz. 1990. Activation of protein kinase C by phorbol ester induces downregulation of the Na+/K+-ATPase in oocytes of Xenopus laevis. Journal of Membrane Biology. 118:131–142.

L. A. Vasilets' permanent address is Institute of Chemical Physics, Chernogolovka, 142432, Moscow Region, USSR.

Regulation of Expression of Na,K-ATPase in MDCK Cells

Christopher P. Cutler and Gordon Cramb

Department of Biology and Preclinical Medicine, University of St. Andrews, Fife, United Kingdom

Introduction

Na,K-ATPase, an abundant enzyme in Madin-Darby canine kidney (MDCK) epithelial cells, is located in the basolateral membrane domain (Lamb et al., 1981). The isoform composition of the enzyme units expressed has not been reported. The presence of Na,K-ATPase α1 and β1 isoform mRNAs has been demonstrated (Bowen and McDonough, 1987); however, the expression of other known Na,K-ATPase isoforms is so far undetermined.

Large differences in the number of Na,K-ATPase units expressed in MDCK cells have been reported, with densities ranging from 2.33 × 10^5 to 8 × 10^7 sites per cell (Lamb et al., 1981; Kennedy and Lever, 1984). Ouabain binding and Na,K-ATPase activity decline as MDCK cell cultures approach confluence (Kennedy and Lever, 1984). In addition, the level of [3H]ouabain binding to cells grown on plastic has been reported to be lower than that of cells grown on permeable filter supports. (Cereijido et al., 1981; Kennedy and Lever, 1984), possibly resulting from the formation of tight junctions and the restricted access of ouabain to the basolateral membranes of cell monolayers. This explanation is questionable, as it has been shown that lactoperoxidase labels the same set of proteins in MDCK monolayers in the presence or absence of tight junctions (Cereijido et al., 1978, 1981; Richardson et al., 1981). Likewise, the effect of various hormones such as mineralocorticoids, glucocorticoids, and thyroid hormones on the Na,K-ATPase expression have not been extensively studied, although mineralocorticoids are known to stimulate transepithelial Na transport in MDCK cells (Simmons, 1977).

This study examines the presence of Na,K-ATPase isoform mRNAs in the MDCK cell line in relation to the cell surface expression of the enzyme during growth under different environmental conditions.

Methods

MDCK strain I cells of 66–69 serial passages were normally grown in a 5-d culture cycle before passaging and reseeding at a density of ~ 4 × 10^4 cells/cm^2 with 0.57 ml of MEME medium/cm^2. Specific ouabain binding to MDCK cultures was assessed using a ouabain concentration of 2–3 × 10−7 M in the absence and presence of potassium, as described by Lamb et al. (1981). Calcium-free buffer was similar to that used by Gumbiner and Simons (1986).

Total RNA and poly A+ mRNA was extracted from cultured cells and rat tissues by the method outlined in Cutler et al. (1988). Northern blots (Maniatis et al., 1982) were then hybridized with Na,K-ATPase isoform-specific DNA probes isolated from rat α1, α2, α3, and β1 cDNA-containing plasmids. Northern blots were hybridized as described in Anderson and Young (1985), except that hybridization was performed at 33°C, the optimum temperature for hybridization of the rat α1 probe to MDCK RNA (data not shown). The low stringency conditions used during hybridization should allow hybridization of all the isoform-specific probes to their complementary mRNAs in MDCK RNA, if present. Dot blots of MDCK total RNA were produced essentially by the technique of Davis et al. (1986) and were hybridized with a partial human α1 cDNA. MDCK cells used for experiments involving hormone treatments were grown for 3 d on collagen supports, followed by 24 h incubation with hormone (300 nM, except T3 at 10 nM).

Figure 1. A series of Northern blots showing the presence of mRNAs for the isoforms (α1, α2, α3, β1) of Na,K-ATPase in 4 μg of rat brain total RNA (R) or 5 μg of MDCK poly A+ mRNA (M). Blots were hybridized at 33°C in 50% formamide and washed for 10 min in 5 × SSC, 0.1% SDS at 50°C followed by washing in 0.1 × SSC, 0.1% SDS at 30°C for 5 min.

Figure 2. Specific ouabain binding sites present on MDCK cells during growth after seeding at high density. Specific ouabain binding sites were determined by the difference between the amount of ouabain bound in the presence and absence of 15 mM potassium. The error bars represent standard deviations where n = 6.

Results and Discussion

Only specific signals for α1 and β1 isoform mRNAs could be detected (Fig. 1) on Northern blots of MDCK poly A+ mRNA, with no expression of α2 and α3 mRNAs. As previously reported (Bowen and McDonough, 1987), the α1 mRNA was ~ 3.65 kb; however, in contrast to previous results, the β1 isoform showed multiple mRNA bands (four or more).

To determine the expression of Na,K-ATPase enzyme during growth, MDCK cells were seeded on plastic plates and assayed for specific ouabain binding at daily intervals (Fig. 2). Ouabain binding site number increased during the first 2 d of growth before decreasing back to a low level after cells reached maximum density (4 d). Cells were grown on permeable Millipore HA filters to remove any restriction of access of ouabain to the basolateral membrane (Fig. 3). Although cells exhibited a greater number of ouabain binding sites when grown on filters, the number of sites declined after 2 d in culture by an equivalent amount compared with cells grown on plastic (a reduction of ~ 8 × 10^5 sites per cell after 5 d). No significant increase in specific ouabain binding sites was found in the absence of calcium ions (reported to dissociate cellular tight junctions; Gumbiner and Simons, 1986; data not shown), suggesting that there was no restriction of access of ouabain to the basolateral membrane. This result supports the conclusions of Cereijido et al. (1981).

Figure 3. The abundance of specific ouabain binding sites present on the surface of MDCK cells during growth on Millipore filters. Specific ouabain binding sites were determined as the difference between the amount of ouabain bound to cells both apically and basally in the presence or absence of 15 mM potassium. The error bars represent standard deviations where n = 3.

Cell growth was then investigated on permeable collagen supports. The effect of alterations in cell density on Na,K-ATPase abundance was also investigated by seeding cells at lower density (~5 × 10^3 cells/cm^2). MDCK cells grown on collagen at normal (high) seeding densities exhibited a larger number of specific binding sites than cells grown on Millipore or plastic (1.6 × 10^6 sites per cell after 1 d; see Fig. 4); however, the number of sites also declined during growth (~ 7 × 10^5 sites per cell being lost after 5 d). Cells seeded on collagen at low density exhibited a highly significant increase in specific ouabain binding, which was approximately fourfold higher than high density seeded cultures after 2 d of growth. Cells viewed at up to 2 d of growth in low density seeded cultures were predominantly single cells, compared with high density seeded cultures where cells were associated into groups or “islands” with similar morphologies to confluent monolayers. The differences in Na,K-ATPase expression between low and high density seeded cultures appeared to be associated with the differences in cell–cell interactions.

Figure 4. Specific ouabain binding during the growth of MDCK strain I cells on collagen inserts after seeding at high (○) or low (●) density. The error bars represent standard deviations where n = 9 (three experiments) for high and n = 6 (two experiments) for low density seeded cells, with the exception that n = 6 (two experiments) for the day 1 time point of high density seeded cells. Statistical significance was tested by the use of the Student's t-test. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001. The results at each time point were assumed to be normally distributed.

Total RNA was extracted from cell cultures grown on plastic and seeded at different densities. Hybridization of dot blots of total RNA with a Na,K-ATPase α1 isoform DNA probe indicated a small (~50%) but insignificant increase in α1 isoform mRNA abundance in low density compared with high density seeded cultures (data not shown). The increase in α1 isoform mRNA abundance was, however, not specific, as the abundance of actin mRNA showed a similar increase (~50%). The abundance of total RNA per cell was also increased in cultures after 24 h of growth (data not shown); however, this was not responsible for differences in Na,K-ATPase expression, as equivalent increases were found in both high and low density seeded cultures. These results suggest that Na,K-ATPase expression in MDCK cells is influenced by several factors, including cell–substrate and cell–cell interactions, and that such regulation was mediated by predominantly posttranscriptional processes.

The effect of various hormone agonists on Na,K-ATPase expression was also briefly investigated. Only treatment of MDCK cell cultures with aldosterone and corticosterone produced small (~ 17 and ~ 15%, respectively) and significant increases in specific ouabain binding (data not shown). None of the hormone treatments were found to induce the expression of any significant levels of α2 or α3 mRNAs. Only dexamethasone-treated cultures showed increases in the abundance of the α1 and β1 isoform mRNAs (by 70 and 65%, respectively) when abundances were expressed per microgram of total RNA. However, these increases were almost entirely lost if mRNA abundances were expressed per cell. Aldosterone treatment produced a small increase in the abundance in β1 mRNA but no increase in α1 mRNA abundance per cell. Corticosterone, deoxycorticosterone, or T3 treatment did not induce any increase in either α1 or β1 mRNA abundance per cell. These results indicate that mineralocorticoid, glucocorticoid, and thyroid hormones have limited actions on Na,K-ATPase expression in MDCK cells.

Acknowledgments

DNA probes were kindly provided by Dr. J. Lingrel, University of Cincinnati College of Medicine, and Dr. R. Levenson, Yale University School of Medicine.

This work was supported by BHF grants 87/46 and 89/56. C. P. Cutler is in receipt of an MRC studentship. We also gratefully acknowledge the receipt of a Wellcome Trust travel grant.

References

Anderson, M. L. M., and B. D. Young. 1985. Quantitative fiber hybridisation. In Nucleic Acid Hybridisation: A Practical Approach. B. D. Hames and S. J. Higgins, editors. IRL Press Ltd., Oxford, UK. 73–111.

Bowen, J. W., and A. McDonough. 1987. Pretranslational regulation of Na-K-ATPase in cultured canine kidney cells by low K+. American Journal of Physiology. 252:C179–C189.

Cereijido, M., J. Ehrenfeld, S. Fernandez-Castelo, and I. Meza. 1981. Fluxes, junctions, and blisters in cultured monolayers of epithelioid cells (MDCK). Annals of the New York Academy of Sciences. 372:422–441.

Cereijido, M., E. S. Robbins, W. J. Dolan, C. A. Rotunno, and D. D. Sabatini. 1978. Polarized monolayers formed by epithelial cells on a permeable and translucent support. Journal of Cell Biology. 77:853–880.

Cutler, C. P., G. Cramb, and J. F. Lamb. 1988. Quantitative analysis of sodium pump-specific mRNA from human endothelial (HeLa) and canine kidney (MDCK) cell cultures. Progress in Clinical and Biological Research. 268B:59–64.

Davis, L. G., M. D. Dibner, and J. F. Battey. 1986. Basic Methods in Molecular Biology. Elsevier Science Publishing Co., Inc., New York. 147–149.

Gumbiner, B., and K. Simons. 1986. A functional assay for proteins involved in establishing an epithelial occluding barrier. Identification of a uvomorulin-like polypeptide. Journal of Cell Biology. 102:457–468.

Kennedy, B. G., and J. E. Lever. 1984. Regulation of Na+,K+-ATPase activity in MDCK kidney epithelial cell cultures. Role of growth state, cyclic AMP, and chemical inducers of dome formation and differentiation. Journal of Cell Physiology. 121:51–63.

Lamb, J. F., P. Ogden, and N. L. Simmons. 1981. Autoradiographic localisation of [3H]-ouabain bound to cultured epithelial cell monolayers of MDCK cells. Biochimica et Biophysica Acta. 644:333–340.

Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. 202–204.

Richardson, J. C. W., V. Scalera, and N. L. Simmons. 1981. Identification of two strains of MDCK cells which resemble separate nephron tubule segments. Biochimica et Biophysica Acta. 673:26–36.

Simmons, N. L. 1977. Hormone stimulation of net transepithelial Na transport in cell culture. Journal of Physiology. 28P–29P.

Enzyme Isoforms

Identification of Cis Elements Regulating the β2-Subunit Gene of the Na+,K+-ATPase

Kiyoshi Kawakami, Yuriko Yagawa, Hiroshi Okamoto, and Kei Nagano

Department of Biology, Jichi Medical School, Tochigi, 329-04, Japan

Introduction

Na+,K+ -ATPase β2-subunit, which has been revealed to be the adhesion molecule on glia (AMOG), has a dual function as the subunit of the sodium pump and the adhesion molecule between neurons and glia (Gloor et al., 1990). The expression of this subunit gene is restricted mainly to brain and is regulated developmentally (Martin-Vasallo et al., 1989). In situ, AMOG is expressed in the developing cerebellum by Bergmann glia cells at the critical stages of granule neuron migration, which appears to involve surface contacts between migrating neurons and Bergmann glia (Antonicek and Schachner, 1988). The gene regulation of AMOG is very important in the neural migration process and in the development of the neuronal network. For the purpose of understanding the molecular mechanism of regulation of the gene expression, we have isolated a 5'-flanking region of the β2-subunit gene, determined the sequence, and identified the transcription initiation point (Kawakami et al., 1990). In this report we describe the regions that are responsible for the modulation of the promoter activity using a transient expression assay of systematic deletion mutants of the gene.

Materials and Methods

We subcloned a fragment of genomic DNA spanning the region from −2,308 to +534 into pMLCAT or pSVOA/L 5'. Construction of deletion mutants is described in the figure legends. Transfection and assays of luciferase and CAT are as described in Yagawa et al. (1991).

Results and Discussion

Cell Type–specific Expression of the β2-Subunit Gene

The pAlCAT-1 containing α1-subunit gene fragment and pB2CAT-l containing β2-subunit gene fragment were transfected into three typical cell lines of B103 (rat neuroblastoma), MDCK (canine kidney), and 3Y1 (rat embryo fibroblast). The CAT activities are expressed as percentages of pSV2CAT (Table I). Consistent with the ubiquitous expression of the α1-subunit gene, the activity of pAlCAT-1 is roughly the same among these cell lines. In contrast, the activity of pB2CAT-l is prominent in B103 cells, while only at the background level in MDCK, indicating cell type-specific expression of the β2-subunit gene.

TABLE I
Cell Type–specific Expression of Na, K-ATPase β2-Subunit Gene

pA1CAT-1 is the reporter plasmid containing a gene fragment of the α1-subunit. pB2CAT-l is the reporter plasmid containing a gene fragment of the β2-subunit. The CAT activities are shown relative to the activities of pSV2CAT.

Analysis of 5' Sequential Deletion Mutants

To identify upstream cis regulatory elements of the β2 gene, we have constructed a series of 5' deletion mutants and transfected into B103 cells (Fig. 1). In these mutants, an approximately sevenfold decrease in the activity was observed from pB2U-118 to pB2U-76, suggesting that the positive regulatory elements reside between −118 and −76.

Figure 1. Reporter gene assays of 5' sequential deletion mutants. The β2-subunit gene fragment from −2308 to +534 was ligated into pSVOA/LAΔ 5' (luciferase gene vector). The following restriction sites were used for the construction of the deletion mutants. Eco 47III (−1777), Nco I (−792), Dra I (−341), Rsa I (−204), Nde I (−118), Eco RII (−76), Mae III (−38), and Ban II (+39). Each terminal nucleotide of the deletions is indicated. The bottom line shows the location of common sequence elements between β2 and three α isoform genes and consensus sequences of SRE, SP1, and TATA box. 16 μg of each of the plasmids was cotransfected with 4 μg of pSV2CAT into B103 cells. The luciferase activity was normalized with CAT activity in the same cell lysate.

Figure 2. Reporter gene assays of linker substitution and deletion mutants. The Bgl II linker was introduced into the positions indicated and ligated into pMLCAT (CAT gene vector). The positions of the linker and the terminal nucleotides are indicated. 17 μg of the plasmids and 3 μg of pSV2A/LΔ 5' were cotransfected into B103 cells. The CAT activity was normalized with the luciferase activity in the same cell lysate.

Analysis of Linker Substitution and Deletion Mutants

To determine the precise location of the positive regulatory elements, we constructed three linker scanning mutants of pB2LSl, pB2LS2, and pB2LS3, which have a Bgl II linker substitution in −115 to −106, −101 to −92, and −87 to −78, respectively (Fig. 2). None of these mutants showed a marked decrease in the promoter activity. This may be due to the presence of multiple positive regulatory elements from −118 to −76, the functioning of the Bgl II linker sequence as a positive regulatory element, or the presence of other compensating positive regulatory elements between −204 and −118. To clarify these possibilities, we made linker deletion mutants of pB2LSΔ1-2, pB2LSΔ1-3, and pB2LSΔ2-3 and tested for the promoter activity. None of these showed a marked decrease in activity (Fig. 2). We do not know the reason for the apparent higher activity in pB2LSΔ1-2. When we removed the region from −204 to −118 as in the constructs pB2LS2 (−118) and pB2LS3 (−118), an approximately fivefold reduction of the activity was observed in pB2LS3 (−118) (Fig. 2). These results suggest that one positive regulatory element is between −91 and −76, and another positive regulatory element which compensates for this resides between −204 and −118.

Figure 3. Reporter gene assays of 3' sequential deletion mutants. The 3' deletion mutants were constructed with use of Bal 31 nuclease and ligated into pMLCAT (CAT gene vector). The positions of terminal nucleotides are shown. Transfections are as described in the legend to Fig. 2.

Analysis of 3' Deletion Mutants

Looking for regulatory elements in the downstream region, we made a series of 3' deletion mutants shown in Fig. 3. About a fourfold increase in the activity was observed between pB2D+99 and pB2D+39, suggesting that a negative regulatory element resides between +39 and +99.

Figure 4. Regulatory regions of rat ATP1B2 gene expression in B103 cells.

Conclusion

Among the cells examined by transfection assay, the promoter activity of the β2-subunit gene was highest in B103, a rat neuroblastoma cell line, indicating cell type-specific transcription of the gene.

The presence of the positive regulatory element (PRE1) between −118 and −76 was suggested from the results of reporter gene assays of 5' deletion mutants. The precise position of the element was from −91 to −76 judging from the result of reporter gene assays of linker substitution and deletion mutants (Fig. 4). Another positive regulatory element (PRE2) that compensates for the activity of PRE1 was suggested to be from −204 to −118 based on the result of the linker substitution mutant analysis (Fig. 4).

Acknowledgments

This work was supported in part by a grant from the Ministry of Education, Science and Culture of Japan (01617001, 02258219)

References

Antonicek, H., and M. Schachner. 1988. The adhesion molecule on glia (AMOG) incorporated into lipid vesicles binds to subpopulations of neurons. Journal of Neuroscience. 8:2961–2966.

Gloor, S., H. Antonicek, K. J. Sweadner, S. Pagliusi, R. Frank, M. Moos, and M. Schachner. 1990. The adhesion molecule on glia (AMOG) is a homologue of the β-subunit of the Na,K-ATPase. Journal of Cell Biology. 110:165–174.

Kawakami, K., H. Okamoto, Y. Yagawa, and K. Nagano. 1990. Regulation of Na,K-ATPase genes. I. Cloning and analysis of 5' flanking region of NKAB2 gene encoding β2 subunit. Gene. 91:271–274.

Martin-Vasallo, P., W. Dackowski, J. R. Emanuel, and R. Levenson. 1989. Identification of a putative isoform of the Na,K-ATPase beta subunit: primary structure and tissue-specific expression. Journal of Biological Chemistry. 264:4613–4618.

Yagawa, Y., K. Kawakami, and K. Nagano. 1991. Cloning and analysis of the 5'-flanking region of rat Na,K-ATPase α1-subunit gene. In The Sodium Pump: Recent Developments. J. H. Kaplan and P. De Weer, editors. The Rockefeller University Press, New York. 1–4.

Alternately Spliced cDNAs Encode the Chicken Sarco/Endoplasmic Reticulum Slow Ca2+ ATPase

A. Malcolm Campbell, Paul D. Kessler, and D. M. Fambrough

Department of Biology, The Johns Hopkins University, Baltimore, Maryland 21218

Two similar forms of the cardiac/slow Ca2+ ATPase (SERCA2a and SERCA2b), differing in mobility on SDS-PAGE, are expressed in chicken heart and brain (Kaprielian et al., 1989). However, when the two subtypes of chicken SERCA2 were analyzed by two-dimensional peptide mapping, there was no discernible difference between Ca2+ ATPase isolated from brain or heart. In this study, cDNAs encoding each subtype were cloned and sequenced. Chicken SERCA2a is very similar to a variety of Ca2+ ATPases (Table I; MacLennan et al., 1985; Burk et al., 1989; Karin et al., 1989; Palmero and Sastre, 1989). The encoding sequence of SERCA2b is identical to SERCA2a except for the portion encoding the carboxyl terminus, where the final four amino acids of SERCA2a have been replaced with 49 amino acids. This extended carboxyl terminus of chicken SERCA2b is conserved in a variety of mammalian species (Table II; Gunteski-Hamblin et al., 1988; Lytton and MacLennan, 1988; Lytton et al., 1989). The extended terminus of SERCA2b accounts for the 5-kD shift in apparent Mr as seen on SDS-PAGE. A Kyte/Doolittle hydropathy plot analysis of the SERCA2b extended terminus suggests that SERCA2b may span the sarco/endoplasmic reticulum membrane an additional time.

TABLE I
A Comparison of the Deduced Chicken SERCA2a Amino Acid Sequence against Four Other Ca2+ ATPases

The numbers are the percentage of amino acid identity for each Ca2+ pump.

Only SERCA2a mRNA is detectable in embryonic and adult heart, while SERCA2b and SERCA2a messages are expressed in adult and embryonic brain. Chicken SERCA2a- and SERCA2b-encoding cDNAs have been cloned into expression vector pRc/RSV (Invitrogen) and transfected into mouse L cells. Using a monoclonal antibody specific for chicken SERCA2 (Kaprielian and Fambrough, 1987), it appears by immunofluorescence microscopy that each Ca2+ ATPase subtype is expressed and correctly targeted to the endoplasmic reticulum (Fig. 1). Additional studies are in progress to characterize enzymatic differences between chicken SERCA2a and SERCA2b.

TABLE II
A Comparison of the Carboxyl-terminal 49 Amino Acids of Chicken SERCA2b against the Homologous Mammalian Termini

The fraction is the number of identical amino acids out of the 49 or 50 residues of the extended terminus.

The mRNA encoding SERCA2b contains within its 3' untranslated region the sequence encoding the carboxyl terminus of SERCA2a. Therefore, it is inferred from the nucleotide sequence of avian cDNAs that the two subtypes of chicken SERCA2 result from alternate splicing of a common primary transcript. Two different RNA splicing mechanisms (cassette type splicing or internal donor site splicing; Breitbart et al., 1987) could account for the production of mRNAs encoding both subtypes of SERCA2, as shown in Fig. 2. Further analysis of chicken genomic sequence is underway to determine which of these two splicing mechanisms is utilized in chicken SERCA2 RNA processing.

Figure 1. Mouse L cells were transfected with SERCA2a-(A) and SERCA2b-(B) encoding cDNAs cloned into the pRc/RSV (Invitrogen) expression vector or vector alone (C). The cells were fixed with paraformaldehyde, permeabilized with saponin, and stained with a monoclonal antibody specific for chicken SERCA2 and a rhodamine-conjugated secondary antibody.

Figure 2. Diagrams depict the two possible alternate splicing mechanisms that might be utilized in chicken SERCA2 primary transcript processing. Boxes represent exons and lines are introns. The black boxes are the common coding portions of SERCA2 and the shaded boxes represent the subtype-specific carboxyl termini, while white boxes represent 3' UT regions.

References

Breitbart, R. E., A. Andreadis, and B. Nadal-Ginard. 1987. Alternative splicing: ubiquitous mechanism for the generation of multiple protein isoforms from single genes. Annual Review of Biochemistry. 56:467–495.

Burk, S. E., J. Lytton, D. H. MacLennan, and G. E. Shull. 1989. cDNA cloning, functional expression and mRNA tissue distribution of a third organellar Ca+2 pump. Journal of Biological Chemistry. 264:18561–18568.

Gunteski-Hamblin, A.-M., J. Greeb, and G. E. Shull. 1988. A novel Ca+2 pump expressed in brain, kidney and stomach is encoded by an alternative transcript of the slow-twitch muscle sarcoplasmic reticulum Ca+2 ATPase gene. Journal of Biological Chemistry. 263:15032–15040.

Kaprielian, Z., A. M. Campbell, and D. M. Fambrough. 1989. Identification of a Ca+2 ATPase in cerebellar Purkinje cells. Molecular Brain Research. 6:55–60.

Kaprielian, Z., and D. M. Fambrough. 1987. Expression of fast and slow isoforms of the Ca+2 ATPase in developing chick skeletal muscle. Developmental Biology. 124:490–503.

Karin, N. J., Z. Kaprielian, and D. M. Fambrough. 1989. Expression of avian Ca+2 ATPase in cultured mouse myogenic cells. Molecular and Cellular Biology. 9:1978–1986.

Lytton, J., and D. H. MacLennan. 1988. Molecular cloning of cDNAs from human kidney coding for two alternatively spliced products of the cardiac Ca+2 ATPase gene. Journal of Biological Chemistry. 263:15024–15031.

Lytton, J., A. Zarain-Herzberg, M. Periasamy, and D. H. MacLennan. 1989. Molecular cloning of the mammalian smooth muscle sarco(endo)plasmic reticulum Ca+2 ATPase. Journal of Biological Chemistry. 264:7059–7065.

MacLennan, D. H., C. J. Brandl, B. Korczak, and M. Green. 1985. Amino-acid sequence of a Ca+2 + Mg+2 dependent ATPase from rabbit muscle sarcoplasmic reticulum, deduced from its complementary DNA sequence. Nature. 316:696–700.

Palermo, I., and L. Sastre. 1989. Complementary DNA cloning of a protein highly homologous to mammalian sarcoplasmic reticulum Ca+2 ATPase from the crustacean Artemia. Journal of Molecular Biology. 210:737–748.

Deletions in the 3' Untranslated Region of the β1-Subunit mRNA Affect mRNA Stability

Joseph P. Taormino and Douglas M. Fambrough

Department of Biology, The Johns Hopkins University, Baltimore, Maryland 21218

Introduction

We have postulated that the decrease in the decay rate of β1-subunit mRNA after veratridine-mediated upregulation of the (Na+ + K+)-ATPase in tissue cultured muscle cells might be mediated by highly conserved regions within the 3' untranslated region (3' UTR) of the β1-subunit mRNA (Taormino and Fambrough, 1990). Sequence motifs in the 3' UTR of apolipoprotein II mRNA (Ratnasabapathy et al., 1990), transferrin mRNA (Casey et al., 1988), and PEPCK mRNA (Petersen et al., 1989) have been shown to affect mRNA decay rates. The 3' UTR of the β1-subunit mRNA contains two large regions (400 and 150 bp) that are > 80% conserved in β1-subunit cDNAs. Within the 400-bp region of homology, there are a 71-bp and several 10–20-bp regions that exhibit > 90% conservation of sequence (Takeyasu et al., 1987). We have constructed a set of β1-subunit minigenes that delete portions of the highly conserved regions in the 3' UTR of the β1-subunit mRNA. These altered constructs were transfected into a rat liver cell line, ARL 15, or a mouse fibroblast cell line, Ltk-, to examine the effects of these deletions in the 3' UTR on β1-subunit mRNA stability. We have found that the half-life of the β1-subunit is dramatically increased by a 552-bp deletion in the 3' UTR.

Methods

Gene Constructs

The β1-subunit gene construct, β1-WT, was made by fusing a 1.2-kb Eco RI–Nco I β1-subunit promoter fragment to a plasmid that contained the 0.9-kb Nco I–Spe I coding region fused to a 2.2-kb Spe I–Hind III 3' genomic fragment containing the 3' UTR and 3' genomic sequences. This construct encodes the entire wild-type β1-subunit mRNA. The deletions β1-L2 and β1-L5 were made by changing the unique Spe I site to a Hind III site and cloning genomic Exo III deletions into the Hind III sites. The 0.8-kb β1-subunit probe used for RNAse protection assays was generated by polymerase chain reaction amplification of cloned β1-subunit cDNA, and the 0.2-kb LGP120 probe was amplified from a rat brain λ gt-11 cDNA library.

Cell Culture, RNA Isolation, and Analysis

ARL 15 cells were maintained in Williams' Medium E/10% fetal bovine serum (WME), and WME supplemented with 8 μg/ml actinomycin D for mRNA decay experiments. Ltk- cells were cultured in Dulbecco's modified Eagle's medium/10% fetal bovine serum (DMEM), and DMEM containing 4 μg/ml actinomycin D for mRNA decay experiments. ARL 15 cells were electroporated according to Potter (Ausubel et al., 1988), and Ltk- cells were transiently transfected using DEAE-dextran according to Seldman (Ausubel et al., 1988). RNA isolation was performed according to Chomczynski and Sacchi (1987) using RNAzol B (Cinna/Biotecx, Friendswood, TX) RNAse protection assays were done according to Gilman (Ausubel et al., 1988) except the RNA hybridization step, which was done at 25°C, and the RNAse digestion, which was done at 25°C using 5 U/ml RNAse T^1 and 1 μg/ml RNAse A.

Figure 1. (A) The chicken β1-subunit minigene plasmid, pβ1-WT. This construct encodes the entire wild-type chicken β1-subunit mRNA. (B) A map of the 3' UTR showing the location of the different deletions in the 3' UTR and regions of high conservation between β1-subunit cDNAs. The heavy black line under the deletions depicts the location of the synthetic RNA probe used for RNAse protection assays.

Results

A Deletion in the 3' UTR of the Chicken Affects β1-Subunit mRNA Stability in Rat Liver Cells

To ascertain the role of the highly conserved region in the 3' UTR of the β1-subunit, we constructed a chicken β1-subunit minigene that encodes a wild-type β1-subunit mRNA, as well as β1-subunit minigenes that delete various portions of the 3' UTR (Fig. 1). Deletion β1-L2 removes 119 bp from the 3' UTR. This deletion excises a 71-bp region that is 90% identical in all known β1-subunit cDNAs. Deletion β1-L5 deletes 552 bp of the 3' UTR, including those nucleotides deleted by β1-L2.

Figure 2. The β1-subunit minigenes were electroporated into the rat liver ARL 15 cells to establish stable transfected cell lines expressing each minigene. Cells were incubated in medium with or without 8 μg/ml actinomycin D for 12 h and total cell RNA was isolated, hybridized with 32 P-labeled antisense RNA probes specific for the 3' UTR of the chicken β1-subunit and the coding region of the rat lysosomal membrane protein, LGP120, digested with RNAse, and electrophoresed in denaturing 6% Polyacrylamide gels. Gels were dried and exposed to x-ray film or analyzed using a Phospho-imager (Molecular Dynamics, Sunnyvale, CA). Lanes 1, 3, and 5: no DNA, β1-WT, and β1-L5. Lanes 2, 4, and 6: no DNA, β1-WT, and β1-L5 in the presence of actinomycin D.

Stable ARL 15 rat liver cell lines that express the various chicken β1-subunit deletions were established by cotransfecting pSVneo and the different β1-subunit plasmids. Colonies were selected for G-418 resistance, and between 30 and 70 resistant colonies for each deletion were pooled. To determine the effects of deletions in the 3' UTR on mRNA decay rates, duplicate 100-mm tissue culture dishes for each deletion were grown to confluence and subsequently cultured in WME or WME supplemented with the transcriptional inhibitor, actinomycin D, for 12 h. RNAse protection assays were performed using an antisense 0.8-kb RNA fragment complementary to a portion of the 3' UTR of the chicken β1-subunit mRNA, as well as a 0.2-kb antisense RNA fragment complementary to a portion of the coding region of the rat lysosomal protein, LGP120. Since the amount of LGP120 mRNA is not significantly decreased after 12 h in actinomycin D and LGP120 mRNA has a relatively long half-life compared with the β mRNA, the signal from LGP120 mRNA was used to control for the amount of total RNA included in each hybridization. The ratio of the signal from LGP120 to β1-subunit mRNA was used to determine the relative expression of mutated β1-subunit mRNAs with respect to the full-length chicken β1-subunit mRNA (β1-WT) in each of the cell lines, and to directly measure the decay rate of the β1-subunit mRNA in the presence or absence of actinomycin D. As shown in Fig. 2 (lanes 1 and 2), mRNA from nontransfected ARL 15 cells grown in the presence or absence of actinomycin D does not hybridize to the chicken β1-subunit probe. The slowest migrating band that appears in Fig. 2 just above the 775-bp marker and is present in all lanes is nonspecific and should be ignored. In contrast, a band at ~ 150 bp indicative of the rat LGP120 mRNA is easily detected. In the cell lines transfected with the various β1-subunit constructs (lanes 3, 5, and 7), a discrete band matching the expected size of each mRNA is observed. In the actinomycin D–treated cells (lanes 4 and 6) the 775-bp band corresponding to the β1-WT mRNA is absent, and the 650-bp band corresponding to the deleted β1-L2 mRNA is greatly reduced compared with nontreated control cells (lanes 3 and 5). In contrast, the 220-bp band corresponding to deletion β1-L5 mRNA is not significantly affected by actinomycin D treatment (lanes 7 and 8). The sequences deleted in β1-L2 do not significantly affect β1-subunit mRNA stability, whereas the sequences removed in the β1-L5 construct greatly increase mRNA stability in ARL 15 cells. The sequences between the 3' endpoint of β1-L2 and the 5' endpoint of β1-L5 might be responsible for destabilizing the wild-type β1-subunit mRNA in ARL 15 cells. Alternatively, since β1-L2 and β1-L5 both delete the 71-bp highly conserved region, and only β1-L5 affects mRNA stability, the deletion of just the 71-bp region might be insufficient, but necessary, to alter β1-subunit mRNA stability.

Figure 3. Mouse fibroblast Ltk- cells were transfected with β-WT, β-L5, or β-WT plus β-L5. After 3 d cells were either harvested for RNA or grown in the presence of 4 μg/ml actinomycin D for 12 h and then harvested for RNA. Total RNA was hybridized to a β1-subunit-specific, 32P-labeled antisense β1-subunit probe, digested with RNAse, electrophoresed, and exposed to x-ray film or analyzed using a Phospho-imager. Lane 1, β1-WT; lane 2, β1-L5; lane 3, β-WT plus β-L5; lane 4, β-WT plus β-L5 in the presence of actinomycin D.

Stability of β1-Subunit mRNA Is Not Affected by the Deletion β1-L5 in Mouse Fibroblast Ltk- Cells

To test whether β1-subunit mRNA stability is affected in other cell types, the mouse fibroblast cell line, Ltk-, was transiently cotransfected with equal amounts of β1-WT and β1-L5, the deletion that alters β1-subunit mRNA stability in ARL 15 cells. The results are shown in Fig. 3. In lanes 1 and 2, cells were transfected with β1-WT or β1-L5 and hybridized with chicken-specific β1-subunit RNA probe, and the 775- and 220-bp protected fragments characteristic of the two respective mRNAs can be seen. In cells cotransfected with β1-WT and β1-L5, the ratio of β1-WT to β1-L5 mRNA is not altered in the presence of actinomycin D (lanes 3 and 4, respectively). Thus, it seems that the deletion β1-L5 affects β1-subunit mRNA stability in a cell type-specific manner, since β1-L5 dramatically increases the stability of β1-subunit mRNA in rat liver ARL 15 cells but has no effect on β1-subunit mRNA stability in the fibroblast cell line, Ltk-.

Discussion and Conclusions

We have constructed a series of deletions in the β1-subunit 3' UTR to examine the possibility that the highly conserved regions contained within the 3' UTR play a role in the stability of chicken β1-subunit mRNA. We have found that a deletion of 552 bp in the 3' UTR can inhibit the degradation of the chicken β1-subunit mRNA in a rat liver cell line. Presumably, the nucleotides that are deleted are responsible for increasing the degradation rate of the wild-type β1-subunit mRNA. In contrast, β1-WT and β1-L5 mRNAs are degraded at equal rates in mouse fibroblasts. Although the possibility exists that intrinsic differences between the mouse and rat can account for the differential degradation rates, we believe that these differences are cell-type specific. Interestingly, Pressley et al. (1988) have shown that β1-subunit mRNA is expressed at a level ~ 30-fold less than the endogenous α1-subunit mRNA in ARL 15 cells. In addition, Gick and Ismail-Beigi (1990) have shown that β1-subunit mRNA levels are 35-fold less than α1-subunit mRNA levels in “nontransformed” rat liver cells grown in medium containing hypothyroid serum. These apparent discrepancies in the ratio of α1- to β1-subunit mRNAs could be due to unequal transcription rates of the α1- and β1-subunit genes, nuclear processing of the nascent α1- and β1-subunit hnRNAs, or the expression of an uncharacterized β-isoform in the liver. Alternatively, the discrepancy between the mRNA expression levels in liver cells could be due, in part, to a tissue-specific difference in the inherent stability of the α1- and β1-subunit mRNAs.

References

Ausubel, F. M., R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, and K. Struhl. 1988. Current Protocols in Molecular Biology. Vol. 2. John Wiley & Sons, Inc., New York.

Casey, J. L., M. W. Hentze, D. M. Koeller, S. W. Caughman, T. A. Rouault, and R. D. Klausner. 1988. Iron-responsive elements: regulatory RNA sequences that control mRNA levels and translation. Science. 240:924–928.

Chomczynski, P., and N. Sacchi. 1987. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Analytical Biochemistry. 162:156–159.

Gick, G. G., and F. Ismail-Beigi. 1990. Thyroid hormone induction of (Na+ + K+)-ATPase and its mRNAs in a rat liver cell line. American Journal of Physiology. 258 (Cell Physiology 27):C544–C551.

Petersen, D. D., S. R. Koch, and D. K. Granner. 1989. 3' noncoding region of phosphoenolpyruvate carboxykinase mRNA contains a glucocorticoid-responsive mRNA-stabilizing element. Proceedings of the National Academy of Sciences, USA. 86:7800–7804.

Pressley, T. A., F. Ismail-Beigi, G. G. Gick, and I. S. Edelman. 1988. Increased abundance of (Na+ + K+)-ATPase mRNAs in response to low K+. American Journal of Physiology. 255 (Cell Physiology 24):C252–C260.

Ratnasabapathy, R., S.-P. L. Hwang, and D. L. Williams. 1990. The 3' untranslated region of apolipoprotein II mRNA contains two independent domains that bind distinct cytosolic factors. Journal of Biological Chemistry. 265:14050–14055.

Takeyasu, K., M. M. Tamkun, N. R. Siegel, and D. M. Fambrough. 1987. Expression of hybrid (Na+ + K+)-ATPase molecules after transfection of mouse Ltk- cells with DNA encoding the β-subunit of an avian brain sodium pump. Journal of Biological Chemistry. 262:10733–10740.

Taormino, J. P., and D. M. Fambrough. 1990. Pre-translational regulation of the (Na+ + K+)-ATPase in response to demand for ion transport in cultured skeletal muscle. Journal of Biological Chemistry. 265:4116–4123.

Analysis of Isoform-specific Regions within the α- and β-Subunits of the Avian Na,K-ATPase

Victor Lemas, Jayson Rome, Joe Taormino, Kunio Takeyasu, and Douglas M. Fambrough

Department of Physiology, The University of Virginia School of Medicine, Charlottesville, Virginia 22903; and Department of Biology, The Johns Hopkins University, Baltimore, Maryland 21218

Introduction

The Na,K-ATPase is an electrogenic ion pump responsible for maintaining the high internal potassium and low internal sodium levels characteristic of most animal cells. The enzyme has been shown to consist of two subunits. The α-subunit contains the ouabain binding site and the catalytic site for ATP hydrolysis. The β-subunit is a glycoprotein believed to be involved in α-subunit folding and targeting to plasma membrane, but with no known function in ion transport.

Three distinct α-isoforms (Shull et al., 1986a; Takeyasu et al., 1990) and two β-isoforms (Martin-Vasallo et al., 1989; Gloor et al., 1990) have been isolated. We have analyzed the encoding DNA for the α1-, α2-, and α3-isoforms of chicken Na,K-ATPase with comparable data for the rat α-subunit isoforms (Shull et al., 1986a) to assess α-subunit isoform diversity among vertebrates (Takeyasu et al., 1990). Within the combined sets of chicken and rat α-isoform sequences, about half of the sequence diversity represents strictly isoform-specific, but not species-specific, variation. We anticipate that these regions may contain structural features unique to the function or regulation of the individual α-isoforms.

In this report we also present an analysis of the β-isoforms, comparing the avian β1-isoform (Takeyasu et al., 1987) and a newly isolated avian β2-isoform with previously published β-isoforms (Kawakami et al., 1986; Noguchi et al., 1986; Ovchinnikov et al., 1986; Shull et al., 1986b; Brown et al., 1987; Young et al., 1987; Martin-Vasallo et al., 1989; Verrey et al., 1989; Gloor et al., 1990) of the Na,K-ATPase. Because there is greater diversity within the coding sequence among β-isoforms than for the α-isoforms, we aligned all available sequences, anticipating several isoform-specific regions that may educe structural features with potential functional properties.

Methods

The construction of cDNA libraries, cloning, and sequence of the α1-, α2-, and α3-isoforms have been described elsewhere (Takeyasu et al., 1990). One of these same cDNA libraries, from adult chicken brain (lambda-ZAP phage vector), was used to recover the cDNA encoding the avian β2-isoform. Briefly, oligonucleotides, [5'TT(T/C)ACI(T/C)TIACIATGTGGGTIATG(T/C)TICA(A/G)AC 3'] and [5'TTICC(A/G)TA(A/G)TAIGG(A/G)(T/A)A(A/G)TA 3'], which were designed to match conserved regions within the mammalian β2-isoforms, were used for polymerase chain reaction (PCR) to amplify part of avian β2-cDNA from the avian brain cDNA library, according to methods provided by Perkin-Elmer. A single fragment of ~ 500 nucleotides was amplified, random-primer 32P-labeled, and used as a probe to screen the same library at high stringency. A clone containing the entire coding sequence and a poly-A tail was isolated (Fig. 1). DNA sequencing was performed both on double-stranded plasmid DNA according to Maniatis et al. (1982) and on single-stranded DNA by the dideoxy method (Sanger et al., 1977) with [35S]deoxyadenosine 5'-(α-thio)triphosphate and Sequenase (United States Biochemical Corp., Cleveland, OH). Primers for sequencing were synthesized on a PCR-Mate (391 DNA synthesizer; Applied Biosystems, Inc., Foster City, CA) and OPC purified.

For Northern analysis, 15 μg of total RNA extracted from both embryonic (embryo day 13) and adult tissues (brain, gizzard, heart, liver, and leg skeletal muscle) was run on a denaturing formaldehyde gel of 1% agarose, followed by staining with ethidium bromide. The RNA blot was hybridized with a 32P-labeled cDNA fragment from a putative β2-subunit clone.

Percent identity among the isoforms was calculated by the GCG-software package program ALIGN. Bar graphs for homology comparisons were assembled with Cricket Graphics software. Alignments were performed by eye and are subject to individual interpretation.

Figure 1. Graphic map of avian putative β2-subunit obtained from adult chick brain lambda-ZAP II cDNA library. Coding region is ~ 850 nucleotides in length.

Results and Discussion

Pairwise comparisons between amino acid sequences of avian and mammalian α-isoforms reveal a very high level of sequence identity: 93% for the corresponding α1- and α2-isoforms and 96% for the α3-isoforms (Table I). In contrast, in comparisons made among the avian α-isoforms, each differs from the others by 14% (Takeyasu et al., 1990). The marked cross-species sequence identities among α-isoforms of the NH_2-terminal region are displayed by maximally aligning the three rodent and three avian sequences and labeling (bold type) all the regions in which five of the six sequences have identical amino acid residues (Fig. 2). Within the full amino acid sequences, 171 positions fall outside the boxed regions of sequence identity. Of these, 77 are positions at which each pair of chicken and rat isoforms has a common amino acid (in some cases two of the isoforms share a common amino acid). We have defined these as isoform-specific positions. At many of the 77 isoform-specific positions the amino acid residue differs markedly from one isoform to the next. Charge differences, for example, can be found at 20 of these residues. The nature of the isoform-specific variations is such that different physiochemical properties may result in the regions of the α-subunit where these variations occur.

TABLE I
Cross-species α-Isoform Identities

Pairwise comparisons between amino acid sequences of avian and mammalian α-isoforms. Percent identities were determined with the ALIGN software program.

The avian putative β2-isoform amino acid sequence differs significantly from published sequences of mammalian β2-isoforms (Martin-Vasallo et al., 1989; Gloor et al., 1990). The mammalian β2-isoforms have > 90% identity among themselves. However, the avian β2 has much less identity with mammalian β2-isoforms (~50%). When amino acid sequences of the mammalian and avian β2-isoforms (Fig. 3) are compared with those of the β1-isoforms, they are ~40% identical. Interestingly, the rat β-subunit of the H,K-ATPase (Shull, 1990) is ~35% identical to all the Na,K-ATPase β1-isoforms and ~37% identical to the Na,K-ATPase β2-isoforms (including avian). In other words, the avian and mammalian β2-isoforms are similarly divergent from the β1-isoforms and from the H,K-ATPase β-subunit. However, the avian β2-isoform diverges almost as much from the mammalian β2-isoforms as does the H,K-ATPase β-subunit.

It is conceivable that the large sequence difference between avian and mammalian β2-isoforms does not simply reflect a sizable evolutionary divergence within the β2 family, but rather indicates that avian β2 is actually a new type of β-isoform of the Na,K-ATPase, hitherto unknown. Supportive evidence can be given for both proposals. That the avian isoform is the β2 type is suggested by Northern analysis where the mRNA distribution corresponds with that of the mammalian β2-isoforms (Martin-Vasallo et al., 1989). The avian message is primarily expressed in embryonic and adult brain tissue. No significant expression of the message can be detected in kidney or liver. In addition, only one type of PCR fragment was ever amplified over a wide range of reaction conditions. The oligonucleotide primers used were specifically targeted toward the mammalian β2-isoforms and there was never any crossreaction with the avian β1-isoform (determined by shifts in the size of the PCR fragment).

The possibility that this avian β-isotherm is of another class of β-isoforms (following the convention, we will refer to this next class as β3) is suggested by more recent evidence that a β-isoform, cloned from a Xenopus laevis embryonic cDNA library, shows a similar amino acid sequence divergence from the β1- and β2-isoforms (Good et al., 1990). In fact, at many positions where the avian β2 sequence diverges, from a mammalian β2-isoform consensus, the same divergence is observed in the Xenopus β3-isoform. The avian β2 shows 15% more identity with the Xenopus β3 than with the mammalian β2-isoforms. However, a β2 has not been isolated from Xenopus and therefore it remains possible that the Xenopus β3 is actually the β2-isoform.

Figure 2. Isoform-specific positions (asterisks) within the NH_2-terminal region of the avian and mammalian amino acid sequences. Regions of homology (at least five identical residues of the six sequences) are shaded. Residue numbers are according to the chicken αl sequence.

Figure 3. β2-subunit isoform sequence diversity. Bar graph representation of the percent identity among the β-isoforms of Na,K-ATPase and H,K-ATPase. Percent identities were determined with the ALIGN software program.

Figure 4. Isoform-specific positions (open circles) within the transmembrane region (A) and near the COOH terminus. (B) Regions of homology (at least 11 identical residues of the 12 sequences) are shaded. Residue numbers are according to the human β1 sequence.

Alignment of amino acid sequences of the 11 published β-subunits and the avian β2 elucidate at least two well-conserved regions (Fig. 4). Shaded positions represent residues shared by all β1- and β2-subunits, whereas open circles indicate identical amino acid residues belonging to one isoform or the other (referred to as isoform-specific residues throughout the text). If all residues must be identical within a particular isoform, then there are 16 isoform-specific positions. However, when the stringency is lowered (11 of 12 residues must be similar), ~40 more positions are revealed. Of the 16 isoform-specific positions, there are two where the charge is altered and three where an aromatic group is found in one isoform and not in the other. Clusters of isoform-specific residues are located at the transmembrane region and near the COOH-terminus when the stringency is lowered.

In summary, we have attempted to define positions within the α- and β-subunits of Na,K-ATPase that represent isoform-specific but not species-specific variation. At many of these isoform-specific positions, residue variation is such that different physiochemical properties may result in the regions where these variations occur. Studies focused on these isoform-specific regions within both α- and β-subunits may disclose structural features unique to the function or regulation of the individual isoforms.

Acknowledgments

This work was supported by NIH grant NS-23241.

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Shull, G. E. 1990. cDNA cloning of the β-subunit of the rat gastric H,K-ATPase. Journal of Biological Chemistry. 265:12123–12126.

Shull, G. E., J. Greeb, and J. B. Lingrel. 1986a. Molecular cloning of three distinct forms of the Na+,K+-ATPase α-subunit from rat brain. Biochemistry. 25:8125–8132b.

Shull, G. E., L. K. Lane, and J. B. Lingrel. 1986b. Amino acid sequence of the β-subunit of the Na+,K+-ATPase deduced from cDNA. Nature. 321:429–431.

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Takeyasu, K., M. M. Tamkun, N. R. Siegel, and D. M. Fambrough. 1987. Expression of hybrid (Na+ + K+)-ATPase molecules after transfection of mouse Ltk- cells with DNA encoding the β-subunit of an avian brain sodium pump. Journal of Biological Chemistry. 262:10733–10740.

Verrey, F., P. Kairouz, E. Schaerer, P. Fuentes, and K. Geering. 1989. Primary sequence of Xenopus laevis Na+ + K+-ATPase and its localization in A6 kidney cells. American Journal of Physiology. 256:F1034–F1043.

Young, R. M., G. E. Shull, and J. B. Lingrel. 1987. Multiple mRNAs from rat kidney and brain encode a single Na+,K+-ATPase β-subunit protein. Journal of Biological Chemistry. 262:4905–4910.

Expression and Assembly of Different α- and β-Subunit Isoforms of Na+,K+ ATPase in Yeast

Kurt A. Eakle, Burton Horowitz, Kyu Seong Kim, Robert Levenson, and Robert A. Farley

Department of Physiology and Biophysics, University of Southern California School of Medicine, Los Angeles, California 90033; and Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06510

Introduction

Recent results demonstrate that expression of both the α- and β-subunits of the Na+,K+ ATPase in yeast (Saccharomyces cerevesiae) leads to their assembly into functional enzyme complexes as judged by the appearance of [3H]ouabain binding sites and ouabain-inhibitable ATPase activities (Horowitz et al., 1990). The lack of endogenous Na+,K+ ATPase in yeast cells makes this an ideal system for testing the assembly of the different isoforms of the α-subunit with the recently cloned putative second isoform of the β-subunit (β2) (Martin-Vasallo et al., 1989; Gloor et al., 1990) and for determining the structural features that are required for assembly of the holoenzyme. The β-subunit genes cloned from a number of mammalian species (β1's) have traditionally shown a high degree of sequence conservation (>90% identical). However, the putative β2-subunit is remarkably divergent from previously identified β genes with only 35% identical residues. In addition, a β-subunit for the rat H+, K+ ATPase (HKβ) has recently been cloned and sequenced (Shull, 1990). The HKβ protein has ~30% identity when compared with the β1 family and ~35% identity when compared with the rat β2. Additional β-subunits may also be found in association with other ion-motive ATPases of this class. The existence of different β isoforms and their developmentally regulated expression could have important consequences for the expression and function of the sodium pump in various tissues. The expression of a number of different α- and β-subunits for various ion-motive ATPases within the same cell raises the question of whether the association of particular α- and β-subunits is a specific process, and if so, what structural features dictate the specificity of assembly for different ion-motive ATPases. Specifically, it has been suggested that the β2-subunit may only assemble with the α2 or α3 isoforms (Gloor et al., 1990). These results imply that methods must be found to test the association of new β isoforms with the α-subunit isoforms of Na+,K+ ATPase, or α-subunits of other ion-motive ATPases, in order to confirm their assignments as subunits of the different enzymes.

Our goal in this study has been to demonstrate unequivocally that the β2 protein is a subunit of Na+,K+ ATPase by its expression in yeast, since this system requires the heterologous expression of both subunits to form an active complex. Using a yeast strain with multiple auxotrophic markers, we can select for transformants that have stably incorporated two plasmids, one for the expression of the α-subunit and another for the β-subunit. This system allows us the flexibility to quickly combine different isoforms of α and β, and to test their assembly in yeast. In addition, the vector we've chosen for expression of the β-subunit in yeast uses the GAL1 promoter which is tightly repressed when yeast are grown in glucose and is induced in the presence of galactose. This feature may allow us to examine the kinetics of assembly in yeast. The structural features that mediate the specific assembly of different β-subunits with the different α-subunits of Na+,K+ ATPase, or with the α-subunits of other ion-motive ATPases remain to be elucidated. Mutagenesis and the construction of chimeric molecules between the different isoforms of the α- and β-subunits will allow us to delineate the portions of the molecules important for assembly and subunit interaction.

Figure 1. Plasmid vector for expression of β-subunits in yeast.

Methods

Plasmid Constructions

A clone of the sheep α1-subunit in the vector YEplPT (YEpαNKA) has been described previously (Horowitz and Farley, 1988). A plasmid encoding the rat α2 cDNA was the kind gift of E. Benz (Washington University, St. Louis, MO). An Eco RI fragment that contained ~30 bp of 5' untranslated sequence and ~200 bp of 3' untranslated sequence was cloned into the unique Eco RI site of YEp1PT (Hitzeman et al., 1984). This plasmid was designated YEpRα2.

The vector pG1T (Fig. 1) was constructed for the expression of β-subunits by the insertion of a 400-bp Xho I-Hind III PGK fragment providing a transcription termination function into the Sal I–Hind III sites behind the GAL1 promoter of pSEY18G (Vida et al., 1990). A plasmid encoding the rat β1 cDNA was the kind gift of E. Benz. The gene was removed by digesting with Nco I, which cuts at the initiating ATG codon, and Spe I, which cuts ~ 65 bp past the termination codon. The rat β2 cDNA was removed from plasmid pBI3 using Bss HII, which cuts ~ 30 bp 5' of the initiating ATG codon and an Eco RI site, which is right at the termination codon. The ends of these fragments were filled in with Klenow and ligated to pG1T plasmid that had been opened with Bam HI and treated with mung bean nuclease to create blunt ends. These plasmids were designated pGlT-Rβ1 and pG1T-Rβ2, respectively.

[3H] Ouabain Binding

A microsomal fraction of yeast cell membranes was isolated and assayed for [3H]ouabain binding as previously described (Horowitz et al., 1990) with the following modifications. For YEpαNKA (sheep α1), [3H]ouabain (NEN, 15.4 Ci/mmol) was added to a final concentration of 20 nM, while for YEpRα2 (rat α2) [3H]ouabain was added to a final concentration of 100 nM.

Figure 2. [3H]Ouabain binding by yeast microsomal membranes. Bars represent the mean of duplicate points, with error bars representing the variance.

Results

Fig. 2 shows the results of [3H]ouabain binding measurements done on membranes prepared from yeast transformed with various combinations of α and β expression plasmids. Fig. 2 A shows the results of binding to membranes from yeast transformed with YEpαNKA (encoding sheep α1) and either pG1T-Rβ1 (encoding rat β1, bars 1 and 2) or pG1T-Rβ2 (encoding rat β2, bars 3 and 4). Membranes from yeast transformed with YEpαNKA and pG1T-Rβ1 (bars 1 and 2) show a significant level of specific [3H]ouabain binding which can be competitively inhibited with 1 mM unlabeled ouabain. This indicates the production and assembly of a Na+,K+ ATPase complex composed of the sheep α1- and rat β1-subunits. Fig. 2 B shows the results of binding to membranes from yeast transformed with YEpRα2 (encoding rat α2) and either pG1T-Rβ1 (encoding rat β1, bars 5 and 6) or pG1T-Rβ2 (encoding rat β2, bars 7 and 8). Because of the difference in ouabain affinity of rat α2 (Kd 10−7 M) (Sweadner, 1985) and sheep α1 (Kd 10−8 M) (Horowitz et al., 1990), the concentration of [3H]ouabain was increased in these assays, resulting in an increase in the background level of [3H]ouabain binding. Here too, membranes from cells with rat α2 and rat β1 expression plasmids show a significant level of specific [3H]ouabain binding. This indicates the production and assembly of a Na+,K+ ATPase complex composed of the rat α2- and rat β1-subunits. Control experiments (not shown) indicated that the appearance of a [3H]ouabain binding complex was dependent on growth in media using galactose as a carbon source. Growth in glucose, which represses the GAL1 promoter and thus β-subunit expression, showed only background levels of [3H]ouabain binding, indicating that a Na+,K+ ATPase complex is not formed in this case.

The rat β2 expression plasmid is unable to give rise to [3H]ouabain binding when combined with either a sheep α1 (Fig. 2, bars 3 and 4) or rat α2 (Fig. 2, bars 7 and 8) expression plasmids. The simplest explanation for this observation is that yeast is unable to properly transcribe and translate the β2 cDNA from the β2 expression plasmid. However, Northern blots of yeast strains transformed with the β2-subunit expression plasmid revealed that a full-length mRNA for rat β2 is produced in these cells in response to galactose induction, indicating that transcription is occurring properly (data not shown). Because a similar expression construct containing the rat β1 cDNA does yield functional Na+,K+ ATPase, it is likely that the β2 mRNA is also translated. Experiments are in progress to directly demonstrate production of the β2 protein in yeast using a variety of anti-β2 antisera.

Conclusions

We are in the process of developing a system to examine the features required for assembly of α- and β-subunits of Na+, K+ ATPase. Using heterologous expression in yeast, which has no endogenous Na+,K+ ATPase or [3H]ouabain binding proteins, we have shown that expression of the sheep α1- or rat α2-subunits along with the rat β1-subunit leads to assembly of Na+,K+ ATPase molecules that are capable of specific [3H]ouabain binding.

A recently discovered β-subunit isoform, the putative Na+,K+ ATPase β2 gene, is markedly different from previously characterized β genes from a number of different species (Martin-Vasallo et al., 1989). We have sought to confirm the identity of β2 as a subunit of Na+,K+ ATPase, and test its assembly in yeast with different α-subunit isoforms. A full-length mRNA is produced in yeast cells harboring the rat β2 expression plasmid. However, in parallel experiments to those with the β1-subunit, we have so far been unable to detect the assembly of rat β2 with either the sheep α1 or rat α2 into a [3H]ouabain binding complex. A number of explanations for this failure are possible, including a rapid turnover of β2 protein in yeast which would not allow for detectable levels of the assembled proteins, the absence in yeast of other proteins required to mediate either the translation of β2 mRNA or the assembly of α and β2, or an altered ouabain affinity for complexes of α/β2 making it undetectable with our current assays. Currently it is thought that β2 may assemble with only a subset of Na+,K+ ATPase α-subunit isoforms, specifically the α2 and/or α3 isoforms (Gloor et al., 1990). We are currently working on cloning a yeast expression plasmid for the α3-subunit of Na+,K+ ATPase to test its assembly with both the β1- and β2-subunits, as well as exploring other assays to detect the presence of an α/β2 complex of Na+, K+ ATPase in yeast.

Acknowledgments

This work was supported by NIH grants GM-28673 and HL-39295, NSF grant DMB-8919336, and American Heart Association, Greater Los Angeles Affiliate award 926 F1-1.

References

Gloor, S., H. Antonicek, K. J. Sweadner, S. Pagliusi, R. Frank, M. Moos, and M. Schachner. 1990. The adhesion molecule on glia (AMOG) is a homologue of the β subunit of Na,K-ATPase. Journal of Cell Biology. 110:165–174.

Hitzeman, R. A., D. W. Leung, L. J. Perry, W. J. Kohr, H. L. Levine, and D. V. Goeddell. 1984. Secretion of human interferon by yeast. Science. 219:188–192.

Horowitz, B., K. A. Eakle, G. Scheiner-Bobis, G. R. Randolph, C. Y. Chen, R. A. Hitzeman, and R. A. Farley. 1990. Synthesis and assembly of functional mammalian Na,K-ATPase in yeast. Journal of Biological Chemistry. 265:4189–4192.

Horowitz, B., and R. A. Farley. 1988. Development of a heterologous gene expression system for the Na,K-ATPase subunits in the yeast Saccharomyces cerevisiae. In The Na+, K+-Pump, Part B: Cellular Aspects. J. C. Skou, J. G. Nørby, A. B. Maunsbach, and M. Esmann, editors. Alan R. Liss, Inc., New York. 85–90.

Martin-Vasallo, P., W. Dackowski, J. R. Emanuel, and R. Levenson. 1989. Identification of a putative isoform of the Na,K-ATPase β subunit: primary structure and tissue-specific expression. Journal of Biological Chemistry. 264:4613–4618.

Shull, G. E. 1990. cDNA cloning of the β-subunit of the rat gastric H,K-ATPase. Journal of Biological Chemistry. 265:12123–12126.

Sweadner, K. J. 1985. Enzymatic properties of separated isozymes of the Na,K-ATPase. Journal of Biological Chemistry. 260:11506–11513.

Vida, T., T. R. Graham, and S. Emr. 1990. In vitro reconstitution of intercompartmental protein transport to the yeast vacuole. Journal of Cell Biology. 111:2871–2884.

Functional Properties of the Na,K-ATPase α2-Subunit Isoform Expressed in Mammalian Cell Lines

Andrew W. Shyjan, Victor A. Canfield, and Robert Levenson

Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06510

Introduction

The existence of multiple Na,K-ATPase α- and β-subunit isoforms has made it inherently difficult to study the enzymatic parameters of individual Na,K-ATPase isoenzymes. To date, purification of Na,K-ATPase α2 or α3 isoenzymes has not been achieved. One approach to the characterization of individual α-subunit isoforms is to identify tissues expressing a limited combination of the isoforms. We have recently shown that the rat pineal gland expresses high levels of the α3-subunit and lower levels of the α1-subunit (Shyjan et al., 1990). No α2-subunits were detected in this tissue. The difference in ouabain sensitivity between the rodent α1- and α3-subunits allowed us to characterize the properties of the α3 isoform. The α3 isoenzyme expressed in pineal gland was found to have a higher affinity for Na+ than the α1-subunit expressed in kidney. In adipose tissue only α1- and α2-subunits appear to be expressed (Lytton, 1985). The α2 isoform of adipose was found to have a markedly lower affinity for Na+ than the α1-subunit. Further, the affinity of the α2-subunit for Na+ appears to increase in response to insulin.

An alternative approach for characterizing individual α-subunit isoforms is to express each isoform separately in cultured cells. We have previously shown that cDNA encoding the rat α1-subunit can be analyzed in a transfection protocol by virtue of its ability to confer ouabain resistance to ouabain-sensitive primate cells (Emanuel et al., 1988). We have also shown that the ouabain-sensitive rat α2-subunit can be converted to ouabain resistance by site-specific mutation of two amino acids located at the termini of the H1-H2 extracellular domain (Canfield et al., 1990). The ability to convert a cDNA encoding a ouabain-sensitive α-subunit to ouabain resistance provides a strategy for analyzing functional differences among α-subunit isoforms. By converting ouabain-sensitive α-subunit cDNAs to ouabain resistance and expressing these cDNAs in primate cells, the enzymatic parameters of each α-subunit can be characterized free of interference from endogenous Na,K-ATPase activity. Here we describe the application of gene transfer techniques to the analysis of the enzymatic parameters of the rat α2-subunit.

Results

Na,K-ATPase Expression in Transfected Cells

To compare the enzymatic properties of the rat Na,K-ATPase α1- and α2-subunits, CV-1 cells were transfected with cDNAs encoding these ATPase subunits. The generation of plasmids containing mutagenized ouabain-resistant rat Na,K-ATPase α2-subunit cDNA and ouabain-resistant rat α1-subunit cDNA (pSV2α1) have been described previously (Emanuel et al., 1988; Canfield et al., 1990). The cDNA encoding the ouabain-resistant rat α2-subunit was inserted into the expression vector pSV2neo to yield the plasmid pSV2α2or. The plasmids pSV2α1 and pSV2α2or were introduced into ouabain-sensitive CV-1 cells by the calcium phosphate coprecipitation procedure. Transfected cells were selected for the ability to proliferate in media containing 1 μM ouabain, a concentration of drug that is cytotoxic to CV-1 cells. Ouabain-resistant colonies generated by each plasmid were expanded and assayed for expression of α1- and α2-subunits. Microsomes prepared from transfected cell lines were analyzed by Western blotting with fusion protein-derived antisera specific for the rat Na,K-ATPase α1- and α2-subunits (Shyjan and Levenson, 1989). As shown in Fig. 1A, antisera specific for the α1-subunit detected a polypeptide of ~ 100 kD in parental CV-1 cells and CV-1 cells transfected with pSV2α1 and pSV2α2or. This indicates that antisera raised against rat α1-subunit fusion protein cross-reacts with the monkey α1-subunit. Antisera specific for the α2-subunit reacts with a 105-kD polypeptide in two independent cell lines derived by transfection with pSV2α2or (Fig. 1 B). The α2-specific antisera failed to detect any α2-subunits in parental CV-1 cells and CV-1 cells transfected with pSV2α1. Taken together these results demonstrate that expression of the α2-subunit is sufficient to support the viability of CV-1 cells.

Figure 1. Expression of rat Na,K-ATPase α1- and α2-subunits in CV-1 cells tranfected with pSV2α1 or pSV2α2or. Microsomes were prepared from CV-1 cells (lane 5) and CV-1 cell lines transfected with pSV2α1 (lanes 1 and 2) or pSV2α2or (lanes 3 and 4). Solubilized microsomal proteins (50 μg/Lane) were fractionated by electrophoresis through an SDS-containing 7.5% Polyacrylamide gel, transferred to a nitrocellulose filter, and probed with antisera specific for the rat Na,K-ATPase α1- or α2-subunits. The positions of the molecular weight markers are indicated on the left. The α1-subunit antisera (A) reacts with polypeptides of ~ 100 kD in the parental CV-1 cell line and the pSV2α1 and pSV2α2or transfectant cell lines. The α2-subunit antisera (B) reacts with a polypeptide of ~ 105 kD in the pSV2α2or transfectant cell lines. No α2-subunits were detected in the parental CV-1 cells or pSV2α1 transfectant cell lines.

Expression of Ouabain-resistant 86Rb+ Uptake in Transfectant Cells

To further analyze Na,K-ATPase α1- and α2-subunit gene expression we measured the ouabain sensitivity of monovalent cation uptake in transfectant clones grown in 1 μM ouabain and parental CV-1 cells. 86Rb+ was used to monitor K+ flux. As shown in Fig. 2, CV-1 cells exhibited a rate of 86Rb+ uptake of 12 nmol/min per 10^6 cells. Ouabain inhibited this rate of uptake by 76% with an apparent Ki(ouabain) between 10−7 and 10−6 M. This suggests that 76% of 86Rb+ uptake under these conditions was mediated by the CV-1 cell Na,K-ATPase. Transfectants expressing the rat α1- and α2-subunits exhibited lower maximal initial uptake rates than CV-1 cells (8 nmol/min per 10^6 cells). The ouabain-sensitive 86Rb+ uptake in the transfectants was 100-fold more resistant to ouabain than that of CV-1 cells. Approximately 64% of 86Rb+ uptake in the transfectant clones was ouabain inhibitable with an apparent Ki(ouabain) of 30 mM. In transfectants, the 86Rb+ uptake catalyzed by the endogenous CV-1 cell Na,K-ATPase cannot be assessed under these conditions since ouabain (present during the culturing of the cells) remains associated with the endogenous ATPase throughout the course of the experiment.

Figure 2. Ouabain-resistant 86Rb+ uptake in CV-1 cells tranfected with pSV2α1 or pSV2α2or. 86Rb+ uptake was assayed in CV-1 cells and ouabain-resistant transfectants. Each uptake measurement was initiated by the addition of 20 μl of DMEM/HEPES containing 2–5 μCi of 86Rb+ to 2.0 ml of a cell suspension that had been preincubated for 30 min at 37°C with the concentration of ouabain indicated on the abscissa. Aliquots of the cell suspension were removed and uptake was determined at 0, 3, and 5 min after isotope addition. Uptake was linear for at least 5 min. Values represent the mean from four separate experiments, each done in duplicate.

Figure 3. Na+ requirement of the ouabain-resistant 86Rb+ uptake in CV-1 cells transfected with pSV2α1 or pSV2α2or Cells were preloaded with the concentrations of Na+ indicated on the abscissa according to the following procedure. Trypsinized cells were cleared of Na+ and K+ by incubation at 37°C in medium lacking Na+ and K+ (all Na+- and K+-free media were osmotically balanced with choline Cl). Na+ -depleted cells were loaded with a given concentration of Na+ by incubation in K+ free media supplemented with the indicated concentrations of Na+. 86Rb+ uptake was initiated by the addition of 20 μl of a solution containing 500 mM KCl and 2–5 μCi 86Rb+ to 2.0 ml of a suspension of Na+ loaded cells. 86Rb+ uptake was measured at all Na+ concentrations in the presence of 1 μM and 1 mM ouabain. Ouabain-resistant 86Rb+ uptake was determined by subtracting the difference in 86Rb+ uptake at 1 μM and 1 mM ouabain.

Na+ Dependence of Na,K-ATPase α1- and α2-Subunits Expressed in CV-1 Cells

The Na+ requirement of the rat Na,K-ATPase α1- and α2-subunit isotypes was determined by measuring 86Rb+ uptake in cells preloaded with varying amounts of Na+ (0–150 mM). Na,K-ATPase activity contributed by the transfected rat α1- and α2-subunits was determined by subtracting 86Rb+ internalized in the presence of 1 μM ouabain from total 86Rb+ uptake. The results are shown in Fig. 3. Cells expressing α1- and α2-subunits internalized similar amounts of 86Rb + at each concentration of Na+ tested. These results suggest that the α1 and α2 isoenzymes expressed in CV-1 cells exhibit similar apparent Km's for internal Na+.

Conclusions

We have used site-directed mutagenesis to convert the ouabain-sensitive rat Na,K-ATPase α2-subunit to ouabain resistance. Western blot analysis shows that CV-1 cells transfected with mutated α2-subunit cDNA express high levels of α2-subunit polypeptide. When cells transfected with pSV2α2or are cultured in the presence of 1 μM ouabain, the only functional Na,K-ATPases are those containing mutated α2-subunits. These results suggest that the α2 isoenzyme can support the viability of CV-1 cells, and that α2-subunits can associate with endogenous monkey β1-subunits to form functional Na,K-ATPase holoenzymes. The development of cell lines expressing ouabain-resistant α2-subunits has allowed us to study the functional properties of the α2-subunit in a cellular context free of contaminating activity from other α-subunits (in this case the endogenous monkey α1-subunit). Comparison of Na+-dependent, ouabain-resistant 86Rb+ uptake in pSV2α1 and pSV2α2or transfectant cell lines indicates that the α1- and α2-subunits have virtually identical requirements for Na+. In contrast, Lytton (1985) reported that the α2 isoenzyme expressed in adipose cells exhibits a lower affinity for Na+ compared with the α1 enzyme and that the affinity of the α2 isoenzyme for Na+ increases in response to insulin treatment. This discrepancy may reflect the fact that the α2-subunit gene is not normally expressed in CV-1 cells. Tissue-specific modifications and/or other factors may contribute to the Na+ affinity of the α2 isoform in adipocytes. It is possible that such modifications or associated factors could also account for alterations in Na+ affinity of the α2 enzyme in response to insulin. It would therefore be of interest to express the pSV2α2or construct in an adipose cell line and determine the Na+ affinity of the ouabain-resistant α2 isoenzyme. A further issue that should be considered in this context is the possibility that mutations introduced into α2-subunit cDNA alter the Na+ affinity of the α2 isoenzyme. Finally, it is possible that the α2-subunit expressed in adipose cells and synapses may be associated with a β-subunit isoform different from the β-subunit present in CV-1 cells (β1-subunit), and that association of the α2-subunit with an alternative β-subunit may affect the Na+ affinity of the transfected α2-subunit. Further experiments will clearly be required to resolve these issues.

Acknowledgments

This work was supported by PHS grant CA-38992 and NIH grant HL-39263 to R. Levenson, who is an Established Investigator of the American Heart Association.

References

Canfield, V., J. R. Emanuel, N. Spickofsky, R. Levenson, and R. F. Margolskee. 1990. Ouabain-resistant mutants of the rat Na,K-ATPase α2 isoform identified by using an episomal expression vector. Molecular and Cellular Biology. 10:1367–1372.

Emanuel, J. R., J. Schulz, X.-M. Zhou, R. B. Kent, D. Housman, L. C. Cantley, and R. Levenson. 1988. Expression of an ouabain-resistant Na,K-ATPase in CV-1 cells after transfection with a cDNA encoding the rat Na,K-ATPase α1 subunit. Journal of Biological Chemistry. 263:7726–7733.

Lytton, J. 1985. Insulin affects the sodium affinity of the rat adipocyte (Na,K)-ATPase. Journal of Biological Chemistry. 260:10075–10080.

Shyjan, A. W., V. Cena, D. C. Klein, and R. Levenson. 1990. Differential expression and enzymatic properties of the Na,K-ATPase α3 isoenzyme in rat pineal glands. Proceedings of the National Academy of Sciences, USA. 88:1178–1182.

Shyjan, A. W., and R. Levenson. 1989. Antisera specific for the α1, α2, α3 and β subunits of the Na,K-ATPase: differential expression of α and β subunits in rat tissue membranes. Biochemistry. 28:4531–4535.

Distribution of Isoforms of the α-Subunit of the Na, K-ATPase in Adult Rat

Paul M. Rowe, W. Timothy Link, Charlene P. Osborn, and R. Wayne Albers

Laboratory of Neurochemistry, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892

Introduction

It has long been known that Na, K-ATPases of many species have biphasic cardiac glycoside sensitivity, the difference between the two components being most pronounced in rodents, but also present in other species. About 12 years ago evidence began to accumulate that there were two forms of the α-subunit of the sodium pump that could account for the different cardiac glycoside sensitivities (for review see Sweadner, 1989). Not two but three different mRNAs coding for α-subunits were identified in rat brain (Shull et al., 1986), and it has been established that there are three forms of the α-subunit in other mammals and in birds (Fambrough et al., 1991). The similarity between isoforms within one species is less than the similarity of a single isoform between different species, and this pattern of conservation extends to the flanking regions of the gene as well as the coding sequences. This suggests that there are highly conserved specialized functions for the various isoforms.

Isoform-selective antibodies have been previously generated by the monoclonal technique (Urayama et al., 1989), by raising antisera against α-subunit fusion proteins expressed in bacteria (Shyjan and Levenson, 1989), and by immunizing rabbits with synthetic peptides (Hsu and Guidotti, 1989; Aristarkhova et al., 1991). We have raised antibodies to synthetic peptides that correspond to conserved sequences and to isoform-specific sequences of those proteins, and we have used these antibodies to study the tissue distribution of the α isoforms.

Methods

Antisera to four synthetic peptides were used in this study. All peptides were synthesized to include a cysteine residue (for coupling purposes), which is bracketed if not in the natural sequence. The sequences are taken from Shull et al. (1986). We refer to the peptides and the corresponding antibodies by the first four letters of the peptide sequences.

Conserved site peptide:

VMKGAPERILDRC Residues 501 to 513 of α1.

Isoform-specific peptides:

AVSEHGDKKS[C] Residues 9–18 of α1, counting the initial met as −5

ATTAENGGGK[C] Residues 8–17 of α2, counting the initial met as −5

KDDKSSPKKS[C] Residues 5–14 of α3, counting the initial met as 1.

These four antisera have been affinity purified (except the ATTA [α2-specific] serum used on the blots in Fig. 1, D–G, which was not affinity purified). Antisera to VMKG, AVSE, and KDDK were purified using the synthetic peptides as affinity ligands. Antiserum to ATTA was purified using a partially purified preparation of rat brain α-subunit as the affinity ligand. Purification resulted in lower background and better signal-to-noise ratio, but the pattern of bands stained in the 100-kD region of the blots was not changed. For more details see Rowe et al. (1988).

Membrane fractions of tissues from 200–250-g male Sprague-Dawley rats were prepared by homogenization in hypotonic buffer, followed by centrifugation for 45 min at 40,000 g. Epididymal fat pad membranes were prepared by a modification of the method of Belsham et al. (1980). These membrane preparations were subjected to SDS-PAGE by Laemmli's method on gels of 6% acrylamide in lanes 2 cm wide and transblotted to PVDF membrane, and the blots were sliced into multiple strips for immunostaining. Goat anti-rabbit/alkaline phosphatase conjugate was used as the second antibody.

Results

Our findings agree with the general view that the α1 isoform is present in all tissues (Fig. 1). Staining of a brain blot (Fig. 1 C) with the conserved site antibody is broad and, in our hands, often does not show separation into clearly defined upper and lower bands. The isoform-specific antibodies stain overlapping but distinct bands, with the α1 band migrating the fastest, the α2 band migrating at an intermediate rate, and the α3 band migrating the slowest. The presence of all three proteins in brain, and the order of their electrophoretic migration, is in agreement with the results of others (Hsu and Guidotti, 1989; Urayama et al., 1989).

On kidney blots (Fig. 1A), the α1-specific antibody stains strongly. Weak reactivity with the ATTA (α2) antibody appears at exactly the same position as the AVSE (α1) band. Hence, we believe it represents crossreactivity, and not authentic α2. Although Orlowski and Lingrel (1988) found α2 mRNA in kidney and Emanuel et al. (1987) found both α2 and α3 mRNA, we conclude, as did Shyjan and Levenson (1989), that only the α1 protein was detectable. Aristarkhova et al. (1991) found α3 in kidney, but at ~ 1% of the level of α1. If α2 or α3 were present as 1% of the total Na,K-ATPase in our samples, it would be below the sensitivity of detection of our method.

In epididymal fat pad membranes (Fig. 1 B), we find both α1 and α2. This agrees with the results of Lytton et al. (1985), who found α and “α+”protein in adipocytes, and those of Young and Lingrel (1987), who found α1 and α2 mRNA.

Figure 1. Western blots of rat tissues using specific antibodies against α-subunit isoforms. For each tissue sample, the four Western blot strips are immunostained with (from left to right): VMKG (conserved site antibody); AVSE (αl specific); ATTA (α2 specific); KDDK (α3 specific). See Methods for the complete sequences of the immunogenic peptides. Kidney, 20 μg protein/lane; epididymal fat pad and brain, 50 μg protein/lane; spinal cord, ventral and dorsal roots, and dorsal root ganglion, 2 mg wet wt/lane; liver, 200 μg protein/lane; whole adrenal, gastric mucosa and neurointermediate lobe, 50 μg protein/lane; anterior pituitary, 6.3 mg wet wt/lane; neural lobe, 4.2 mg wet wt/lane; intermediate lobe, 19 intermediate lobes/lane.

Emanuel et al. (1987) found all three mRNAs in liver; however, our results (Fig. 1 H) agree with those of Young and Lingrel (1987), who found only α1 mRNA, and Shyjan and Levenson (1989), who detected only α1 protein. The faint reactivity with the α2 antibody is probably crossreactivity with α1 (see also kidney), because the band appears at the same position as the α1 band.

In anterior pituitary (Fig. 1 L), α1 is the predominant form; the faint reactivity for α3 is ambiguous because it does not exactly correspond to the migration of α3 in other samples on this gel.

The neuro-intermediate lobe of the pituitary (Fig. 1 M) shows the presence of all three isoforms, and when the neural and intermediate lobes are separated (Fig. 1, N and P), both show all three isoforms. Note that the pattern of bands in the intermediate lobe sample includes an extra band of higher apparent Mr that stains for α1, and a band of lower apparent Mr that stains for α3. The origin of these bands is not known, but there is often variation in the banding pattern of the isoforms, sometimes including well-defined doublets of each of the three.

In all the neural tissues (Fig. 1, C-G and N), we find both α1 and α3, and in all neural tissues with the exception of the spinal nerve roots (Fig. 1, E and F) we detect α2. In the spinal nerve roots, the relative predominance of α1 may reflect the abundant Schwann cells, while axolemma, known to be rich in α2 and α3 (Sweadner, 1989), may represent a minor component of the preparation.

On the gel represented in Fig. 1, D-G, α1 and α3 ran nearly identically. Image analysis (not shown) shows that in both the spinal cord sample and the dorsal root ganglion sample, the α2 and α3 bands are almost completely overlapping. We have previously found variation in the migration pattern of the three α's (Rowe et al., 1989). While α1 is always the fastest-moving band, and α3 the slowest, the position of the α1 immunoreactivity varies such that it may migrate nearly as fast as α1, or nearly as slow as α3. In membranes from whole adrenal gland (Fig. 1 J), only α1 is detectable. In gastric mucosa (Fig. 1 K), only the α1 isoform of the sodium pump is detectable; the second heavy band detected by the conserved site antibody, at a slightly lower Mr than the sodium pump, is presumably the H,K-ATPase. Young and Lingrel (1987) reported both α1 and α3 mRNA in whole stomach.

Our results confirm and extend a growing consensus about the distribution of the three known isoforms of the α-subunit of the Na,K-ATPase. What was once known as “α+”is now clearly seen to be α2 or α3 or both. In the nervous system, the expression of α3 is widespread; we detect it in every nervous tissue we examine. There are other reports of α3 in nonneural tissues (Sweadner, 1989), and we may have detected a trace of it in the anterior pituitary; nevertheless, α3 is most prominent in nervous tissues. While α3 may be found only at low levels outside of the nervous system, we find α2 in fat at substantial levels, and others find it plentiful in muscle (Sweadner, 1989).

It is not yet certain whether the isoforms have different intrinsic kinetic properties, respond differently to hormones, exist for purposes of differential intracellular localization, or reflect the mechanism for specific tissue expression that multiple genes afford. In any case, the availability of these specific antibodies should allow us to study the cellular distributions of the specific isoforms in more detail.

References

Aristarkhova, E. A., N. M. Vladimirova, N. A. Potapenko, and N. N. Modyanov. 1991. Structural Analysis of Na,K-ATPase isoforms in mammalian tissues. In The Sodium Pump: Recent Developments. J. H. Kaplan and P. De Weer, editors. The Rockefeller University Press, New York. 249–252.

Belsham, G. J., R. M. Denton, and M. J. Tanner. 1980. Use of a novel rapid preparation of fat-cell plasma membranes employing Percoll to investigate the effects of insulin and adrenaline on membrane protein phosphorylation within intact fat cells. Biochemical Journal. 192:457–467.

Emanuel, J. R., S. Garetz, L. Stone, and R. Levenson. 1987. Differential expression of Na,K-ATPase α- and β-subunit mRNAs in rat tissues and cell lines. Proceedings of the National Academy of Science, USA. 84:9030–9034.

Fambrough, D. M., B. A. Wolitzky, J. P. Taormino, M. M. Tamkun, K. Takeyasu, D. Somerville, K. J. Renaud, M. V. Lemas, R. M. Lebovitz, B. C. Kone et al. 1991. A cell biologist's perspective on sites of Na,K-ATPase regulation. In The Sodium Pump: Structure, Mechanism, and Regulation. J. H. Kaplan and P. De Weer, editors. The Rockefeller University Press, New York. 17–30.

Hsu, Y.-M., and G. Guidotti. 1989. Rat brain has the α3 form of the Na,K-ATPase. Biochemistry. 28:569–573.

Lytton, J., J. C. Lin, and G. Guidotti. 1985. Identification of two molecular forms of Na,K-ATPase in rat adipocytes. Journal of Biological Chemistry. 260:1177–1184.

Orlowski, J., and J. B. Lingrel. 1988. Tissue-specific and developmental regulation of rat Na,K-ATPase catalytic α isoform and β subunit mRNAs. Journal of Biological Chemistry. 263:10436–10442.

Rowe, P. M., W. T. Link, and R. W. Albers. 1989. Demonstration of three isoforms of Na,K-ATPase α-subunit in rat brain. Transactions of the American Society of Neurochemistry. 20:156.

Rowe, P. M., W. T. Link, A. K. Hazra, P. G. Pearson, and R. W. Albers. 1988. Antibodies to synthetic peptides as probes of the structure of the α subunit of the Na,K-ATPase. Progress in Clinical and Biological Research. 268A: 115–120.

Shull, G. E., J. Greeb, and J. B. Lingrel. 1986. Molecular cloning of three distinct forms of the Na,K-ATPase α subunit from rat brain. Biochemistry. 25:8125–8132.

Shyjan, A. W., and R. Levenson. 1989. Antisera specific for the α1, α2, α3 and β subunits of the Na,K-ATPase: differential expression of α and β subunits in rat tissue membranes. Biochemistry. 28:4531–4535.

Sweadner, K. J. 1989. Isozymes of the Na+/K+-ATPase. Biochimica et Biophysica Acta. 988:185–220.

Urayama, O., H. Shutt, and K. J. Sweadner. 1989. Identification of three isozyme proteins of the catalytic subunit of the Na,K-ATPase in rat brain. Journal of Biological Chemistry. 264:8271–8280.

Young, R. M., and J. B. Lingrel. 1987. Tissue distribution of mRNAs encoding the α isoforms and β subunit of rat Na,K-ATPase. Biochemical and Biophysical Research Communications. 145:52–58.

α1 and α3 Isoforms of (Na,K)-ATPase Are Both Produced in Neurons and Show Unique Distributions in the Rat CNS and PNS

George J. Siegel, Marina Mata, Virginia Hieber, and David J. Fink

GRECC Neurology Research Laboratory, Veterans Administration Medical Center, and Neurochemistry Laboratory, Department of Neurology, University of Michigan, Ann Arbor, Michigan 48105

Introduction

To test hypotheses regarding presumed special cellular functions of the (Na, K)-ATPase α isoforms, it is necessary to determine the regional and cellular distribution of both mRNAs and the peptides. There are very few data regarding distribution of isoform-specific mRNAs in brain (Schneider et al., 1988; Filuk et al., 1989; Hieber et al., 1989). There are no previous studies of peripheral nerve, spinal cord, or brain stem.

The three different α isoforms in rat have substantial homology (Shull et al., 1986; Herrera et al., 1987). However, the untranslated regions of the mRNAs are unique. We have exploited this feature to create a 35S-labeled riboprobe from the 3' untranslated region of α1 cDNA. We have also created a riboprobe to a relatively distinct region of the translated α3 cDNA. We have used these specific probes to study the differential distribution of α1 and α3 isoform mRNAs in the central and peripheral nervous system of the rat.

Methods

Production of Riboprobes

Cloned rat α isoform cDNAs, graciously provided to us by Dr. Robert Levenson (Yale University, New Haven, CT) and by Dr. Jerry Lingrel (University of Cincinnati College of Medicine, Cincinnati, OH), were used to prepare subclones containing the 3' untranslated region of the α1 isoform cDNA, and a segment of the α3 isoform cDNA containing a translated region. A 203-bp Bam H1–Pst 1 restriction fragment from the 3' end of the α1 cDNA and a 342-bp Pst 1-Eco R1 fragment containing a portion of the translated region of the α3 cDNA were subcloned into the vector pGEM4.35S-Labeled riboprobes were prepared for in situ hybridization studies and 32P-labeled probes for use in dot blot studies (Mata et al., 1990; Hieber et al., 1991).

Dot Blotting

RNA was isolated from nervous system and kidney and spotted on GeneScreen membranes as described (Hieber et al., 1989, 1991; Mata et al., 1990). Hybridization to 32P-labeled RNA probes was carried out as described previously (Bevilacqua et al., 1988), except that the temperature was maintained at 70°C. Autoradiograms of the filters were produced on Kodak XAR film exposed at −80°C.

In Situ Hybridization

In situ hybridization was performed as described (Mata et al., 1990; Hieber et al., 1991). Slides were initially exposed to Kodak X-AR film for 1–10 d before being dipped in emulsion and developed. The x-ray film was analyzed using an Imaging Resources Inc. (London, Ontario, Canada) image analyzer and digitized images were photographed. Developed slides were counterstained with cresyl violet and photographed under bright field and dark field optics.

Results

The in situ hybridization gave distinct autoradiographic patterns for the two different isoforms. In the cerebral hemispheres the α1 probe labeled predominantly cortex, hippocampus, and habenula, while the α3 probe labeled all these structures and the deep nuclei including striatum, thalamus, and amygdala with similar intensity. In the cerebral cortex the α1 probe labeled layers 1, 2, and 3 more densely than deeper layers, while the α3 probe labeled layer 5 more intensely than the more superficial layers. In the hippocampus α1 labeling was more intense in the dentate gyrus than in the pyramidal cells, while α3 labeling was equally intense in both dentate gyrus and pyramidal cells (Fig. 1 A). In the brainstem most neurons were strongly labeled with the α3 probe. Some structures such as the substantia nigra pars compacta, the red nucleus, and the motor nucleus of III were particularly intensely labeled with this probe. In contrast, dense α1 labeling was seen in neurons of only a few isolated brainstem nuclei including the locus ceruleus, the motor nucleus of V, and the VII nerve nucleus.

In the cerebellum the α3 probe labeled Purkinje cells most strongly. Basket and stellate cells were also labeled, while little label was seen in the granule cell layer. In comparison, the α1 probe preferentially labeled the granule cell layer (Fig. 1 B). In all of the brain regions examined, in situ hybridization signal was localized predominantly over neuronal perikarya with both probes. The only strongly labeled nonneuronal structure was the ependyma of the choroid plexus, which was labeled exclusively with the α1 probe (Fig. 1 A).

In situ hybridization of the dorsal root ganglia (DRG) with both α1 and α3 showed intense labeling of DRG neuronal perikarya with both probes. Silver grains representing binding of the riboprobe were found over neuronal cell bodies but not over satellite cells in the ganglion. Many neurons contained both α1 and α3, while some neurons contained only α3 but not α1. We were unable to identify any neurons that contained α1 but not α3. In the lumbar spinal cord α1 mRNA was found in a limited set of motor neuron cell bodies in the ventral horn. α3 mRNA, in contrast, was found in neurons throughout the dorsal and ventral gray matter of the spinal cord (Fig. 1 C). 1-μm serial semi-thin sections showed that all of the neurons producing α1 also contained α3 mRNA, while many individual cells contained only α3 but not α1.

In the peripheral nerve Schwann cells showed α1 mRNA in a patchy distribution around most Schwann cell nuclei. This reaction product was seen after 4 wk of exposure, in contrast to the DRG α1 signal which was clear after 3 d of exposure. In agreement with the dot blot analysis, no α3 hybridization signal was seen in peripheral nerve. A similar pattern was seen in the white matter tracts of the spinal cord, where α1 but not α3 hybridization signal was found after prolonged exposure.

Figure 1. Distribution of αl (left) and α3 (right) mRNAs by in situ hybridization. (A) Film autoradiogram of cerebral hemispheres. 1, 2, 3, and 5, layers of cortex; cp, caudate–putamen; ch, choroid plexus; a, amygdala; th, thalamus; p, pallidum. (B) Dark field of cerebellum. g, granule cell layer; solid arrows, Purkinje cells; arrowheads, basket cells; open arrows, stellate cells. (C) Dark field of spinal cord. Arrows, motor neurons in lateral aspect of ventral horn.

Dot blot analysis of cortex, brain stem, DRG, and spinal cord showed the presence of α1 and α3. α1 but not α3 was found in dot blots of kidney and peripheral nerve.

Discussion

There are three major findings of this study:

(1)Both α1 mRNA and α3 mRNA have distinct distributions in the central and peripheral nervous system. Our results with the longer riboprobes confirm the main observations in rat cerebrum obtained with the synthetic oligonucleotide probes for α1 and α3 (Filuk et al., 1989). As reported in the earlier study, α1 is expressed mainly in the more superficial layers of cortex and in the granule cells of the dentate gyrus, while α3 is expressed at high levels in deeper cortical laminae, thalamic nuclei, striatum, and hippocampal granule and pyramidal cells. In addition, our results demonstrate a marked separation of isoform distribution in the hindbrain, with α3 in most brain stem nuclei and al in only a few isolated nuclei.

(2)Individual neurons in spinal cord and DRG may contain abundant α3 and little or no al mRNAs, while some neurons contain an abundance of both α1 and α3. So far, no neurons containing abundant α1 without α3 have been identified. It remains to be seen whether neurons in other regions of the nervous systems of rodents contain α1 without α3, and if so, to compare the two types of neurons.

(3)α1 mRNA but not α3 mRNA is found in Schwann cells and oligodendroglia. Before the identification of the three isoforms of (Na,K)-ATPase, Sweadner reported that the kidney isoform (called α, but identical to the currently labeled al isoform) was the predominant glial isoform, based on biochemical identification of (Na,K)-ATPase α1 polypeptide in cultured neonatal astrocytes (Sweadner, 1979). A recent immunohistochemical study of optic nerve suggested the presence of α1 and α2 polypeptides in glial cells of the optic nerve (McGrail and Sweadner, 1989). The present in situ mRNA data for peripheral nerve and spinal cord white matter, where α1 but not α3 mRNA is expressed in Schwann cells and oligodendroglial cells, confirm the isoform distribution in oligodendroglia and are the first evidence for α1 mRNA in Schwann cells.

Acknowledgments

This work was supported by VA Merit Review grants (to M. Mata and D. J. Fink), the NINDS (NS-27771-01 to D. J. Fink), and the MDRTC (G. J. Siegel).

References

Bevilacqua, A., R. P. Erickson, and V. Hieber. 1988. Antisense RNA inhibits endogenous gene expression in mouse preimplantation embryos: lack of double-stranded RNA “melting” activity. Proceedings of the National Academy of Sciences, USA. 85:831–835.

Filuk, P. E., M.A. Miller, D. M. Dorsa, and W. L. Stahl. 1989. Localization of messenger RNA encoding isoforms of the catalytic subunit of Na,K-ATPase in rat brain by in situ hybridization histochemistry. Neuroscience Research Communications. 5:155–162.

Herrera, V. L., J. R. Emanuel, N. Ruiz Opazo, R. Levenson, and B. Nadal Ginard. 1987. Three differentially expressed Na,K-ATPase alpha subunit isoforms. Structural and functional implications. Journal of Cell Biology. 105:1855–1865.

Hieber, V., G. J. Siegel, T. Desmond, J. Lee-Hwa Liu, and S. A. Ernst. 1989. Na,K-ATPase: comparison of the cellular localization of alpha-subunit mRNA and polypeptide in mouse cerebellum, retina, and kidney. Journal of Neuroscience Research. 23:9–20.

Hieber, V., G. J. Siegel, D. J. Fink, M. W. Beaty, and M. Mata. 1991. Differential distribution of (Na,K)-ATPase alpha isoforms in the central nervous system. Cellular and Molecular Neurobiology. 11:253–262.

Mata, M., G. J. Siegel, V. Hieber, M. W. Beaty, and D. J. Fink. 1990. Differential distribution of (Na,K)-ATPase alpha isoforms in the peripheral nervous system. Brain Research. In press.

McGrail, K. M., and K. J. Sweadner. 1989. Complex expression patterns for Na+,K+ ATPase isoforms in retina and optic nerve. European Journal of Neuroscience. 2:170–176.

Schneider, J. W., R. W. Mercer, M. Gilmore-Hebert, M. F. Utset, C. Lai, A. Greene, and E. J. Benz. 1988. Tissue specificity, localization in brain, and cell-free translation of mRNA encoding the A3 isoform of Na+,K+-ATPase. Proceedings of the National Academy of Sciences, USA. 85:284–288.

Shull, G. E., J. Greeb, and J. B. Lingrel. 1986. Molecular cloning of three distinct forms of the Na+, K+-ATPase alpha-subunit from rat brain. Biochemistry. 25:8125–8132.

Sweadner, K. J. 1979. Two molecular forms of (Na+ + K+)-stimulated ATPase in brain: separation and difference in affinity for strophanthidin. Journal of Biological Chemistry. 254:6060–6067.

Expression of α- and β-Subunit Isoforms of the Na,K-ATPase in the Nervous System

William R. Anderson, Peter E. Filuk, Auday A. Maki, and William L. Stahl

Departments of Physiology and Biophysics and Medicine (Neurology), University of Washington School of Medicine, and the Veterans Affairs Medical Center, Seattle, Washington 98108

Introduction

The functional Na,K-ATPase is composed of two subunits, the catalytic (α) subunit containing both the ATP and ouabain binding sites, and a β-subunit, the function of which is currently under investigation. Three distinct isoforms of the α-subunit (α1, α1, and α3) and two β-subunit isoforms (β1 and β2) have been identified (see Stahl, 1986; Sweadner, 1989). The functional significance of multiple subunit isoforms is unclear. In an attempt to gain insight into this question we used in situ hybridization to examine distribution of the mRNAs coding for these subunits in the adult rat brain (Filuk et al., 1989; Stahl and Baskin, 1990). These techniques have enabled us to identify at the regional and cellular levels the presence of message for a given subunit isoform throughout the brain. Our results show a differential but overlapping distribution of mRNAs for the isoforms in many regions of the brain.

Methods

Oligonucleotide antisense probes directed to either translated or untranslated regions of their corresponding subunit mRNAs were synthesized and 3' end-labeled with 35S-dATP. The α1, α2, α3, and β1 probes and hybridization techniques were described by Filuk et al. (1989) and by Stahl and Baskin (1990). The β2 probe sequence is (5'-3') GGGAGACTCCGATCACAGATATGCAGCTGT, corresponding to the 5' untranslated mRNA nucleotides –137 to –108 (Martin-Vasallo et al., 1989). In brief, 15-μm sagittal tissue sections from adult Sprague-Dawley rat brains were hybridized overnight with a 35S-labeled probe. Sections were washed to remove nonspecifically bound probe under high stringency conditions. The tissue sections were dried and exposed to film for 3–6 d to generate images for film autoradiography. Slides were dipped in emulsion and developed after 1–3 wk. Grains associated with cell bodies were counted using an imaging system. Specificity of the probes was assessed using Northern blots, cold displacement studies with excess unlabeled probe, and comparison of hybridization using probes targeted to both translated and untranslated regions of the same mRNA species. Each putative probe was initially converted to its sense sequence and screened against known mRNA species in the GENEPRO 4.1 database to assure specificity.

Results and Discussion

We found a characteristic pattern of distribution for each probe that showed considerable overlap with that of each of the other probes examined (Fig. 1) in the major areas of interest (identified in Fig. 1f). The α1 probe showed a diffuse pattern of binding throughout the rat brain (Fig. 1 a) with regions of higher labeling in the cerebral cortex (laminae 2 and 3), hippocampal formation (pyramidal cell layer), and the dentate gyrus (granule cell layer). The highest levels of binding were found in the cerebellum, with approximately equal distribution between granule and Purkinje cell layers. Low binding was seen in thalamic nuclei.

Figure 1. Localization of mRNAs to isoforms of the Na,K-ATPase subunits in rat brain using 35S-labeled oligonucleotide probes. Labeled tissue sections were exposed to Hyperfilm βMax. (a) α1 probe (arrow, DG granule cells); (b) α2 probe; (c) α3 probe (arrow, DG granule cells; arrowhead, CA3 pyramidal cells); (d) β1 probe; (e) β2 probe; (f) schematic drawing of rat brain showing selected regions of interest (CC, cerebral cortex; CB, cerebellum; HC, hippocampus; DG, dentate gyrus; TN, thalamic nuclei).

The α2 probe exhibited a very diffuse distribution throughout the brain (Fig. 1 b). Binding in the cerebral cortex was uniformly low across all laminae. More intense labeling was seen in the pyramidal cell layer of the hippocampus and the granular cell layer of the dentate gyrus. Specific binding was also found in all of the thalamic nuclei examined. Binding in the cerebellum appeared to be at levels comparable to those seen in the rest of the brain, with somewhat higher concentrations in the Purkinje cell layer.

The α3 probe showed high levels of binding in most areas of the brain (Fig. 1 c). In cerebral cortex the heaviest areas of labeling were found in laminae 4 and 5. Hippocampal formation labeling was concentrated throughout the pyramidal cell layer and this heavy labeling extended to include the granular cell layer of the dentate gyrus. Intense binding was associated with all of the thalamic nuclei. In the cerebellum labeling was seen in laminae containing cell bodies, with the highest labeling corresponding to the Purkinje cell layer.

Figure 2. Emulsion autoradiogram showing distribution of α3-specific mRNA in CA3 pyramidal cells of hippocampus. (a) Brightfield view. (b) Darkfield view of same section with selected pyramidal cells outlined for grain counting. Slides were exposed for 18 d, developed, and stained with cresyl violet. Grain counting was performed with an MCID image analysis system linked to a microscope. Note arrows for orientation.

The probe to β1 mRNA labeled all of the regions of rat brain where labeling by α-specific probes was observed (Fig. 1 d). The β2 probe showed a distribution (Fig. 1 e) similar to that seen for the α1 probe. In general there was a diffuse level of binding in all brain regions examined, with the very high levels in the cerebellum. Binding in the cerebellum appeared to label all of the layers associated with cell bodies at comparable levels.

We have begun to study the distribution of different α mRNAs in cells of the nervous system using emulsion autoradiography. We examined the frequency distribution of grains per cell in neurons in hippocampus and a typical autoradiogram is shown in Fig. 2, a and b. Most hippocampal neurons studied in CA1, CA3, and the dentate gyrus express all three α isoforms, but the level of expression varied considerably between the populations of cells examined.

In conclusion, we have demonstrated differences in the distribution of mRNAs coding for isoforms of the Na,K-ATPase. Each subunit isoform shows a characteristic distribution pattern in the brain regions studied, a pattern that also shows much overlap with that of the other subunit isoforms (see also Schneider et al., 1988; Filuk et al., 1989; Hieber et al., 1989; Stahl and Baskin, 1990). While most cell groups studied showed specific labeling for all of the probes used, quantitative differences were obvious. Different cell groups have dissimilar types and levels of firing activity and therefore have specialized requirements for ion homeostasis. An attractive hypothesis would find these specialized requirements directly reflected in relative concentrations of the mRNA and the relevant expressed Na,K-ATPase molecules within specific cell types. Little is known, however, about the functional differences between these subunit isoforms.

References

Filuk, P. E., M.A. Miller, D. M. Dorsa, and W. L. Stahl. 1989. Localization of messenger RNA encoding isoforms of the catalytic subunit of the Na,K-ATPase in rat brain by in situ hybridization histochemistry. Neuroscience Research Communications. 5:155–162.

Hieber, V., G. J. Siegel, T. Desmond, J. Lee-Hwa Liu, and S. A. Ernst. 1989. Na,K-ATPase: comparison of the cellular localization of α-subunit mRNA and polypeptide in the mouse cerebellum, retina, and kidney. Journal of Neuroscience Research. 23:9–20.

Martin-Vasallo, P., W. Dackowski, J. R. Emanuel, and R. Levenson. 1989. Journal of Biological Chemistry. 264:4613–4618.

Schneider, J. W., R. W. Mercer, M. Gilmore-Herbert, M. F. Utset, C. Lai, A. Greene, and E. J. Benz, Jr. 1988. Tissue specificity, localization in brain, and cell-free translation of mRNA encoding the A3 isoform of Na +,K+-ATPase. Proceedings of the National Academy of Sciences, USA. 85:284–288.

Stahl, W. L. 1986. The Na,K-ATPase of nervous tissue. Neurochemistry International. 8:449–476.

Stahl, W. L., and D. G. Baskin. 1990. Histochemistry of ATPases. Journal of Histochemistry and Cytochemistry. 38:1099–1122.

Sweadner, K. J. 1989. Isozymes of the Na+/K+-ATPase. Biochimica et Biophysica Acta. 988:185–220.

Diversity of Na,K-ATPase Isoform Distribution in the Nervous System

Kevin M. McGrail, Joseph M. Phillips, and Kathleen J. Sweadner

Neurosurgical Research, Massachusetts General Hospital, Boston, Massachusetts 02114

Introduction

The existence of three different isoforms of the catalytic subunit of the Na,K-ATPase was deduced by cDNA cloning of α gene homologues from rat brain libraries (reviewed in Sweadner, 1989). Only in the nervous system are all three isoforms expressed in high abundance. The crucial function of the Na,K-ATPase in maintaining and possibly modulating electrical excitability has made it attractive to determine whether each isoform is associated with particular cell types with characteristic physiological roles.

In early work (Sweadner, 1979; Specht and Sweadner, 1984), only two isoforms were known. The presence of one [“α(+)”] in axolemma and its selective axonal transport in optic nerve suggested that it might have a particular role in either the ion transport function or the subcellular localization of the Na,K-ATPase in myelinated axons. In contrast, glia in culture and in the optic nerve expressed al. Even in the earliest studies, however, it was shown that it was an oversimplification to call one isoform a “neuronal form” and the other a “glial form”: cultured sympathetic neurons were found to express only α1 (Sweadner, 1979), and a small proportion of α1 was cotransported with α(+) in the optic nerve (Specht and Sweadner, 1984). It was also shown that glia can express a Na,K-ATPase with a high affinity for ouabain, presumably α2 or α3 (Atterwill et al., 1984). Nonetheless, the simplicity of the hypothesis that there are neuronal and glial types of Na,K-ATPase (analogous to intermediate filaments) was very attractive, and the idea has been repeated often enough to take on a life of its own.

We have taken advantage of isoform-specific antibodies to track the expression of the three α gene products (McGrail and Sweadner, 1989; McGrail et al., 1991).

Methods

Immunofluorescent localization was performed with 6–12-μM cryostat sections of tissue that had been fixed with periodate-lysine-paraformaldehyde (McLean and Nakane, 1974). Anesthetized rats were perfused transcardially with the fixative, and fixed tissues were soaked in 25% sucrose, 0.1 M phosphate buffer before sectioning. Sections on gelatin-subbed slides were incubated in primary antibody for 1 h, washed, and stained with rhodamine-conjugated goat anti-mouse or-rabbit Ig antisera, and then examined with fluorescence microscopy.

The antibodies were as follows: early work was performed with polyclonal antisera (K1 and Ax2) raised against purified rat kidney and axolemma Na,K-ATPases (Sweadner and Gilkeson, 1985). Most results obtained with these antisera were similar to those obtained later with monoclonal antibodies specific for α1 and α3, respectively. More recent experiments were performed with mouse monoclonal antibodies specific for α1 (McK1) (Felsenfeld and Sweadner, 1988), α2 (McB2), and α3 (McBX3) (Urayama et al., 1989), all of which were raised against rat Na,K-ATPases purified from kidney or brainstem axolemma.

Figure 1. Immunofluorescent stain for α2 and α3 in a myelinated tract in the pons, cut in cross-section. Photographed through a 100× objective; scale bar is 10μm.

Results and Discussion

α1 appeared to be the predominant isoform found in cerebellar granule cell somas, and it was very abundant in the cerebellar glomeruli, a multicellular synaptic structure. It was also present in hippocampal pyramidal neurons and in many cell bodies (presumably amacrine cells as well as Müller glia) in the inner nuclear layer of the retina. Fine vertical processes stained for α1 in the cerebral cortex. Several regions were seen with very little stain for α1, including the stratum oriens of the hippocampus and certain white matter tracts in the brainstem and spinal cord.

α2 staining tended to appear more diffuse than staining for α1 and α3, possibly due to a higher proportion in diffusely distributed glia than in neurons. There were several clear instances of neuronal stain for α2, however, including a subclass of amacrine cells in the retina, and spinal motoneurons.

α3 staining was frequently seen in the large-diameter neurons that produce long, myelinated axons. Examples of this were seen in the retinal ganglion cells, hippocampal and cortical pyramidal cells, cerebellar Purkinje cells, and spinal motoneurons. The presence of α3 in interneurons was also indicated by its abundance in the synaptic layers of the retina, where there are no myelinated axons, and in fact where there are no Na+-dependent action potentials. α3 was the only isoform that was not explicitly identified in glia.

Fig. 1 illustrates the very different staining patterns obtained with antibodies specific for α2 and α3 in a white matter tract. In this case, it was a bundle of myelinated axons, some very large, in the ventral tegmentum of the pons. There was little or no staining for α1 (not shown). The α2 and α3 immunofluorescence was photographed at high magnification, permitting a close examination of the differences in cellular distribution. The α3 antibody ring-stained the myelinated axons, apparently at the axolemma. Myelin was unstained. The α2 antibody, in contrast, stained the astroglial processes between the myelinated axons. This stain has been shown to colocalize with GFAP (glial fibrillary acidic protein), an astroglial marker (McGrail et al., 1991). At this level of staining intensity, we could not identify the isoform expressed in oligodendrocytes with any certainty. It should be stressed that other myelinated axon tracts, notably the corpus callosum and some tracts in the pons, had α1 stain as abundant as α3, and in some cases it was clear that the α1 was axonal. Thus, it cannot be stated that all myelinated axons express only α3. Finally, the ring-staining of axons cut in cross-section (Fig. 1) implies that the Na,K-ATPase cannot be exclusively located to nodes of Ranvier.

Acknowledgments

This work was supported by NIH grant NS-27653.

References

Atterwill, C. K., V. J. Cunningham, and R. Balazs. 1984. Characterization of Na+,K+-ATPase in cultured and separated neuronal and glial cells from rat cerebellum. Journal of Neurochemistry. 43:8–18.

Felsenfeld, D. P., and K. J. Sweadner. 1988. Fine specificity mapping and topography of an isozyme-specific epitope of the Na,K-ATPase catalytic subunit. Journal of Biological Chemistry. 263:10932–10942.

McGrail, K. M., J. M. Phillips, and K. J. Sweadner. 1991. Immunofluorescent localization of three Na,K-ATPase isozymes in the rat central nervous system: both neurons and glia can express more than one Na,K-ATPase. Journal of Neuroscience. 11:381–391.

McGrail, K. M., and K. J. Sweadner. 1986. Immunofluorescent localization of two different Na,K-ATPases in the rat retina and in identified dissociated retinal cells. Journal of Neuroscience. 6:1272–1283.

McGrail, K. M., and K. J. Sweadner. 1989. Complex expression patterns for Na,K-ATPase isoforms in retina and optic nerve. European Journal of Neuroscience. 2:170–176.

McLean, I. W., and P. K. Nakane. 1974. Periodate-lysine-paraformaldehyde fixative: a new fixative for immunoelectron microscopy. Journal of Histochemistry and Cytochemistry. 22:1077–1083.

Specht, S.C., and K. J. Sweadner. 1984. Two different Na,K-ATPases in the optic nerve: cells of origin and axonal transport. Proceedings of the National Academy of Sciences, USA. 81:1234–1238.

Sweadner, K. J. 1979. Two molecular forms of (Na+ + K+)-stimulated ATPase in brain. Separation, and difference in affinity for strophanthidin. Journal of Biological Chemistry. 254:6060–6067.

Sweadner, K. J. 1989. Isozymes of the Na+/K+-ATPase. Biochimica et Biophysica Acta. 988:185–220.

Sweadner, K. J., and R. C. Gilkeson. 1985. Two isozymes of the Na,K-ATPase have distinct antigenic determinants. Journal of Biological Chemistry. 260:9016–9022.

Urayama, O., H. Shutt, and K. J. Sweadner. 1989. Identification of three isozyme proteins of the catalytic subunit of the Na,K-ATPase in rat brain. Journal of Biological Chemistry. 264:8271–8280.

Cellular Distribution and Differential Expression of the Na,K-ATPase α Isoform (α1, α2, α3), β1, and β2/AMOG Genes in the Ocular Ciliary Epithelium

Miguel Coca-Prados, Pablo Martin-Vasallo, Nati Hernando, and Sikha Ghosh

Departments of Ophthalmology and Visual Science, Yale University Medical School, New Haven, Connecticut 06510; and Department of Biochemistry and Molecular Biology, University de La Laguna, Tenerife, Spain

Introduction

The ciliary epithelium (CE) is a neuroepithelial bilayer composed of cells from the pigmented (PE) and the nonpigmented epithelium (NPE). A major function of the CE is the formation and secretion of aqueous humor in the mammalian eye. The CE forms a barrier between the blood and aqueous humor which prevents the free passage of serum proteins from the stroma into the posterior chamber of the eye. It is known that the Na,K-ATPase plays an important role in determining the ionic composition of the aqueous humor. However, the mechanism by which NPE and PE cells regulate Na,K-ATPase to produce the aqueous humor has not yet been discovered.

Previous studies from this laboratory have shown that there is a differential cellular distribution and gene expression of the three α-subunit isoforms of the Na,K-ATPase in the CE (Martin-Vasallo et al., 1989b; Ghosh et al., 1990). These studies support the notion that the NPE cells provide the bulk of the ion-transporting activity of the CE and that there are cellular (PE versus NPE) and regional (pars plicata versus pars plana) differences within the CE that might reflect functional differences in aqueous humor secretion.

We describe the localization and the differential gene expression of the β1-subunit of the Na,K-ATPase and its homologue, the adhesion molecule on glia (β2/AMOG) (Martin-Vasallo et al., 1989b; Gloor et al., 1990) in the pars plicata and pars plana regions of the CE.

Materials and Methods

Tissue Source

Eyes from 2–5-d-old calves were obtained from a local slaughterhouse and the ciliary epithelium was dissected in two regions, the pars plicata and pars plana, as described previously (Ghosh et al., 1990) (see Fig. 1).

Immunocytochemistry

Immunolocalization of Na,K-ATPase α (α1, α2, α3) and β (β1, β2) isoforms in intact ciliary processes was carried out by indirect immunofluorescence in 0.5-μm cryostat sections as previously described (Ghosh et al., 1990).

Western Blots

Microsomal fractions were prepared from intact ciliary epithelium, pars plicata, pars plana, and sorted cells. Protein samples were treated with dithiothreitol and iodoacetamide and subjected to electrophoresis in 10% Polyacrylamide SDS gels (50 μg protein/lane). Proteins were transferred to nitrocellulose sheets as described (Ghosh et al., 1990). Polyclonal antibodies to dog kidney α1-subunit were used as described previously (Ghosh et al., 1990). Monoclonal antibodies to the α2- (McB2), and α3-subunit (McB-X3) isoforms (Urayama et al., 1989) were generous gifts from Dr. K. Sweadner (Massachusetts General Hospital, Boston, MA). Polyclonal antibodies to a TrpE-rat α3 fusion protein (Shyjan and Levenson, 1989) were provided by Dr. A. Shyjan (Yale University, New Haven, CT). Monoclonal antibodies to the β1-subunit (IEC 1/48) (Marxer et al., 1989) were from Dr. A. Quarone (Cornell University, Ithaca, NY). Polyclonal antibodies to the AMOG/β2 subunit isoform (Gloor et al., 1990) were provided by Dr. M. Schachner (University of Heidelberg, Heidelberg, Germany).

Figure 1. Scheme of ocular ciliary epithelium and the two regional zones (pars plicata and pars plana) presented in this work. The differential immunolocalization of Na,K-ATPase α (α1, α2, α3) and β (β1, β2) isoforms in NPE and PE cells in the pars plicata and pars plana regions is summarized in the upper drawing.

Northern Blots

Samples of total cellular RNA (20 μg) were electrophoresed on 1% agarose/formaldehyde gels and capillary blotted onto Zytabind membranes (AMF Cuno, Meriden, CT). Membranes were prehybridized and hybridized as described previously (Ghosh et al., 1990). cDNA probes for the human Na,K-ATPase α1-, α2-, α3-, β1-, and β2-subunit isoforms (Martin-Vasallo et al., 1989a, b) were provided by Dr. R. Levenson (Yale University).

Results and Discussion

A schematic drawing of the pars plicata and pars plana regions of the ciliary epithelium in relation to the iris and the retina is shown Fig. 1. The PE layer in the ciliary epithelium is a continuation of the retina pigment epithelium (RPE), while the ciliary NPE is related to the sensory layers of the retina. The apical plasma membrane (a.m.) of the NPE cell layer apposes the apical plasma membrane of the PE cell layer. The basal plasma membrane (b.m.) of the NPE cells faces the aqueous humor, while the basal plasma membrane of PE faces the stroma. Tight junctions are only present between the NPE cells near their apical plasma membranes forming the blood-aqueous barrier.

When antisera to each of the three α- and two β-subunit isoforms were applied to semi-thin cryostat sections of the pars plicata and pars plana regions of the ciliary epithelium, distinct patterns of staining were observed for NPE and PE under the fluorescence microscope (Fig. 2). Differences in staining were noted at the regional, cellular, and subcellular levels.

At the regional level, the most remarkable difference was the failure of McBX3 antibodies to stain the α3 isoform in NPE cells from the pars plana region, whereas the same antibody labeled NPE cells from the pars plicata. However, expression of the α3 isoform by NPE cells in the pars plana was demonstrated by Western blot analysis, suggesting that the epitope recognized by McBX3 in the pars plana might not be as readily accessible as in the pars plicata. Whether the differential immunolocalization patterns of α3 in these two regions might reflect a functional conformation change on α3 remains to be determined (Ghosh et al., 1990).

At the cellular level, the staining for each of the α and β isoforms of Na,K-ATPase in NPE and PE cells was strikingly different. The α1, α2, α3, and β2 isoforms were clearly immunodetected on NPE cells from the pars plicata and restricted preferentially to their basolateral plasma membrane. PE cells from the pars plicata and pars plana stained brightly for α1 in the basolateral membrane and for β1 in the basolateral and apical plasma membranes. However, no staining was detected on PE cells with α2, α3, and β2 antibodies. These results indicate a clear difference in expression of Na,K-ATPase α and β isoforms between NPE and PE cells.

At the subcellular level, β1 antibodies (IEC 1/48) gave bright staining of basolateral PE cytoplasm and weak staining of the apical plasma membrane of PE cells. The relative lack of labeling for β1 on the basolateral membrane of NPE cells is puzzling. One possible explanation is that IEC 1/48 may recognize β1 on PE cells in a posttranslational processing form different from the β1 form present on NPE cells. The presence of β1 isoforms on NPE cells cannot be ruled out.

Previously we have shown (Ghosh et al., 1990) the presence of α1, α2, and α3 Na,α-ATPase polypeptide isoforms in FACS®-sorted NPE cells, but only α1 polypeptide isoform in sorted PE cells (Fig. 3 A, lanes 1–3). The present study includes an investigation of tissue expression for β1 and AMOG/β2. Microsomes from the ciliary epithelium were subjected to reduction-alkylation (Charlemagne et al., 1987) before separating proteins in a 10% SDS-polyacrylamide slab gel. Proteins were transferred to nitrocellulose filters and probed with antibodies to Na,K-ATPase β1 and AMOG/β2. IEC1/48 monoclonal antibody (Marxer et al., 1989) was used as a probe for the β1 isoform of the Na,K-ATPase. Previous work has demonstrated that IEC 1/48 is specific for the β- (probably β1) subunit of the Na,K-ATPase (Marxer et al., 1989). IEC 1/48 antibodies reacted with a sharp band of ~45 kD in the rat ciliary epithelium (Fig. 3 A). Antisera to AMOG stained two bands: one at 105 kD corresponding to the Na,K-ATPase α2- and/or α3-subunit isoforms, and a broader band ranging from 45 to 50 kD corresponding to the β2-subunit (Fig. 3 A).

Figure 2. Immunolocalization of Na,K-ATPase α1-, α2-, α3-, β1-, and β2-subunit isoforms in the pars plicata region of the ciliary epithelium. Semithin frozen sections of bovine ciliary epithelium were incubated with antisera against α1, α2, α3, and AMOG/β2. Cryostat sections from an albino rat were incubated with antisera against β1 (IEC 1/48). Specific bound antibodies were visualized with rhodamine-conjugated secondary antibodies. PE, pigmented cell; NPE, nonpigmented cell; S, stroma; am, apical membrane.

Figure 3. (A) Immunoblotting analysis of Na,K-ATPase α (α1, α2, α3) and β (β1, β2) subunit isoforms in the ciliary epithelium and cell-sorted ciliary epithelial cells. Microsomes were prepared and proteins (50 μg of protein per lane) separated by SDS-PAGE, transferred into a nitrocellulose filter, and probed with antisera against Na,K-ATPase α1, α2 (McB2), TrpE-rat α3 fusion protein, β1 (IEC 1/48), and AMOG/β2. Lane 1, intact ciliary epithelium from the pars plicata; lane 2, sorted NPE cells; lane 3, sorted PE cells. (B) Northern blot analysis of mRNA encoding the α (α1, α2, α3) and β (β1, β2) subunit isoforms of human Na,K-ATPase in the ciliary epithelium. Five identical Northern blots containing 20 μg/per lane of total RNA from the pars plicata of a human eye were hybridized with three specific human α (α1, α2, α3) and two β (β1, β2) subunit cDNAs as indicated in Materials and Methods. The positions of the 28S and 18S rRNA markers are indicated.

To analyze the expression of Na,K-ATPase α- and β-subunit mRNAs in the ciliary epithelium, cDNA probes specific for the human α1-, α2-, α3-, β1-, and β2-subunit isoforms were hybridized to five identical Northern blots containing 20 μg of total RNA from the pars plicata region of the ciliary epithelium of a human eye. The α1 and α3 probes hybridized to a single mRNA of 4.5 kb, while the α2 probe hybridized to two mRNAs of 4.5 and 6 kb. The β1 probe hybridized to mRNA of 2 and 2.5 kb, and the β2 probe hybridized to a single mRNA of 3.4 kb (see Fig. 3 B). These results suggest that transcripts for the three α and two β isoforms are expressed in the ocular ciliary epithelium.

In summary, the ocular ciliary epithelium expresses mRNA and polypeptide for the α- (α1, α2, α3) and β- (β1, β2) subunit isoforms of the Na,K-ATPase. There is a marked cellular (NPE versus PE) and regional (pars plicata versus pars plana) difference in the distribution and gene expression of the α and β isoforms in the ciliary epithelium. The α2, α3, and β2 isoforms are restricted to the NPE cells in the pars plicata. The β1 isoform is predominantly expressed in PE cells. The α (α1, α2, α3) and β (β1, β2) isoforms are distributed along the basolateral domains of ciliary epithelial cells. In addition, β1 also appears on the apical domain of PE cells.

Acknowledgments

We thank Dr. K. Sweadner for McB2 and McB-X3 antisera, Dr. A. Shyjan for a TrpE-rat α3 fusion protein antisera, Dr. A. Quaroni for IEC 1/48 antibodies, and Dr. M. Schachner for AMOG antibodies. We thank Dr. R. Levenson for the human cDNA probes.

This work was supported by NIH grant EY-08672 (to M. Coca-Prados). Additional support was from the Alcon Research Institute and Allergan, Inc. P. Martin-Vasallo was supported by a fellowship from Consejeria de Educacion del Gobierno Autonomo Canario, Spain.

References

Charlemagne, D., E. Mayoux, M. Poyard, P. Oliviero, and K. Geering. 1987. Identification of two isoforms of the catalytic subunit of Na,K-ATPase in myocytes from adult rat heart. Journal of Biological Chemistry. 262:8941–8943.

Ghosh, S., A. C. Freitag, P. Martin-Vasallo, and M. Coca-Prados. 1990. Cellular distribution and differential gene expression of the three α subunit isoforms of the Na,K-ATPase in the ocular ciliary epithelium. Journal of Biological Chemistry. 265:2935–2940.

Gloor, S., H. Antonicek, K. J. Sweadner, S. Pagliusi, R. Frank, M. Moos, and M. Schachner. 1990. The adhesion molecule on glia (AMOG) is a homologue of the β subunit of the Na,K-ATPase. Journal of Cell Biology. 110:165–174.

Martin-Vasallo, P., W. Dackowski, J. R. Emanuel, and R. Levenson. 1989a. Identification of a putative isoform of the Na,K-ATPase β subunit. Journal of Biological Chemistry. 264:4613–4618.

Martin-Vasallo, P., S. Ghosh, and M. Coca-Prados. 1989b. Expression of Na,K-ATPase alpha subunit isoforms in the human ciliary body and cultured ciliary epithelial cells. Journal of Cellular Physiology. 141:243–252.

Marxer, A., B. Stieger, A. Quaroni, M. Kashgarian, and H.-P. Hauri. 1989. (Na+ + K+) -ATPase and plasma membrane polarity of intestinal epithelial cells: presence of a brush border antigen in the distal large intestine that is immunologically related to β subunit. Journal of Cell Biology. 109:1057–1069.

Shyjan, A. W., and R. Levenson. 1989. Antisera specific for the α1, α2, α3 and β subunits of the Na,K-ATPase: Differential expression of α and β subunits in rat tissue membranes. Biochemistry. 28:4531–4535.

Urayama, O., H. Shutt, and K. J. Sweadner. 1989. Identification of three isozyme proteins of the catalytic subunit of the Na,K-ATPase in rat brain. Journal of Biological Chemistry. 264:8271–8280.

Na,K-ATPase α1, α2 Isoform and β-Subunit Are Differentially Expressed in Different Regions of the Colonic Circular Muscle Layer

Burton Horowitz and Edmund Burke

Department of Physiology, University of Nevada School of Medicine, Reno, Nevada 89557

Introduction

Resting membrane potential (RMP) is one of the determinants of excitation-contraction coupling in gastrointestinal (GI) smooth muscle (Casteels, 1981). While reports vary on the magnitude of the contribution of electrogenic sodium pumping to resting membrane potential in GI smooth muscle (Casteels, 1969; Brading and Widdecombe, 1974), it is clear that this ion pump is important to the generation and maintenance of RMP. Rhythmic oscillations in membrane potential, termed slow waves, are responsible for initiating contractions or generating action potentials that lead to contractions (Szurszewski, 1987). In the colon, slow waves originate at the submucosal surface of the circular muscle and conduct in a nonregenerative manner through the thickness of the circular layer (Smith et al., 1987a, b). Recent reports have demonstrated a large potential gradient through the thickness of the circular layer, such that cells near the submucosal surface are polarized to approximately −78 mV, whereas cells near the myenteric border are polarized only to about −40 mV (Smith et al., 1987a, b).

Burke et al. (1988) suggested that a gradient in the contribution of the sodium pump to RMP causes the gradient in membrane potential. The molecular and biochemical bases for gradient in RMP have not been determined. Several interrelated hypotheses might explain the gradient in RMP. (a) The form of the sodium pump, reflected in the affinity of the pump for Na+ or K+, may vary with distance through the circular layer. This would affect pump activity. (b) The pump density (amount of pump protein per unit area of membrane) may vary through the circular layer. (c) The concentration of a substrate for pump activity, such as [Na+]i, may vary in cells through the circular layer, perhaps due to a gradient in sodium permeability.

This study investigated the possibility that differential expression of Na,K-ATPase genes through the thickness of the circular muscle layer contributes to the gradient in membrane potential.

Results

Relative Na,K-ATPase abundance in strips of muscle taken from the submucosal and myenteric borders was determined. Since these regions exhibit the greatest disparity in membrane potential, we reasoned that in preliminary experiments these muscle strips should be examined first.

Figure 1. Schematic diagram representing tissue dissection.

A thin band of circular muscle lying adjacent to the submucosal border was dissected free with fine iris scissors. These strips are referred to as submucosal strips. Another thin strip of muscle bordering the myenteric region was dissected in a similar manner. These strips are referred to as myenteric strips (Fig. 1). Na,K-ATPase α1-, α1-, and β-subunit mRNA abundance was determined in these muscle regions. Na,K-ATPase α3 isoform mRNA has not been detected in colonic smooth muscle. Northern blots containing equal amounts of poly A+ RNA prepared from submucosal and myenteric muscle strips and brain tissue were hybridized with 32P-labeled Na,K-ATPase subunit-specific cDNA probes. The resulting autoradiograms are shown in Fig. 2. A cDNA of CHO-B, a Chinese hamster ovary cell mRNA expressed at equivalent levels in all tissue examined (Harpold et al., 1979), was used as an internal standard and is also shown in Fig. 2. Relative mRNA abundance was then quantitated for submucosal and myenteric muscle strips using the slot blot technique. The data shown in Fig. 3 were compiled from two independent poly A+ RNA preparations. Each preparation was derived from tissue obtained from four or five animals; therefore, animal variability was not in question. The results demonstrate that submucosal muscle strips express approximately twice as much Na,K-ATPase α1 isoform mRNA as muscle strips dissected from the myenteric border. Na,K-ATPase α2 isoform mRNA, however, appears to be more abundant in myenteric muscle strips, β-subunit mRNA expression levels demonstrate an even more pronounced difference between submucosal and myenteric strips than does α1. The nonstoichiometric differences in β-subunit expression relative to differences in α1 have been described in other tissues (McDonough et al., 1990).

Figure 2. Autoradiograms of Northern blots containing mRNA from submucosal and myenteric muscle strips and canine brain tissue. S, Submucosal muscle strips; M, myenteric muscle strips; B, brain tissue. The positions of 18S and 28S ribosomal RNAs are indicated.

Figure 3. Na,K-ATPase α isoform and β-subunit mRNA levels in submucosal and myenteric muscle strips. An arbitrary value of 1.0 is given to submucosal mRNA abundance. mRNA abundance in myenteric muscle strips is normalized to that of submucosal muscle strips.

Isoform distribution in submucosal and myenteric muscles was determined by Western blot analysis using isoform-specific antibodies. Quantitation was performed by densitometry of filters from slot blots of graded amounts of microsomal protein from each sample. The data shown in Fig. 4 were compiled from three independent microsomal protein preparations. Again, each preparation was derived from tissue obtained from four or five animals. Na,K-ATPase α1 polypeptide demonstrates a similar distribution to α1 mRNA. The amount of α1 in submucosal muscle strips is significantly greater than that found in the myenteric border. α2 polypeptide, however, unlike mRNA abundance, appears to be equally distributed in submucosal and myenteric muscle strips. The discrepancy between α2 mRNA and polypeptide abundance may be due to posttranscriptional regulatory events.

Figure 4. Na,K-ATPase α isoform polypeptide levels in submucosal and myenteric muscle strips. An arbitrary value of 1.0 is given to submucosal polypeptide abundance. Polypeptide abundance in myenteric muscle strips is normalized to that of submucosal muscle strips.

Sodium pump activity was assayed using the K+ -stimulated phosphatase activity assay. A K+ plus ouabain negative control was included in the assays to supplement the Na+ negative control. The data are shown in Fig. 5 as units of pNPPase activity and as activity relative to that of submucosal muscle strips. Sodium pump activity distribution directly correlates with the relative abundance of α1 mRNA and protein in submucosal and myenteric muscle strips.

These results suggest that the gradient in membrane potential through the thickness of the circular muscle layer is at least partially dependent upon a similar gradient in sodium pump activity and Na,K-ATPase α1 genetic expression. These results provide evidence of differential gene expression in circular smooth muscle cells apparently based on their position in the circular muscle layer. This emphasizes the need to determine the factors affecting Na,K-ATPase gene expression in colonic smooth muscle in relation to changes in electrical activity and pathological conditions. Another of the implications of this phenomenon is the possibility of differential expression of ion channel proteins involved in slow wave formation. This would provide more evidence of a molecular basis for GI motility.

Figure 5. Na,K-ATPase-specific, K+ -dependent phosphatase activity in submucosal and myenteric muscle strips and canine heart and kidney microsomes. Data are given in terms of both units of activity (micromoles Pi per milligram per hour) and activity relative to the activity in submucosal muscle strips. Microsomal membranes prepared from myenteric muscle strips display Na,K-ATPase activity that is 57.8% that of microsomes prepared from submucosal muscle strips.

Acknowledgments

This work was partially supported by a program project grant from the NIDDKD (DK-41315).

References

Brading, A. F., and J. H. Widdecombe. 1974. An estimate of the sodium-potassium pump activity and the number of pump sites in the smooth muscle of the guinea pig taenia coli, using [3H]ouabain. Journal of Physiology 238:235–249.

Burke, E. P., J. B. Reed, and K. M. Sanders. 1988. Role of sodium pump in membrane potential gradient of canine proximal colon. American Journal of Physiology. 254:C475–C483.

Casteels, R. 1969. Calculation of the membrane potential in smooth muscle cells of the guinea pig's taenia coli by the Goldman equation. Journal of Physiology. 205:193–208.

Casteels, R. 1981. Membrane potential in smooth muscle cells. In Smooth Muscle: An Assessment of Current Knowledge. E. Bulbring, A. F. Brading, A. W. Jones, and T. Tomita, editors. University of Texas Press, Austin. 105–126.

Harpold, M. M., R. M. Evans, M.G. Salditt, and J. E. Darnell. 1979. Production of mRNA in Chinese hamster cells: relationship of the rate of synthesis to the cytoplasmic concentration of nine specific mRNA sequences. Cell. 17:1025–1035.

McDonough, A. A., K. Geering, and R. A. Farley. 1990. The sodium pump needs its β subunit. FASEB Journal. 4:1598–1605.

Smith, T. K., J. B. Reed, and K. M. Sanders. 1987a. Interaction of two electrical pacemakers in muscularis of canine proximal colon. American Journal of Physiology. 252:C290–C299.

Smith, T. K., J. B. Reed, and K. M. Sanders. 1987b. Origin and propagation of electrical slow waves in circular muscle of canine proximal colon. American Journal of Physiology. 252:C215–C224.

Szurszewski, J. H. 1987. Electrical basis for gastrointestinal motility. In Physiology of the Gastrointestinal Tract. 2nd ed. L. R. Johnson, editor. Raven Press, New York. 383–422.

Regulation of Na,K-ATPase α1 and α2 Isoforms in Rat Myocardial Cells by Thyroid Hormone

Charles B. Hensley, Ming-Jer Tang, Kay K. Azuma, and Alicia A. McDonough

Department of Physiology and Biophysics, University of Southern California School of Medicine, Los Angeles, California 90033

Introduction

Thyroid hormone effects on cardiac contractility are well established (increased heart rate, increased velocity of contraction, and enhanced rate of diastolic relaxation). In adult cardiac myocytes, two isoforms of the Na,K-ATPase α-subunit are expressed. The α2 isoform is distinguished from α1 isoform by its much higher affinity for cardiac glycosides (Sweadner, 1979). Recently, it has been shown that T3 regulates the expression of the α1 and α2 isoforms in primary cultures of neonatal heart cells (Orlowski and Lingrel, 1990) and adult rat ventricle (Gick et al., 1990; Horowitz et al., 1990). Furthermore, we have shown that in hypokalemia the α2 isoform is greatly decreased in heart and muscle relative to α1 (Azuma et al., 1991). The present study aimed to determine the effect of thyroid status on α isoform and β1 mRNA and protein levels in isolated adult rat cardiac myocytes from euthyroid, hypothyroid, and hyperthyroid rats.

Methods

Rats were rendered hypothyroid by feeding them a low iodine test diet and 0.02% methimazole (MMI) in their drinking water for 15 d. Hyperthyroid rats were hypothyroids given a daily subcutaneous dose of T3 (1 μg/g body weight). Hypothyroid and euthyroid animals received a daily dose of diluent. Animals were killed by anesthesia with metofane and then decapitated, and cardiac myocytes were prepared as described by Piper et al. (1982).

Total RNA was isolated from cardiomyocytes according to Chomczynski and Sacchi (1987). Relative amounts of α- and β-mRNA were measured by the dot-blot method as described by McDonough et al. (1988). Hybridization was performed as previously described (Horowitz et al., 1990) using isoform-specific probes prepared according to Orlowski and Lingrel (1988). Abundance of Na,K-ATPase subunits was detected by immunoblot analysis as described by Horowitz et al. (1990). Immunoblots were probed with either α1 (McK1) or α2 (McB2) specific anti-rat monoclonal antibodies (Urayama and Sweadner, 1988) provided by K. Sweadner (Harvard Medical School), or with polyclonal rabbit anti-rat β1 antibody (Shyjan and Levenson, 1988) provided by R. Levenson (Yale Medical School). Na,K-ATPase activity was assessed in heart homogenates by the measurement of K+ -dependentp-nitrophenyl phosphatase reaction of Murer et al. (1976). Protein was measured by the method of Lowry et al. (1951).

Results and Discussion

Transition from Euthyroid to Hypothyroid

The α1, α2, and β mRNAs and protein levels in cardiomyocytes respond as reported previously for whole ventricle (Horowitz et al., 1990). These results are summarized in Table I. The α1, α2, and β mRNA did not change from that of the euthyroid controls. In the hypothyroid state, α1 protein levels were decreased to 0.57 of euthyroid, α2 protein levels to 0.34 of euthyroid, and β protein levels to 0.57 of euthyroid. The Na,K-ATPase activity levels were decreased to 0.52 of euthyroid. These results support our previous observation that T3 regulates the euthyroid level of expression of both isoforms of the α- and β-subunit. Our findings indicate that in the transition from euthyroid to hypothyroid, the drop in T3 levels affects NaK-ATPase levels translationally or post-translationally. One potential step is at the level of αβ assembly. In recent work on kidney cells from this laboratory, pulse-chase experiments demonstrated that 50% of nascent Na,K-ATPase β-subunits are degraded within 90 min of synthesis, whereas no degradation of the nascent α-subunit proteins occurred within 120 min of synthesis (Lescale-Matys and McDonough, 1991). If these results are extended to heart cells, they suggest that a decrease in β mRNA translatability coupled to the degradation of newly synthesized β may indeed render β rate limiting for assembly to α1 and α2 in hypothyroid heart cells, which may lead to an increase in the rate of degradation of unassembled subunits.

TABLE I
Na,K-ATPase mRNA Abundance in Cardiomyocytes from Euthyroid, Hypothyroid, and Hyperthyroid Hearts

All values are relative to hypothyroid. The data are given as the mean ± SD. Significance (*) is accepted at P ≤ 0.05.

Transition from Hypothyroid to Hyperthyroid

In the hyperthyroid state, the mRNAs for α1, α2 and α3 were increased well above euthyroid levels (Table I). The time course of mRNA accumulation induced by T3 was distinct for the three mRNAs. The abundance of α1 mRNA increased to the maximum of 2.5-fold by 6 h of treatment, whereas the α2 and β mRNA abundance continued to increase during 48 h of treatment to 6.2- and 10.9-fold, respectively. These results are in good agreement with our previous findings (Horowitz et al., 1990) and those of Gick et al. (1990). These results suggest that the turnover times for these mRNAs may be quite different, or that the mechanisms of activation, whether direct or indirect, have different kinetics.

The increased α1 mRNA abundance was reflected by a similar twofold change in α1 protein abundance (Table II). In the hyperthyroid state, α2 protein increased to a maximum of 15.4-fold above hypothyroid levels, reaching a new steady state after 8 d of treatment, far higher than the 6.2-fold increase in α2 mRNA. In contrast, the 3.1-fold induction of the β-subunit protein was substantially less than the 11-fold increase in β mRNA. We suggest that newly synthesized β is in excess of combined α1 and α2-subunits and that unassembled β-subunits are degraded quickly, as suggested in kidney cells (Lescale-Matys and McDonough, 1991).

A rationale for the large increase in β mRNA in the hyperthyroid state lies in the inherent complexity of differentially regulating multiple isoforms. So far, only β1 has been shown to be expressed in myocardial cells (Shyjan et al., 1990). It follows that α1 and α2 proteins must compete for assembly with β1. The large increase in the β mRNA we observe could indicate a mechanism in which the cell differentially upregulates both isoforms of the α-subunit heterodimer while avoiding competition for a rate-limiting β-subunit. In this model, T3 induces enough α1 and α2 mRNAs to drive the synthesis of the respective proteins. Concomitantly, a large excess of β mRNA is accumulated by increasing transcription rate and/or increasing the stability of the mRNA. As the nascent β proteins are translated, they are either assembled with a nascent α1 or α2 protein or rapidly degraded. Thus, in contrast to the hypothyroid state where we hypothesize that β-subunits may be rate limiting for αβ assembly, we hypothesize that in the hyperthyroid state α isoform proteins are rate limiting for assembly, allowing for a tight pretranslational regulation of the ratio of α1 to α2 heterodimers. The large increase in α2 protein relative to the change in α2 mRNA may reflect a decrease in the rate of degradation of nascent α2 protein as a consequence of assembly with the increased supply of nascent β.

TABLE II
Na,K-ATPase Protein Abundance and Enzyme Activity in Cardiomyocytes from Euthyroid, Hypothyroid, and Hyperthyroid Hearts

All values are relative to hypothyroid. The data are given as the mean ± SD. Significance (*) is accepted at P ≤ 0.05.

Previous studies have indicated that T3 increases myocardial Na,K-ATPase activity twofold or less (Klein et al., 1984; Norgaard et al., 1985; Chaudhury et al., 1987; Gick et al., 1990). In the present study we find the Na,K-ATPase activity was increased 1.7-fold by the second day, increasing to a maximum of 2.3-fold by the fourth day of T3 treatment. We hypothesize that the difference between the T3-induced changes in the α isoforms, β-subunit, and enzyme activity reflects differences in the relative abundance of the α1 and α2 isoforms in the cardiac myocytes. We calculate that in the transition from hypothyroid to hyperthyroid, the α1 to α2 protein ratio is shifted from 12:1 to 2:1 as a consequence of the 15.4-fold change in α2 relative to the 2.1-fold change in α1.

This study demonstrates that T3 administered in vivo differentially regulates the expression of the Na,K-ATPase α1-, α2-, and β-subunit mRNA and protein levels in myocardial cells, primarily influencing the protein abundance of the high ouabain affinity α2 isoform. The relationship between the change in the α isoform ratios and myocardial function has yet to be established. While the increases in the α2 protein could reflect a mechanism for increasing overall Na,K-ATPase activity, alternatively there may be a specific and specialized responsibility for the myocardial α2 isoform. For example, the α2 isoform has been suggested to be responsible for the inotropic effect of digitalis in vivo (Maixent et al., 1987). By regulating the sodium concentration at the cytoplasmic face of Na/Ca exchange proteins, the α2 protein could influence the flux of calcium through the exchanger. The resulting changes in cytosolic calcium are reflected by a change in the amount of releasable calcium stored in the sarcoplasmic reticulum and consequently an increase in the force of myocardial contractions. The possibility of an asymmetric distribution of the α2 isoform is intriguing. Given the close proximity of the transverse tubule system to the sarcoplasmic reticulum, alteration in either the abundance or activity of the Na,K-ATPase α2 isoform protein in this region should have a large impact on calcium dynamics and therefore myocardial performance.

References

Azuma, K. K., C. B. Hensley, D. S. Putnam, and A. A. McDonough. 1991. Differential regulation of Na,K-ATPase α1, α2, and β in heart, skeletal muscle, and brain of hypokalemic rats. In The Sodium Pump: Recent Developments. J. H. Kaplan and P. De Weer, editors. The Rockefeller University Press, New York. 177–181.

Chaudhury, S., F. Ismail-Beigi, G. G. Gick, R. Levenson, and I. S. Edelman. 1987. Effect of thyroid hormone on the abundance of Na,K-adenosine triphosphate α-subunit messenger ribonucleic acid. Molecular Endocrinology. 1:83–89.

Chomczynski, P., and N. Sacchi. 1987. Single step method of RNA isolation by acid guanidinium thiocyanate phenol chloroform extraction. Analytical Biochemistry. 162:156–159.

Gick G. G., J. Melikian, and F. Ismail-Beigi. 1990. Thyroidal enhancement of rat myocardial Na,K-ATPase: preferential expression of α2 activity and mRNA abundance. Journal of Membrane Biology. 115:273–282.

Horowitz, B., C. B. Hensley, M. Quintero, K. K. Azuma, D. Putnam, and A. A. McDonough. 1990. Differential regulation of Na,K-ATPase α1, α2 and β mRNA and protein levels by thyroid hormone. Journal of Biological Chemistry. 265:14308–14314.

Klein, L. E., M. S. Bartolomei, and C. C. Lo. 1984. Corticosterone and triiodothyronine control of myocardial Na,K-ATPase activity in rats. American Journal of Physiology. 247:H570-H575.

Lescale-Matys, L., M.-J. Tang, and A. A. McDonough. 1991. Low K+ stimulates differential regulation of Na,K-ATPase subunits in kidney cells. In The Sodium Pump: Recent Developments. J. H. Kaplan and P. De Weer, editors. The Rockefeller University Press, New York. 83–87.

Lowry, O. H., N.J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry. 193:265–275.

Maixent, J. M., D. Charlemagne, B. de la Chapelle, and L. G. Lelievre. 1987. Two Na, K-ATPase isoenzymes in canine cardiac myocytes: molecular basis of inotropic and toxic effects of digitalis. Journal of Biological Chemistry. 262:6842–6848.

McDonough, A. A., T. A. Brown, B. Horowitz, R. Chiu, J. Schlotterbeck, J. Bowen, and C. A. Schmitt. 1988. Thyroid hormone coordinately regulates Na,K-ATPase α and β mRNA levels in kidney. American Journal of Physiology. 254:C323–C329.

Murer, H., E. Ammann, J. Biber, and U. Hopfer. 1976. The surface membrane of the small intestinal epithelial cell I. Localization of adenyl cyclase. Biochimica et Biophysica Acta. 433:509–519.

Norgaard, A., K. Kjeldsen, and O. Hansen. 1985. K-dependent 3-O-methylfluorescein phosphatase activity in crude homogenate of rodent heart ventricle: effect of K depletion and changes in thyroid status. European Journal of Pharmacology. 113:373–382.

Orlowski, J., and J. Lingrel. 1988. Tissue-specific and developmental regulation of rat Na, K-ATPase catalytic alpha isoform and beta subunit mRNAs. Journal of Biological Chemistry. 263:10436–10442.

Orlowski, J., and J. Lingrel. 1990. Thyroid and glucocorticoid hormones regulate the expression of multiple Na,K-ATPase genes in cultured neonatal rat cardiac myocytes. Journal of Biological Chemistry. 265:3462–3470.

Piper, H. M., I. Probst, P. Schwartz, F. J. Hutter, and P. G. Spieckermann. 1982. Culturing of calcium stable adult cardiac myocytes. Journal of Molecular and Cellular Cardiology. 14:397–412.

Shyjan, A. W., C. Gottardi, and R. Levenson. 1990. The Na,K-ATPase β_2 subunit is expressed in rat brain and copurifies with Na,K-ATPase activity. Journal of Biological Chemistry. 265:5166–5169.

Shyjan, A. W., and R. Levenson. 1988. Antisera specific for the α1, α2, α3, and β subunits of the Na,K-ATPase: differential expression of α and β subunits in rat tissue membranes. Biochemistry. 28:4531–4535.

Sweadner, K. J. 1979. Two molecular forms of (Na + K)-stimulated ATPase in brain. Journal of Biological Chemistry. 254:6060–6067.

Urayama, O., and K. J. Sweadner. 1988. Ouabain sensitivity of α3 isozyme of rat Na, K-ATPase. Biochemical and Biophysical Research Communications. 156:796–800.

Differential Regulation of Na,K-ATPase α1, α2, and β in Heart, Skeletal Muscle, and Brain of Hypokalemic Rats

Kay K. Azuma, Charles B. Hensley, Daun S. Putnam, and Alicia A. McDonough

Department of Physiology and Biophysics, University of Southern California School of Medicine, Los Angeles, California 90033

Introduction

Na,K-ATPase maintains electrical excitability in the heart, skeletal muscle, and brain by restoring resting levels of Na+ and K+ after depolarization. K+ deficiency induced in rats by the administration of a K+ -deficient diet leads to a rapid decrease in plasma K+ known as hypokalemia. This is associated with a progressive loss of K+ (K+ depletion) from the skeletal muscle and to a minor extent from heart muscle (Norgaard et al., 1984, 1985; Nagaoka et al., 1989). In contrast, the brain maintains normal K+ levels during hypokalemia (Bradbury and Kleeman, 1967). Clausen and his collaborators observed a decrease in 3H-ouabain binding capacity in rat heart and skeletal muscle in vivo in response to hypokalemia associated with a decrease in Na,K-ATPase activity (reviewed in Kjeldsen, 1987). The aim of this study was to quantitate the Na,K-ATPase α and β isoform-specific responses to hypokalemia in vivo in heart, skeletal muscle, and brain and to determine if the level of regulation is pre- or post-translational.

Methods

Male Sprague-Dawley rats (250–275 g) were fed a low K+ diet (ICN #904665) or a matched control diet (ICN #902487) for 2 wk. Rats were killed by metofane anesthesia and decapitation. Serum ion concentrations were measured by flame photometry. Total RNA was isolated from rat tissues by the method of Chomczynski and Sacchi (1987). RNA electrophoresis, blotting, and hybridization were performed as previously described (Horowitz et al., 1990) using isoform-specific ~300-bp probes prepared according to Orlowski and Lingrel (1988). Tissue homogenization and immunoblot analysis have been described by Horowitz et al. (1990). Na,K-ATPase activity was assessed in the tissue homogenates after the addition of deoxycholate to 0.1% final concentration as the measurement of K+ -dependent p-nitrophenyl phosphatase (K+ -pNPPase) reaction of Murer et al. (1976) or the ouabain-sensitive ATPase activity of Lo et al. (1976). Skeletal muscle homogenates were freeze-thawed three times to permeabilize vesicles before assaying. Immunoblots were incubated with either anti-α1 (McKl, 1:200 dilution) or anti-α2 (McB2, 1:100 dilution) specific anti-rat monoclonal antibodies provided by K. Sweadner (Harvard Medical School) (Urayama et al., 1989) or with polyclonal rabbit anti–rat β1 antisera provided by R. Levenson (Yale Medical School) (Shyjan and Levenson, 1989). Significance was determined by unpaired Student's t test and accepted at P < 0.05.

Results and Discussion

During the 2-wk low K+ dietary period, serum [K+] fell from 6.0 ± 0.3 mM to 3.1 mM, confirming the hypokalemic state. The [Na+] remained unchanged throughout the course of the study at 141.0 ± 2.3 mM.

Both heart and skeletal muscle respond similarly to hypokalemia with decreases in α2 mRNA (Table I) to 0.60 and 0.65 and in α2 protein abundance (Table II) to 0.38 and 0.18 of control, respectively. The decrease in α2 protein is greater than the decrease in mRNA, suggesting translational and/or posttranslational mechanism(s) of regulation as well as pretranslational regulation in response to hypokalemia. There were no significant changes in α1 or β mRNA or protein levels in heart or skeletal muscle. K+ -pNPPase activity (Table II) decreased in heart and skeletal muscle to 0.67 and 0.58, respectively. The decrease in the ouabain-sensitive ATPase activity in the hypokalemic heart was 0.69 ± 0.03 relative to control but could not be measured in crude homogenates of skeletal muscle.

Our results in skeletal muscle are consistent with those reported by Kjeldsen et al. (1984), who observed a 34% decrease in ouabain binding in rats fed a low K+ diet for 4 wk and also by Norgaard et al. (1984), who reported a 38% decrease in K+ -dependent 3-O-methylfluorescein phosphatase activity in 1-wk K+-deficient rats. Norgaard et al. (1985) also report a 43% reduction in 3H-ouabain binding in the hypokalemic heart, consistent with our results if measurements of 3H-ouabain binding overlook the low affinity, α1 binding sites and measure only the α2 isozyme (a problem addressed by Kjeldsen [1988]). Our results are also consistent with reported changes in intracellular K+ levels in these tissues with hypokalemia. In the rat, intracellular K+ is maintained to a greater degree in cardiac than in skeletal muscle. In hypokalemic rat studies there was a loss of 13% cardiac tissue K+ (Norgaard et al., 1985), whereas in skeletal muscle K+ was reduced by 40–50% (Norgaard et al., 1984; Nagaoka et al., 1989). Because the Na,K pumps in heart are primarily α1 (Sweadner and Farshi, 1987) and because α1 in hypokalemia remains unchanged, there appear to be sufficient cardiac membrane pumps to maintain intracellular K+ closer to control levels despite a decrease in α2 expression. In contrast, in skeletal muscle where the α2 isoform predominates, the decrease of the α2 isoform in hypokalemia is reflected in a significant decrease in total Na,K pumps, which appear to be insufficient for maintaining intracellular K+.

TABLE I
Na,K-ATPase mRNA Abundance in Hypokalemic Heart, Skeletal Muscle, and Brain Relative to Control

* Amounts are reported relative to control which is assigned an arbitrary value of 1. The data are presented as mean ± SEM and significance (‡) is accepted at P ≤ 0.05.

TABLE II
Na,K-ATPase Protein Abundance and Enzymatic Activity in Hypokalemic Heart, Skeletal Muscle, and Brain Relative to Control

* Amounts are reported relative to control which is assigned an arbitrary value of 1. The data are presented as mean ± SEM and significance (‡) is accepted at P ≤ 0.05.

In the hypokalemic brain, similar to heart and skeletal muscle, there was no change in α1 protein abundance and a relatively small decrease in the α2 protein level to 0.72 of control. In contrast to results in heart and skeletal muscle, β1 protein abundance decreases in the hypokalemic brain to 0.75 of control. Na,K-ATPase activity did not change significantly in the hypokalemic rat brains measured by both K+-pNPPase and ouabain-sensitive ATPase activities. These results are consistent with the fact that brain maintains almost normal [K+] during hypokalemia (Bradbury and Kleeman, 1967), in keeping with the concept of the blood–brain barrier where, despite changes in plasma [K+], K+ flux into brain shows little change when compared with other tissues. Due to the cellular heterogeneity of brain tissue, we cannot exclude the fact that greater changes in expression in α-isoforms, β, or Na,K-ATPase activity are occurring in certain brain cell types during hypokalemia.

Conclusions

Hypokalemia differentially regulates the in vivo expression of Na,K-ATPase isoform expression, primarily effecting a large and specific decrease in the expression of the α2 isoform without a significant change in either α1 or β1 in heart and skeletal muscle, and with a small change in β1 in brain. The decrease in α2 expression in the muscle could conceivably decrease K+ reuptake, allowing net K+ exit from the ICF and thus buffering the plasma [K+] during hypokalemia.

The decrease in α2 protein is dramatically greater than the decrease in the isoform-specific mRNA, suggesting translational and/or posttranslational regulation (in brain, heart, and skeletal muscle) as well as pretranslational regulation (in heart and skeletal muscle) in response to hypokalemia.

The large decrease in the α2:α1 ratio in both heart and skeletal muscle provides a molecular explanation for the development of digitalis toxicity in hypokalemia. The α2 isoform is distinguished from the α1 isoform by its 200–300-fold higher affinity for cardiac glycosides. Because the predominate isoform expressed in skeletal muscle is α2, this tissue contains the largest pool of digitalis receptors in the rat, 12 times more than in the heart (Norgaard et al., 1985). In dog heart, the α2 isoform has been suggested to be responsible for the inotropic effect of digitalis in vivo, while the α1 is associated with digitalis toxicity (Maixent et al., 1987). The loss of the inotropic α2 digitalis sites and the increased contribution of the low affinity, α1 toxic sites may therefore account for the increased incidence of digitalis toxicity in the hypokalemic heart. A similar situation has been reported for the ischemic heart (Maixent and Lelievre, 1987).

Acknowledgments

This work was supported by NIH grants HL-39295 and DK-34316. K. K. Azuma was supported by NRSA fellowship HL0874; C. B. Hensley was supported by NRSA fellowship DK08141.

References

Bradbury, M. W. B., and C. R. Kleeman. 1967. Stability of the potassium content of cerebrospinal fluid and brain. American Journal of Physiology. 213:519–528.

Chomczynski, P., and N. Sacchi. 1987. Single step method of RNA isolation by acid guanidinium thiocyanate phenol chloroform extraction. Analytical Biochemistry. 162:156–159.

Horowitz, B., C. B. Hensley, M. Quintero, K. K. Azuma, D. S. Putnam, and A. A. McDonough. 1990. Differential regulation of Na,K-ATPase alpha 1, alpha 2 and beta mRNA and protein levels by thyroid hormone. Journal of Biological Chemistry. 265:14308–14314.

Kjeldsen, K. 1987. Regulation of the concentration of 3H-ouabain binding sites in mammalian skeletal muscle-effects of age, K-depletion, thyroid status and hypertension. Danish Medical Bulletin. 34:15–46.

Kjeldsen, K. 1988. Homogeneity of 3H-ouabain-binding sites in rat soleus muscle. Biochemical Journal. 249:481–485.

Kjeldsen, K., A. Norgaard, and T. Clausen. 1984. Effect of K-depletion on 3H-ouabain binding and Na-K contents in mammalian skeletal muscle. Acta Physiologica Scandinavica. 122:103–117.

Lo, C. S., T. R. August, U. A. Lieberman, and I. S. Edelman. 1976. Dependence of renal Na,K-adenosine triphosphatase activity on thyroid status. Journal of Biological Chemistry. 251:256–275.

Maixent, J. M., D. Charlemagne, B. de la Chapelle, and L. G. Lelievre. 1987. Two Na,K-ATPase isoenzymes in canine cardiac myocytes. Journal of Biological Chemistry. 262:6842–6848.

Maixent, J. M., and L. G. Lelievre. 1987. Differential inactivation of inotropic and toxic digitalis receptors in ischemic dog heart. Journal of Biological Chemistry. 262:12458–12461.

Murer, H., E. Ammann, J. Biber, and U. Hopfer. 1976. The surface membrane of the small intestinal epithelial cell. I. Localization of adenylcyclase. Biochimica et Biophysica Acta. 433:509–519.

Nagaoka, R., S. Yamashita, and N. Akaike. 1989. Change of intracellular K+ activity in rat soleus muscle during hypokalemia. Brain Research Bulletin. 22:1009–1013.

Norgaard, A., K. Kjeldsen, and O. Hansen. 1984. Na,K-ATPase activity of crude homogenates of rat skeletal muscle as estimated from their K+ -dependent 3-O-methylfluorescein phosphatase activity. Biochimica et Biophysica Acta. 770:203–209.

Norgaard, A., K. Kjeldsen, and O. Hansen. 1985. K+ -dependent 3-O-methylfluorescein phosphatase activity in crude homogenate of rodent heart ventricle: effect of K+ depletion and changes in thyroid status. European Journal of Pharmacology. 113:373–382.

Orlowski, J., and J. B. Lingrel. 1988. Tissue-specific and developmental regulation of rat Na,K-ATPase catalytic alpha isoform and beta subunit mRNAs. Journal of Biological Chemistry. 263:10436–10442.

Shyjan, A. W., and R. Levenson. 1989. Antisera specific for the alpha 1, alpha 2, alpha 3, and beta subunits of the Na,K-ATPase: differential expression of alpha and beta subunits in rat tissue membranes. Biochemistry. 28:4531–4535.

Sweadner, K. J., and S. K. Farshi. 1987. Rat cardiac ventricle has two Na,K-ATPases with different affinities for ouabain: developmental changes in immunologically different catalytic subunits. Proceedings of the National Academy of Sciences, USA. 84:8404–8407.

Urayama, O., H. Shutt, and K. J. Sweadner. 1989. Identification of three isozyme proteins of the catalytic subunit of the Na,K-ATPase in rat brain. Journal of Biological Chemistry. 264:8271–8280.

A Highly Ouabain-sensitive Isoform of Na,K-ATPase in Nervous Cells: Functional Evidence for a Multigenic Expression of Its Catalytic Subunit

Gustavo Blanco and Luis Beaugé

Instituto de Investigación Médica M. y M. Ferreyra, 5000 Córdoba, Argentina

Introduction

It is now well established that the α-subunit of Na,K-ATPase exists as multiple isoforms in many tissues and species (Sweadner, 1989). In brain, two forms of the enzyme, known at present as α1 and α2, were determined on the basis of their electrophoretic mobility and sensitivity to cardiotonic steroids (see Sweadner, 1989). The existence of a third isozyme, termed α3, arose from cDNA cloning studies (Shull et al., 1986) and thereafter its translational product was recognized in axolemma membranes by means of polyclonal (Hsu and Guidotti, 1989) and monoclonal antibodies (Urayama et al., 1989). The additional finding that there is a family of related genes for the Na,K-ATPase catalytic subunit raises the possibility of the existence of more forms of the enzyme (Shull and Lingrel, 1987; Sverdlov et al., 1987). In the rat, α2 was shown to have a high and α1 a low affinity for ouabain (Sweadner, 1989), while it has been reported that α3 has a ouabain sensitivity similar to (Urayama and Sweadner, 1988) or slightly higher than α2 (Hara et al., 1988). On the basis of strophanthidin inhibition of ATP hydrolysis, we have previously reported that, in the rat hippocampus, there are at least three Na,K-ATPase isoforms, one of which has a Ki for the inhibitor around 10−9 M (Blanco and Beaugé, 1988). This report is an attempt to further characterize this isoform in rat brainstem and in cultured rat nervous cells.

Results

The existence of various forms of Na,K-ATPase in rat brainstem enzyme, partially purified following the method of Jorgensen (1974), is seen in the heterogeneous dose–response curve for ouabain inhibition of Na,K-ATPase activity (Fig. 1). It is worthwhile to note that preincubation with the inhibitor was 180 min for ouabain concentrations below 10−8 M and either 20 or 180 min for 10−8 M or higher since these times were seen to be more than sufficient to achieve the equilibrium for the binding of the inhibitor to the enzyme (data not shown). The experimental points were fitted with a nonlinear regression computing program using equations that considered the eventuality of two or three forms of the enzyme with different affinities for ouabain. The best fits were obtained by assuming three (1.8% fitting error) rather than two (5.5% error) isoforms. A further analysis with the Snedecor F test showed that the introduction of a third component to the fitting model is significant (P < 0.01) and that the results were not due simply to an increased flexibility of the equation with a larger number of parameters. The calculated inhibition constants (Ki) and fractional concentrations of each isoform are depicted in Table I. The Ki's of the order of 10−4 M and 10−6 M agree with those reported for the α1 and α2 isoforms, respectively (Sweadner, 1989), whereas the one around 10−9 M represents an additional highly ouabain-sensitive form of the enzyme (termed αH throughout this report).

The three Na,K-ATPase populations could also be exposed by the dose–response curve for ouabain inhibition of the Na-dependent steady-state phosphorylation of rat brainstem enzyme from [γ^32P]ATP. Aliquots of 25 μg protein were phosphorylated for 15 s at 0°C in media containing 3 mM MgCl_2, 150 mM NaCl, 30 mM imidazole (pH 7.5), 10 μM [γ^32P]ATP, and different ouabain concentrations. Again, the best fits of the data were achieved using a three (3% fitting error) rather than a two (7.2% error) component model (F test, P < 0.01). The computed ouabain-enzyme dissociation constants and fractional composition of enzyme forms appear in Table I.

Figure 1. Ouabain inhibition of rat brainstem Na,K-ATPase activity. ATPase activity of 1 μg protein samples was assayed either by the colorimetric determination of Pi (Fiske and SubbaRow, 1925) using the amidol reagent, or by the release of 32Pi from [γ^32P]ATP (Beaugé and Campos, 1983) at 37°C in the presence of 120 mM NaCl, 30 mM KCl, 3 mM MgCl_2, 3 mM ATP, 30 mM imidazole (pH 7.5), and different ouabain concentrations. Specific Na,K-ATPase activity corresponded to the Na + K-dependent ATP hydrolysis inhibitable by 1 mM ouabain. Symbols represent the mean ± SE of two to four different experiments. The curves are the best fits corresponding to heterogeneous enzyme populations of three (solid line) or two (dashed line) isoforms.

Further demonstration of the highly ouabain-sensitive form of Na,K-ATPase was obtained by the phosphorylation of rat brainstem enzyme from 32Pi. In this case, phosphorylation of 50 μg protein was carried out for 10 min at 37°C with 1 mM [32P]Pi in 3 mM MgCl_2, 150 mM Tris-HCl (pH 7.4 at 37°C), and different concentrations of ouabain. Interaction of rat brainstem enzyme with Pi proved to be dependent on ouabain concentration and the E-P formed was stabilized by the presence of the steroid. Once more the best fit of the experimental points favored the existence of three isoforms (3.8% fitting error, compared with 5.8% for two forms; F test, P < 0.01). In this case, as in the phosphorylation from [γ^32P]ATP, in which the amounts of protein used were high, ouabain binding to the enzyme reduced its free concentration in the solution and [total ouabain] > [free ouabain]. This was taken into account by replacing [total ouabain] with the expression leading to [free ouabain] obtained from the equation for fractional occupancy of binding sites by a ligand. As shown in Table I, the kinetic parameters obtained with this method are similar to those obtained from ouabain inhibition of ATP hydrolysis and ouabain inhibition of ATP phosphorylation. From the mean values of the kinetic parameters determined for each isoform by the different methods used (see Table I), the theoretical curves resulting from the interaction of each Na,K-ATPase form with ouabain can be obtained (Fig. 2).

TABLE I
Ki Values and Fractional Amounts of Rat Brainstem Na,K-ATPase lsoforms Estimated on the Basis of Ouabain Effects

We also investigated the presence of the high ouabain affinity form of Na,K-ATPase in dissociated rat hippocampal cell cultures, prepared by the method of Bartlett and Banker (1984). These cultures are highly enriched in a particular type of neurons (hippocampal pyramidal cells). Two stages of development were studied: 1 and 10 d in vitro, periods that significantly differ and correspond, respectively, to neurons that have initiated the formation of undifferentiated neuronal processes (neurites) and to mature cells that have developed well-defined axons and dendrites. In this case, a microsomal preparation, obtained as previously described (Blanco and Beaugé, 1988) was used, and isoforms were detected on the basis of the dose–response curve for ouabain inhibition of Na,K-ATPase activity (Fig. 3). Once more, the fitting of the experimental data with the three component model turned out to be more accurate. Enzyme populations with affinities of 1.3 × 10−9, 0.6 × 10−6, and 2.0 × 10−4 M resulted from the interaction of Na,K-ATPase of 1-d in vitro cultures with ouabain, and their proportions were 16.2, 25.7, and 58.1%, respectively. For the enzyme of 10-d in vitro cultures the Ki's were 9.8 × 10−10, 0.2 × 10−6, and 1.3 × 10−3 M, with relative amounts of 10.0, 54.0, and 36.0%, respectively.

Figure 2. Kinetic behavior of isoforms of rat brainstem Na,K-ATPase with ouabain. Theoretical curves were obtained from the average Ki values and fractional amounts for each isoform shown in Table I. Dot-dashed line, α1; dashed line, α2; solid line, αH.

Figure 3. Ouabain inhibition of Na, K-ATPase activity of rat dissociated hippocampal cell cultures of 1 (○) or 10 (●) d in vitro. ATPase activity was determined as explained in Fig. 1. The curves are the best fits corresponding to heterogeneous enzyme populations of three (solid lines) or two (dashed lines) isoforms with different affinity for ouabain.

Discussion

The data presented in this report indicate the existence of a functionally competent isoform of Na,K-ATPase in rat nervous cells which is extremely sensitive to ouabain. Despite the different experimental conditions used in its identification and the fact that exact Kd values for the less abundant sites are difficult to obtain by curve fitting, its dissociation constant of the order of 10−9 M is clearly distinct from those observed for the α2 and α1 (see also Sweadner, 1989). The fractional abundance of αH ranged from 10 to 14% with the three methods used (ouabain inhibition of ATP hydrolysis, ouabain inhibition of ATP phosphorylation, and ouabain stabilization of Pi phosphorylation); similar values were obtained in the whole hippocampus of prenatal and adult rats on the basis of strophanthidin inhibition of the Na, K-dependent ATP hydrolysis (Blanco and Beaugé, 1988), which suggests that this form could be widely distributed in the nervous system. In addition, in this work αH was found to be expressed at early stages of the in vitro development of neuronal hippocampal cells. Cultured pyramidal neurons also displayed isoforms corresponding to α2 and α1, and a relative increase in the α2 at the expense of α1 accompanied cell differentiation, a finding that concurs with a previous observation made in a heterogeneous population of nerve cells (Inoue et al., 1988). A question that arises is whether the high ouabain affinity form described here represents the α3 Na,K-ATPase isozyme. The available experimental evidence is conflicting; on the one hand, the same ouabain affinity for α2 and α3 has been reported (Urayama and Sweadner, 1988); on the other hand, there is evidence that α3 ouabain sensitivity is slightly higher than that of α2 (Hara et al., 1988). An interesting alternative regarding our results is that the third isoform we encountered does not represent α3, but is the translational product (α4 ?) encoded by one of the additional genes that have been described for the Na,K-ATPase (Shull and Lingrel, 1987; Sverdlov et al., 1987). Although any of these alternatives require further demonstration, this report gives functional evidence for the existence of a multigenic expression of the catalytic subunit of the Na,K-ATPase in rat nervous system.

Acknowledgments

This work was supported by grants from CONICET, CONICOR, and Fundación Antorchas.

References

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Blanco, G., and L. Beaugé. 1988. Pools of Na,K-ATPase isoforms in unborn and adult rats can be detected by their sensitivity to strophanthidin, but have similar reactivity to Na,K and ATP. Progress in Clinical and Biological Research. 268A:271–278.

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Hara, Y., A. Nikamoto, T. Kojima, A. Matsumoto, and M. Nakao. 1988. Expression of sodium pump activities in Balb/C 3T3 cells transfected with cDNA encoding α3 subunit of rat brain Na,K-ATPase. FEBS Letters. 238:27–30.

Hsu, Y. M., and G. Guidotti. 1989. Rat brain has the α3 form of the Na,K-ATPase. Biochemistry. 28:569–573.

Inoue, N., H. Matsui, H. Tsukui, and H. Hatanaka. 1987. The appearance of a highly digitalis-sensitive isoform of Na,K-ATPase during maturation in vitro of primary cultured rat cerebral neurons. Journal of Biochemistry. 104:349–354.

Jorgensen, P. L. 1986. Purification and characterization of Na,K-ATPase. III. Purification from the outer medulla of mammalian kidney after selective removal of membrane components by sodium dodecylsulphate. Biochimica et Biophysica Acta. 256:36–52.

Shull, G. E., J. Greeb, and J. B. Lingrell. 1986. Molecular cloning of three different forms of the alpha subunit from rat brain. Biochemistry. 25:8125–8132.

Shull, M., and J. B. Lingrell. 1987. Multiple genes encode the human Na,K-ATPase catalytic subunit. Proceedings of the National Academy of Sciences, USA. 84:4039–4043.

Sverdlov, E. D., G. S. Monastyrskaya, N. E. Broude, Y. A. Ushkaryou, R. L. Alliknets, A. M. Melkov, Y. V. Smirnov, I. V. Malishe, I. E. Dulobova, K. E. Petrukhin et al. 1987. The family of human Na,K-ATPase genes. FEBS Letters. 217:275–278.

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Differential Expression of Three Na,K-ATPase Catalytic Subunit Isoforms in Human Tissues, Organs, and Cell Lines

N. S. Akopyanz, N. E. Broude, N. G. Vinogradova, Yu. A. Balabanov, G. S. Monastyrskaya, and E. D. Sverdlov

M. M. Shemyakin Institute of Bioorganic Chemistry, USSR Academy of Sciences, Moscow, USSR; All-Union Scientific Research Institute of Biotechnology, USSR Ministry of Medical and Biological Industry, Moscow, USSR; and Institute of Experimental Medicine, USSR Academy of Sciences, Leningrad, USSR

Introduction

Use of molecular cloning methods in recent years has shown the Na,K-ATPase catalytic subunit to exist at least in mammalian cells as several isoforms whose structure features high homology (Shull et al., 1986; Sverdlov et al., 1987b). Expression of three isoforms of this family was investigated in various rat (Emanuel et al., 1987; Herrera et al., 1987; Young and Lingrel, 1987) and human (Sverdlov et al., 1987a, 1988) tissues. On all occasions, expression was shown to be tissue specific.

The problem of the biological role of various isoforms remains open. The revealed tissue specificity of their expression supports the suggestion that different ratios of the isoforms in various tissues have functional effects. One of the approaches to solving this problem is to compile a catalogue of tissue distribution of the isoforms and to determine the dependence of their content on the stage of development of the organism or, at the cell level, on cell differentiation. To solve these problems one needs adequate, rapid, and sensitive methods of analysis.

The purpose of this work was to investigate expression of three Na,K-ATPase α-subunit isoforms in a wide range of normal human tissues and cell lines.

Materials and Methods

All tissues and organs were obtained by autopsy 4–6 h after death from 25–40-yr-old donors who died in accidents. Human cell line HL60 and neuroblastoma cell line IMR32 were from the cell culture museum, Institute of Cytology, USSR Academy of Sciences, Leningrad. The immortalized astrocyte cell line from human fetal spinal cord was obtained using simian adenovirus oncogen SA-7 (Balabanov et al., 1990). Neuron cell line was the primary culture obtained from the cortex of 10–12-wk-old human fetus. Selection was carried out on medium N-1.

The total cell RNA was isolated from 1 g of tissue or from 5 × 10^6–10^7 cells according to the method of Feramisco et al. (1982) using guanidine isothiocyanate to inhibit RNAses and hot phenol for deproteination. Amplification was done in a Perkin-Elmer-Cetus thermal cycler. Activity of reverse transcriptase was 1.5–3.5 U/ml (Ferment, Vilnius, USSR). Conditions of the reaction were as described (Sambrook et al., 1989). The products of amplification were analyzed by gel electrophoresis on 2% agarose containing 0.5 mg/ml of ethidium bromide.

Figure 1. A scheme of gene amplification of Na,K-ATPase α-subunit. The sizes of amplification products of mRNA and genome fragments are given below.

Figure 2. PCR analysis of gene transcription of the Na,K-ATPase α1, α2, and α3 isoforms in various human organs. The results of electrophoresis of the amplification mixture on 2% agarose gels are presented. The bands above the marked size represent the products of DNA amplification which is present in minute amounts in RNA preparations.

Figure 3. PCR analysis of transcription of the Na,K-ATPase α3 isoform in various renal sections. (1) Cortex; (2) medulla; (3) juxtamedullary part; (4) total kidney; (5) brain (control).

Results and Discussion

We used the PCR method as the most sensitive and suitable for work with total RNA preparations. A gene region between exons 9 and 10 was chosen for amplification. The exon 10 region corresponds to the variable protein domain; therefore, the primers specific for each isoform were chosen from that region. The primer to synthesize the second chain corresponded to the most conservative region in exon 9 and was common for all three isoforms. A scheme of polymerase chain reaction and the sizes of the fragments obtained as a result of amplification are given in Fig. 1.

Fig. 2 presents the results of expression of the three Na,K-ATPase α-subunit isoforms in human tissues. The α1 mRNA was found in the kidney, thyroid gland, uterus, aorta intima, lung, and, in lesser amounts, in the skeletal muscle, aorta media, and heart. The α2 isoform mRNA was present in the brain, heart, and muscles. The α3 was found in the brain and heart. Thus, using the PCR method, we failed to detect the transcripts of the α3 isoform mRNA in the kidneys, which contradicts the data we obtained from Northern blotting with the NH_2-terminal probe specific to the α3 isoform (Sverdlov et al., 1987a, 1988). To examine this issue in more detail, we isolated RNA from three kidney sections: the cortex, medulla, and juxtamedullary region. The results indicate (Fig. 3) that minor amounts of the α3 isoform are present in the juxtamedullary region. These data are consistent with those of Arystarkhova et al. (1989a, b) on the presence of α3 in minor amounts in the pig and human kidneys. The problem of what is the cause of cross-hybridization to the Na,K-ATPase α3 isoform-specific probe in the kidneys is currently being examined. Table I summarizes the results of expression of the three Na,K-ATPase α-subunit isoforms in the human tissues obtained by the PCR method.

TABLE I
Expression of Three Na,K-ATPase α-Subunit Isoforms in Various Human Organs

In the lymphoid cell line HL60, both induced and noninduced with DMSO, as well as in normal human lymphocytes, the transcripts of only the α1 isoform were found (data not shown). Fig. 4 presents the results of expression of three Na,K-ATPase catalytic subunit isoforms in human neurons and glial cells. The results indicate that the α3 isoform is common to all types of cells tested. The α2 was present in neuroblastoma cells and neurons but was not found in astrocytes. At the same time, the α1 isoform was detected in large amounts in astrocytes but not in neurons and neuroblastoma cells. Thus, these results support the differential expression of the catalytic subunit isoforms in various types of brain cells and raise the issue of functional significance of each isoform.

It should be stressed that the cell lines used in this study are only simplified models of different cells of the nervous system and do not represent the complexity of this tissue. Nevertheless, these simplified models could be of great value in studying mechanisms of tissue-specific expression. Besides, a comparison of the results obtained on the simplified models with those obtained on in situ hybridization might give new information on the role of different Na,K-ATPase isoforms.

Figure 4. PCR analysis of gene transcription of the α1, α2, and α3 isoforms in human neuronal cells. (1) Astrocytes; (2) neurons; (3) neuroblastoma IMR32, nondifferentiated; (4) neuroblastoma IMR32, differentiated; (5) control (reverse transcriptase was omitted from the reaction mixture).

Acknowledgments

The authors sincerely thank Dr. V. M. Rostapshov and Dr. S. A. Gryaznov for oligonucleotide synthesis, and Dr. E. P. Bekman for participation in some experiments.

References

Arystarkhova, E. A., O. E. Lakhtina, N. B. Levina, and N. N. Modyanov. 1989a. Immunoaffinity isolation of Na,K-ATPase α3-isoform from pig kidney. FEBS Letters. 257:24–26.

Arystarkhova, E. A., O. E. Lakhtina, and N. N. Modyanov. 1989b. Immunodetection of Na,K-ATPase α3-isoform in renal and nerve tissues. FEBS Letters. 250:545–548.

Balabanov, Yu. V., B. A. Zavizion, and I. N. Trakht. 1990. The immortalized astrocyte cell line from human spinal cord. Doklady Akademii Nauk SSSR. 315:1238–1241.

Emanuel, J. R., S. Garetz, L. Stone, and R. Levenson. 1987. Differential expression of Na+,K+,-ATPase α- and β-subunit mRNAs in rat tissues and cell lines. Proceedings of the National Academy of Sciences, USA. 84:9030–9034.

Feramisco, J. R., D. M. Helfman, J. E. Smart, K. Burridge, and G. P. Thomas. 1982. Coexistence of vinculin-like protein of higher molecular weight in smooth muscle. Journal of Biological Chemistry. 257:11024–11031.

Herrera, V. L., J. R. Emanuel, N. Ruiz-Opazo, R. Levenson, and B. Nadal-Ginard. 1987. Three differentially expressed Na,K-ATPase α subunit isoforms. Structural and functional implications. The Journal of Cell Biology. 105:1855–1864.

Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. 14.0–14.35.

Shull, G. E., J. Greeb, and J. B. Lingrel. 1986. Molecular cloning of three distinct forms of the Na,K-ATPase α-subunit from rat brain. Biochemistry. 25:8125–8132.

Sverdlov, E. D., N. S. Akopyanz, K. E. Petrukhin, N. E. Broude, G. S. Monastyrskaya, and N. N. Modyanov. 1988. Na,K-ATPase: tissue-specific expression of genes coding for α-subunit in diverse human tissues. FEBS Letters. 239:65–68.

Sverdlov, E. D.,N. E. Broude, V. E. Sverdlov, G. S. Monastyrskaya, K. E. Petrukhin, N. S. Akopyanz, N. N. Modyanov, and Yu. A. Ovchinnikov. 1987a. Family of Na,K-ATPase genes: intra-individual tissue-specific restriction fragment length polymorphism. FEBS Letters. 221: 129–133.

Sverdlov, E. D., G. S. Monastyrskaya, N. E. Broude, Yu. A. Ushkaryov, R. L. Allikmets, A. M. Melkov, Yu. V. Smirnov, I. V. Malyshev, I. E. Dulubova, K. E. Petrukhin et al. 1987b. The family of human Na,K-ATPase genes: no less than five genes and/or pseudogenes related to the α-subunit. FEBS Letters. 217:275–278.

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Transfection of Na,K-ATPase α1- and β1-Subunit cDNAs into Clone 9 Cells: Analysis of Na,K-ATPase mRNA Abundance and Enzyme Activity

Mary Ann Hatala, Isidore S. Edelman, and Faramarz Ismail-Beigi

Department of Biochemistry and Molecular Biophysics, and Department of Medicine, Columbia University, New York 10032

Introduction

In rat liver and rat liver–derived cell lines the abundances of mRNAα1 and mRNAβ1encoding the subunits of Na,K-ATPase are markedly unequal, with mRNAα1 exceeding mRNAβ1 content several-fold (Gick et al., 1988; Pressley et al., 1988; Gick and Ismail-Beigi, 1990). Given the heterodimeric composition of the enzyme and isomolar ratios of α: β, the above inequality in subunit-mRNA abundance suggests translational and/or post-translational regulatory events in Na,K-pump biogenesis. In the present study we investigated whether selective increases in Na,K-ATPase subunit-mRNA abundance resulted in enhanced enzyme activity in Clone 9 cells, a “nontransformed” rat liver cell line that expresses only the α1 and β1 mRNA isoforms of the enzyme (Gick and Ismail-Beigi, 1990). We transfected Clone 9 cells with a constitutive expression vector containing either cDNAα1 or cDNAβ1, and compared the subunit-mRNA content of each stable transfectant with its Na,K-ATPase activity as a measure of the abundance of mature enzyme.

Methods

Plasmid constructs were prepared by individually cloning full-length rat kidney cDNAα1 and cDNAβ1 into a retroviral-derived constitutive expression vector possessing LTR promoters and the neomycin resistance gene. The constructs or the vector alone were transfected into cells by calcium phosphate precipitation (Davis et al., 1986) and stable transfectants were selected by G418 resistance. 9 cDNAα1-construct transfectants, 17 cDNAβ1-construct transfectants, and 4 vector transfectants were analyzed.

Na,K-ATPase activity was determined in whole-cell lysates of confluent cells according to previously described methods after “permeabilization” of membrane vesicles (Gick and Ismail-Beigi, 1990). Protein, RNA, and DNA of the cell lysates were also measured (Gick and Ismail-Beigi, 1990). Previously described methods for the isolation and Northern blot analysis of cytoplasmic RNA were used without modification (Bhutada et al., 1990). For each experimental set examining the role of α1 or β1 mRNA abundance, the cDNA-containing construct transfectants, as well as vector transfectants and nontransfectants, were analyzed. P values were calculated by analysis of variance, and values < 0.05 were considered significant (Alder and Roessler, 1968).

Results

cDNAα1-Construct Transfectants

The mRNAα1 content of nine cDNAα1-construct transfectants increased moderately relative to vector transfectants and nontransfected cells; the abundances ranged from 1.2 to 3.1 times that of nontransfected cells. Transfection of cells with vector alone did not alter mRNAα1 abundance. No qualitative changes in the appearance of the mRNAα1 band were noted, and mRNAα1 migrated as a single band in both nontransfectants and transfectants at ~27S (Shull et al., 1986a; Gick and Ismail-Beigi, 1990).

To determine whether the enhanced mRNAα1 content of the cDNAα1-construct transfectants increased Na,K-ATPase activity, the enzyme activity of confluent cells of each cDNAα1-construct transfectant, vector transfectant, and nontransfectant was measured. Na,K-ATPase activity (expressed per unit protein) of cDNAα1-construct transfectants remained unchanged when compared with vector transfectants or nontransfected cells. Expression of data per unit DNA also yielded no significant changes. Mg2+-ATPase activity (expressed per unit protein or DNA), as well as the protein, RNA, and DNA content of the cDNAα1-construct transfectants, vector transfectants, and nontransfectants were not different from each other.

cDNAβ1-Construct Transfectants

Transfection of the cDNAβ1-construct into Clone 9 cells increased their mRNAβ1abundance over a wide range as compared with vector transfectants and nontransfected cells. Among 17 cDNAβ1-construct transfectants mRNAβ1 abundance ranged from 1.5 to 6.1 times that of nontransfectants, whereas mRNAβ1 content of vector transfectants was unchanged. mRNAβ1 bands migrated at ~22S, 21S, and 17S in nontransfectants and transfectants as previously documented (Shull et al., 1986b; Gick and Ismail-Beigi, 1990).

To determine whether the enhanced mRNAβ1 content of the cDNAβ1-construct transfectants increased Na,K-ATPase activity, the enzyme activity of confluent cells of each cDNAβ1-construct transfectant, vector transfectant, and nontransfectant was measured. No significant change in Na,K-ATPase activity was noted in cDNAβ1-construct transfectants (expressed per unit protein) when compared with vector transfectants and nontransfectants. Expression of data per unit DNA also yielded no significant changes. Mg2+-ATPase activity expressed per unit protein or per unit DNA was unchanged in all three cell populations. Protein, RNA, and DNA content of cDNAβ1-construct and vector transfectants were not different from each other, although a slight increase of RNA in vector transfectants proved significant.

In Vitro Translation of cRNAα1 and cRNAβ1

The translational activity of the subunit-cDNA constructs was examined in a cell-free system. cDNAα1 and cDNAβ1 were cloned into the pSPT18 plasmid and, after in vitro transcription, the cRNA products were translated in a rabbit reticulocyte lysate system containing L-[35S]methionine. cRNAα1 and cRNAβ1 were translated to their respective subunit peptides, thereby verifying the translational competency of the cDNA products.

Conclusion

Transfection of Clone 9 cells with Na,K-ATPase cDNAα1 or cDNAβ1 constructs results in increased subunit-mRNA abundance. These increases, however, are not associated with enhanced enzyme activity, suggesting a predominant role for translational and/or post-translational regulatory processes in Na,K-pump biogenesis.

Acknowledgments

This work was supported by NIH grant RO1 GM-39835 and New York State Health Research Council Postdoctoral Fellowship (Diabetes Program).

References

Alder, H. L., and E. B. Roessler. 1968. The analysis of variance, one criterion of classification. In Introduction to Probability and Statistics. W. H. Freeman and Company, San Francisco. 260–288.

Bhutada, A., C. Perez, D. Y. Chon, and F. Ismail-Beigi. 1990. Induction of Na+-K+-ATPase and its subunit mRNAs by serum in a rat liver cell line. American Journal of Physiology. 258 (Cell Physiology 27):C1044–C1050.

Davis, L. G., M.D. Dibner, and J. F. Battey. 1986. Transfection of mammalian cells in culture. In Basic Methods in Molecular Biology. Elsevier Science Publishing Co., Inc., New York. 285–300.

Gick, G. G., and F. Ismail-Beigi. 1990. Thyroid hormone induction of Na+-K+-ATPase and its mRNAs in a rat liver cell line. American Journal of Physiology. 258 (Cell Physiology 27):C544–C551.

Gick, G. G., F. Ismail-Beigi, and I. S. Edelman. 1988. Thyroidal regulation of rat renal and hepatic Na,K-ATPase gene expression. Journal of Biological Chemistry. 263:16610–16618.

Pressley, T. A., F. Ismail-Beigi, G. G. Gick, and I. S. Edelman. 1988. Increased abundance of Na+-K+-ATPase mRNAs in response to low external K+. American Journal of Physiology. 255 (Cell Physiology 24):C252-C260.

Shull, G. E., J. Greeb, and J. B. Lingrel. 1986a. Molecular cloning of three distinct forms of the Na +,K +-ATPase α-subunit from rat brain. Biochemistry. 25:8125–8132.

Shull, G. E., L. K. Lane, and J. B. Lingrel. 1986b. Amino-acid sequence of the β-subunit of the (Na+ + K+)ATPase deduced from a cDNA. Nature. 321:429–431.

Structure

Esterification of a Carboxyl Residue in the Na Pump α-Subunit Abolishes K Sensitivity

José M. Argüello and Jack H. Kaplan

Department of Physiology, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104

Introduction

In this work chemical modification has been used to try to identify amino acids involved in the interaction of the Na,K-ATPase with Na+ and/or K+ ions. One hypothesis is that carboxyl moieties of glutamic or aspartic acids might interact as local counterions of Na+ and K+. Previous studies have used carbodiimides to modify the Na,K-ATPase, looking for carboxyl groups involved in cation binding (Yamaguchi et al., 1983; Gorga, 1985; Pedemonte and Kaplan, 1986a, b; Shani-Sekler et al., 1988). However, although cation prevention of inactivation by carbodiimides was reported, because of their propensity to form intra- and intermolecular iso-peptide bonds, there is also uncertainty as to whether or not carboxyl group modification or cross-linking is the basis of inactivation by carbodiimides (Pedemonte and Kaplan, 1986a, b, 1990). We decided to re-examine the involvement of carboxyl residues in the interaction of the sodium pump with monovalent cations. To this end we used 4-(diazomethyl)-7-(diethylamino)-coumarin (DEAC) to modify the Na,K-ATPase. DEAC is a relatively stable fluorescent diazomethane analogue which reacts with carboxyl groups producing the corresponding ester (Ito and Maruyama, 1983). An advantage of this compound lies in the fluorescence of the coumarin residue which is then attached to the protein and allows the quantitation and tracking of the modified residues during their localization. We have found that the modification of one (or two) carboxyl residue(s) of the Na,K-ATPase α-subunit by DEAC disrupts the K+ binding by the enzyme.

Methods

Na,K-ATPase was purified from dog kidney outer medulla by the method of Jorgensen (1974) with the modifications of Liang and Winter (1976). The Na,K-ATPase activity of the enzyme was ~ 17–23 µmol Pi mg−1 min−1. The enzyme (0.1–0.5 mg/ml) was treated with DEAC in 50 mM imidazole, pH (20°C) 6.5, and 1 mM EDTA during 2 h at 37°C. In most experiments 3 mM MgC1_2 and 3 mM P¡ were included in the treatment media, and the modified protein obtained in these media will be referred to as DEAC-enzyme. DEAC in dimethylsulfoxide (DMSO) was added at the desired concentration. The final DMSO concentration was always 10% in the treatment medium and <0.25% in the ATPase assay medium. The reaction was stopped by dilution and centrifugation.

DEAC binding stoichiometry determinations were performed after treating the enzyme with 250 µM DEAC in the media described above, including Mg2+ and Pi. 40 mM K+ was included in the treatment media to obtain K-protected enzyme. DEAC not covalently bound to protein was removed by cold acetone extraction. The protein pellets were resuspended in 50 mM Tris-acetate, pH (20°C) 7, 1.66 M urea, and 0.2% SDS and treated with proteinase K (1:5, wt/wt; protease protein) and the absorbance of the samples at 390 nM was determined. DEAC-benzoate was used as a standard.

Na,K-ATPase activity, phosphorylation by ATP, Rb+ binding and ADP binding determinations, and trypsin treatment of the enzyme were performed as previously described (Pedemonte and Kaplan, 1986b; Argiiello and Kaplan, 1990). Na+ binding in the presence of oligomycin (Matsui and Homareda, 1982) was determined using ion exchange columns according to the procedure of Shani-Sekler et al. (1988). Protein concentration was determined by the method of Lowry et al. (1951). SDS-PAGE was performed according to Laemmli (1970) in 10% acrylamide gels.

Results

Treatment of isolated Na,K-ATPase with DEAC leads to enzyme inactivation. Fig. 1 shows the inactivation kinetics during the DEAC treatment. The inactivation rate was significantly increased by the presence of 3 mM Mg2+ plus 3 mM Pi in the DEAC treatment media. On the other hand, the presence of K+ ions protects the enzyme against the inactivation by DEAC. This protective effect of K+ is observed in the presence or absence of 3 mM Mg2+ plus 3 mM Pi. The effects of other enzyme ligands on the inactivation by DEAC were also studied (Fig. 2). No significant effect is observed when ATP, Mg2+, or Pi are separately included in the treatment media; the presence of Na+ ions has a small protective effect.

Figure 1. Na,K-ATPase inactivation by DEAC. The enzyme was treated at 37°C with 250 µM DEAC, 50 mM imidazole, pH 6.5, and 1 mM EDTA in the absence (, ) or presence (, ) of 3 mM Mg_2Cl and 3 mM Pi, and in the absence (, ) or presence (, ) of 40 mM KCl. At the indicated times, aliquots were taken to determine Na,K-ATPase activity. The values are the means of four experiments.

Fig. 3 shows the results of SDS-PAGE of native, DEAC-treated and DEAC-treated, K-protected enzyme observed under UV light. The DEAC fluorescence is associated with the α-subunit of the enzyme and the labeling is partially prevented by K+ (as is the inactivation). The amount of DEAC labeling responsible for the inactivation was 2.05 ± 0.18 nmol DEAC per mg protein. This was estimated as the difference between DEAC bound to inactivated Na,K-ATPase (remaining activity 10–15% of the native enzyme) and DEAC bound to K-protected enzyme (activity 80–90% of the native enzyme). Assuming the ADP binding site number to be the number of active enzyme molecules per milligram protein, the previous result suggests that the inactivation would be associated with the modification of one carboxyl group.

Figure 2. Influence of the enzyme ligands on the Na,K-ATPase inactivation by DEAC. The enzyme was treated with 250 µM DEAC, 50 mM imidazole, pH 6.5, and 1 mM EDTA, plus the indicated ligand at 37°C for 120 min and the Na,K-ATPase activity was determined afterward. The values are the means of four experiments. ChCI, choline chloride.

The prevention of inactivation by K+ suggests that DEAC binding may take place in the K+ binding locus on the enzyme. In keeping with this notion, after inactivation with DEAC, Rb+ binding and occlusion by the enzyme are greatly reduced. Modified enzyme has ~ 10% of the Rb+ (or K+) binding and occlusion capacity of native enzyme. The binding of Na+ ions is also affected. Modified enzyme has ~ 20% of the Na binding capacity of native enzyme. The inactivated enzyme exhibited normal levels of high affinity ATP binding so that the carboxyl group modified is evidently not located in the ATP binding domain. When 32P-phosphoenzyme is produced by incubating enzyme with [γ^32P]ATP and Na+ ions, the DEAC-modified enzyme shows a reduced level (37%) of phosphorylation compared with native enzyme. The DEAC-modified phosphoenzyme obtained is markedly unaffected by the addition of K+ ions, which cause the rapid (< 5 s) and complete (< 5%) disappearance of native phosphoenzyme.

Figure 3. SDS-PAGE in a 10% acrylamide gel of control, DEAC-enzyme, and K-protected enzyme. Enzyme was treated as indicated in Methods. 40 mM KCI was included in the treatment media to obtain the K-protected enzyme. Protein bands were observed by staining the gels with Coomassie brilliant blue. Coumarin groups were detected by their fluorescent emission under UV lamp (360 nm).

Partial tryptic digestion and electrophoretic analysis of DEAC-enzyme reveals normal degradation patterns in the presence of ADP (E_1 form), but the typical pattern seen with K+ ions in native enzyme (E_2K) is absent.

Discussion

We have shown that the modification of carboxyl residues of the Na,K-ATPase by DEAC produces inactivation of the enzyme. This reaction is not affected by the presence of ATP, Mg2+, or Pi in the treatment media, but it is accelerated by Mg2+ plus Pi prevented by K+, and to a lesser extent by Na+.

The modification removes the K+ binding and occlusion capacity of the DEAC-treated enzyme, and the K-associated characteristics of the enzyme (such as the E_2K tryptic pattern, E_2P dephosphorylation by K+) are abolished. Na+ only slightly protects against the inactivation and the Na+ binding is reduced to almost the same extent as the K+ binding. These results suggest that the modified carboxyl residue may be directly involved in the K+ (and perhaps Na+) binding site on the enzyme.

It is difficult to predict what properties might be expected in an Na,K-ATPase molecule in which the K+ (and Na+) binding sites have been disrupted after covalent modification. It is interesting that although the nucleotide binding capacity is unaltered, there is a decrease in the phosphorylation level. Thus an effect is transferred to the phosphorylation domain. A trivial cause of this might be that asp 369 is esterified, and in some (unspecified) way esterification of the residue, which is the site of the normal acyl phosphate intermediate, affects cation binding. This seems unlikely since the modified site is apparently more reactive toward DEAC under conditions where the enzyme is substantially in the phosphorylated state (in the presence of Mg2+ and Pi ions). It has recently been reported that other modifications that inactivate the enzyme and alter the K+ binding site also result in a loss of phosphorylating capacity (Fukushima et al., 1991; Krämer et al., 1991).

In summary, our results suggest that a K-protectable carboxyl residue on the α-subunit of the Na,K-ATPase is essential for Na,K-ATPase activity and K+ binding and occlusion. This same residue is also critical to the K+ sensitivity of the Na,K-ATPase phosphoenzyme intermediate. Esterification of this carboxyl residue by DEAC inactivates the enzyme.

Acknowledgments

This work was supported by NIH grant GM-39500.

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Liang, S. M. L., and C. G. Winter. 1976. The measurement of ouabain binding and some related properties of digitonin-treated (Na+,K+)-ATPase. Biochimica et Biophysica Acta. 452:552–565.

Lowry, O. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry. 193:265–275.

Matsui, H., and H. Homareda. 1982. Interaction of sodium and potassium ions with Na+,K+-ATPase. I. Ouabain-sensitive alternative binding of three Na+ or two K+ to the enzyme. Journal of Biochemistry. 92:193–217.

Pedemonte, C. H., and J. H. Kaplan. 1986a. Carbodiimide inactivation of Na,K-ATPase, via intramolecular cross-link formation, is due to inhibition of phosphorylation. Journal of Biological Chemistry. 261:3632–3639.

Pedemonte, C. H., and J. H. Kaplan. 1986b. Carbodiimide inactivation of Na,K-ATPase. A consequence of internal cross-linking and not carboxyl group modification. Journal of Biological Chemistry. 261:16660–16665.

Pedemonte, C. H., and J. H. Kaplan. 1990. Chemical modification as an approach to elucidation of sodium pump structure-function relations. American Journal of Physiology. 258 (Cell Physiology 27):C1–C23.

Shani-Sekler, M., R. Goldshleger, D. M. Tal, and S. J. D. Karlish. 1988. Inactivation of Rb+ and Na+ occlusion on (Na+,K+)-ATPase by modification of carboxyl groups. Journal of Biological Chemistry. 263:19331–19341.

Yamaguchi, M., J. Sakamoto, and Y. Tonomura. 1983. Binding of monovalent cations to the Na+,K+-ATPase. Current Topics in Membranes and Transport. 19:203–217.

Lysine 480 Is an Essential Residue in the Putative ATP Binding Site of Lamb Kidney (Na,K)-ATPase

Terence L. Kirley and Hellmuth R. Hinz

Department of Pharmacology and Cell Biophysics, University of Cincinnati, Cincinnati, Ohio 45267-0575

Introduction

The (Na,K)-ATPase consists of two subunits, α and β, although the function of the β-subunit in the mature enzyme has yet to be defined. The α-subunit contains all of the known functional sites, including the ouabain binding site, the phosphorylation site, and the ATP binding site(s). Several probes have been used as affinity ligands for the ATP site, including fluorescein isothiocyanate (FITC) (Farley et al., 1984; Kirley et al., 1984), 8-azido ATP (Tran et al., 1988), fluorosulfonylbenzoyl adenosine (FSBA) (Ohta et al., 1986), CIR-ATP (Ovchinnikov et al., 1987), and the site of phosphorylation (Bastide et al., 1973). All of the sites are located in the large cytoplasmic domain between the putative fourth and fifth membrane spanning helices of the α-subunit (Shull et al., 1985). This study involves the use of pyridoxal phosphate (PLP) and the adenosine derivative of pyridoxal phosphate (AP_2PL) as ATP site-directed probes to further define the boundaries of the nucleotide binding site in (Na,K)-ATPase.

Experimental Procedures

(Na,K)-ATPase was purified from lamb kidneys as described previously (Lane et al., 1979). (Na,K)-ATPase activity was measured by the linked enzyme spectrophotometric assay (Schwartz et al., 1969). Pyridoxal-5′-phosphate monohydrate (PLP) was obtained from Chemical Dynamics Corp. (South Plainfield, NJ). ATP was from Boehringer Mannheim GmbH (Mannheim, Germany). Adenosine 5′-phosphomorpholidate was from Sigma Chemical Co. (St. Louis, MO). Tributylphosphine (TBP) was from Fluka AG (Buchs, Switzerland). The two Bio-Sil TSK-250 size exclusion HPLC columns, as well as urea and SDS, were from Bio-Rad Laboratories (Richmond, CA). The 300-Å pore size 15 cm C_18, C_4, and phenyl reverse phase HPLC columns were from VYDAC (Hesperia, CA). TPCK-treated trypsin was from Worthington Biochemical Corp. (Freehold, NJ). Centricon 30 microconcentrators were from Amicon (Beverly, MA). The HPLC system used was described previously (Kirley et al., 1984) with the modification of a zinc lamp, a 308-nm excitation filter, and a 370–700-nm emission filter incorporated into the fluorometer used to detect the pyridoxal fluorescence. Purified peptides were analyzed using the Pico-Tag method of amino acid analysis after acid hydrolysis and 470A sequencer (Applied Biosystems, Inc., Foster City, CA) was used to determine the sequence of each peptide. These analyses were done by the Protein Core Facility of the Department of Pharmacology and Cell Biophysics of the University of Cincinnati (Cincinnati, OH). The synthesis of AP_2PL was performed according to the published procedure (Tamura et al., 1986). Protein concentrations were determined by the method of Lowry (Lowry et al., 1951), using BSA as the standard. All PLP and AP_2PL reactions were performed at 22°C in 50 mM HEPES (pH 7.8), 5 mM MgCl_2, 100 mM NaCl, and 10 mM KCl. Reactions were quenched and the Schiff base formed was converted to a stable secondary amine by the addition of 0.5 mM NaBH_4. ATP protection was accomplished by adding 5 mM ATP 1 min before addition of the pyridoxal compound. After labeling, the α-subunit was purified (Ball et al., 1988) and digested with trypsin as described (Hinz and Kirley, 1990). The peptides were then purified by reverse phase chromatography using a gradient of 0.1% trifluoroacetic acid (TFA) in water to 0.1% TFA in acetonitrile. The digest was applied first to a C_18 column, and the pyridoxal-containing peptides identified by fluorescence were repurified sequentially on C_4 and phenyl columns. Amino acid analyses were obtained on portions of each purified peptide, and the remainder sequenced.

Figure 1. Tryptic peptide maps of the pyridoxal labeled (Na,K)-ATPase α-subunit. (A) The tryptic peptides derived from AP_2PL labeling of 9.0 mg of (Na,K)-ATPase in either the absence (top chromatogram) or the presence (bottom chromatogram) of 5 mM ATP were mapped on a C_18 column. (B) The tryptic peptides derived from 1.5 mg of (Na,K)-ATPase labeled with PLP in the absence (top chromatogram) or the presence (bottom chromatogram) of 5 mM ATP were mapped on a C_18 column. The peptides indicated by an arrow in each panel were repurified and sequenced.

Results

The treatment with either PLP or AP_2PL resulted in a concentration- and time- dependent inhibition of (Na,K)-ATPase activity which was largely reversible in the absence of NaBH_4 and protectable by the presence of ATP (Hinz and Kirley, 1990). The tryptic peptide maps of the PLP and the AP_2PL modified α-subunits are shown in Fig. 1. It can be seen in both cases that there is a single peptide protected from labeling (presumably resulting in inactivation) by the presence of ATP. This suggests that the site of labeling is located in or very near the ATP binding site. The peptides indicated by an arrow in Fig. 1 were repurified and sequenced. Surprisingly, the same sequence was obtained for both peptides and the same lysine residue was labeled by both pyridoxal compounds, corresponding to lysine 480 in the cDNA sequence of lamb kidney (Na,K)-ATPase (Shull et al., 1985). The sequences obtained along with the homology of this region with other (Na,K)-ATPases and (H,K)-ATPases are shown in Table I.

TABLE I
Homology of the AP_2PL and PLP Binding Site

Discussion

The specificity of the reaction of PLP with the (Na,K)-ATPase was somewhat unexpected, and the adenosine derivative was synthesized to improve upon that specificity and utilized before the PLP labeling experiments were begun. However, as is evident from Fig. 1, given the short incubation times used in this study (3 min with 0.5 mM pyridoxal compound), sufficient specificity is attained. It is interesting to note that the concentration necessary to inhibit the (Na,K)-ATPase activity was approximately the same for both probes (not shown). This relatively high concentration used (0.5 mM) suggests that the AP_2PL may not be a particularly good affinity reagent compared with other reagents such as FITC. It appears as if the short time of reaction results in the specificity of these reagents. The pyridoxal probe labeled site resides in the large cytoplasmic domain of the α-subunit, which is located between the fourth and fifth postulated membrane spanning helices (Shull et al., 1985). A summary of our current knowledge of this region of the α-subunit, which has been the result of labeling with many different reagents, is shown in Fig. 2. The citations for the elucidations for the various sites are given in the Introduction. The site of labeling of 5-iodoacetamidofluorescein (5-IAF), (cysteine 457 [Tyson et al., 1989]) is also included to give an example of a site that has been localized which is not in the ATP binding/hydrolysis site, since reaction at that site does not result in inactivation, but instead is useful as a reporter site for the active enzyme. It should be noted that it was recently shown that FITC, if used to label the (Na,K)-ATPase under different conditions than originally used to label lysine 501 (Farley et al., 1984; Kirley et al., 1984), will also label lysine 480, apparently resulting in inactivation (Xu, 1989). It is speculated that this is the result of the different reaction conditions and longer incubation time used in that study which may have resulted in the FITC molecule “wobbling” in its binding site before covalent attachment. Also, in the pig gastric (H,K)-ATPase, pyridoxal phosphate was demonstrated to label lysine 497 (Tamura et al., 1989), which corresponds to lysine 480 in the lamb kidney (Na,K)-ATPase, again showing the striking similarity between these related enzymes. Interestingly, there is no such region of homology in the Ca2+-ATPase of sarcoplasmic reticulum. Apparently, the recognition of these two pyridoxal reagents and the resulting specificity of reaction is not determined by the presence or absence of the adenosine moiety. The results reported here suggest that the recognition of the probes is dependent on the phosphate and/or the pyridoxal moiety, and not on the adenosine ring.

Figure 2. Model of the ATP binding site of (Na,K)-ATPase. The region shown is the large cytoplasmic domain of the α-subunit. The references for the sites of labeling are given in the text.

Acknowledgments

This work was supported by grants HL-07382 (to H. R. Hinz), AR-38576 (to T. L. Kirley), and P01 HL-22619 (Core 2) from the National Institutes of Health.

References

Ball, W. J., Jr., T. L. Kirley, and L. K. Lane. 1988. Preparation of antibodies to Na+,K+-ATPase and its subunits. Methods in Enzymology. 156:87–101.

Bastide, F., G. Meissner, S. Fleischer, and R. L. Post. 1973. Similarity of the active site of phosphorylation of the adenosine triphosphatase for transport of sodium and potassium ions in kidney to that for transport of calcium ions in the sarcoplasmic reticulum of muscle. Journal of Biological Chemistry. 248:8385–8391.

Farley, R. A., C. M. Tran, C. T. Carilli, D. Hawke, and J. E. Shively. 1984. The amino acid sequence of a fluorescein-labeled peptide from the active site of (Na,K)-ATPase. Journal of Biological Chemistry. 259:9532–9535.

Hinz, H. R., and T. L. Kirley. 1990. Lysine 480 is an essential residue in the putative ATP site of lamb kidney (Na,K)-ATPase. Journal of Biological Chemistry. 265:10260–10265.

Kirley, T. L., E. T. Wallick, and L. K. Lane. 1984. The amino acid sequence of the fluorescein isothiocyanate reactive site of lamb and rat kidney Na+- and K+-dependent ATPase. Biochemical and Biophysical Research Communications. 125:767–773.

Lane, L. K., J. D. Potter, and J. H. Collins. 1979. Large-scale purification of (Na,K)-ATPase and its protein subunits from lamb kidney medulla. Preparative Biochemistry. 9:157–170.

Lowry, D. H., N. J. Rosebrough, A. L. Farr, and R. G. Randall. 1951. Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry. 193:265–275.

Ohta, T., K. Nagano, and M. Yoshida. 1986. The active site structure of Na+/K+-transporting ATPase: location of the 5′-(p-fluorosulfonyl)benzoyl adenosine binding site and soluble peptides released by trypsin. Proceedings of the National Academy of Sciences, USA. 83:1–5.

Ovchinnikov, Y. A., K. N. Dzhandzugazyan, S. V. Lutsenko, A. A. Mustayev, and N. N. Modyanov. 1987. Affinity modification of E_1-form of Na+,K+-ATPase revealed Asp-710 in the catalytic site. FEBS Letters. 217:111–116.

Schwartz, A., J. C. Allen, and S. Harigaya. 1969. Possible involvement of cardiac Na+,K+-adenosine triphosphatase in the mechanism of action of cardiac glycosides. Journal of Pharmacology and Experimental Therapeutics. 168:31–41.

Shull, G. E., A. Schwartz, and J. B. Lingrel. 1985. Amino-acid sequence of the catalytic subunit of the (Na+,K+)-ATPase deduced from a cDNA. Nature. 316:691–695.

Tamura, J. K., R. D. Rakov, and R. L. Cross. 1986. Affinity labelling of nucleotide binding sites on kinases and dehydrogenases by pyridoxal 5′-diphospho-5′-adenosine. Journal of Biological Chemistry. 261:4126–4133.

Tamura, S., M. Tagaya, M. Maeda, and M. Futai. 1989. Pig gastric (H+ + K+)-ATPase: lys-497 conserved in cation transporting ATPases is modified by pyridoxal 5′-phosphate. Journal of Biological Chemistry. 264:8580–8584.

Tran, C. M., G. Scheiner-Bobis, W. Schoner, and R. A. Farley. 1988. The amino acid sequence of a peptide from the ATP-binding site of Na,K-ATPase labeled by 8-N_3-ATP. Biophysical Journal. 53:344a. (Abstr.)

Tyson, P. A., M. Steinberg, E. T. Wallick, and T. L. Kirley. 1989. Identification of the 5-iodoacetamidofluorescein receptor site on the (Na,K)-ATPase. Journal of Biological Chemistry. 264:726–734.

Xu, K.-Y. 1989. Any of several lysines an react with 5′-isothiocyanato-fluorescein to inactivate sodium and potassium ion activated adenosine triphosphatase. Biochemistry. 28:5764–5772.

Disulfide Bonds in the (Na,K)-ATPase: Are They Important?

Terence L. Kirley

Department of Pharmacology and Cell Biophysics, University of Cincinnati, Cincinnati, Ohio 45267-0575

Introduction

This study used a relatively new, very specific sulfhydryl reagent, 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (ABD-F), for the determination of the number, location, and importance of the disulfides in the β-subunit, as well as the number of the disulfides in the α-subunit. ADB-F becomes fluorescent after reaction with sulfhydryls, a reaction that can be performed under the harsh reaction conditions necessary for quantitative reaction and yet maintain its specificity due to the superior properties of the reagent (Toyo'oka and Imai, 1985). A single disulfide was demonstrated earlier in the β-subunit (Ohta et al., 1986). However, no systematic study of all seven cyst(e)ine residues derived from the cDNA sequence (Shull et al., 1986) has previously been reported. It has been reported (Kawamura and Nagano, 1984) that the loss of (Na,K)-ATPase activity observed upon treatment with high concentrations of β-mercaptoethanol (β-ME) at elevated temperatures was accompanied by disulfide bond reduction in the β-subunit, and that this effect was sensitive to the concentrations of NaCl and KCl used to induce different conformations in the enzyme, presumably indicating some interaction between the α- and β-subunits (Kawamura et al., 1985). No disulfide bonds were previously reported to exist in the α-subunit, although the number of free sulfhydryls determined was always less than the total number of cyst(e)ine residues (23) derived from the cDNA sequence (Shull et al., 1985).

Experimental Procedures

(Na,K)-ATPase was purified from lamb kidneys as described previously (Lane et al., 1979). (Na,K)-ATPase activity was measured by the linked enzyme spectrophotometric assay (Schwartz et al., 1969). Protein was measured as described (Lowry et al., 1951). The α- and β-subunits were separated from each other and from excess reagents by two TSK-250 size exclusion HPLC columns (Bio-Rad Laboratories, Richmond, CA) connected in series (Ball et al., 1988). Reduction with tributylphosphine (TBP) and alkylation with ABD-F was carried out as described (Kirley, 1989, 1990). TBP was used as the reducing agent since it does not react directly with ABD-F, unlike β-ME and DTT. Samples were concentrated using Centricon 30 microconcentrators and were precipitated with acetone before trypsin digestion. Most samples were alkylated with iodoacetamide as described (Kirley, 1990) to inhibit the possibility of disulfide interchange. ABD-F was from Wako Chemical Co. (Dallas, TX). TBP was from Aldrich Chemical Co. (Milwaukee, WI). Peptides were purified on Vydac C_18, C_4, and phenyl columns using the HPLC system described previously (Kirley et al., 1984). Purified peptides were analyzed by the Protein Core Facility of the Department of Pharmacology and Cell Biophysics at the University of Cincinnati (Cincinnati, OH). Amino acid analysis was accomplished using the Pico-Tag system, and peptides were sequenced on a 470A sequencer (Applied Biosystems, Inc., Foster City, CA). Other experimental details for this study have been previously published (Kirley, 1989, 1990).

Results

When the purified β-subunit was treated with ABD-F in the presence and absence of a reducing agent (TBP) and then digested with trypsin and mapped on a C_18 reverse-phase HPLC column, the results shown in Fig. 1 were obtained. In both cases a very hydrophobic peptide was observed (peak D). It was also evident that there were multiple disulfide bonds present in that subunit, as indicated by the fluorescent peaks seen in B. The peaks labeled A, B, and C in Fig. 1 B were very reproducibly generated and were used to evaluate the extent of reduction of the three disulfide bonds (see below). By separation of peptides on the C_18 column with intact disulfide bonds (before reduction and alkylation), and subsequent reduction and alkylation with TBP and ABD-F, rechromatography of the peptides (the peptides previously joined by a disulfide bond are now fluorescently labeled), and sequencing, it was possible to determine the location of all three disulfide bonds (Kirley, 1989). The hydrophobic peptide containing the lone free sulfhydryl (peptide D in Fig. 1) was also sequenced and found to begin at residue 27 and contain the single predicted membrane-spanning helix. The identified disulfides were incorporated into a secondary structural model of the β-subunit based on the PREDICT program, and the results are shown in Fig. 2. To test the hypothesis that the reduction of a single disulfide in the β-subunit was responsible for the inactivation observed upon treatment of (Na,K)-ATPase with β-ME at elevated temperatures, the enzyme was treated with 200 mM β-ME at 40°C at 2.0 mg/ml in 50 mM imidazole-Cl and 1 mM EDTA, pH 7.5, for various time periods with and without either 200 mM NaCl or 15 mM KCl. Aliquots were withdrawn and the samples were analyzed for ATPase activity and for the reduction of each of the three β-subunit disulfide bonds by purifying the β-subunit and using ABD-F to fluorescently label both halves of the disulfide bonds reduced by this treatment. The amount each of the three disulfides reduced was quantitated by comparison with identical amounts of the fully reduced (TBP) β-subunit by using ratios of the fluorescent peak areas. The peaks used to quantitate are labeled A, B, and C in Fig. 1. These three peaks represent one of each pair of the peptides involved in the three β-subunit disulfide bonds. Table I summarizes the results obtained for both time course experiments and NaCl and KCl protection experiments. Although it is obvious that one of the β-subunit disulfides is more susceptible to reduction than the other two (cys125–cys148), the reduction of this bond does not quantitatively correlate with the loss of activity. In addition, the two events can be even more convincingly uncoupled by the addition of protecting salts: the (Na,K)-ATPase activity is completely protected from inactivation, while the disulfide bond connecting cys125–cys148 is reduced ~ 50%. The α-subunit was also evaluated for the presence of disulfide bonds, even though it appeared that the β-ME treatment used to inactivate the enzyme did not reduce any disulfides in that subunit. The purified subunit was alkylated with iodoacetamide twice to block all the free sulfhydryls and then treated with ABD-F in the presence and absence of a reducing agent (TBP), and the carboxymethyl cysteine (derived from the free sulfhydryls) and the ABD-cysteine (derived from the disulfide bonds) were quantitated by amino acid analysis and absorbance at 385 nm, respectively. It was determined that there were 16.8 ± 0.6 (n = 10) free sulfhydryls and 2.93 ± 0.02 (n = 4) disulfides, accounting for all 23 cyst(e)ine residues from the cDNA sequence (Shull et al., 1985).

Figure 1. HPLC tryptic peptide maps of the ABD-F-treated β-subunit. β-Subunit was isolated from 1.5 mg of (Na,K)-ATPase and labeled with ABD-F in either the absence (A) or the presence (B) of a reductant (TBP). The samples were then digested with trypsin and mapped on a C_18 column. The relative fluorescence due to ABD-cysteine is shown (excitation with a 370-nm lamp, using a 370-nm excitation filter and a 450–700-nm emission filter). The peaks labeled A, B, and C were used in the quantitation of the extent of individual disulfide bond reduction. The peak labeled D, which is present regardless of reduction, contains the single free sulfhydryl (cys44) located in the single membrane-spanning region of the β-subunit.

Figure 2. Model of the secondary structure of the β-subunit. The disulfide bond locations are given and the location of the speculative hinge region is indicated.

TABLE I
Integrated Fluorescence Peak Areas (Expressed as Percent of Totally Reduced β-Subunit Sample) of the β-ME-treated Samples

Discussion

The knowledge of the oxidation states of all seven cyst(e)ine residues in the sheep kidney (Na,K)-ATPase will undoubtedly aid in our understanding of that subunit. Since all of the β_1 sequences obtained thus far contain the seven cyst(e)ine residues, and because the β-subunits of all species are resistant to sulfhydryl-directed chemical modification reagents, it is speculated that all of these proteins might contain the same three disulfides and the lone, unreactive, free sulfhydryl in the single membrane-spanning region of the subunit. This conservation of structural elements might connote functional significance, although it is clear from this work that the reduction of a single disulfide bond in β is not the sole factor causing the loss of (Na,K)-ATPase upon treatment with β-ME. However, it is speculated here that the disulfide bond most susceptible to reductive cleavage joining cysteine 125 to cysteine 148 may serve as a type of “hinge.” When this hinge is broken by reduction in the absence of other protein stabilization agents such as salts, then the COOH-terminal half of the β-subunit may change its interaction with the NH_2-terminal half of the β-subunit and/or the extracellular part of the α-subunit. These interactions are probably important for function. However, in the presence of stabilizing Na or K salts, even after this disulfide is broken, the β-subunit tertiary structure would remain largely unchanged, accounting for the protection of loss of ATPase activity afforded by the salts. It is interesting to note in this regard that the kinetics of inhibition of (Na,K)-ATPase activity by β-ME are identical to the kinetics of inhibition of ouabain binding and p-nitrophenyl phosphatase (pNPPase) activity (data not shown). This is not the case for most chemical modification reagents affecting the (Na,K)-ATPase, where the order of inhibition is typically ATPase > pNPPase > ouabain binding inhibition. The fact that β-ME inhibits all three to the same extent suggests a general denaturation of structure rather than the reduction of a single disulfide bond.

The observation that there may be three disulfide bonds in the α-subunit also has important structural and functional implications. The fact that the location of these bonds has not been deciphered by this laboratory using the same techniques described above for the β-subunit may indicate that these bonds are located in the hydrophobic membrane-spanning regions (and therefore not recoverable from reverse-phase HPLC columns), or that some cysteine residues exist in the α-subunit that cannot be alkylated due to oxidation, acylation, or the presence of mixed disulfides with glutathione or the free amino acid cysteine.

Acknowledgments

This work was supported by grants AR-38576 and P01 HL-22619 (core 2) from the National Institutes of Health.

References

Ball, W. J., Jr., T. L. Kirley, and L. K. Lane. 1988. Preparation of antibodies to Na+,K+-ATPase and its subunits. Methods in Enzymology. 156:87–101.

Kawamura, M., and K. Nagano. 1984. Evidence for essential disulfide bonds in the β-subunit of (Na++K+)-ATPase. Biochimica et Biophysica Acta. 774:188–192.

Kawamura, M., K. Ohmizo, M. Morohashi, and K. Nagano. 1985. Protective effect of Na+ and K+ against inactivation of (Na++K+)-ATPase by high concentrations of 2-mercaptoethanol at high temperatures. Biochimica et Biophysica Acta. 821:115–120.

Kirley, T. L. 1989. Determination of three disulfide bonds and one free sulfhydryl in the β subunit of (Na,K)-ATPase. Journal of Biological Chemistry. 264:7185–7192.

Kirley, T. L. 1990. Inactivation of (Na+,K+)-ATPase by β-mercaptoethanol: differential sensitivity of the three β-subunit disulfide bonds. Journal of Biological Chemistry. 262:4227–4232.

Kirley, T. L., E. T. Wallick, and L. K. Lane. 1984. The amino acid sequence of the fluorescein isothiocyanate reactive site of lamb and rat kidney Na+- and K+-dependent ATPase. Biochemical and Biophysical Research Communications. 125:767–773.

Lane, L. K., J. D. Potter, and J. H. Collins. 1979. Large-scale purification of (Na,K)-ATPase and its protein subunits from lamb kidney medulla. Preparative Biochemistry. 9:157–170.

Lowry, D. H., N. J. Rosebrough, A. L. Farr, and R. G. Randall. 1951. Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry. 193:265–275.

Ohta, T., M. Yoshida, K. Nagano, H. Hirano, and M. Kawamura. 1986. Structure of the extra-membranous domain of the β-subunit of (Na,K)-ATPase revealed by the sequences of its tryptic peptides. FEBS Letters. 204:297–301.

Schwartz, A., J. C. Allen, and S. Harigaya. 1969. Possible involvement of cardiac Na+,K+-adenosine triphosphatase in the mechanism of action of cardiac glycosides. Journal of Pharmacology and Experimental Therapeutics. 168:31–41.

Shull, G. E., L. K. Lane, and J. B. Lingrel. 1986. Amino-acid sequence of the β-subunit of the (Na+,K+)-ATPase deduced from a cDNA. Nature. 321:429–431.

Shull, G. E., A. Schwartz, and J. B. Lingrel. 1985. Amino acid sequence of the catalytic subunit of the (Na++K+)ATPase deduced from a complementary DNA. Nature. 316:691–695.

Toyo'oka, T., and K. Imai. 1985. Isolation and characterization of cysteine-containing regions of proteins using 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole and high performance liquid chromatography. Analytical Chemistry. 57:1931–1937.

Three Proline Residues in the Fifth Transmembrane Segment of Na+,K+-ATPase α-Subunit Inhibit Its Membrane Insertion

Haruo Homareda, Kiyoshi Kawakami, Kei Nagano, and Hideo Matsui

Department of Biochemistry, Kyorin University School of Medicine, Mitaka, Tokyo 181, Japan; and Department of Biology, Jichi Medical School, Minamikawachi, Tochigi 329-04, Japan

Na+,K+-ATPase α-subunit has at least six transmembrane segments, which are designated M1–M6 from the NH_2 terminus (Kawakami et al., 1986). We have studied the mechanism of membrane insertion of the Na+,K+-ATPase α-subunit. In previous papers we demonstrated that the fragment containing M5 to the COOH terminus of the α-subunit (FM56C) is not stably inserted into microsomal membranes, though two other fragments, one containing M1 and M2 and the other containing M3 and M4, are inserted in a translation system in vitro (Homareda et al., 1988, 1989). These results suggest that the insertion signals are contained in M1 and M3 but not in M5. Comparing the amino acids in M5 with those in M1 and M3, a striking difference is that three prolines, which are known to disturb α-helix formation, are contained in M5 but not in M1 and M3 of the α-subunits of Drosophila, Torpedo, rat, sheep, and chicken, as well as human used in our study (cf. Baxter-Lowe et al., 1989). Proline is not contained in the transmembrane segment of the Na+,K+-ATPase β-subunit (Kawakami et al., 1986; Noguchi et al., 1986; Shull et al., 1986). Since the prolines in M5 are suggested to have some effects on the membrane insertion of FM56C, we prepared the mutants of FM56C and α-subunit, in which the prolines were replaced by leucines, and examined the membrane insertion of the mutants. The present data indicate that the FM56C mutant with three leucines instead of prolines acquired the capability of membrane insertion.

Methods

Preparation of DNA Mutants

Construction of glycosylation site. The Eco RI fragment (2353–2823) of plasmid pSNα-100, which codes whole α-subunit of human Na+,K+-ATPase (Kawakami et al., 1986), was ligated into RFM13mp19 as shown in Fig. 1. The single-stranded M13mp19 containing the Eco RI cut fragment was prepared and used as a template for site-directed mutagenesis. To construct a glycosylation site between M5 and M6, G^2479 and C^2480 were displaced to A and A by the Amersham mutagenesis system (Amersham Corp., Arlington Heights, IL). These displacements changed Ala^827-Glu-Ser to Asn^827-Glu-Ser which was a potential sequence for glycosylation.

Displacement of proline to leucine in M5. The single-stranded M13mp19 with the mutant DNA fragment coding a glycosylation site was used as a template. (a) C^2366 was displaced by T in order to change Pro^789 to Leu. (b) C^2396 and C^2402 were displaced by T for the exchange of Pro^799 and Pro^801 to Leu and Leu, respectively. (c) C^2366, C^2396, and C^2402 were displaced by T for the exchange of Pro^789, Pro^799, and Pro^801 to Leu, Leu, and Leu, respectively. The displacement of the bases was confirmed by the dideoxy sequencing method. The mutant DNA fragments were put back into pSNα-100 and pSNα56C, which codes FM56C, as shown in Fig. 1.

Figure 1. Summary of preparation of DNA templates for in vitro mRNA synthesis.

Transcription and Translation In Vitro

Procedures were previously described (Kawakami and Nagano, 1988; Homareda et al., 1989). Translation products, in which [35S]methionine was incorporated, were analyzed by SDS-PAGE and fluorography at –80°C as previously described (Kawakami and Nagano, 1988).

Results and Discussion

Preparation of pSNα56C Mutants

We prepared cDNAs coding four kinds of mutants of FM56C; i.e., mutants containing (a) a glycosylation site between M5 and M6 (GFM56C), (b) a glycosylation site and Leu^789 (GL1FM56C), (c) a glycosylation site and Leu^799 and Leu^801 (GL23FM56C), and (d) a glycosylation site and Leu^789, Leu^799, and Leu^801 (GL123FM56C). The glycosylation site was induced to be used as a criterion for membrane insertion of the translation products.

Identification of Peptides Synthesized In Vitro

Linearized plasmid coding wild-type or mutant FM56C was transcribed and the synthesized RNA was translated in the rabbit reticulocyte lysate system with microsomal membranes and [35S]methionine (Amersham Corp.). The translation products were immunoprecipitated by the method of Anderson and Blobel (1983) with an anti-α serum which is different from the one used in a previous paper (Homareda et al., 1989). The synthesized FM56C's, wild-type and all mutants, were recognized with the antibody against the α-subunit as a peptide with molecular mass of 39 kD. In addition to the 39-kD peptide, 43-kD peptide was observed on SDS-PAGE of GL123FM56C (Fig. 2, lanes 2 and 5).

Figure 2. SDS-PAGE of alkali-extracted or Endo H–digested GL123FM56C. After the incubation for translation, the mixture containing GL123FM56C was kept on ice (lanes 1 and 2), incubated in 0.1 N NaOH for 10 min at 0°C (lanes 4 and 5), or treated with 1 mU of Endo H (cloned preparation; Genzyme Corp., Cambridge, MA) in 0.25 M sodium acetate (pH 6.0) containing 0.2% SDS for 2 h at 37°C (lane 3). Then the mixtures were separated into supernatant (s) and precipitate (p) through neutral (lanes 1 and 2) or alkali (lanes 4 and 5) sucrose cushion by centrifugation except for that digested with Endo H.

Evidence for Membrane Insertion of GL123FM56C

The 43-kD peptide disappeared after digestion with endoglycosidase H (Endo H) (Fig. 2, lane 3) and was precipitated by centrifugation through sucrose cushion after the alkali treatment of translation mixture (lane 5). These results indicate that the peptide is the glycosylated FM56C and is stably inserted into membranes. In other words, the mutant FM56C in which three prolines were displaced by leucines acquired the capability of membrane insertion.

Failure in Glycosylation of α-Subunit Mutants

The pSNα-100 mutants were prepared as shown in Fig. 1. When the RNAs transcribed from the cDNA mutants were translated in the presence of the membranes, the apparent increase in molecular mass by glycosylation was not observed with any α-subunit mutants. Therefore, M5 of the α-subunit was judged not to be inserted into the membranes in the present translation system, even though the prolines were displaced by leucines. It is likely that a specific conformation of the large hydrophilic peptide region between M4 and M5 is necessary for the contact and insertion of M5 to the membranes after the insertion of M1–M4.

Acknowledgments

This research was supported in part by a grant provided by the Ichiro Kanahara Foundation.

References

Anderson, D. J., and G. Blobel. 1983. Immunoprecipitation of proteins from cell-free translation. Methods in Enzymology. 96:111–120.

Baxter-Lowe, L. A., J. Z. Guo, E. E. Bergstrom, and L. E. Hokin. 1989. Molecular cloning of the Na,K-ATPase α-subunit in developing brine shrimp and sequence comparison with higher organisms. FEBS Letters. 257:181–187.

Homareda, H., K. Kawakami, K. Nagano, and H. Matsui. 1988. Membrane insertion of α and β subunits of human Na+,K+-ATPase. In The Na+,K+-Pump, part B. J. C. Skou, J. G. Nørby, A. B. Maunsbach, and M. Esmann, editors. Alan R. Liss, Inc., New York. 77–84.

Homareda, H., K. Kawakami, K. Nagano, and H. Matsui. 1989. Location of signal sequences for membrane insertion of the Na+,K+-ATPase α subunit. Molecular and Cellular Biology. 9:5742–5745.

Kawakami, K., and K. Nagano. 1988. The transmembrane segment of the human Na,K-ATPase β-subunit acts as the membrane incorporation signal. Journal of Biochemistry. 103:54–60.

Kawakami, K., H. Nojima, T. Ohta, and K. Nagano. 1986. Molecular cloning and sequence analysis of human Na,K-ATPase β-subunit. Nucleic Acids Research. 14:2833–2844.

Kawakami, K., T. Ohta, H. Nojima, and K. Nagano. 1986. Primary structure of the α-subunit of human Na,K-ATPase deduced from cDNA sequence. Journal of Biochemistry. 100:389–397.

Noguchi, S., M. Noda, H. Takahashi, K. Kawakami, T. Ohta, K. Nagano, T. Hirose, S. Inayama, M. Kawamura, and S. Numa. 1986. Primary structure of the β-subunit of Torpedo californica (Na++K+)-ATPase deduced from the cDNA sequence. FEBS Letters. 196:315–320.

Shull, G. E., L. K. Lane, and J. B. Lingrel. 1986. Amino-acid sequence of the β-subunit of the (Na++K+)ATPase deduced from a cDNA. Nature. 321:429–431.

Determination of the Four Disulfide Bond Locations Contained in Na,K-ATPase Inhibitor (SPAI)

Kengo Araki, Manabu Kuwada, Osamu Ito, Jun Kuroki, and Shinro Tachibana

Tsukuba Research Laboratories, Eisai Co., Ltd., 5-1-3 Tokodai, Tsukuba, Ibaraki 300-26, Japan

Introduction

Diarrhea is sometimes seen as an adverse gastrointestinal side-effect of digitalis therapy. This led us to investigate whether the intestine not only had receptors for digitalis but also endogenous digitalis-like factors. The hypothesis has been proposed that endogenous digitalis-like factors may play an important role in sodium homeostasis, and consequently in the pathophysiology of primary hypertension. However, many researchers have tried in vain to identify these endogenous factors from blood, urine, and other organs (Haddy, 1987).

We have recently succeeded in isolating three unique peptides, SPAI-1, -2, and -3 from porcine duodenal extract based on Na,K-ATPase inhibitory activity and have reported their primary amino acid sequences (Araki et al., 1989). This was the first time that endogenous peptide inhibitors of the Na,K-ATPase had been isolated from mammalian organs. As shown in Fig. 1, SPAI-2 and -3 have almost the same amino acid sequence except for two amino acid substitutions at positions 22 and 30, while SPAI-1 lacked the NH_2-terminal dodecapeptide sequence of SPAI-2. These three peptides contained four disulfide bonds in common and showed significant homology to human mucous proteinase inhibitor (HUSI), whose 20 amino acid residues, including 8 Cys, occupy the same positions as those of SPAI (Grutter et al., 1988). It is of some interest to know the location of the four disulfide linkages of SPAI, since SPAI does not show proteinase inhibitory activity (Araki et al., 1989). We now report the four disulfide linkage locations of SPAI and their determination.

Results and Discussion

To locate the four disulfide linkages, we attempted to obtain suitable proteolytic fragments of SPAI without modification of the disulfide bonds, so that the combination of their amino acid analyses with their direct sequencing results would allow us to estimate the individual disulfide bridge locations. At first SPAI-1 was digested with trypsin, and four tryptic fragments (T-l to T-4) could be separated. From the amino acid analyses and the enzyme specificity, the structures of T-l, T-3, and T-4 could be easily deduced to be Leu-Leu-Ser-Lys, Ile-Leu-Phe-Arg, and undigested starting material SPAI-1, respectively. Although T-2 was estimated not to be homogeneous from the results of the amino acid analysis and its HPLC pattern, T-2 was further digested with protease V8(EC 3.4.21.19, sequence grade; Boehringer Mannheim Corp., Indianapolis, IN) without further purification. SPAI-1 contained only one Glu susceptible to this enzyme. Protease V8 proteolytic fragments (V8-1 to -4) of the T-2 fraction were separated by reverse phase HPLC on an ODS column.

Figure 1. The amino acid sequences of SPAIs and the second domain of HUSI. Dotted lines indicate the common amino acids among the four peptides. Cystine residues are boxed.

About one-tenth of each fragment was oxidized by performic acid and then used for amino acid analysis. The residual material was used for sequencing. Stepwise Edman degradation was performed by a gas phase semiautomated sequencer (model 470A; Applied Biosystems, Inc., Foster City, CA). Disulfide bonds were not modified before sequencing and reducing reagents were omitted from direct contact with the peptide on the solid support during sequencing. Cystine in the sequence was identified by the detection of the phenylthiohydantoin (PTH) derivative of cystine and also the dithiothreitol adduct of the PTH derivative of dehydroalanine (Marti et al., 1987). V8-1 and V8-2 each contained two disulfide bonds, while V8-3 and V8-4 each had only one disulfide bond. The amino acid compositions of both V8-1 and V8-2 were almost the same except that V8-1 contained one additional Lys. Edman degradation of V8-2 yielded three sequences in approximately equal amounts, which were assigned as follows: Gly-His-Cys-Pro-Arg, Cys-Cys-Glu, and Cys-Trp-Arg. DiPTH-cystine was significantly detected only when the second half-cystine residue of a disulfide bond was split off. The direct sequencing of V8-1 gave no clear sequences due to its limited availability (200 pmol). However, V8-1 could be deduced to contain the tetrapeptide sequence Lys-Cys-Cys-Glu from the two enzyme specificities used and from the comparison of the amino acid compositions between V8-1 and V8-2.

Edman degradation of V8-3 (1.1 nmol) yielded two sequences and DiPTH-cystine at the third cycle, while the sequencing of V8-4 (4 nmol) gave two sequences and diPTH-cystine at the fourth cycle. The alignment of all the proteolytic fragments is summarized in Fig. 2. These results revealed that SPAI-1 has the same disulfide linkage pattern as that of the recently determined HUSI (Grutter et al., 1988). It can be reasonably inferred that both SPAI-2 and -3 have the same disulfide pattern as SPAI-1 because these three peptides can be regarded as members of the same family.

Figure 2. Alignment of all proteolytic fragments and disulfide linkages of SPAI-1. T, tryptic fragment of SPAI-1; V8, protease V8 fragment of T-2.

Figure 3. Four disulfide connective pattern of SPAI-2.

The three-dimensional structure of HUSI has been determined by x-ray crystallography on the complex formed between bovine alpha-chymotrypsin and a recombinant HUSI (Grutter et al., 1988). It is interesting that SPAI has no inhibitory activities on proteases, though it has not only the same amino acids in the twenty positions but also the same disulfide linkages as those of the second domain of HUSI. The primary binding site of HUSI to the enzyme is suggested to be the eight residues from Thr(67) to Leu(74) (Fig. 1). This octapeptide sequence of HUSI corresponds to the sequence Ile(23) to Ser(30) of SPAI. However, there are no identical amino acids occupied in the positions of both octapeptides except for the Cys residue. This fact could explain the difference in the activities of SPAI and HUSI.

Conclusion

The disulfide linkage pattern of SPAI was determined by the combination of amino acid analyses with the direct application to a gas phase sequencer of its limited proteolytic fragments as shown in Fig. 3. The pattern of these four disulfide bonds was the same as that of the recently reported HUSI, though SPAI showed no proteinase inhibitory activity. We are very interested in learning whether HUSI shows inhibitory activity on Na,K-ATPase. Physiological as well as pathological roles for SPAI have not been proposed, but the disclosure of the secondary structure of SPAI should assist research on the biological diversity of the four disulfide core proteins.

References

Araki, K., J. Kuroki, O. Ito, M. Kuwada, and S. Tachibana. 1989. Novel peptide inhibitor (SPAI) of Na,K-ATPase from porcine intestine. Biochemical and Biophysical Research Communications. 164:496–502.

Grutter, M. G., G. Fendrich, R. Huber, and W. Bode. 1988. The 2.5 A X-ray crystal structure of the acid-stable proteinase inhibitor from human mucous secretions analysed in its complex with bovine alpha-chymotrypsin. EMBO Journal. 7:345–351.

Haddy, F. J. 1987. Endogenous digoxin-like factors. ISI Atlas of Science Pharmacology. 1:119–123.

Marti, T., S. J. Rosselet, K. Titani, and K. A. Walsh. 1987. Identification of disulfide-bridged substructures within human von Willebrand factor. Biochemistry. 26:8099–8109.

Site-directed Mutagenesis of Cys in the β-Subunit of the Torpedo californica Na,K-ATPase

Shunsuke Noguchi, Yoshinobu Mutoh, and Masaru Kawamura

Department of Biology, University of Occupational and Environmental Health, Yahatanishi, Kitakyushu, 807 Japan

Introduction

The Na,K-ATPase consists of two subunits, α and β. The function of the β-subunit is unknown, although it may play an important role in the biosynthesis or assembly of functional enzyme molecules (Noguchi et al., 1990a, b). The amino acid sequence of the β-subunit contains seven cysteine residues which are highly conserved (for Torpedo californica β-subunit, Cys^46, Cys^127, Cys^150, Cys^160, Cys^176, Cys^215, and Cys^278). Recently, Kirley (1989) and Miller and Farley (1990) reported that six of these seven cysteine residues are crosslinked by a disulfide bridge and, hence, that there are three disulfide bonds in the β-subunit (for T. californica, Cys^127–Cys^150, Cys^160–Cys^176, and Cys^215–Cys^278). Here we examine the effects of the mutations of these cysteine residues. Point mutations were introduced to replace Cys with Ser for several cysteine residues in the β-subunit of T. californica Na,K-ATPase and the mutant cDNAs were expressed in Xenopus laevis oocytes.

Figure 1. Construction of template plasmids for mutagenesis. Coding sequence for the β-subunit of T. californica Na,K-ATPase (open boxes) and Nde I linker (closed box) are indicated.

Figure 2. Mutagenesis of cysteine residues in the β-subunit of Na,K-ATPase. Amino acid (top) and cDNA (middle) sequences of the wild-type β-subunit and mutagenic oligonucleotide sequences (bottom) are shown. In mutagenic oligonucleotide sequences, the changed nucleotide is underlined and the amino acid that is mutated is shown below the changed codons. The number of the cysteine residue is given above.

Materials and Methods

Site-directed Mutagenesis

Template plasmids for Cys^46 mutation (Fig. 1 a) and for mutations of the other Cys residues (Fig. 1 b) were constructed as shown in Fig. 1. The oligonucleotide primers used for mutagenesis are shown in Fig. 2. In the mutagenesis of double mutants (βC127,150S and βC215,278S), mutagenesis reactions were performed twice. The gene fragments carrying the mutations were cut off from the pUC119 and used to replace the corresponding segments of the β-subunit gene that had been inserted into pSP65.

Figure 3. Fluorogram of translation products from Na,K-ATPase subunit-specific mRNAs in Xenopus oocytes. Either mRNA for wild-type β-subunit (lanes 19–21) or βC46S (lanes 1–3), βC127S (lanes 4–6), βC127,150S (lanes 7–9), βC215S (lanes 10–12), βC278S (lanes 13–15), or βC215,278S (lanes 16–18), was injected together with mRNA for wild-type α-subunit. Microsomes were solubilized and immunoprecipitated with antiserum against the α-subunit (lanes 2, 5, 8, 11, 14, 17, and 20), with antiserum against the β-subunit (lanes 3, 6, 9, 12, 15, 18, and 21), or with a mixture of both sera (lanes 1, 4, 7, 10, 13, 16, and 19) and analyzed by gel electrophoresis followed by fluorography. Positions of the α-subunit (α), core glycosylated (βc), and fully glycosylated β-subunit (βm) are indicated.

Expression in Xenopus Oocytes

In vitro transcription and injection into Xenopus laevis oocytes were performed as in Noguchi et al. (1990a). The concentration of mRNA was 0.5 μg/μ1 for each mRNA and ~20 nl, on average, was injected per oocyte. For labeling translation products, oocytes were incubated in the presence of L-[4,5-3H]leucine (140 Ci/mmol; final concentration, 0.6 mCi/ml). Microsome preparation, measurement of Na,K-ATPase activity, and immunoprecipitation of the α- and β-subunits were carried out as in Noguchi et al. (1990a).

Results and Discussion

Immunoprecipitation of Translation Products from Na,K-ATPase Subunit-specific mRNAs in Xenopus Oocytes

As shown in Fig. 3, in βC46S mutant the amounts of the α- and β-subunit expressed in microsome (lanes 1–3) were similar to those of wild-type (lanes 19–21). The α-subunit was also immunoprecipitated with antiserum against the β-subunit (lane 3), and vice versa (lane 2), which indicates that the βC46S mutant can assemble with the α-subunit. On the other hand, in βC215S, βC278S mutants, and βC215,278S double mutant no detectable amounts of the labeled α-subunit were immunoprecipitated with antiserum against the β-subunit (lanes 12, 15, and 18), and vice versa (lanes 11, 14, and 17). These results indicate that the βC215S, βC278S mutants, and the βC215,278S double mutant cannot assemble with the α-subunit. Significant decrease of the amounts of α-subunit expressed in microsomes (lanes 10, 11, 13,14, 16, and 17) may have been caused by the lack of αβ interaction (Noguchi et al., 1990b). Futhermore, in βC127S mutant and βC127,150S double mutant, the α-subunit was immunoprecipitated with antiserum against the β-subunit (lanes 6 and 9), and vice versa (lanes 5 and 8). The amounts of the α-subunit expressed in microsomes decreased slightly (lanes 4, 5, 7, and 8) from that of the wild type (lanes 19 and 20). These results indicate that the βC127S mutant and βC127,150S double mutant can assemble with the α-subunit, although the efficiency of assembly may be decreased.

Na,K-ATPase Activity in Microsomes from Oocytes Injected with mRNAs

As shown in Table I, the ATPase activity in the microsome-expressed βC46S mutant was higher than that of the control. This indicates that the Cys^46 is not involved in the formation of αβ complex and ATPase activity. The increments of ATPase activity from the control level in βC215S, βC278S mutants, and βC215,278S double mutant were very slight. These results are due to the loss of the ability to form αβ complex, and indicate that the Cys^215–Cys^278 disulfide bond is essential for the formation of αβ complex. On the other hand, the ATPase activity of microsomes from oocytes injected with mRNA for βC127S mutant or βC127,150S double mutant was definitely higher than that of the control, although it was lower than that of microsomes from oocytes injected with mRNA for wild-type β-subunit. This indicates that the Cys^127–Cys^150 disulfide bond is not essential for ATPase activity. These results are consistent with the observation that the reduction of the Cys^127–Cys^150 disulfide bond did not terminate ATPase activity (Kirley, 1990). From the decrease in amounts of the α-subunit expressed in microsomes (Fig. 3, lanes 4, 5, 7, and 8), it can be seen that mutation at Cys^127 and/or Cys^150 may cause reduction in the efficiency of αβ complex formation, and this reduction may cause lower increments of ATPase activity.

TABLE I
Na,K-ATPase Activity of Mutants Expressed in Xenopus Oocytes

Na,K-ATPase activity

μmol Pi/mg per h

Control*

2.33

Wild type

6.48

βC46S

7.52

βC127S

4.11

βC127,150S

3.99

βC215S

2.66

βC278S

2.70

βC215,278S

2.87

Xenopus oocytes were injected with one of the mutated mRNA listed in the table together with the mRNA for the wild-type α-subunit.

* No mRNAs were injected.

References

Kirley, T. L. 1989. Determination of three disulfide bonds and one free sulfhydryl in the β subunit of (Na,K)-ATPase. Journal of Biological Chemistry. 264:7185–7192.

Kirley, T. L. 1990. Inactivation of (Na+,K+)-ATPase by β-mercaptoethanol. Differential sensitivity to reduction of the three β subunit disulfide bonds. Journal of Biological Chemistry. 265:4227–4232.

Miller, R. P., and R. A. Farley. 1990. β subunit of (Na++K+)-ATPase contains three disulfide bonds. Biochemistry. 29:1524–1532.

Noguchi, S., K. Higashi, and M. Kawamura. 1990a. Assembly of the α-subunit of Torpedo californica Na+/K+-ATPase with its preexisting β-subunit in Xenopus oocytes. Biochimica et Biophysica Acta. 1023:247–253.

Noguchi, S., K. Higashi, and M. Kawamura. 1990b. A possible role of the β-subunit of (Na,K)ATPase in facilitating correct assembly of the α-subunit in the membrane. Journal of Biological Chemistry. 265:15991–15995.

NMR Studies on Intermediate States of SR Ca2+-ATPase Using Co(III), Cr(III), and Rh (III) Nucleotides

Theresa A. Kuntzweiler and Charles M. Grisham

Department of Chemistry, University of Virginia, Charlottesville, Virginia 22901

Introduction

SR Ca2+-ATPase forms a phosphorylated intermediate that undergoes conformational changes upon active transport of calcium ions across the membrane. Ca2+ occlusion resulting from intermediate steps in the transport of Ca2+ has been correlated with enzyme phosphorylation. This work focuses on the structural changes that accompany phosphorylation and Ca2+ occlusion.

In the following studies, nucleotide or substrate conformations with respect to metal binding site(s) of SR Ca2+-ATPase in both its native and phosphorylated forms have been characterized by NMR spectroscopy. The NMR experiments described here require: (a) a paramagnetic probe that can be placed uniquely at the active site, (b) a stable substrate that forms a Michaelis complex with the enzyme, and (c) a single enzyme conformation in each experiment. In these experiments Gd3+ serves as a paramagnetic analogue of Ca2+. Metal ATP complexes with Co(NH_3)4-, Rh(H_2O)4-, and Cr(H_2O)4- are used as substitutes for MgATP, the naturally occurring substrate. These complexes are inert to enzyme hydrolysis at low temperatures and neutral-to-acidic pH, but inhibit the ATPase competitively with respect to MgATP and form a Michaelis complex with Ca2+-ATPase. At high temperatures (25 or 37°C), incubation of Ca2+-ATPase with Cr(H_2O)4ATP and Rh(H_2O)4ATP slowly inactivates the enzyme by formation of a highly stable E-P(M) phosphoenzyme intermediate, with subsequent dissociation of ADP.

This E-P(M) phosphoenzyme is a stable model for the native E-P-Mg complex.

The paramagnetic metal (Gd3+ or Cr3+) affects substrate 1H and 31P longitudinal relaxation rates and allows distances between the metal and the nuclei to be calculated. Substrate and metal conformations are different in the phosphorylated and native forms of Ca2+-ATPase.

Experimental Methods

SR Ca2+-ATPase was prepared in two steps. SR vesicles were prepared by the procedure of deMeis and Hasselbach (1971). Ca2+-ATPase was purified by method 2 of Meissner et al. (1973). Protein concentration was determined by the microbiuret procedure of Goa (1953). Barnett's (1970) continuous assay was used in the determination of Ca2+-ATPase activity.

MgATP analogues were synthesized by the procedures of Cornelius et al. (1978) with slight modifications for each metal complex according to the metal complex stability. The Rh(H_2O)nATP prepared by this method was 50:50 Rh(H_2O)4ATP:Rh(H_2O)3ATP. This mixture was purified to 99.5% Rh(H_2O)4ATP via Sephadex G-10 and C_18 reverse phase HPLC.

Proton and phosphorus spectra were obtained on two General Electric spectrometers, a QN-300 and a GN-500. The spectra were obtained at 4°C. Longitudinal relaxation times (1/T_1) were determined from an optimized inversion recovery experiment. T_1 values and their standard deviations were calculated using a three-parameter fit. This fitting routine corrects for inhomogeneities in the magnetic field which produce incomplete inversion during the 180° pulse.

Based on the distances calculated from the NMR data, a conformational analysis was done on a Silicon Graphics computer using the program MMS (copyright UCSD). The basic structure of the MgATP analogues was constructed from the x-ray data of the rubidium adenosine 5′-diphosphate monohydrate (Viswamitra et al., 1976) and Co(NH_3)4H_2P_3O_10H_2O (Merritt et al., 1978). A correction for the oxygen to methylene change between ATP and AMPPCP was made (Merritt et al., 1978; Kuntzweiler, 1986).

Theory of NMR Distance Calculations

The distances reported are based on Solomon and Bloembergen's (1956) theory of paramagnetic effects on nuclear relaxation rates. The paramagnetic contribution to longitudinal relaxation, 1/T1p, is described by the following equation: 1/T1p = pq/(T1M + Tm), where 1/T1M is the longitudinal relaxation rate in the first coordination sphere of the paramagnet, pq is the mole fraction of the nuclei in the coordination sphere of the paramagnetic ion, and τm is the mean residence time for nuclei in the first coordination sphere.

When τm T1M (fast exchange), the distance (r) between nuclei and paramagnetic probes can be calculated from the dipolar effect of the paramagnet on the nucleus: 1/T1M = [qC^6f (τc)]/r^6, where q is the coordination number and C depends on the nucleus and paramagnet. C for Gd3+-H is 896 and C for Gd3+-P is 664. The correlation time for the metal-nucleus dipolar interaction, (τc) is dependent on τm, τs (electron spin lattice relaxation time), and τr (time constant for the rotation of the paramagnetic complex).

Results and Discussion

Co(NH_3)4AMPPCP Conformation on Native Ca2+-ATPase

The conformation of Co(NH_3)4AMPPCP in fast exchange with the Ca2+-ATPase active site can be seen in Fig. 1. This conformation is based on 1H-Gd3+ and 31P-Gd3+ distances calculated from NMR data (Table I). The computer fit of the distances indicates the adenine torsional angle (χ) to be 110° as defined by 0_4,-C_1,-N_9-C_4. The torsional angle of the ribose ring (δ) corresponds to a puckered conformation with a value of 120°. γ, the orientation angle of O_5 relative to the ribose ring, is 160°. All of these angles are in good agreement with a nucleotide bound to adenylate kinase (Kuntzweiler, 1986). When Co(NH_3)4ATP was used as the MgATP analogue, a similar conformation for the active site was observed.

Figure 1. Conformation of CoAMPPCP on native Ca2+-ATPase (E).

Co(NH_3)4ATP Conformation on Phosphorylated Ca2+-ATPase

The conformation of Co(NH_3)4ATP in fast exchange with the phosphorylated intermediates E-P-Rh(III) and E-P-Cr(III) can be seen in Fig. 2. The intermediate E-P-Rh(III) was used to measure 1H-Gd3+ and 31P-Gd3+ distances similar to those mentioned above. The intermediate E-P-Cr(III) contains Cr(III), which is a paramagnetic ion. 31P-Cr(III) distances were measured in E-P-Cr(III). The largest alteration in the two conformations (i.e., native versus phosphorylated ATPase) occurs in the phosphate chain of the Co(NH_3)4ATP. The β and γ phosphorous atoms have folded back due to spatial interference of the metal and/or phosphate attached to the ATPase intermediate. The adenine and ribose angles of the substrate on the phosphoenzyme are approximately the same as those of the native enzyme conformation.

TABLE I
Distances Used to Construct Conformations of Ca2+-ATPase Active Site

Figure 2. CoATP conformation on Ca2+-ATPase intermediate (E-P(M)).

Gd3+ Movement into an Occluded State upon Phosphorylation

It has been shown previously by several groups that Gd3+ supports enzyme phosphorylation (Squier et al., 1990) and that Gd3+ is occluded by the ATPase (Stephens and Grisham, 1979) (Klemens and Grisham, 1988). To characterize the effects of phosphoenzyme formation on Gd3+ (i.e., Ca2+) sites on the enzyme, we have formed stable E-P complexes with Rh(H_2O)4ATP in a Gd3+-dependent fashion:

The influence of Gd3+ on the relaxation rate of 1H and 31P of the nucleotide were measured in this E-P complex (Fig. 3) and compared with the results described above for the native ATPase. The paramagnetic effects were small, which means the 1H-Gd3+ and 31P-Gd3+ distances are longer than those in the two previous conformations. This change in the Gd3+ position is consistent with previous observations of occlusion of Ca2+ (Vilsen and Andersen, 1987) or Gd3+ (Klemens and Grisham, 1988) upon formation of stable E-(M(III)ATP) and/or E-P complexes. The movement of the Gd3+ ion by ~4 Å upon formation of E-P may be related to the events of cation translocation by SR Ca2+-ATPase.

Figure 3. Gd3+ at the active site of phosphorylated Ca2+-ATPase. “Occluded Gd3+” is associated with the (Gd)E-P(Rh) complex.

References

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Cornelius, R., P. Hart, and W. Cleland. 1977. Phosphorus-31 NMR studies of complexes of adenosine triphosphate, adenosine diphosphate, tripolyphosphate and pyrophosphate with cobalt (III) ammines. Inorganic Chemistry. 16:2799–2805.

deMeis, L., and W. Hasselbach. 1971. Acetyl phosphate as substrate for Ca2+ uptake in skeletal muscle microsomes. Journal of Biological Chemistry. 246:4759–4763.

Goa, M. 1953. A micro biuret method for protein determination. Scandinavian Journal of Clinical and Laboratory Investigation. 5:218–222.

Klemens, M. R., and C. M. Grisham. 1988. NMR relaxation measurements detect four intermediate states of ATPase and transport cycle of SR Ca2+-ATPase. Biochemical and Biophysical Research Communications. 155:236–242.

Kuntzweiler, T. A. 1986. 1H NMR studies of the conformation of a substrate analogue bound to SR Ca2+-ATPase. Masters Thesis. University of Virginia, Charlottesville, VA.

Meissner, G., G. Conner, and S. Fleischer. 1973. Isolation of sarcoplasmic reticulum by zonal centrifugation and purification of Ca2+-pump and Ca2+-binding proteins. Biochimica et Biophysica Acta. 298:246–269.

Merritt, E., M. Sundaralingam, R. Cornelius, and W. Cleland. 1978. X-ray crystal and molecular structure and absolute configuration of Co(NH_3)4H_2P_3O_10·H_2O: a model for a metal-nucleoside polyphosphate complex. Biochemistry. 17:3274–3278.

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Vilsen, B., and J. P. Andersen. 1987. Characterization of CrATP-induced calcium occlusion in membrane-bound and soluble monomeric SR Ca2+-ATPase. Biochimica et Biophysica Acta. 898:313–322.

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Expression of Functional Sodium and Calcium Pump Chimeric Molecules

Douglas B. Luckie, Kimberly L. Boyd, Atsushi Mizushima, Zhifeng Shao, Andrew P. Somlyo, and Kunio Takeyasu

Department of Physiology, University of Virginia, Charlottesville Virginia 22908

Introduction

Recent cDNA cloning and sequencing studies have shown that a variety of E_1-E_2 type ATPases (ion pumps) share common structural characteristics, including a general structure with 7–10 transmembrane regions as well as highly conserved specific domains for ATP binding and phosphorylation. The hydrophobicity plots obtained from the deduced amino acid sequences of the cloned cDNAs are virtually identical for the ~100-kD catalytic polypeptides, including the α-subunit of the plasma membrane (PM) (Na+ + K+)-ATPase (sodium pump) and the gastric (K+ + H+)-ATPase, and the sarcoplasmic reticulum (SR) Ca2+-ATPase (calcium pump), while the primary amino acid sequences of these ATPases are rather divergent from each other (e.g., reviewed by Jorgensen and Andersen, 1988). On the other hand, each member of this ion pump family shows distinct specificity for particular ions, inhibitors, and cofactors (subunits). One way to identify such specific functional domains is to construct chimeric molecules between distinct functional proteins with very similar structures. In this study we have constructed and functionally expressed chimeric molecules between the SR Ca2+-ATPase and the α-subunit of the PM (Na+ + K+)-ATPase. Analysis of the expressed chimeric proteins has identified ouabain-binding domains in the sodium pump α-subunit and Ca2+-sensitive domains in the calcium pump, as well as domains possibly involved in assembly with the β-subunit and targeting to the plasma membrane.

Expression of Sodium and Calcium Pump Chimeric Molecules

The conserved and functionally important amino acid sequences for the ATP-binding and phosphorylation domains reside between the fourth and fifth potential transmembrane domain (M4 and M5). In addition, the region just before M5 is very well conserved between distinct ion pumps. We have taken advantage of this fact to construct and express a set of chimeric chicken cDNAs in mouse L cells. This avian/mouse hybrid system allows us to monitor enzymatic functions of the relatively ouabain-sensitive chicken sodium pump in relatively ouabain-insensitive mouse cells (Takeyasu et al., 1988).

Fig. 1 is a schematic representation of the chimeric molecules produced. The first four transmembrane domains reside in the NH_2-terminal third of the molecule, and the rest in the COOH-terminal third. A unique restriction site, Eco NI, which is endogenous to both the SR calcium pump cDNA and the PM sodium pump α1-subunit cDNA, was used as an exchange point for recombination. This exchange occurred at nucleotide 2,134 on the α1-subunit cDNA and at nucleotide 2,170 on the calcium pump cDNA, both of which encode lysine in identical regions just before M5 of the two proteins. Therefore, the overall topological structure of the encoded sodium and calcium pump chimeric proteins as ion pumps should be preserved. These chimeric cDNAs, as well as wild-type cDNAs, were cloned into an expression vector derived from a plasmid pSV2CAT (Takeyasu et al., 1987) and stably introduced into mouse L cells which had been transfected with chicken β1-subunit cDNA and expressing the avian protein on the cell surface (Takeyasu et al., 1987).

Fig. 2 illustrates the subcellular localization of the chimeric pumps imaged with a laser scanning confocal microscope. Chimera I (containing the NH_2-terminal two-thirds of the sodium pump α1-subunit) was only found internally, while chimera II (containing the NH_2-terminal two-thirds of the calcium pump) was found both on the plasma membrane and inside the cell. These results suggest that the COOH-terminal third of the α-subunit is most important in the sorting of the sodium pump to the plasma membrane.

Figure 1. Schematic representation of the structures of the sodium/calcium pump chimeric molecules. Shaded regions indicate the potential transmembrane domains. Mn represents the nth transmembrane domain.

Identification of Ouabain-binding Domains

Fig. 3 shows the effects of varying concentrations of ouabain on the ATPase activity (measured at 120 mM NaCl and 15 mM KCl without Ca2+) of mouse cells transfected with various chicken cDNAs. The inhibition of the ATPase activity of both wild-type and chimera I-transfected cells by ouabain is biphasic; ~60% show IC_50's of < 5 μM and the rest show IC_50's of ~300 μM. On the other hand, ouabain has a single phase inhibitory effect (IC_50 ~300 μM) on cells transfected with chimera II cDNAs as well as on untransfected mouse L cells. Since the endogenous mouse sodium pumps do not have a high affinity for ouabain, this new presence of a high affinity ouabain site must come from the transfected chicken α1-subunit and chimeric pump.

Figure 2. Laser confocal micrographs of cloned mouse L cells that express the chicken sodium/calcium pump chimeric molecules. By the calcium phosphate precipitate method, chimeric cDNAs were cotransfected with a plasmid containing a neomycin-resistant gene into mouse Ltkβ3 cells (Takeyasu et al., 1987). Cells were selected against resistance of G418-sulfate and screened against chicken-specific monoclonal antibody binding. (Left) Cells transfected with chimera I cDNAs were fixed, permeabilized, and stained with TRITC-labeled monoclonal IgG 7C specific to the chicken α1-subunit (Takeyasu et al., 1988). (Right) Cells transfected with chimera II cDNAs were fixed, permeabilized, and stained with TRITC-labeled monoclonal IgG 5D2 specific to the chicken SR calcium pump (Kaprielian and Fambrough, 1987). Scale bar, 10 μm.

The α-subunit is the major determinant for ouabain sensitivity as demonstrated in gene-transfer experiments (Kent et al., 1987; Noguchi et al., 1987; Takeyasu et al., 1988; Emanuel et al., 1988). A recent mutagenesis experiment demonstrated that the extracellular domain between M1 and M2, consisting of 11 amino acids, is the major ouabain binding region (Price and Lingrel, 1988). However, it remains to be determined whether ouabain binds only to this site or to additional site(s) in order to exert its action. The results from the inhibitory effect of ouabain on chimera I suggest that ouabain does not have to bind to the ectodomains after M5.

Identification of Ion-specific Domains

Calcium ions (1 μL of 0.5 M CaCl_2) added to the 1 mL assay system consisting of 120 mM NaCl, 15 mM KCl, and 0.5 mM EGTA stimulated chimera I ATPase activity greater than twofold above that of the control cell line. Under the same conditions, chimera II was not stimulated by any level of calcium addition, and in some runs appeared to be inhibited up to 15% by physiological concentrations of calcium. Some of the potential transmembrane segments, including M4, M5, and M6 of both sodium and calcium pumps have glutamic and asparatic acids. Mutagenesis experiments have demonstrated that these charged amino acids are critical for both sodium pump (Lingrel et al., 1991) and calcium pump activities (Clarke et al., 1989). In our chimeric molecules, these amino acids are preserved. These data suggest that the COOH-terminal third of the calcium pump (present in chimera I) is a calcium-specific domain and is actually functioning in chimera I. The existence of the COOH-terminal third of the calcium pump molecule may be critical for the calcium-stimulated ATPase activity of the calcium pump. This domain of the calcium pump appears to undergo the proper protein folding required for at least the calcium pump (and also possibly for other ion pumps), because incorporation of this domain into the COOH-terminal third of the sodium pump α1-subunit did not interfere with high affinity ouabain binding or ion-stimulated ATPase activity.

Figure 3. Dose–response relationships for ouabain inhibition of the ATPase activity in untransfected and transfected mouse cells. The ATPase activity was measured by an enzyme-coupled assay system (Kutchai and Campbell, 1989) in which, for each ADP produced in the assay, a molecule of NADH was changed to NAD+ and this was monitored spectroscopically by a decrease in absorbance at 340 nm. In the enzyme-coupled assay system, the ATPase activities of cell homogenate were measured in the presence of 0.2% Triton X-100, which allow ouabain access to both sides of the membrane. With appropriate inhibitors present (0.5 mM EGTA, 2 mM sodium azide), we could remove all background except that of the endogenous sodium pumps and transfected mutant pumps.

Speculation on the Subunit Assembly Sites

Extensive studies on the expression of sodium pump subunits in a variety of systems have suggested that the β-subunit is critically involved in stabilization and transport of the α-subunit to the plasma membrane (Noguchi et al., 1987; Takeyasu et al., 1989; Geering, 1990; McDonough et al., 1990). A recent study, using in vitro expression of sodium/calcium pump chimeric molecules, has demonstrated that the COOH-terminal third of the α-subunit contains the assembling domain(s) with the β-subunit (see Kawamura and Noguchi, 1991). Chemical cross-linking modification followed by amino acid sequencing of the modified fragments of the (H+ + K+)-ATPase α-subunit has also suggested that the ectodomain between M5 and M6 in the COOH-terminal third of the molecule is situated very close to the β-subunit (Sachs et al., 1990). Subcellular distributions of chimeras I and II (Fig. 2) are consistent with these results. Our interpretation is that chimera I lacks the assembling domains with the β-subunit and thus is not transported to the plasma membrane, and that chimera II acquires the assembling domains and is transported to the cell surface.

The (Na+ + K+)-ATPase is supposed to exist as an αβ-subunit complex in order to exert its full activity, while the SR Ca2+-ATPase does not require any additional subunits for its full functioning. In this sense, it is intriguing that chimera I (NH_2-terminal sodium/COOH-terminal calcium pump) exhibits ATPase activity. Our approach using construction and expression of functional chimeric molecules between different ion pumps will provide a practical way to further understand the structure, conformational changes, and functional relationships of the E_1-E_2ATPases.

Acknowledgments

This work was supported by NIH grant ROl-GM-44373 (K. Takeyasu), NIH Training grant HL-07284 (D. Luckie, K. Boyd), and a Postdoctoral Fellowship from the American Heart Association (A. Mizushima).

References

Clarke, D. M., T. W. Loo, G. Inesi, and D. H. MacLennan. 1989. Location of high affinity Ca2+-binding sites within the predicted transmembrane domain of the sarcoplasmic reticulum Ca2+-ATPase. Nature. 339:476–478.

Emanuel, J. R., J. Shultz, X.-M. Zhou, R. B. Kent, D. Housman, L. Cantley, and R. Levenson. 1988. Expression of an ouabain-resistant Na,K-ATPase in CV-I cells after transfection with a cDNA encoding the rat Na,K-ATPase α1 subunit. Journal of Biological Chemistry. 263:7726–7733.

Geering, K. 1990. Subunit assembly and functional maturation of Na,K-ATPase. Journal of Membrane Biology. 115:109–121.

Jorgensen, P. L., and J. P. Andersen. 1988. Structural basis for E1-E2 conformational transitions in Na,K-pump and Ca-pump proteins. Journal of Membrane Biology. 103:95–120.

Kaprielian, Z., and D. M. Fambrough. 1987. Expression of fast and slow isoforms of the Ca2+-ATPase in developing chick skeletal muscle. Developmental Biology. 124:490–503.

Kawamura, M., and S. Noguchi. 1991. Possible role of the β-subunit in the expression of the sodium pump. In The Sodium Pump: Structure, Mechanism, and Regulation. J. H. Kaplan and P. DeWeer, editors. The Rockefeller University Press, New York. 45–61.

Kent, R. B., J. R. Emanuel, Y. B. Neriah, R. Levenson, and D. E. Housman. 1987. Ouabain resistance conferred by expression of the cDNA for a murine Na+,K+-ATPase α-subunit. Science. 237:901–903.

Kutchai, H., and K. P. Campbell. 1989. Calcium transport by sarcoplasmic reticulum of skeletal muscle is inhibited by antibodies against the 53-kilodalton glycoprotein of the sarcoplasmic reticulum membrane. Biochemistry. 28:4830–4839.

Lingrel, J. B., J. Orlowski, E. M. Price, and B. G. Pathak. 1991. Regulation of the α-subunit genes of the Na,K-ATPase and determinants of cardiac glycoside sensitivity. In The Sodium Pump: Structure, Mechanism, and Regulation. J. H. Kaplan and P. De Weer, editors. The Rockefeller University Press, New York. 1–16.

McDonough, A. A., K. Geering, and R. A. Farley. 1990. The sodium pump needs its β-subunit. FASEB Journal. 4:1598–1605.

Noguchi, S., M. Mishina, M. Kawamura, and S. Numa. 1987. Expression of functional (Na+ + K+)-ATPase from cloned cDNAs. FEBS Letters. 225:27–32.

Price, E. M., and J. B. Lingrel. 1988. Structure-function relationships in the Na,K-ATPase α-subunit: site-directed mutagenesis of glutamine-111 to arginine and asparagine-122 to aspartic acid generates a ouabain-resistant enzyme. Biochemistry. 27:8400–8408.

Sachs, G., B. Wallmark, K. Hall, and K. Munson. 1990. A functional region of the gastric H, K-ATPase. Biophysical Journal. 57:17a. (Abstr.)

Takeyasu, K., K. Renaud, J. Taormino, B. A. Wolitsky, A. Barnstein, M. M. Tamkun, and D. M. Fambrough. 1989. Differential subunit and isoform expression involved in regulation of sodium pump in skeletal muscle. Current Topics in Membranes and Transport. 34:143–165.

Takeyasu, K., M. M. Tamkun, K. J. Renaud, and D. M. Fambrough. 1988. Oaubain-sensitive (Na+ + K+)-ATPase activity expressed in mouse L cells by transfection with DNA encoding the α-subunit of an avian sodium pump. Journal of Biological Chemistry. 263:4347–4354.

Takeyasu, K., M. M. Tamkun, N. R. Siegel, and D. M. Fambrough. 1987. Expression of hybrid (Na+ + K+)-ATPase molecules after transfection of mouse Ltk- cells with DNA encoding the β-subunit of an avian brain sodium pump. Journal of Biological Chemistry. 262:10733–10740.

Three-dimensional Structure of CO(NH_3)4ATP-induced Membrane Crystals of Na,K-ATPase

Elisabeth Skriver, Hans Hebert, Urban Kavéus, and Arvid B. Maunsbach

Department of Cell Biology, Institute of Anatomy and Biomembrane Research Center, University of Aarhus, DK-8000 Aarhus C, Denmark; and Structural Biochemistry Unit, Center for Nutrition and Toxicology, Novum, Karolinska Institutet, S-14152 Stockholm, Sweden

Introduction

Membrane-bound Na,K-ATPase purified from kidney outer medulla forms two-dimensional crystals during incubation with vanadate and magnesium (Skriver et al., 1981). The crystals as observed by electron microscopy are of the p21 and p1 types and have been analyzed in projections perpendicular to the membrane by image processing (Hebert et al., 1982). The three-dimensional (3-D) structures have been determined from tilt series (Hebert et al., 1985, 1988, 1990; Maunsbach et al., 1988). In the present work a new 3-D structure has been reconstructed from membrane crystals with p4 symmetry that were formed during incubation with cobalt-tetrammine-ATP, CO(NH_3)4ATP (Skriver et al., 1989). This is a MgATP complex analogue that binds to the low-affinity ATP-binding site of the enzyme, thus forming a cobalt-tetrammine-phosphoenzyme in an E_2 conformational state (Scheiner-Bobis et al., 1987, 1989).

Materials and Methods

Membrane-bound Na,K-ATPase purified from pig kidney outer medulla treated with phospholipase A_2, was incubated in 0.16 mM Co(NH_3)4ATP and 0.1 mM CoCl_2 in 10 mM Tris-HCl buffer, pH 7.3, at 37°C for 1 h and then placed at 4°C for up to 28 d. Tilt series of crystalline membrane fragments negatively stained with 1% uranyl acetate were recorded in a Jeol 100CX electron microscope at angles from –60° to +60° with increments of 6°. Electron micrographs of the tilt series were digitized into 1,024 × 1,024 pixel squares using an EIKONIX 1412 scanner and image processing of each projection was performed using correlation averaging methods (Saxton and Baumeister, 1982). The 3-D data set in Fourier space was collected from the aligned averages and an inverse Fourier transformation yielded the 3-D model with an estimated resolution of 20–25 Å in the x and y planes.

Figure 1. Tetragonal membrane crystal of purified Na,K-ATPase negatively stained with 1% uranyl acetate and recorded at nominal tilt angle 0°. The surface particles (αβ-units) are paired and form dimers which in turn are arranged in a square-mesh pattern. Bar, 0.1 μM.

Figure 2. Gallery of experimental projections from tilted micrographs after correlation averaging. The increment between consecutive micrographs is 6°.

Results and Discussion

During incubation of purified membrane-bound Na,K-ATPase with Co(NH_3)4ATP, two-dimensional crystalline arrays of the p21 and p4 types are formed (Fig. 1). The building blocks of both types correspond to (αβ)2 dimers of the enzyme. The tetragonal p4 type of membrane crystal has a relatively large unit cell (a = b = 138 Å) with very small variations of the cell parameters. This makes p4 crystals more suitable for a high resolution 3-D structural analysis of the enzyme than the previously studied p1 and p21 crystals.

Figures 3–5. (3) Three-dimensional surface model of four pairs of enzyme units reconstructed from a membrane crystal with p4 symmetry. The viewing direction is almost parallel to the plane of the membrane. Bar, 20 Å. (4) 3-D model of four pairs of Na,K-ATPase molecules observed along the c axis, running perpendicular to the membrane and probably from the exoplasmic side of the membrane. (5) The four pairs of enzyme units observed from the proposed cytoplasmic side at an angle of ~30° relative to the c axis.

The 3-D model of the dimer calculated from a tilt series (Fig. 2) consists of two symmetrically related rodlike structures which are arranged perpendicular to the membrane and have a volume of 2.2 × 10^5 Å^3 suggesting that it constitutes an (αβ)2 dimer of Na,K-ATPase (Figs. 3–5). The height of the model is ~90 Å. The two protein units show a connecting domain, which is located at the level where the model has a constriction and which may correspond to the position of the lipid bilayer (Fig. 3). This observation is consistent with an asymmetric distribution of the Na,K-ATPase protein relative to the bilayer and shows most of the mass of the protein extending into the cytoplasm. The cavity between the two rods extends toward the suggested plane of the membrane. The width of the cavity is ~15 Å. Three models reconstructed from independent data sets show the same structural characteristics.

The overall structure obtained for Na,K-ATPase dimers in the Co(NH_3)4ATP-stabilized form is similar to our previous reconstruction of the enzyme from vanadate-induced p21 crystals (Hebert et al., 1985). However, the connecting domain within the dimer is more constricted and shifted toward the center in the Co(NH_3)4ATP-induced crystals, which may imply a different conformation of the cobalt-tetrammine-phosphoenzyme.

Acknowledgments

We thank Prof. W. Schoner and Dr. G. Scheiner-Bobis for providing the cobalt-tetrammine-ATP. This investigation was supported by grants from the Danish Biomembrane Research Center, the Danish Medical Research Council (12-8501), the Research Foundation of the University of Aarhus, and the Swedish Medical Research Council (03X-00144).

References

Hebert, H., P. L. Jørgensen, E. Skriver, and A. B. Maunsbach. 1982. Crystallization patterns of membrane-bound Na,K-ATPase. Biochimica et Biophysica Acta. 689:571–574.

Hebert, H., E. Skriver, U. Kavéus, and A. B. Maunsbach. 1990. Coexistence of different forms of Na,K-ATPase in two-dimensional membrane crystals. FEBS Letters. 268:83–87.

Hebert, H., E. Skriver, and A. B. Maunsbach. 1985. Three-dimensional structure of renal Na,K-ATPase determined by electron microscopy of membrane crystals. FEBS Letters. 187:182–186.

Hebert, H., E. Skriver, M. Söderholm, and A. B. Maunsbach. 1988. Three-dimensional structure of renal Na,K-ATPase determined from two-dimensional membrane crystals of the p1 form. Journal of Ultrastructure and Molecular Structure Research. 100:86–93.

Maunsbach, A. B., E. Skriver, M. Söderholm, and H. Hebert. 1988. Three-dimensional structure and topography of membrane-bound Na,K-ATPase. In The Na+,K+-Pump. Part A: Molecular Aspects. J. C. Skou, J. G. Nørby, A. B. Maunsbach, and M. Esmann, editors. Alan R. Liss, Inc., New York. 39–56.

Saxton, W. O., and W. Baumeister. 1982. The correlation averaging of regular arranged bacterial cell envelope protein. Journal of Microscopy. 127:127–138.

Scheiner-Bobis, G., M. Esmann, and W. Schoner. 1989. Shift to the Na+ form of Na+,K+-transporting ATPase due to modification of the low-affinity ATP-binding site by Co(NH_3)4ATP. European Journal of Biochemistry. 183:173–178.

Scheiner-Bobis, G., K. Fahlbusch, and W. Schoner. 1987. Demonstration of cooperating α subunits in working (Na+ + K+)-ATPase by the use of MgATP complex analogue cobalt tetrammine ATP. European Journal of Biochemistry. 168:123–131.

Skriver, E., A. B. Maunsbach, H. Hebert, G. Scheiner-Bobis, and W. Schoner. 1989. Two-dimensional crystalline arrays of Na,K-ATPase with new subunit interactions induced by cobalt-tetrammine-ATP. Journal of Ultrastructure and Molecular Structure Research. 102:1891–95.

Skriver, E., A. B. Maunsbach, and P. L. Jørgensen. 1981. Formation of two-dimensional crystals in pure membrane-bound Na,K-ATPase. FEBS Letters. 131:219–222.

Structural Analysis of Na,K-ATPase Isoforms in Mammalian Tissues

Elena A. Arystarkhova, Natalia M. Vladimirova, Natalia A. Potapenko, and Nikolai N. Modyanov

Shemyakin Institute of Bioorganic Chemistry, USSR Academy of Sciences, 117871 Moscow, USSR

Introduction

The functional interpretation of multiple isoforms of the Na,K-ATPase is still unclear. Isozymes may be expressed by different cells in various tissues or there could be simultaneous coexistence of different isoforms with their own specific functions. The analysis of tissue distribution is the first step in the elucidation of isozyme structural and functional diversity. Identification of the different isoforms with specific antibodies is commonly used, but direct sequence analysis is more convincing and can be semiquantitative. Here we present data on isoform distribution in mammalian kidney and brain based on both immunochemical detection and direct sequence analysis.

Materials and Methods

Isolation of microsomes and Na,K-ATPase purification from kidney, human brain, and calf brain were performed as in Arystarkhova et al. (1989b).[1]

Production, purification of antibodies, indirect solid-surface adsorption-binding assay, and Western blot procedures have been previously described in Arystarkhova et al. (1989b). A freshly prepared solution of dodecyloctaethylene glycol monoether (C_12E_8, Calbiochem Corp., La Jolla, CA) was dropped into a suspension of pig kidney microsomes to a final concentration of 1 mg/ml in buffer containing 10% sucrose, 0.1 M KCl, 30 mM histidine, pH 7.2, and 3 mM EDTA, up to the detergent/protein ratio 3:1 (5 min, 20°C). The separation of solubilized proteins on an Ultro Pak TSK G-3000 SWG (21.5 × 600 mm; LKB, Sweden) and final purification of the α3 protein on immunosorbent were made as in Arystarkhova et al. (1989a).

Electrophoresis and electroblotting onto PVDF membranes were performed.[1] The protein bands were cut into thin strips and applied directly to a gas phase sequencer (model 470A) with an on-line PTH analyzer (model 120A; Applied Biosystems, Inc., Foster City, CA).

Results and Discussion

The comparison of the α1, α2, and α3 primary structures deduced from nucleotide sequences revealed large difference in two regions (reviewed in Sweadner, 1989). One of them adjoins the FITC-binding site; the second is located in the NH_2-terminal part of the molecule. The polyclonal sera or affinity-purified monospecific antibodies against the latter (GDKKDDKSSPK) sequence unique for α3, were used in Western blot analysis with the avidin–biotin amplification procedure (Arystarkhova et al., 1989b). For unknown reasons, these antibodies do not detect intact α3 in rat brain membrane preparations (Arystarkhova, E. A., and K. J. Sweadner, unpublished observations), although they do react with the synthetic peptide used for immunization. A polypeptide with a molecular mass of 100 kD (or sometimes its dimer) was detected in microsomal fractions as well as in preparations of the purified Na,K-ATPase from pig kidney medulla or cortex, human kidney (total homogenate), and human brain (cortex). However, these data were in disagreement with Hsu and Guidotti (1989), where the α3 protein was completely undetected in rat kidney. To seek other evidence for α3 gene expression in animal kidneys, we attempted to isolate the protein and analyze its structure. Immunoaffinity chromatography with the antibodies raised against the synthetic peptide was performed. Pig kidney microsomes were solubilized with C_12E_8 at a 3:1 detergent/protein ratio (Arystarkhova et al., 1989a). The solubilizate was first chromatographed on TSK-gel and the fraction containing 100-kD proteins was applied directly to the affinity column just after removing SDS. Analysis of the eluted protein immobilized on PVDF membrane revealed that the sequence unambiguously belonged to the α3 isoform and was Met-Gly-Asp-Lys-Lys-Asp-Asp (Table I). The α1-subunit, the so-called renal form, lacks this sequence. The ratio between α3 and α1 isoforms in kidney is estimated to be ~ 1:100 (20 mg of microsomes gives ~ 10 µg of the α3 protein with possible protein loss at various stages of its isolation). However, the α3 isoform content in kidney could be higher if its greater sensitivity to proteolysis is taken into account.[1] We propose that this molecule is expressed selectively in specific regions of the mammalian nephron, namely in the collecting tubules, which have a different embryologic origin from other nephron segments. This could be a reasonable explanation for the higher affinities for ouabain and sodium ions detected in rabbit collecting tubules (Barlet-Bas et al., 1990).

TABLE I
Microsequence Analysis of the Protein Eluted from the Immunosorbent, Electrotransferred onto PVDF Membranes

Sequence cycle no.

Amino acid

Yield

pmol

1

Met

8.32

2

Gly

2.08

3

Asp

3.64

4

Lys

2.42

5

Lys

3.08

6

Asp

2.04

7

Asp

2.50

The transfer yield was ~ 70–80%. The sequence yields at each cycle of Edman degradation are shown.

Since mammalian brain is supposed to contain all three isoforms of Na,K-ATPase catalytic subunit (Sweadner, 1989), we attempted to isolate the functionally active enzymes from different parts of calf brain, namely, gray matter, brainstem, and brainstem axolemma. The protein components were separated by SDS-PAGE electrophoresis and electroblotted onto PVDF membranes. The NH_2-terminal sequences of isoforms α1, α2, α3, β1, and β2 were determined.[1]

α1 Gly ArgAspLysTyr GluPro AlaAlaValSer

α2 Gly ArgGluTyrX* Pro AlaAlaThrThr

α3 MetGly AspLysLys AspAspLys X* Ser

β1 Ala ArgGly LysAlaLys GluGluGlySer X* LysLysPhelle

β2 Val Ile X* LysGluLys Lys Ser X* X* X* ValVal (*X, amino acid that was not identified).

The NH_2-terminal sequences of the α3 isoform from pig kidney and calf brain coincide completely with that deduced from the α3 gene nucleotide sequence. This means that mature α3 can be expressed without proteolytic processing, α1- and α2-subunits lack the first five amino acids (Sweadner, 1989).

The NH_2-terminal sequence of β2 is identical to the β2 (AMOG) sequence derived from mouse (Gloor et al., 1990), rat, and human (Martin-Vassalo et al., 1989) cDNAs beginning with the second residue. As in the case of the β1-subunit, methionine cleavage occurs here as well.

Interpretation of the results of electrophoretic analysis of Na,K-ATPase from brainstem and axolemma microsomes is rather difficult. Preliminary treatment of the samples with the proteinase inhibitor, diisopropylfluorophosphate (DIFP), was shown to be obligatory for the identification of intact α3. Otherwise, specific fragmentation yielding a rather stable NH_2-terminal polypeptide (Mr 50 kD) was observed. The identity of this fragment was determined by direct sequencing as well as through its interaction with monoclonal antibodies against the synthetic peptide corresponding to the α3 NH_2 terminus. Carboxypeptidase treatment of this fragment immobilized on the PVDF membrane allowed us to deduce the site of the endogenous proteolysis. It occurs in a region unique for α3 sequence:

Pro-Asn-Asp-Asn-Arg492 ↓ (Tyr^493-Leu-Leu-Val-Met).

Evidently, the higher lability of α3 isoform to proteolysis made our previous attempts at its isolation from brain unsuccessful.

Assuming a ratio α:β of 1:1, the type of αβ complexes could be estimated from the sequence data. α_1β_1 and α_3β_1 are likely to be the predominant forms in gray matter and axolemma, respectively. Brainstem membranes could also contain α_2β_1 complexes.[1]

Acknowledgments

We would like to thank O. E. Lakhtina for the production of antibodies and N. B. Levina for performing the sequence.

References

Arystarkhova, E. A., O. E. Lakhtina, N. B. Levina, and N. N. Modyanov. 1989a. Immunoaffinity isolation of Na,K-ATPase α3 isoform from pig kidney. FEBS Letters. 257:24–26.

Arystarkhova, E. A., O. E. Lakhtina, and N. N. Modyanov. 1989b. Immunodetection of Na,K-ATPase α3 isoform in renal and nerve tissues. FEBS Letters. 250:545–548.

Barlet-Bas, C., L. Cheval, C. Khadoury, S. Marsy, and A. Doucet. 1990. Difference in the Na-affinity of Na,K-ATPase along the rabbit nephron: modulation by K+. American Journal of Physiology. 259:F246–F251.

Gloor, S., H. Antonicek, K. J. Sweadner, S. Pagliusi, R. Frank, M. Moos, and M. Schachner. 1990. The adhesion molecule on glia is a homolog of the β subunit of the Na,K-ATPase. Journal of Cell Biology. 110:165–174.

Hsu, Y.-M., and G. Guidotti. 1989. Rat brain has the α3 form of the Na,K-ATPase. Biochemistry. 28:569–573.

Martin-Vassalo, P., W. Dackowski, J. R. Emanuel, and R. Levenson. 1989. Identification of a putative isoform of the Na,K-ATPase β-subunit. Primary structure and tissue-specific expression. Journal of Biological Chemistry. 264:4613–4618.

Sweadner, K. J. 1989. Isozymes of the Na/K-ATPase. Biochimica et Biophysica Acta. 988:185–220.

[1] See also Vladimirova et al., manuscript submitted for publication.

Function and Orientation of the COOH-terminal Domain of the α-Subunit

T. Krämer, R. Antolovic, H.-J. Brüller, U. Richter, G. Scheiner-Bobis, and W. Schoner

Institut für Biochemie und Endokrinologie, Justus-Liebig Universität, D-W6300 Giessen, Germany; and Institut für Molekularbiologie und Tumorforschung, D-W3550 Marburg, Germany

Introduction

According to Karlish et al. (1985) voltage-dependent Na+ translocation through animal cell membranes is achieved by two negative charges within the cation binding sites. Furthermore, the capacity of cation occlusion sites for Na+ and Rb+ has been seen to decrease by exposure to N,N ′-dicyclohexylcarbodiimide (DCCD) (Shani-Sekler et al., 1988). Inactivation of Na+/K+-ATPase by DCCD, which specifically labels the 439–1,016 COOH-terminal part of the α-subunit (Richter et al., 1987), has been found to be protected by Na+,K+ as well as by Mg2+ (Schoner and Schmidt, 1969). This indicates that carboxyl groups may be involved in functions other than cation binding. The exact location of the DCCD-modified groups is unclear, however. Acidic amino acids in the hydrophobic environment of the transmembrane part of the enzyme such as Glu 953 and 954 (Ovchinnikov et al., 1986) are candidates for the ionophoric role. But folding of the catalytic α-subunit in the COOH-terminal portion is a matter of dispute. The COOH-terminal end has been located on the extracellular side (Ovchinnikov et al., 1986) as well as on the cytoplasmic side (Ball and Loftice, 1987). We therefore tried to characterize the orientation of the COOH terminus with peptide sequence antibodies and test the role of carboxyl groups in the overall Na+/K+-ATPase activity and its partial reactions with DCCD.

Materials and Methods

Na+/K+-ATPase from pig kidney (sp act 15–32 U/mg) was incubated at 37°C in 10 mM imidazole-HCl (pH 7.25) at a concentration of 0.1–1.0 mg protein/ml in 75 nM–3 mM DCCD (ratio of DCCD/mg protein: 50–200 nmol DCCD/mg protein). Modification was started by adding DCCD in DMSO (final concentration of 1% DMSO). At the times indicated in the figures aliquots of the incubation mixture were transfered to the respective assays. Assays have been described (Gorga, 1985; Scheiner-Bobis et al., 1987; Vilsen et al, 1987; Shani-Sekler et al., 1988). The peptide IFVYDEVRKLIIRRR (991–1,006) was obtained by solid phase synthesis and the protective groups were removed. It was injected subcutaneously into rabbits on a weekly basis to raise antibodies. The IgG was purified by affinity chromatography on Sepharose A to which the purified peptides had been coupled. Amino acid sequences of purified peptides were verified by gas phase sequencing.

Figure 1. Effect of inactivation of Na+/K+-ATPase by DCCD on partial activities of the E_1 and E_2 conformations. (A) Effects on 22Na+occlusion (Vilsen et al., 1987) and phosphorylation from [γ-32P]Cr[NH_3]4ATP. (B) Effects on 86Rb+ occlusion and backwards phosphorylation.

Results and Discussion

Studies on the Modification of the Overall and Partial Activities of Na+/K+-ATPase by DCCD

Studies of the partial activities of Na+/K+-ATPase with varying extent of inactivation by DCCD reveal (Fig. 1) that DCCD affects accessible groups in the E_2 conformation. Partial activities of this conformation like 86Rb+ occlusion or backwards phosphorylation in the presence of ouabain decrease in parallel with the overall reaction (Fig. 1 B), but partial activities of the E_1 conformation like occlusion of 22Na+ or the formation of a Na+-dependent phosphointermediate from [γ-32P]-Cr(NH_3)4ATP (Fig. 1 A) remain unaltered. Furthermore, DCCD did not affect binding of CO(NH_3)4ATP to the low affinity ATP binding site (data not shown). We conclude that DCCD does not modify groups in the ATP binding sites in the E_1 or E_2 conformation. This finding is consistent with a Repke-Stein model of interacting catalytic subunits (Buxbaum et al., 1991). It confirms Gorga's observation (1985), but not that of Shani-Sekler et al. (1988).

TABLE I
Protective Effect of Cations against the Incorporation of [14C]DCCD into the Transmembranal Part of Na+/K+-ATPase

Cations present during
modification with 67 µM
DCCD for 60 min at 37°C

Protective effect
(mol DCCD/mol α-subunit;
± SD of three experiments)

100 mM NaCl

3.47 ± 0.37

100 mM KCl

0.08 ± 0.01

10 mM MgCl_2

8.80 ± 0.68

The transmembranal part was obtained by extensive trypsinolysis for 16 h at 37°C and measurement of the radioactivity in the sediment. The transmembranal part contained 11.2 mol DCCD/mol α-subunit.

Labeling of the 56-kD COOH-terminal part of Na+/K+-ATPase by [14C]DCCD is protected by Na+and K+ (Richter et al., 1987). Since DCCD supposedly interacts with carboxyl groups in a hydrophobic and possibly transmembranal environment, we inactivated Na+/K+-ATPase with 67 µM [14C]DCCD under different ionic conditions and looked for their effects on the labeling of the transmembranal parts. These were obtained after extensive tryptic digestion by centrifugation. This revealed that a high concentration of 11 mol DCCD per mol of α-subunit was found in the membrane part (Table I). Only three carboxyl groups per α-subunit were protected by Na+ in the transmembranal part; K+-ions could not protect at all against the modification of transmembranal carboxyl groups by DCCD. Mg2+ ions were even more effective than Na+.

Figure 2. Interaction of immunoglobulin directed against IFVYDEVRKLIIRRR with closed and open vesicles from pig kidney.

In conclusion, carboxyl groups in a membranal part of the COOH-terminal 56-kD tryptic fragment are accessible for DCCD in the E_2 conformation, but not after Na+- or Mg2+-induced conformational changes.

Study of the Orientation of the COOH-Terminal Part of the α-Subunit by Sequence Antibodies

Cys 964 in the COOH-terminal part of the α-subunit has also been reported to undergo conformational changes (Nagai et al., 1986). It is close to Glu 953 and Glu 954, which like Cys 964 have tentatively been localized to the seventh transmembrane segment (Ovchinnikov et al., 1986) of this subunit. Since the folding of the α-subunit in this region is a matter of debate (Ball and Loftice, 1987), we applied sequence-specific antibodies to closed and SDS-opened rightside-out vesicles (Forbush, 1982) to get more information on the folding. After sedimentation of the vesicles, remaining IgG was determined in the supernatant. As is seen in Fig. 2, antibodies that had been raised against the sequence 991–1,006 and affinity-purified on a Sepharose column containing this sequence reacted with SDS-opened vesicles much better than with closed ones. This means that the sequence 991–1,006 is on the cytoplasmic side of the membrane. It is highly probable, therefore, that Na+/K+-ATPase has eight transmembranal sequences (Ball and Loftice, 1987) and not seven (Ovchinnikov et al., 1986; Bayer, 1990).

Acknowledgments

This work was supported by SFB 249 Giessen Pharmakologie Biologischer Makromoleküle and the Fonds der Chemischen Industrie Frankfurt/M.

References

Ball, J. W., and C. D. Loftice. 1987. Immunochemical studies of (Na++K+)-ATPase using site-specific, synthetic peptide directed antibodies. Biochimica et Biophysica Acta. 916:100–111.

Bayer, R. 1990. Topological disposition of the sequences QRKIVE and KETYY in native (Na++K+)-ATPase. Biochemistry. 29:2251–2256.

Buxbaum, E., E. H. Serpersu, R. Antolovic, E. Hamer, M. Willeke, and W. Schoner. 1991. High and low affinity ATP binding sites as well as Na+ and K+ forms coexist in Na+,K+-ATPase. In The Sodium Pump: Recent Developments. J. H. Kaplan and P. De Weer, editors. The Rockefeller University Press, New York. 405–408.

Forbush, B., III. 1982. Characterization of right-side-out membrane vesicles rich in (Na,K)-ATPase and isolated from dog kidney outer medulla. Journal of Biological Chemistry. 257:12678–12684.

Gorga, F. R. 1985. Inhibition of (Na+,K+)-ATPase by dicyclohexylcarbodiimide: evidence for two carboxyl groups that are essential for enzymatic activity. Biochemistry. 24:6783–6788.

Karlish, S. J. D., A. Rephaeli, and W. D. Stein. 1985. Transmembrane modulation of cation transport by the Na,K-pump. In The Sodium Pump. I. Glynn and C. Ellory, editors. The Company of Biologists, Ltd., Cambridge, UK. 487–499.

Nagai, M., K. Taniguchi, K. Kangawa, H. Matsuo, S. Nakamura, and S. Iida. 1986. Identification of N-[p-(2-benzimidazolyl)phenyl]maleimide-modified residue participating in dynamic fluorescence changes accompanying Na+,K+-dependent ATP hydrolysis. Journal of Biological Chemistry. 261:13197–13202.

Ovchinnikov, Yu. A., N. N. Modyanov, N. E. Broude, K. E. Petrukhin, A. V. Grishin, N. M. Arzamazova, N. A. Aldanova, G. S. Monastyrskaya, and E. D. Sverdlov. 1986. Pig kidney Na+,K+-ATPase: primary structure and spatial organization. FEBS Letters. 201:237–245.

Richter, U., G. Scheiner-Bobis, and W. Schoner. 1987. Carbodiimides inactivate (Na++K+)-ATPase by modifying the catalytic subunit on the 56000 tryptic fragment bearing the ADP subsite. Biological Chemistry Hoppe-Seyler. 386:1267.

Scheiner-Bobis, G., K. Fahlbusch, and W. Schoner. 1987. Demonstration of cooperating α subunits in working (Na++K+)-ATPase by the use of the MgATP complex analogue cobalt tetrammine ATP. European Journal of Biochemistry. 168:123–131.

Schoner, W., and H. Schmidt. 1969. Inhibition of (Na++K+)-activated ATPase by N,N′-dicyclohexylcarbodiimide. FEBS Letters. 5:285–287.

Shani-Sekler, M., R. Goldshleger, D. M. Tal, and S. Karlish. 1988. Inactivation of Rb+ and Na+ occlusion on (Na+,K+)-ATPase by modification of carboxyl groups. Journal of Biological Chemistry. 263:19331–19341.

Vilsen, B., J. P. Andersen, J. Petersen, and P. L. Jøtrgensen. 1987. Occlusion of 22Na+ and 86Rb+ in membrane-bound and soluble protomeric αβ-units of Na,K-ATPase. Journal of Biological Chemistry. 262:10511–10517.

Location of Amino Acid Residues Critical for Ca2+ Transport by Sarcoplasmic Reticulum Ca2+-ATPase in Light of Membrane Protein Folding Proposed for Na+,K+-ATPase

Jesper V. Møller, Marc le Maire, and Philippe Champeil

Institute of Biophysics, University of Aarhus, DK-8000 Aarhus C, Denmark; Centre de Génétique Moléculaire, Laboratoire Propre du Centre National de la Recherche Scientifique, Associé a l'Université Pierre et Marie Curie (Paris VI), 91198 Gif-sur-Yvette Cedex, France; and Service de Biophysique et URA 1290, Centre National de la Recherche Scientifique, Departement de Biologié, Centre d'Etudes Nucléaires de Saclay, 91191 Gif-sur-Yvette Cedex, France

Despite recent progress in the elucidation of the structure of actively transporting ATPases, the mechanism of cation transport remains a major challenge. It is our purpose in this communication to review briefly the status of the cation binding sites of sarcoplasmic reticulum (SR) Ca2+-ATPase and to compare and contrast those results with similar data obtained on Na+,K+-ATPase (le Maire et al., 1990a). Recently we approached this problem by studying the ability of different peptide fragments of Ca2+-ATPase, produced by treatment with staphylococcal V8 or trypsin, to bind Ca2+ (Fig. 1 A). By far the largest Ca2+ binding ability was found to reside in the carboxy-terminal part of the polypeptide chain, corresponding to peptide p31, which is generated by treatment with V8, while there was lesser binding by the NH_2-terminal A_2 tryptic fragment, and the middle portion of the polypeptide chain seemed to have little affinity for Ca2+. Originally, the high affinity Ca2+ binding sites were suggested to be located in the extramembranous stalk region of the enzyme, formed by portions of the NH_2-terminal polypeptide chain (Brandl et al., 1986). Our data suggest a location for Ca2+ binding closer to the COOH-terminal part of the polypeptide chain, which has been suggested to comprise many transmembrane segments (le Maire et al., 1990b). In agreement with a COOH-terminal location of cation transport sites, Karlish et al. (1990) have demonstrated Rb(K) occlusion by a 19-kD COOH-terminal membrane fragment obtained after tryptic digestion of renal Na+,K+-ATPase.

Similar conclusions were reached in MacLennan's laboratory after modification of specific amino acid residues in SR Ca2+-ATPase by oligonucleotide-directed mutagenesis. It was found that single and multiple modifications of acidic amino acid side chains, considered to form part of the stalk region of the enzyme, led only in a few cases to a decrease in Ca2+ transport rates (Clarke et al., 1989b). In contrast, many mutations of single amino acid residues with a potential for Ca2+ binding abolished both Ca2+ transport and Ca2+-dependent phosphorylation with ATP (Clarke et al., 1989a) in supposedly transmembrane segments of the ATPase. These studies provide convincing evidence for the involvement of the COOH-terminal part in high affinity Ca2+ binding and transport, while Ca2+ binding by the ATPase NH_2-terminal part may be nonspecific and caused by the presence of many clustered amino acid residues in this region.

Figure 1. Ca2+ binding regions of Ca2+-ATPase in relation to models proposed for folding of SR Ca2+-ATPase and the homologous Na+,K+-ATPase in the lipid membrane. (A) Ca2+ binding of isolated tryptic (A_1, A_2, B) and V8((p48, p31) fragments as evaluated from both the Ca2+-induced changes in electrophoretic mobility and the Ca2+ overlay technique after transfer to Immobilon membranes (le Maire et al., 1990b). The relative extent of Ca2+ binding by the fragments is indicated by means of a graduated scale from 0 to + + +. Ca2+ binding by the NH_2-terminal V8 fragment is missing, since it could not be isolated by SDS-PAGE, presumably because of extensive fragmentation. The relatively strong binding of Ca2+ by A_2 is in accord with previous data (Lüdi and Hasselbach, 1984). (B) A proposed model for the folding of SR Ca2+-ATPase in relation to the lipid membrane (Clarke et al., 1989a), assuming 10 intramembranous helices (M1–M10) and a helical stalk region (S1–S5) formed from the continuation of the first five intramembranous helices (M1–M5). Filled circles indicate the position on this model of residues found to be critical for Ca2+ transport by oligonucleotide-directed mutagenesis. These are Glu_309 (M4), Glu_771 (M5), Asn_796, Thr_799, Asp_800 (all on M6), and Glu_908 (M8). The open bars indicate the location of soluble peptides isolated during sequencing of the proteins (Allen et al., 1980; Green and Toms, 1985). The main tryptic and V8 peptide bond cleavage positions are also indicated. Note that according to this model all the reported critical residues are located within the membrane and, apart from Glu_309, are contained within the COOH-terminal p31 fragment. (C) Proposed model for the folding of the Na+,K+-ATPase α-subunit (Ovchinnikov, 1987), assuming seven intramembranous helices. Filled circles indicate the position on this model of residues homologous to those found to be critical for Ca2+ transport in the SR Ca2+-ATPase, namely, Glu_327, Glu_779, Asp_804, Thr_807, Asp_808, and Val_920 according to the alignment by Clarke et al. (1989a). Note that, apart from Glu_327, all these residues are located on the outer or inner side of the membrane in the model. The open bars indicate the location of soluble peptides, isolated after extensive trypsinolysis of membranous preparations, within the Na+,K+-ATPase sequence. Hatched bars indicate epitopes of monoclonal antibodies (Ovchinnikov, 1987) reacting with membranous Na+,K+-ATPase. The foldings proposed by Serrano (1988) for Ca2+-ATPase and Na+,K+-ATPase are similar, but contain one additional membranous helix (M8) at amino acid residues 914–934 and 909–930, respectively. The numbering of helices corresponds to that for homologous sequences in Ca2+-ATPase (B).

Nevertheless, some of the detailed conclusions drawn by Clarke et al. (1989a) are debatable. Based on a previous prediction of 10 intramembranous helices in the SR Ca2+-ATPase polypeptide chain (Brandl et al., 1986), the amino acid residues that had been found critical for Ca2+ transport were suggested to be inside the membrane (Fig. 1 B). A remarkable feature of the location of these critical amino acid residues in this model is that they all appear to be at about the same level within the membrane and thus could be clustered, since the predicted 10 α-helices are likely to be close to one another (see Fig. 1 of the review by Green [1989] on these findings). However, other predictions of the structure of Ca2+-ATPase and homologous transport ATPases have resulted in consensus models where 2 of the 10 proposed transmembrane helices (M6 and M10) are deleted (Serrano, 1988). For Na+,K+-ATPase a model of seven helices, based on both theoretical analysis and experimental data of surface exposure, has been proposed (Ovchinnikov, 1987). In this model M6, M8, and M10 are not considered to be transmembrane segments (see Fig. 1 C). For the double helical traverse M5–M6 the dilemma is the presence of a long stretch of predominantly hydrophobic residues, which is nevertheless too short to be readily accommodated within two intramembranous helices. For Na+,K+-ATPase a single transmembrane stretch in this region was therefore proposed, from amino acid residue 780 to 803. In addition, for this ATPase there is experimental evidence against the existence of M6, since a monoclonal antibody binds to an epitope, corresponding to the end of the putative M6 helix (Ovchinnikov, 1987). In a similar way the presence of M8 was questioned on the basis that the sequence is part of water-soluble peptide fragments that are released from Na+,K+-ATPase membranes by treatment with trypsin.

Fig. 1 C shows that amino acid residues in Na+,K+-ATPase, homologous to amino acid residues critical for Ca2+ transport in Ca2+-ATPase, apart from Glu_309 in the M4 helix, are located on both sides of the membrane, rather than being clustered in the middle of the membrane. The same conclusion is reached using the eight helical model (deletion of M6 and M8) proposed by Serrano (1988). In this connection it is of interest to note that both the hydropathy profile (Fig. 2) and pattern of release of water-soluble peptide fragments (Fig. 1 B) of SR Ca2+-ATPase are virtually identical to those of Na+,K+-ATPase in the COOH-terminal half. It thus appears unlikely that the structures of the two transport ATPases are as different as is suggested by Fig. 1, B and C. But if the arrangements are similar, and if immunoreactivity and proteolytic cleavage only take place in parts of the polypeptide chain that are extramembranous in the native structure, then the possibility must be considered that at least three (Asn_796, Thr_799, and Asp_800) of the six proposed Ca2+ binding groups are located in the vesicular space, while two are located on the cytoplasmic side of the membrane (see Fig. 1 C). In agreement with this view, the most hydrophobic 17 residue segment for Ca2+-ATPase in the M5–M6 region runs from residue 772 to 788. Furthermore, both M8 and the major fraction of M6 are part of soluble peptides obtained by cleavage at tryptophan residues (Green and Toms, 1985).

Figure 2. Similarity of hydropathy profile of the COOH-terminal half of slow twitch muscle SR Ca2+-ATPase and Na+,K+-ATPase α-subunit. Numbers and bars indicate the regions that represent transmembrane domains according to Fig. 1, B and C. Taken with permission from Fig. 8 of Shull and Greeb (1988).

How is this analysis and comparison between the two ATPases to be understood? Three different possibilities present themselves, with implications for either the mechanism of Ca2+ transport or membrane protein structure. The first possibility is to take the location of the designated Ca2+ binding groups from the Fig. 1 C model at face value by proposing two different binding loci for the two translocated Ca2+, one on the outside, the other on the inside of the membrane. If so, these might act as entrance and exit points for a Ca2+ channel running through the protein. The second possibility is that while the model for membrane folding, depicted by Fig. 1 C, may be essentially correct, Ca2+ binding sites do have an intramembranous location, involving other groups than previously reported. This would probably imply that some of those groups found critical for Ca2+ transport have an indirect effect via changes in protein conformation or because they are involved in energy transduction. The third possibility is that the membrane protein structure of Ca2+-ATPase and Na+,K+-ATPase do differ. A particular problem when applying the seven helical model to Ca2+-ATPase (Fig. 1 C) is that it implies a different orientation of the NH_2- and COOH-terminal ends. This is not in harmony with a cytoplasmic orientation of both ends of the polypeptide chain, as evidenced by immunoreactivity with antibodies raised against the NH_2-terminal and COOH-terminal peptides (Matthews et al., 1989), but it would conform with, for example, an eight helical model for Ca2+-ATPase. In relation to the location of the putative Ca2+ binding groups, the most important issue concerns the existence of a single or a double membrane traverse in the M5–M6 region. This part of the polypeptide chain is characterized by a broad hydrophobic stretch, but the hydrophobicity of the proposed second traverse (M6) is less pronounced than what is considered typical for a membrane helix. In addition, as mentioned above, susceptibility to proteolytic attack not far from the putative Ca2+ binding groups has been demonstrated in the region corresponding to M6 (Fig. 1 B) which does not fit with its intramembranous location. However, immunological evidence for a luminal exposure of the polypeptide loop between M7 and M8 has been obtained, consistent with the existence of the proposed M6 membrane traverse. In contrast, for H+,K+-ATPase there is evidence, after modification of amino acid residues with membrane impermeable reagents, of an extracellular location of the polypeptide chain between M5 and M7 (Sachs et al., 1989), consistent with the Na+,K+-ATPase folding (Fig. 1 C). It may be, therefore, that Na+,K+-ATPase and H+,K+-ATPase, the two cation pumps that share the highest degree of homology, differ significantly from the structure of Ca2+-transporting ATPases in the COOH-terminal end, perhaps as the result of interaction with their associated β-subunit and other subtle differences. Nonetheless the similarity of the hydropathy plots of Ca2+-ATPase and Na+,K+-ATPase (Fig. 2) and the similar susceptibility to proteolytic attack and water solubility of tryptophan-cleaved fragments are features that favor a common model.

In conclusion, a comparative approach based on more experimental data may illuminate aspects of cation pump mechanisms.

References

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Brandl, C. J., N. M. Green, B. Korczak, and D. H. MacLennan. 1986. Two Ca2+-ATPase genes: homologies and mechanistic implications of deduced amino acid sequences. Cell. 44:597–607.

Clarke, D. M., T. W. Loo, G. Inesi, and D. H. MacLennan. 1989a. Location of high affinity Ca2+-binding sites within the predicted transmembrane domain of the sarcoplasmic reticulum Ca2+-ATPase. Nature. 339:476-478.

Clarke, D. M., K. Maruyama, T. W. Loo, E. Leberer, G. Inesi, and D. H. MacLennan. 1989b. Functional consequences of glutamate, aspartate, glutamine, and asparagine mutations in the stalk sector of Ca2+-ATPase of sarcoplasmic reticulum. Journal of Biological Chemistry. 264:11246–11251.

Green, N. M. 1989. Ions, gates and channels. Nature. 339:424–425.

Green, N. M., and E. J. Toms. 1985. The sequence of two peptides isolated from the Ca2+-transporting ATPase of rabbit sarcoplasmic reticulum after cleavage at tryptophan. Biochemical Journal. 231:425–429.

Karlish, S. J. D., R. Goldshleger, D. M. Tal, and W. D. Stein. 1991. Structure of the cation binding sites of Na,K-ATPase. In The Sodium Pump: Structure, Mechanism, and Regulation. J. H. Kaplan and P. De Weer, editors. The Rockefeller University Press, New York. 129–141.

le Maire, M., P. Champeil, and J. V. Møller. 1990a. Twists and turns. Nature. 346:617.

le Maire, M., S. Lund, A. Viel, P. Champeil, and J. V. Møller. 1990b. Ca2+-induced conformational changes and location of Ca2+ transport sites in sarcoplasmic reticulum Ca2+-ATPase as detected by the use of proteolytic enzyme (V8). Journal of Biological Chemistry. 265:1111–1123.

Lüdi, H., and W. Hasselbach. 1984. Separation of tryptic fragments of sarcoplasmic reticulum ATPase with high performance liquid chromatography: identification of the calcium binding site. FEBS Letters. 167:33–36.

Matthews, I., J. Colyer, A. M. Mata, N. M. Green, P. P. Sharma, A. G. Lee, and J. M. East. 1989. Evidence for the cytoplasmic location of the N- and C-terminal segments of sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPase. Biochemical and Biophysical Research Communications. 161:683–688.

Ovchinnikov, Y. 1987. Probing the folding of membrane proteins. Trends in Biochemical Sciences. 12:434–438.

Sachs, G., K. Munson, V. N. Balagi, D. Aures-Fischer, S. J. Hersey, and K. Hall. 1989. Functional domains of the gastric HK-ATPase. Journal of Bioenergetics and Biomembranes. 21:573–588.

Serrano, R. 1988. Structure and function of proton translocating ATPase in plasma membranes of plants and fungi. Biochimica et Biophysica Acta. 947:1–28.

Shull, G. E., and J. Greeb. 1988. Molecular cloning of two isoforms of the plasma membrane Ca2+ transporting ATPase from rat brain. Journal of Biological Chemistry. 263:8646–8657.

Structure–Function Studies of Na/K-ATPase Isozymes

Bruce C. Kone, Kunio Takeyasu, and Douglas M. Fambrough

Departments of Medicine and Biology, The Johns Hopkins University, Baltimore, Maryland 21218; and Department of Physiology, The University of Virginia School of Medicine, Charlottesville, Virginia 22908

Recent molecular–genetic studies have established that the Na,K-ATPase comprises a multigene family. To date, encoding DNAs for three isoforms of the α-subunit (Shull et al., 1986; Herrera et al., 1987; Takeyasu et al., 1990), and two isoforms of the β-subunit (Martin-Vassallo et al., 1989; Gloor et al., 1990; Lemas et al., 1991) have been cloned and sequenced in mammals and birds. Although the α-subunit isoforms display significant homology in their amino acid sequences, each isoform can be distinguished by unique clusters of amino acids that have been strikingly conserved during evolution (Takeyasu et al., 1990). It has been hypothesized that these structural refinements might endow the different isoforms with specialized functions, offering some biological advantage to the organism. Such functional differences might include subtle differences in ion transport capacity, responses to regulatory factors, or inhibition by cardiac glycosides.

Unfortunately, analysis of the function and the various α-subunit isoforms and of the possible α-β cognate pairs has proven quite difficult. Studies using tissue preparations suffer from the multiplicity of cell types and the possibility of different isoforms being present in the sample. Ideally, each isoform and each α-β pair would be analyzed under comparable conditions; that is, when expressed in a common host cell. In earlier studies we adopted this strategy, using gene transfer to express the avian α1-subunit (Takeyasu et al., 1988) and β1-subunit (Takeyasu et al., 1987) in tissue cultured mouse L cells (which express a ouabain-resistant α1 isoform). This system allows expressed avian sodium pumps to be identified both by binding of avian-specific monoclonal antibodies to the α- and β1-subunits and by the appearance of high-affinity ouabain binding sites and more ouabain-sensitive ion transport. Given the utility of this approach, we sought to establish mouse L cell lines expressing each of the avian α-subunit isoforms together with the avian β1-subunit in order to compare the ouabain binding affinities, ion transport properties, and subunit assembly of these isozymes.

Encoding DNAs for the avian α1-, α2-, and α3-subunit isoforms were cloned into the shuttle vector pSVDF4, and the resulting constructs were linearized with Pvu I or Aat II. These DNAs were mixed with the resistance plasmid pSV_2neo in a calcium precipitate to cotransfect mouse L cells stably expressing the chicken β1-subunit (the result of an earlier transfection; see Takeyasu et al., 1987). G418-resistant clones expressing avian sodium pumps were then identified by (a) production of a ~ 100-kD protein detected with a monoclonal antibody to the avian α-subunit (mAb α5), (b) expression of high-affinity [3H]ouabain binding sites at the cell surface, and (c) increased sensitivity of ion transport to inhibition by ouabain. Cell lines fulfilling these criteria were selected for further study. For all studies, cells were treated with 10 mM sodium butyrate for 48 h to induce expression of avian sodium pumps under control of the SV40 early promoter.

Fig. 1 presents a comparison of the binding of [3H]ouabain to cell lines expressing the avain β1-subunit alone (Ltkβ) or together with each of the avian α-subunit isoforms. The Ltkβ cells showed virtually no specific binding of [3H]ouabain, whereas the cell lines also expressing the avian α1, α2, or α3 isoforms demonstrated high-affinity ouabain binding sites. Scatchard analysis of these data revealed comparable Kd's of 200–600 nM for ouabain binding to the three avian α-subunit isoforms. These results substantiate our earlier observation that ouabain binding is an exclusive feature of the α-subunit (Takeyasu et al., 1988). Moreover, the observed similarity in ouabain binding affinities of the three avian α-subunit isoforms contrasts dramatically with the large differences in ouabain binding affinity displayed by the homologous set of rat α isoforms; this presumably reflects a greater degree of structural relatedness of the ouabain binding sites of the avian isozymes.

Figure 1. High affinity [3H]ouabain binding sites on mouse L cells expressing the avian β1-subunit alone (Ltkβ) or coexpressing avian α-subunit isoforms with the avian β1-subunit. Specific [3H]ouabain was measured as a function of [3H]ouabain concentration for 1 h at room temperature as described in Takeyasu et al. (1988). Values were normalized to cell number.

To prove that the expressed avian sodium pumps were functionally competent, we compared the ouabain-sensitive oxygen consumption (QO_2) of Ltkβ cells with that of the avian α-β-expressing cells. By providing only oxidative substrates (4 mM glutamine and 10 mM sodium butyrate) to promote tight coupling of mitochondrial ATP production to sodium pump–mediated ATP utilization, ouabain-sensitive QO_2 served as a convenient index of sodium pump activity (Harris et al., 1982). As seen in Fig. 2, ~ 10-fold less ouabain was required to inhibit the QO_2 of the α-β-expressing cells compared with the Ltkβ cells. Just as in the [3H]ouabain binding experiments, the ouabain concentration–response curves for the three avian α isoforms were virtually identical. These data probably underestimate the true difference in ouabain inhibition of ion transport between the avian and mouse enzymes because inhibition of the expressed avian sodium pumps by low concentrations of ouabain probably increases intracellular Na+ concentration and thereby stimulates QO_2 mediated by the endogenous mouse enzyme. Nonetheless, these results indicate that the expressed avian sodium pumps are functional.

Figure 2. Dose–response curves demonstrating increased sensitivity of avian α isoform–expressing cells compared with the parent Ltkβ cells. Cells were incubated in a calcium- and magnesium-free buffer and then scraped into oxygenated Ringer's solution. The Qo_2 of the resulting cell suspension was measured in a thermoregulated glass chamber as detailed in Kone et al. (1989). Each data point represents the mean ± SEM (n = 3).

Given the mounting evidence that interactions between nascent α- and β-subunits might regulate sodium pump abundance at the cell surface, and the possibility that different pairings of α and β isoforms might give rise to functionally different holoenzymes, we sought to determine whether each avian α-subunit isoform could stably assemble with the avian β1-subunit. As an initial approach to this question, we designed a sequential immunoprecipitation protocol that identifies the subunit components of sodium pump α-β complexes. We first tested this method on primary cultures of chick skeletal muscle. The cells were metabolically labeled with [35S]methionine, extracted with detergent, and incubated with immunobeads bearing anti–chicken β mAb 24. As seen in Fig. 3 A, mAb 24 precipitates both α and β (i.e., assembled) subunits of chicken sodium pumps. By incubating the mAb 24 precipitate first with SDS sample buffer (to dissociate the subunits) and then with anti–chicken α5 immunobeads, it was possible to identify the avian α-subunit. For the mouse cells coexpressing the avian α isoforms and avian β1 subunit, then, this approach would allow us to distinguish assembled avian sodium pumps from interspecies hybrid molecules (composed of the endogenous mouse α-subunit and the exogenous chicken β-subunit). In each case, the avian α-subunit was immunopurified from the assembled α-β complex (Fig. 3 B). Combined with the above physiological studies, these data suggest that each a isoform can assemble with the β1-subunit to form a functional holoenzyme.

Figure 3. Assembly of avian α-subunit isoforms with the avian β1-subunit coexpressed in mouse L cells. Fluorographs of immunoprecipitates analyzed by SDS-PAGE: (A) Left lane, α- and β-subunits precipitated from cultures of chick skeletal muscle with anti–chicken β-subunit mAb 24. Right lane, Reprecipitation of the mAb 24 precipitate with anti–α-subunit mAb α5. (B) mAb α5 reprecipitation of the mAb 24 precipitate for cell lines expressing the avian β1-subunit alone (left lane) or coexpressing avian α-subunit isoforms with the avian β1-subunit.

In summary, we have demonstrated stable expression of each of the major chicken α isoforms together with the β1-subunit in mouse L cells. The expressed avian sodium pumps exhibit comparable affinities for ouabain binding and sensitivities to ouabain inhibition of ion transport. In addition, each of the α isoforms appears to complex stably with the β1-subunit. These cell lines should prove useful for more detailed analyses of the function and bioregulation of sodium pump isozymes.

Acknowledgments

This work was supported by National Institutes of Health grants R01 NS-23241 (to D. M. Fambrough) and K08 DK-01885 (to B. C. Kone).

References

Gloor, S., H. Antonicek, K. J. Sweadner, S. Pagliusi, R. Frank, M. Moos, and M. Schachner. 1990. The adhesion molecule on glia (AMOG) is a homologue of the β-subunit of the Na,K-ATPase. Journal of Cell Biology. 110:165–174.

Harris, S. I., L. Patton, L. Barrett, and L. J. Mandel. 1982. (Na+,K+)-ATPase kinetics within the intact renal cell. Journal of Biological Chemistry. 257:6996–7002.

Herrera, V. L., J. R. Emanuel, N. Ruiz-Opazo, R. Levenson, and B. Nadal-Ginard. 1987. Three differentially expressed Na,K-ATPase α subunit isoforms. Structural and functional implications. Journal of Cell Biology. 105:1855–1865.

Kone, B. C., D. Kikeri, M. L. Zeidel, and S. R. Gullans. 1989. Cellular pathways of potassium transport in the renal inner medullary collecting duct. American Journal of Physiology. 256 (Cell Physiology 256):C823–C830.

Lemas, V., J. Rome, J. Taormino, K. Takeyasu, and D. M. Fambrough. 1991. Analysis of isoform-specific regions within the α- and β-subunits of the avian Na,K-ATPase. In The Sodium Pump: Recent Developments. J. H. Kaplan and P. De Weer, editors. The Rockefeller University Press, New York. 117–123.

Martin-Vassallo, P., W. Dackowski, J. R. Emanuel, and R. Levenson. 1989. Identification of a putative isoform of the Na,K-ATPase beta subunit: primary structure and tissue specific expression. Journal of Biological Chemistry. 264:4613–4618.

Shull, G. E., J. Greeb, and J. B. Lingrel. 1986. Molecular cloning of three distinct forms of the Na+,K+-ATPase α-subunit from rat brain. Biochemistry. 25:8125–8132.

Takeyasu, K., M. V. Lemas, and D. M. Fambrough. 1990. Stability of the (Na+ + K+)-ATPase α-subunit isoforms in evolution. American Journal of Physiology. 259 (Cell Physiology 28): C619–C630.

Takeyasu, K., M. M. Tamkun, K. J. Renaud, and D. M. Fambrough. 1988. Ouabain-sensitive (Na+ + K+)-ATPase activity expressed in mouse L cells by transfection with DNA encoding the α-subunit of an avian sodium pump. Journal of Biological Chemistry. 263:4347–4354.

Takeyasu, K., M. M. Tamkun, N. R. Siegel, and D. M. Fambrough. 1987. Expression of hybrid (Na+ + K+)-ATPase molecules after transfection of mouse Ltk− cells with DNA encoding the β-subunit of an avian brain sodium pump. Journal of Biological Chemistry. 262:10733–10740.

Posttranslational Processing and Basic Properties of a Putative β3-Subunit of Na,K-ATPase

Philippe Jaunin, Peter Good, Irène Corthesy, and Käthi Geering

Institut de Pharmacologie et de Toxicologie de l'Université de Lausanne, CH-1005 Lausanne, Switzerland; Laboratory of Molecular Genetics, National Institutes of Health, Bethesda, Maryland 20892; and Department of Biochemistry, Stanford University School of Medicine, Stanford, California 94305-5307

Introduction

The catalytic α-subunit of Na,K-ATPase bears all known functional domains of the enzyme, while the functional role of the β-subunit is still ill-defined. Recently, different β-isoforms have been identified in rat (Martin-Vasallo et al., 1989) and mouse (Gloor et al., 1990), which raises the interesting possibility that each isoform might have a distinct function. If this is the case, it remains to be elucidated whether the functional diversity is related to differences in regulatory properties of β-isoforms as elements of the active enzyme, and also whether the ability to participate in cell adhesion described for a mouse β2-subunit (Gloor et al., 1990) is a common feature of all β-isoforms.

Apart from these unresolved questions, it has become increasingly clear that a concomitant synthesis of α- and β-subunits is a prerequisite for the expression of functional pumps at the plasma membrane (for review see Geering, 1990, 1991).

In Xenopus oocytes, which synthesize an excess of structurally immature α-subunits over β-subunits, we could indeed show that Xenopus β1-subunits synthesized from injected cRNA are able to impose a structural change on the immature oocyte α-subunit population. This change is reflected in an increased trypsin resistance of the polypeptides as well as in an increased number of ouabain binding sites at the plasma membrane (Geering et al., 1989). In addition, concomitant synthesis of β1-subunits increases the half-life of the α1-subunits synthesized from injected cRNA, and finally, α- and β-subunits depend on each other for their intracellular transport to the plasma membrane (Ackermann and Geering, 1990).

In this study we have used these functional criteria, previously established for Xenopus β1-subunits, to characterize a putative β3-isoform recently identified in cDNA libraries of Xenopus neurula stages (Good et al., 1990). This polypeptide shows about 43% identity to Xenopus β1-subunits and its expression in adult Xenopus appears to be limited to the brain. To determine whether this putative β3-isoform can assemble with α-subunits, we injected α1 and/or β3 cRNA into Xenopus oocytes and followed the posttranslational processing of the newly synthesized β3-subunit as a glycoprotein, as well as the cellular accumulation and the acquisition of trypsin resistance of concomitantly synthesized α-subunit.

Results and Discussion

Fig. 1 shows the glycosylation processing of both the Xenopus β1-isoform and the putative β3-isoform synthesized in Xenopus oocytes injected with β cRNA alone or with α cRNA. After a 4-h pulse with [35S]methionine, oocytes injected with α1 and β1 (Fig. 1, lane 2) or with α1 and β3 cRNA (Fig. 1, lane 7) express a 40- or 38-kD polypeptide, respectively, which can be revealed with an anti-β1 serum. Both these polypeptides are core glycosylated since they are sensitive to endoglycosidase H (endo H) treatment (Fig. 1, lanes 3 and 8). Partial digestions with endo H reveal that three and four core sugars can be removed in β1- and β3-subunits, respectively. Interestingly, examination of the amino acid sequence of the β3-subunit reveals only three accepted consensus sequences for N-linked glycosylation (NRT at aa 147, NCT at aa 190, and NFT at aa 240). Apparently, an additional unusual asparagine residue (NDS at aa 117) is glycosylated in the β3-isoform.

Figure 1. Glycosylation processing of β1- and β3-subunits in the oocyte. Oocytes were injected with 5 ng of β1 cRNA (lane 1) or β3-cRNA (lane 6), or with 5 ng each of α1 and β1 cRNA (lanes 2–5) or α1 and β3 cRNA (lanes 7–10). cRNA was transcribed in vitro as previously described (Geering et al., 1989) from Xenopus α1 and β1 cDNA (Verrey et al., 1989) or β3 cDNA (Good et al., 1990). Oocytes were labeled for 4 h (lanes 2, 3, 7, and 8) or 48 h (lanes 1, 4–6, 9, and 10) with 1–3 mCi/ml [35S]methionine in MBS medium and extracted with Triton X-100 (Coleman, 1984). Extracts were immunoprecipitated with anti-β1 serum, treated (lanes 3, 5, 8, and 10) or not (lanes 1, 2, 4, 6, 7, and 9) with endo H (Zamofing et al., 1988), and subjected to SDS-PAGE and fluorography, as described (Geering et al., 1985). Dots in lanes 3 and 8 indicate the three and four core sugars in β1- and β3-subunits, respectively, which can be revealed by endo H treatment.

After a 48-h pulse, both β1- (Fig. 1, lane 4) and β3-subunits (Fig. 1, lane 9) are processed into 49- and 59-kD fully glycosylated forms, respectively, which are endo H resistant (Fig. 1, lanes 5 and 10). As previously shown for β1-subunits (Ackermann and Geering, 1990), full glycosylation and thus transport of β3-subunits to a trans-Golgi compartment only occur if they are coexpressed with α-subunits. β3- subunits (Fig. 1, lane 6), like β1-subunits (Fig. 1, lane 1), remain core glycosylated if they are synthesized alone even after a 48-h pulse.

To further strengthen the observation that β3-subunits can associate with α1-subunits, we tested the ability of β3-subunits to influence the membrane organization and thus the stability of newly synthesized α-subunits. Recently we have shown that α1-subunits synthesized alone in the oocyte are rapidly degraded with a half-life of ~ 2 h (Ackermann and Geering, 1990). Association with β1-subunits considerably increases the half-life of α1-subunits and thus leads to their increased cellular accumulation (Fig. 2; cf. lanes 1 and 2). Coinjection of α1- and β3 cRNA (Fig. 2, lane 3) leads to a similar elevation of the cellular expression of the α1-subunit than coinjection of α1 and β1 cRNA, indicating that β3- as β1-subunits are able to associate and impose a stable membrane organization to α1-subunits.

Figure 2. Cellular accumulation of α1-subunits synthesized in the presence or absence of β1- or β3-subunits. Oocytes were injected with α1 cRNA (5 ng; lane 1) or α1 (5 ng) and β1 (10 ng) cRNA (lane 2), or α1 (5 ng) and β3 (10 ng) cRNA (lane 3) and labeled for 4 h with [35S]methionine. Aliquots of oocyte extracts were prepared and immunoprecipitated with anti-α serum and processed as described in Fig. 1. Quantification of immunoprecipitated α-subunits revealed on fluorograms (mean ± SE, n = 3) is shown. The amount of α-subunits found in αcRNA injected oocytes was arbitrarily set to 1.

This conclusion is supported by the fact that β3-subunits also increase the trypsin resistance of α-subunits, a phenomenon characterizing the structural maturation of newly synthesized α-subunits (Geering et al., 1987). Fig. 3 shows that, as previously described (Geering et al., 1989), a large proportion of oocyte α-subunits (Fig. 3, lanes 1 and 2) as well as α1-subunits synthesized from injected α1 cRNA (Fig. 3, lanes 3 and 4) are highly trypsin sensitive. β3-subunits synthesized from injected β3 cRNA render oocyte α-subunit (Fig. 3, lanes 9 and 10) as well as exogenous α1-subunit (Fig. 3, lanes 11 and 12) trypsin resistant to an extent similar to β1-subunits (Fig. 3, lanes 5–8).

In conclusion, our data indicate that the putative Xenopus β3-isoforms can associate with α1-subunits of Na,K-ATPase and induce a conformational change in the enzyme, which probably permits its exit from the ER. The enzyme then reaches a trans-Golgi compartment where β3-subunits acquire complex-type sugars. Thus, β3-subunits have basic properties similar to β1-subunits and it is likely that the β3-polypeptide is a β-isoform of Na,K-ATPase.

Figure 3. Controlled trypsinolysis of α-subunits synthesized in the presence or absence of β1- or β3-subunits. Oocytes were injected with H_2O or 5 ng of α1, β1, α1 and β1, or β3, α1, and β3 cRNA as indicated and labeled for 24 h with [35S]methionine. Oocytes were homogenized and aliquots were treated (lanes 2, 4, 6, 8, 10, and 12) or not (lanes 1, 3, 5, 7, 9, and 11) with trypsin as described (Geering et al., 1989). Quantification of immunoprecipitated α-subunits revealed on fluorograms (mean, n = 2) is shown. The amount of α-subunits found in H_2O-injected oocytes was arbitrarily set to 1. The numbers over the bars represent the percentage change in the amount of α-subunits in trypsin-treated samples compared with nontreated controls for each injection protocol.

Acknowledgments

This work was supported by grant 31-26241.89 from the Swiss National Fund for Scientific Research.

References

Ackermann, U., and K. Geering. 1990. Mutual dependence of Na,K-ATPase α- and β-subunits for correct posttranslational processing and intracellular transport. FEBS Letters. 269:105–108.

Colman, A. 1984. Translation of eukaryotic messenger RNA in Xenopus oocytes. In Transcription and Translation: A Practical Approach. B. D. Hames and S. J. Higgins, editors. IRL Press, Oxford, Washington. 271–302.

Geering, K. 1990. Subunit assembly and functional maturation of Na,K-ATPase. Journal of Membrane Biology. 115:109–121.

Geering, K. 1991. Posttranslational modifications and intracellular transport of sodium pumps: importance of subunit assembly. In The Sodium Pump: Structure, Mechanism, and Regulation. J. H. Kaplan and P. De Weer, editors. The Rockefeller University Press, New York. 31–43.

Geering, K., J. P. Kraehenbuhl, and B. C. Rossier. 1987. Maturation of the catalytic α-subunit of Na,K-ATPase during intracellular transport. Journal of Cell Biology. 105:2613–2619.

Geering, K., D. I. Meyer, M. P. Paccolat, J. P. Kraehenbühl, and B. C. Rossier. 1985. Membrane insertion of α- and β-subunits of Na+,K+-ATPase. Journal of Biological Chemistry. 260:5154–5160.

Geering, K., I. Theulaz, F. Verrey, M. T. Häuptle, and B. C. Rossier. 1989. A role for the β-subunit in the expression of functional Na+-K+-ATPase in Xenopus oocytes. American Journal of Physiology. 257:C851–C858.

Gloor, S., H. Antonicek, K. J. Sweadner, S. Pagliusi, R. Frank, M. Moos, and M. Schachner. 1990. The adhesion molecule on glia (AMOG) is a homologue of the β subunit of the Na,K-ATPase. Journal of Cell Biology. 110:165–174.

Good, P. J., K. Richter, and I. B. Dawid. 1990. A nervous system-specific isotype of the β subunit of Na+,K+-ATPase expressed during early development of Xenopus laevis. Proceedings of the National Academy of Sciences, USA. 87:9088–9092.

Martin-Vasallo, P., W. Dackowski, J. R. Emanuel, and R. Levenson. 1989. Identification of a putative isoform of the Na,K-ATPase β subunit: primary structure and tissue-specific expression. Journal of Biological Chemistry. 264:4613–4618.

Verrey, F., P. Kairouz, E. Schaerer, P. Fuentes, K. Geering, B. C. Rossier, and J. P. Kraehenbuhl. 1989. Primary sequence of Xenopus laevis Na+-K+-ATPase and its localization in A6 kidney cells. American Journal of Physiology. 256:F1034–F1043.

Zamofing, D., B. C. Rossier, and K. Geering. 1988. Role of the Na,K-ATPase β-subunit in the cellular accumulation and maturation of the enzyme as assessed by glycosylation inhibitors. Journal of Membrane Biology. 104:69–79.

Immunodetection and Characterization of the β-Subunit of Na+,K+-ATPase in Mammalian Liver Tissues

Ying Sun and William J. Ball, Jr.

Department of Pharmacology and Cell Biophysics, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-0575

Introduction

The Na+,K+-ATPase (NKA) is an active ion pump which maintains the cellular electrochemical gradient and is responsible for several physiological cell functions, such as the regulation of cell volume, pH, solute uptake in the kidney, liver, and intestines, and electrical responses in nerve and muscle. The enzyme is generally assumed to consist of a catalytic (α) and a glycoprotein (β) subunit. The α-subunit has a molecular weight of ~ 112,000 and contains the cation, ATP, Pi, and ouabain binding sites, while no specific function for β (~ 55,000 mol wt) has yet been established. However, recent studies have provided evidence that the synthesis of α- and β-subunits are separate transcriptional and translational events, but that the β-subunit regulates, through αβ assembly, the number of pumps transported to the plasma membrane (McDonough et al., 1990). In addition, three isoforms of α have been identified and characterized and recently an additional putative β isoform (termed β2 or AMOG) has been sequenced from rat brain (Shyjan et al., 1990) and immunopurified from mouse brain (Gloor et al., 1990). While the β1-subunit of NKA has been identified in most tissues, i.e., brain, colon, heart, and kidney, no clear evidence has shown that a β-subunit exists in either red blood cells (Blostein and Grafova, 1988) or liver (Hubert et al., 1986). Because NKA activity in the liver is so low, the detection of the β protein has remained obscure, even though rat α has been precipitated by a monoclonal antibody (Hubert et al., 1986). Similarly, β mRNA has been nearly undetectable in normal adult rat liver using β1 specific cDNA probes (Young and Lingrel, 1987; Martin-Vasallo et al., 1989). However, β mRNA was found in the liver of hypothyroid and triodothyronine (T_3)-treated rats after exposure of Northern blots to autoradiography for extended times (Gick et al., 1988). Thus, previous data have suggested that the level of β in normal liver is very low or absent. In this study we have used a variety of polyclonal and monoclonal antibodies to determine whether mammalian liver NKA actually lacks β or has a different β isoform.

Materials and Methods

Preparation of Liver NKA Microsomes

The liver microsomes were prepared from Sprague-Dawley male rats (300–350 g) and adult female lambs. The liver tissue was homogenized in buffer A (0.25 M sucrose, 1 mM EDTA, 25 mM imidazole, and PMSF, pH 7.2) after its perfusion with buffer A (without PMSF). The homogenate was first centrifuged for 30 min at 8,000 g, and the recovered supernatant was then centrifuged for 45 min at 48,000 g at 4°C. The pellet was resuspended in buffer A (minus PMSF) and layered onto a discontinuous sucrose gradient (consisting of 44, 42.3, and 10% sucrose [wt/wt] at 1.1:1:0.4 volume ratios), and then centrifuged for 2 h at 90,000 g. The microsomes banding at the 10–42.3% sucrose interface were removed and diluted ninefold with a Tris-buffered 1 mM EDTA solution and subsequently centrifuged for 60 min at 48,000 g to remove the sucrose. The pelleted material was then resuspended in the buffered EDTA. Protein concentrations were determined by the method of Lowry with bovine serum albumin as standard.

Antibody Production and Western Blot Analysis

The monoclonal antibodies were generated and purified using the procedure described by Ball et al. (1982). The polyclonal antibodies were raised in New Zealand rabbits according to Ball and Schwartz (1982). Western blots were made by the electrophoretic transfer of slab-gel (7.5% Laemmli gel) resolved lamb or rat liver microsomes (100 µg) and purified kidney NKA (1 µg) onto nitrocellulose sheets and the immunodetection of antigens by antibodies as described by Towbin et al. (1979).

Enzyme Activity Assays

The NKA activity was determined by using a coupled-enzyme spectrophotometric assay (Schwartz et al., 1969). The ATPase reaction was initiated by the addition of varying concentrations of ATP, Na+, or K+ as indicated. Before initiating the activity assays, the liver microsomes were treated with SDS (0.6 mg/ml SDS to 3 mg/ml protein) in a buffer containing 3 mM ATP, 1 mM EDTA, and 30 mM imidazole at room temperature for 30 min. The microsomal NKA activity was calculated as the difference between the ATPase activity in the presence and absence of 1 mM ouabain (Boehringer Mannheim Biochemicals, Indianapolis, IN) for lamb liver and 3 mM ouabain for rat liver, respectively.

Deglycosylation of β

The protein samples were denatured in 0.15% SDS at 60°C for 10 min, and then the deglycosylation reaction was carried out in 20 mM sodium phosphate buffer (pH 8.0), 8 mM EDTA, 0.6% octylglucoside, and 0.4% 2-mercaptoethanol with 3 U N-glycosidase F (Genzyme Corp., Boston, MA) per 100 µg glycoprotein at 37°C for 19 h.

Results

The Distribution of α and β in Rat Tissues

Nitrocellulose-blotted microsomes from different rat tissues (heart, kidney, liver, brain, colon, and stomach) were initially screened using polyclonal antisera raised to purified rat kidney holoenzyme (designated 222) and to isolated lamb kidney β (designated 517). Western blot analysis with antiserum 222 (Fig. 1 A) demonstrated the presence of the α-subunits in kidney, heart, brain, stomach, and colon (not shown) with a low abundance of α in liver. However, the β-directed serum (Fig. 1 B) showed a strong response with kidney, heart, brain, and colon and only very low levels in the liver and stomach. Therefore, it was difficult to identify any β in these latter two tissues. Given the possibility that β2 rather than β1 might be present in these tissues, a polyclonal antiserum directed against a 12 amino acid long synthetic peptide representing the NH_2-terminal region of β2 (conjugated to KLH) was used to determine the tissue distribution of the putative β2. Although not shown, we found that the β2 isoform was detected only in brain tissue.

Immunodetection of α1 and β1 in Liver Tissue

The ambiguous results regarding the presence of β1 in liver were due to an insufficient quantity of antigen blotted on the sheets. Western blot analysis done with liver microsomes from lamb and rat liver further purified by discontinuous sucrose gradient centrifugation showed that antisera 517 reacted with a 42-kD protein in both lamb and rat liver (Fig. 2). Further, when this protein was deglycosylated by N-glycosidase F (Fig. 3) the remaining βc core protein, with a molecular mass of 32 kD, had identical electrophoretic mobility to that of deglycosylated kidney β1. These data indicated that the 42-kD protein in the liver is a β-subunit of NKA. This was further demonstrated by using the monoclonal antibody M17-P5-F11 which is directed against lamb kidney β1. Figs. 2 and 3 show a reactive, deglycosylatable protein in lamb liver that migrates identically with that identified in both rat and lamb liver using the β-directed polyclonal serum. Unfortunately, monoclonal M17-P5-F11 does not crossreact with any rat β. In more fully characterizing the liver α proteins we found that our monoclonal antibody M12-P7-F11, directed toward the lamb kidney α1-subunit immunoreacted with the α of lamb and rat livers (Fig. 2) and that antibody McK1, a rat α1-specific monoclonal antibody obtained from Kathleen Sweadner (Felsenfeld and Sweadner, 1988) also reacted with the liver enzyme.

Figure 1. Western blot analysis of the distribution of α- and β-subunits in rat tissues. A shows the rat tissue reactivity (toward α) of rabbit serum 222, raised to the rat kidney holoenzyme, and B shows reactivity of a lamb kidney β-subunit-directed serum (517) toward rat tissue β.

The Effect of Ouabain on NKA in Liver Microsomes

The comparison of the Ki50 for ouabain for the liver and kidney enzymes from both rat and lamb was done by measuring the inhibitory effect of varying concentrations of ouabain. In both rat kidney and liver the Ki50 values for ouabain were 1 × 10−5 M, while in lamb kidney and liver the Ki50 values were 1 × 10−7 M. Analysis of the data revealed that only a single ouabain binding component was present in both tissues from the two species. This confirmed that there was a single ouabain binding site for the liver and kidney NKAs and that the α1 isoform is predominant.

Figure 2. Western blot analysis of density-gradient purified lamb and rat liver microsomes and NKA (lamb kidney NKA). The antibodies used were: M12-P7-F11; a maby directed against α of lamb kidney NKA; M17-P5-F11; a maby directed against lamb kidney β; and antiserum 517; a polyclonal antiserum directed against isolated lamb kidney β.

Characterization of the Ligand Dependencies of Lamb Liver NKA

The ligand dependency of the NKA activity in lamb liver microsomes was also compared with that of the purified lamb kidney enzyme. Lineweaver-Burk plots of enzyme activity as a function of varying Na+ or K+ concentrations showed the expected nonlinear relationship for both the kidney and liver enzymes, while the ATP dependence of the NKA activity was linear. The liver and kidney enzymes showed no significant differences in their Hill coefficients for Na+ (1.8) or K+ (1.9) interactions; however, the apparent dissociation constants (K′app) of the liver NKA for Na+ (8 vs. 18 mM) and K+ (1 vs. 2 mM) and the Km of ATP (0.5 vs. 0.8 mM) were all approximately one-half those for the kidney enzyme.

Figure 3. Western blot analysis of deglycosylated lamb liver β-subunit. The deglycosylated β was resolved on 12% SDS-polyacrylamide gels transferred to nitrocellulose sheets, and immunodetected by β-directed monoclonal antibody (M17-P5-F11). Prestained protein molecular weight standards (Bethesda Research Laboratories, Gaithersburg, MD) are shown in the left lane, while lanes a and b represent native and deglycosylated lamb kidney β, and lanes c and d represent native and deglycosylated lamb liver β.

Conclusion

The present study has demonstrated the presence of the NKA β-subunit in mammalian liver; thus, the liver NKA does consist of both the α- and β-subunits. This result agrees with the generally accepted conclusion that the Na+ pump needs its β-subunit. However, the liver enzyme exists at rather low levels compared with most tissues, as reflected by our observation that ouabain-sensitive liver microsomal ATPase activity is 5–10 µmol/mg per h versus 200 for lamb kidney microsomes. The results of ouabain inhibition studies and Western blot analysis have confirmed that α1 and β1 are the isoforms in the liver. The apparent difference in the molecular mass of liver β versus kidney β were found to be due to differences in the N-linked carbohydrates. While our kinetic studies revealed that there were no differences in the heterotropic cooperativities of the Na+ and K+ sites of the lamb liver and kidney enzymes, all three ligands, ATP, Na+, and K+, showed higher binding affinities to the liver than to the kidney enzyme.

Acknowledgments

The authors would like to thank Dr. A. Balasubramaniam for the peptide synthesis, Dr. Kathleen Sweadner for providing the monoclonal antibody McK1, and Purabi Dey and Chuck Loftice for their excellent technical assistance.

This work was supported by NIH grants P01-HL-32214 and P01-HL-22619.

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Martin-Vasallo, P., W. Dackowski, J. R. Emanuel, and R. Levenson. 1989. Identification of a putative isoform of the Na+,K+-ATPase β subunit. The Journal of Biological Chemistry. 264:4613–4618.

McDonough, A. A., K. Geering, and R. A. Farley. 1990. The sodium pump needs its β-subunit. The FASEB Journal. 4:1598–1605.

Schwartz, A., J. C. Allen, and S. Harigaya. 1969. Possible involvement of cardiac Na+,K+-adenosine triphosphatase in the mechanism of action of cardiac glycosides. The Journal of Pharmacology and Experimental Therapeutics. 168:31–40.

Shyjan, A. W., C. Gottandi, and R. Levenson. 1990. The Na+,K+-ATPase β2 subunit is expressed in rat brain and copurifies with Na+,K+-ATPase activity. The Journal of Biological Chemistry. 265:5166–5169.

Towbin, H., T. Staehelin, and J. Gordon. 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets procedure and some applications. Proceedings of the National Academy of Sciences, USA. 76:4350–4354.

Young, R. M., and J. B. Lingrel. 1987. Tissue distribution of mRNAs encoding the α isoforms and β subunit of rat Na+,K+-ATPase. Biochemical and Biophysical Research Communications. 145:52–58.

Association and Dissociation of Solubilized Na+/K+-ATPase by Phosphatidylserine and ATP

Yutaro Hayashi, Kunihiro Mimura, Hideo Matsui, and Toshio Takagi

1st Department of Biochemistry, Kyorin University School of Medicine, Mitaka, Tokyo 181, Japan; and Institute for Protein Research, Osaka University, Suita, Osaka 565, Japan

According to the reaction scheme of Post and Albers (Glynn and Karlish, 1975), the conformational state of Na+/K+-ATPase changes cyclically from E_1 to E_2, and then from E_2 to E_1 during each turnover in the membrane. Post et al. (1972) and Glynn and Richards (1982) have shown that a higher concentration of ATP accelerates the rate of transition from the E_2 conformation bound by K+ (E_2K) to E^1. Hegyvary et al. (1980) have shown that phospholipids are essential for the reaction step from E_1P to E_2P, which are the conformations phosphorylated by ATP. That is, the effects of phospholipid and ATP on the conformational change between E_1 and E_2 are opposite in direction.

We have shown by means of low-angle laser light scattering photometry coupled with high-performance gel chromatography (HPGC/LALLS method) that solubilized Na+/K+-ATPase is in a dissociation–association equilibrium (2αβ (αβ)2) between the αβ-protomer (Mr = 1.5 × 10^5) and (aβ)2-diprotomer (3.0 × 10^5) irrespective of the enzyme turnover, and that the association constant (Ka) of the equilibrium in the E_2 state is ~ 50 times larger than that in the E_1 state (Hayashi et al., 1989b). Thus, it is strongly suggested that the two protomers in E_2 associate more strongly than in E_1. Therefore, using the HPGC/LALLS method, we investigated how ATP and phosphatidylserine (PS) would change the oligomeric structure of the solubilized enzyme.

Methods

Na+/K+-ATPase was purified from dog kidney in membrane-bound form and solubilized with octaethylene glycol n-dodecylether (C_12E_8) as described elsewhere (Hayashi et al., 1989b). A TSKgel G4000SWXL column (7.8 × 300 mm) equipped with a guard column (TSK guard column SWXL, 6.0 × 40 mm) was used for gel chromatography of the solubilized enzyme. The columns were equilibrated and eluted with an elution buffer (pH 7.0) containing 0.3 mg/ml C_12E_8, 0.05 M KCl, 0.05 M NaCl, 4 mM MgCl_2, 1 mM EDTA, 10 mM imidazole, and 13 mM HEPES at a flow rate of 0.40 ml/min and at 20°C unless otherwise stated. PS from bovine brain with 98% purity (Sigma Chemical Co., St. Louis, MO) was added to the elution buffer as described elsewhere (Hayashi et al., 1989b). The solubilized enzyme was chromatographed on the columns and the eluate was monitored successively using three kinds of detector: a LALLS photometer (LS; TSK model LS–8000), a UV spectrophotometer (UV), and a differential refractometer (RI). The three kinds of elution pattern obtained were fractionated at 6-s intervals on the chart paper record. Molecular weight (Mr) for each fraction of the protein component was calculated using essentially the same method as that described elsewhere (Hayashi et al., 1989a). Weight-averaged molecular weight (w)of the protein components eluted during a given time was calculated according to the equation

where the suffix i denotes the value for each 6-s interval component and w is the weight fraction of each component relative to all of the protein components eluted during the observation time, as estimated from the UV absorption pattern.

Figure 1. High-performance gel chromatography of solubilized Na+/K+-ATPase carried out with an elution buffer containing various concentrations of PS. (A) The elution patterns obtained with a LALLS photometer (LS) and a UV-spectrophotometer (UV) are indicated by solid (LS) and dotted (UV) lines, respectively. Mr values of the protein components fractionated at 6-s intervals are indicated by solid circles. The length of the scale bar corresponds to 0.05 absorbance units at 280 nm for the UV pattern. (B) The w values for the protein components eluted between 17 and 26 min (indicated by the bar with arrows in the upper part of A) were calculated as described in Methods and plotted against the concentrations of PS.

Results and Discussion

Reversible Association of the Solubilized Enzyme by PS

When the solubilized enzyme was choromatographed with the elution buffer containing neither PS nor ATP, the w of the protein components eluted between 17 and 26 min was estimated to be 238,000, indicating that they contained mainly the diprotomer and the protomer. The w of the protein components increased as the concentration of PS added to the elution buffer increased (Fig. 1). This result indicated that PS made the solubilized enzyme associate into protein components with an oligomeric structure larger than the diprotomer.

The solubilized enzyme was chromatographed with the elution buffer containing 120 µg/ml PS, and the protein components eluted with a retention time of 20–25.4 min were collected as the associated enzyme, of which w was 1.15 × 10^6. A 400-µ1 aliquot of each of the associated enzymes containing 0.06 mg protein was rechromatographed with the elution buffer containing no PS or 120 µg/ml PS, but otherwise the same ingredients as those for the first chromatography. The w values of the protein components eluted between 17 and 24 min were 2.75 × 10^6 and 3.06 × 10^5 in the case of 120 µg/ml PS and no PS, respectively. In the latter case, 26 and 61% (wt/wt) of the eluted protein were identified as the protomer and diprotomer, respectively. When no PS was added to the elution buffer, it was expected that the exogenous PS included in the enzyme aliquot loaded onto the column would be separated from the enzyme. Therefore, the decrease in w of the associated enzyme upon rechromatography in the absence of PS showed that the association was reversible upon removal of PS.

Figure 2. The solubilized Na+/K+-ATPase was chromatographed with elution buffer containing various concentrations of ATP (), ADP (), or AMP (∆), and 0.20 mg/ml C_12E_8 and 60 µg/ml PS in common at a flow rate of 0.50 ml/min. The w values for the protein components eluted between 14 and 21 min were plotted against the concentration of the nucleotides. The w obtained in the absence of ATP and PS is indicated by a broken line.

Dissociation of the Associated Enzyme by ATP

The PS-induced association of the enzyme was inhibited by ATP and ADP, but not by AMP, when each nucleotide was added to the elution buffer containing 0.20 mg/ml C_12E_8 and 60 µg/ml PS in common (Fig. 2). The concentration required for half-maximal action (K0.5) was 0.44 mM for ATP. The PS-associated enzyme was isolated by chromatography in the presence of 120 µg/ml PS as described above, and then rechromatographed in the presence of various concentrations of ATP, 0.3 mg/ml C_12E_8, and 80 µg/ml PS. The w of the eluted protein components decreased with the increase in ATP concentration, so that the K0.5 for dissociation became 0.16 mM. The K0.5 value for dissociation of the associated enzyme by ATP was comparable to that for the inhibition of the association described above.

It was concluded that PS induced the solubilized enzyme to associate into protein components with a much higher Mr than the diprotomer, and that ATP induced the associated enzyme to dissociate into components comparable to the diprotomer and the protomer. That is, the effects of PS and ATP on oligomerization of the enzyme were opposite in direction, in the same manner as their effects on the conformational change between E_1 and E_2. Therefore, it can be inferred from these results that PS tightens the interaction of protomer-with-protomer and diprotomer-with-diprotomer, and that ATP with low affinity loosens it. This is consistent with our conclusion that the protomers in a pair interact with higher affinity in E_2 than in E_1 (Hayashi et al., 1989b).

Acknowledgments

This work was supported in part by a grant 63580127 from the Ministry of Education, Science and Culture of Japan (to Y. Hayashi).

References

Glynn, I. M., and S. J. D. Karlish. 1975. The sodium pump. Annual Review of Physiology. 37:13–55.

Glynn, I. M., and D. E. Richards. 1982. Occlusion of rubidium ions by the sodium-potassium pump: its implications for the mechanism of potassium transport. Journal of Physiology. 330:17–43.

Hayashi, Y., H. Matsui, and T. Takagi. 1989a. Membrane protein molecular weight determined by low-angle laser light scattering photometry coupled with high-performance gel chromatography. Methods in Enzymology. 172:514–528.

Hayashi, Y., K. Mimura, H. Matsui, and T. Takagi. 1989b. Minimum enzyme unit for Na+/K+-ATPase is the αβ-protomer: determination by low-angle laser light scattering photometry coupled with high-performance gel chromatography for substantially simultaneous measurement of ATPase activity and molecular weight. Biochimica et Biophysica Acta. 983:217–229.

Hegyvary, C., R. Chigurupati, K. Kang, and D. Mahoney. 1980. Reversible alterations in the kinetics of cardiac sodium- and potassium-activated ATPase after partial removal of membrane lipids. Journal of Biological Chemistry. 255:3068–3074.

Post, R. L., C. Hegyvary, and S. Kume. 1972. Activation by ATP in the phosphorylation kinetics of sodium and potassium ion transport ATPase. Journal of Biological Chemistry. 247:6530–6540.

Identification of a 97-kD Protein from Rat Kidney Related to the Gastric H,K-ATPase

Mark D. Okusa and Michael J. Caplan

Departments of Internal Medicine and Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06510; and VA Medical Center, West Haven, Connecticut 06516

The mammalian distal nephron participates in the maintenance of potassium homeostasis by regulating transepithelial movement of potassium ions. Whereas transport processes mediating potassium secretion by the distal nephron have been well described (Wright and Giebisch, 1985), those pathways mediating net potassium absorption have not. Previous studies suggest that the mechanism by which potassium is absorbed is active (Malnic et al., 1964; Okusa et al., 1989) and bears functional characteristics similar to the gastric H,K-ATPase (Doucet and Marsy, 1987; Garg and Narang, 1988; Okusa et al., 1989; Wingo, 1990). The gastric H,K-ATPase, a member of the E_1-E_2 class of cation-transporting ATPases (Sachs, 1988), is found in tubulo-vesicular structures of gastric parietal cells and is the major mechanism by which hydrogen ions are pumped into the stomach lumen (Sachs, 1988; Rabon and Reuben, 1990). The H,K-ATPase mediates an electroneutral counter-transport of hydrogen for potassium ions (Sachs et al., 1976) and is selectively inhibited by omeprazole (Fellenius et al., 1981) and by SCH 28080 (Wallmark et al., 1987).

In addition to functional studies, biochemical studies in rat and rabbit kidney (Doucet and Marsy, 1987; Garg and Narang, 1988) have demonstrated that a K-ATPase activity (believed to represent an H,K-ATPase) is present in the connecting tubule (CNT), initial collecting tubule (ICT), cortical collecting tubule (CCT), and medullary collecting tubule (MCT). This activity is inhibited by omeprazole or SCH 28080 (Wingo, 1990). Recent immunolocalization studies, using monoclonal antibodies generated against the gastric H,K-ATPase, suggest that a protein similar to gastric H,K-ATPase might be present in intercalated cells of rat and rabbit cortical collecting duct and outer medullary collecting duct (Wingo et al., 1990).

The aim of this study was to use a synthetic peptide antibody generated against an epitope of the gastric H,K-ATPase to determine whether a protein related to the gastric H,K-ATPase is present in rat kidney. We find that our antibody specifically cross-reacted with a protein of ~ 97 kD. In addition, this protein appears to be present in a discrete population of cells of the outer medullary collecting duct.

Materials and Methods

Kidneys from adult Sprague-Dawley rats maintained on a regular diet were perfusion-fixed in paraformaldehyde-lysine-periodate (PLP) as described previously (Ellison et al., 1989). Kidney sections were then processed and embedded in paraffin.

A synthetic peptide antibody (HK9) directed against a sequence of 20 amino acids derived from the amino terminus of the gastric H,K-ATPase was developed. Because the sequence of the gastric α-subunit of the H,K-ATPase (Shull and Lingrel, 1986; Maeda et al., 1988) and α1-subunit of the Na,K-ATPase (Shull et al., 1986) are known, we were able to make an antibody to a defined epitope of the H,K-ATPase which shares no homology with the Na,K-ATPase. The peptide corresponding to this 20 amino acid sequence was synthesized by the Yale Protein Chemistry Facility. A cysteine residue was added to the carboxyl terminus to facilitate haptenization of the peptide to keyhole limpet hemocyanin. Coupled peptide was mixed with Freund's adjuvant and standard immunization of rabbits was performed. ELISA assay revealed that this antiserum reacts with the antigenic peptide with a titer > 1:10,000. Studies from our laboratory indicate that HK9 does not cross-react with Na,K-ATPase.

Membrane proteins from rat outer medulla were obtained from anesthetized rats. 100 µg of membrane proteins were denatured in Laemmli sample buffer (Laemmli, 1970), separated by a 10% SDS-PAGE, and transferred electrophoretically to nitrocellulose. Nitrocellulose strips were washed several times in a solution containing PBS/5% powdered milk to quench nonspecific binding sites and then incubated in HK9 at a dilution of 1:150 for 60 min at room temperature. To determine specificity, competition experiments were performed in which excesses of either antigenic or nonantigenic peptides were preincubated with the primary antibody. After several washes the strips were incubated with a goat anti–rabbit secondary antibody conjugated to alkaline phosphatase. In the presence of excess substrate a dense reaction product forms.

After perfusion and fixation, kidneys were cut into 2-mm coronal sections and embedded in paraffin for light microscopy (Zeiss Axiophot microscope). Light microscopy was performed on 4-µm sections. After deparaffinization and hydration the background peroxidatic activity associated with the kidney sections was blocked through incubation with H_2O_2. Nonspecific protein binding sites were blocked by incubation with Tris HCl/1% BSA. After incubation with HK9 and after several washes, kidney sections were incubated with a secondary antibody conjugated to horseradish peroxidase and the reaction was developed in the presence of DAB and H_2O_2.

Results

To determine the specificity of the HK9 antibody, Western blot analysis was performed on pig stomach membrane proteins and immunocytochemistry was performed on rat stomach sections. In the absence of competition, HK9 antibody cross-reacted avidly with a 97-kD polypeptide. In the presence of the antigenic peptide, however, labeling of this 97-kD polypeptide was blocked. Competition experiments were also carried out using two nonantigenic peptides: (a) peptide to a sequence near the FITC binding site, and (b) peptide to a sequence from the α1-subunit of the Na,K-ATPase. Preincubation of the antibody with the two nonantigenic peptides failed to block labeling of the 97-kD polypeptide. Immunocytochemistry of rat stomach sections revealed the presence of dense reaction product in the parietal cells of the gastric mucosa. As would be expected, preincubation with the antigenic peptide successfully preabsorbed specific antibodies and thus blocked labeling of the parietal cells. In keeping with the results of the Western blot analysis, preincubation with a nonantigenic peptide failed to block labeling. These results indicate that our antibody specifically recognizes an epitope associated with a protein which has a molecular weight comparable to that of the gastric H,K-ATPase and is localized to parietal cells of gastric gland.

Western blot analysis of rat outer medulla was performed to determine whether this tissue expresses a protein that cross-reacts with our antibody. In the absence of the antigenic peptide, HK9 detects a polypeptide of ~97 kD in the rat outer medulla. When the experiment is performed in the presence of the antigenic peptide, labeling of this 97-kD polypeptide is blocked. The addition of an unrelated peptide has no effect on the labeling. These results indicate that the rat outer medulla possesses a protein that has closely related immunological characteristics and molecular weight to the gastric H,K-ATPase.

To determine the cellular distribution of this 97-kD protein, immunocytochemistry was performed on rat kidney sections. Dense reaction product was localized diffusely over a discrete population of cells of the outer medullary collecting duct. The identity of these cells, and the degree to which HK9 specifically interacts with cells of other nephron segments, is being determined at this time.

Discussion

The results of these studies suggest that a protein in rat kidney has been identified with molecular and antigenic characteristics similar to those of the gastric H,K-ATPase. Furthermore, immunocytochemical analysis suggests that this protein is localized to a select population of cells of the outer medullary collecting duct.

Previous studies have shown functional (Okusa et al., 1989; Wingo, 1990), biochemical (Doucet and Marsy, 1987; Garg and Narang, 1988), and immunocytochemical evidence (Wingo et al., 1990) for the renal expression of a transporter with characteristics of an H,K-ATPase. In a recent study, investigators made use of two monoclonal antibodies raised against hog gastric H,K-ATPase in order to detect cross-reactivity with proteins of specific cells of the cortical and outer medullary collecting ducts. Double labeling experiments using polyclonal antibodies raised against band 3 protein suggested that an H,K-ATPase-like protein is localized to intercalated cells. Biochemical characterization of the antigen recognized by these probes was not reported in this study.

For the purposes of the current experiments, it was necessary to develop an antibody that recognizes the H,K-ATPase and not other closely related members of the E_1-E_2 family of ATPases. Because of the extensive sequence homology between the H,K-ATPase and α1-Na,K-ATPase (Shull and Lingrel, 1986), we generated a synthetic peptide antibody. Based on comparisons of the sequences of the gastric H,K-ATPase (Shull and Lingrel, 1986; Maeda et al., 1988) and the Na,K-ATPase (Shull et al., 1986) we chose to make an antibody to an epitope consisting of 20 amino acids at the amino terminus of the H,K-ATPase. This region shares no homology with the Na,K-ATPase. Specificity of this antibody for the gastric H,K-ATPase was determined by ELISA assay, Western blot analysis, and light and EM immunocytochemistry. ELISA assay revealed that the antibody reacted with the antigenic peptide with a titer of > 1:10,000. Western blot analysis revealed that HK9 specifically recognizes a 97-kD protein from pig and rat stomach. This 97-kD protein appears to be localized to parietal cells by light and EM microscopy (Caplan et al., 1988). Thus HK9 specifically recognizes an epitope of the gastric H,K-ATPase.

We next sought to determine whether a protein related to the gastric H,K-ATPase is present in kidney. Both Western blot analysis and immunocytochemistry specifically identified a 97-kD protein that appears to be localized to specific cells of the medullary collecting duct. This segment of the nephron is composed of both intercalated cells and principal cells. The results of this study do not permit us to determine the exact cell type to which this protein is localized. However, the previous double labeling experiments using a monoclonal antibody directed against the gastric H,K-ATPase and polyclonal antibody against band 3 protein described above, indicated that a single cell type reacted with both antibodies, suggesting that the H,K-ATPase was localized to α-intercalated cells. It is probable, therefore, that the select population of cells recognized by our antibody may represent α-intercalated cells. Whether similar cell types of the cortical collecting duct as well as other cells (e.g., inner medullary collecting duct cells) are labeled has not been determined at this time.

We conclude that a protein present in rat kidney has molecular and physiological characteristics similar to those of the principal ion pump of the gastric gland. This protein is involved in active potassium absorption through the counter-transport of hydrogen for potassium ions. The precise molecular relationship of the renal 97-kD protein to the gastric H,K-ATPase will await sequencing of a cDNA clone encoding this putative renal H,K-ATPase.

Acknowledgments

This study was supported by funds from the David and Lucille Packard Foundation and grants from the National Institutes of Health to M. J. Caplan. M. D. Okusa is a recipient of a Career Development Award (research associate) from the VA Medical Center.

References

Caplan, M. J., R. Baron, L. Neff, G. Shull, and B. Forbush III. 1988. H,K-ATPase. A close molecular relative of the Na,K-ATPase which is present on the apical membranes of parietal cells. Journal of Cell Biology. 107:783a. (Abstr.)

Doucet, A., and S. Marsy. 1987. Characterization of K-ATPase activity in distal nephron: stimulation by potassium depletion. American Journal of Physiology. 253:F418–F423.

Ellison, D. H., H. Velázquez, and F. S. Wright. 1989. Adaptation of the distal convoluted tubule of the rat. Structural and functional effects of dietary salt intake and chronic diuretic infusion. Journal of Clinical Investigation. 83:113–126.

Fellenius, E., R. Berglindh, G. Sachs, L. Olbe, B. Elander, S.-E. Sjostrand, and B. Wallmark. 1981. Substituted benzimidazoles inhibit gastric acid secretion by blocking (H+ + K+)ATPase. Nature. 290:159–161.

Garg, L. C., and N. Narang. 1988. Ouabain-insensitive K-adenosine triphosphatase in distal nephron segments of the rabbit. Journal of Clinical Investigation. 81:1204–1208.

Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 227:680–685.

Maeda, M., J. Ishizaki, and M. Futai. 1988. cDNA cloning and sequence determination of pig gastric (H+ + K+)-ATPase. Biochemical and Biophysical Research Communications. 157:203–209.

Malnic, G., R. M. Klose, and G. Giebisch. 1964. Micropuncture study of renal potassium excretion in the rat. American Journal of Physiology. 206:674–686.

Okusa, M. D., H. Velázquez, D. H. Ellison, and F. S. Wright. 1989. Presence of a mechanism for potassium absorption in the renal distal tubule. Clinical Research. 37:498A. (Abstr.)

Rabon, E. C., and M. A. Reuben. 1990. The mechanism and structure of the gastric H,K-ATPase. Annual Review of Physiology. 52:321–344.

Sachs, G. 1988. Gastric H,K-ATPase as therapeutic target. Annual Review of Pharmacology and Toxicology. 28:269–284.

Sachs, G., H. H. Chang, E. Rabon, R. Schackman, M. Lewin, and G. Saccomani. 1976. A nonelectrogenic H+ pump in plasma membranes of hog stomach. Journal of Biological Chemistry. 251:7690–7698.

Shull, G. E., J. Greeb, and J. B. Lingrel. 1986. Molecular cloning of three distinct forms of the Na+,K+-ATPase α-subunit from rat brain. Biochemistry. 25:8125–8132.

Shull, G. E., and J. B. Lingrel. 1986. Molecular cloning of the rat stomach (H+ + K+)-ATPase. Journal of Biological Chemistry. 261:16788–16791.

Wallmark, B., C. Briving, J. Fryklund, K. Munson, R. Jackson, J. Mendlein, E. Rabon, and G. Sachs. 1987. Inhibition of gastric H+,K+-ATPase and acid secretion by SCH 28080 a substituted puridyl(1,2a)imidazole. Journal of Biological Chemistry. 262:2077–2984.

Wingo, C. S. 1990. Active proton secretion and potassium absorption in the rabbit outer medullary collecting duct: functional evidence for H-K-ATPase. Journal of Clinical Investigation. 84:361–365.

Wingo, C. S., K. M. Madsen, A. Smolka, and C. C. Tisher. 1990. H-K-ATPase immunoreactivity in cortical and outer medullary collecting duct. Kidney International. 38:985–990.

Wright, F. S., and G. Giebisch. 1985. Regulation of potassium excretion. In The Kidney: Physiology and Pathophysiology. D. W. Seldin and G. Giebisch, editors. Raven Press, New York. 1223–1249.

An Analysis of Digitalis Sugar Binding Site Characteristics

Dwight S. Fullerton, Arthur H. L. From, and Khalil Ahmed

College of Pharmacy, University of Utah, Salt Lake City, Utah 84112; and U. S. Department of Veterans Affairs Medical Center and University of Minnesota, Minneapolis, Minnesota 55417

Introduction

It is generally accepted that the plasma membrane Na+,K+-ATPase (EC 3.6.1.37) is the cellular site of action of cardenolide (“digitalis”) and bufadienolide (“toad poison”) glycosides. These compounds inhibit this ATPase activity and evoke their well-established cardiotonic effects. The intrinsic digitalis sensitivity of a given Na+,K+-ATPase may vary in relation to both tissue and species of origin (Ahmed et al., 1983; Price and Lingrel, 1988). Thus, an implicit assumption has been that the characteristics of the binding sites that determine the activity of one digitalis genin or glycoside in relation to another are independent of the intrinsic digitalis sensitivity of the enzyme.

In a study of three Na+,K+-ATPase preparations isolated from different sources with differing ouabain sensitivites, we have previously presented data that suggest that this assumption is probably correct with regard to digitalis genins (Ahmed et al., 1983). However, it is not known whether this is the case with the sugar binding sites of different Na+,K+-ATPase preparations.

It is generally agreed that the sugars bind to sites separate from the genin binding sites (Yoda, 1974). In previous studies with hog kidney Na+K+-ATPase (Rathore et al., 1986), we have shown: (a) The sugar 4′-OH of digitalis monoglycosides has the greatest role in binding to the sugar site. If equatorial, as with β-D-glucose, binding is generally good; but if axial, as with β-D-galactose, binding is poor. (b) The 2′-OH and 3′-OH of α-L-glycosides apparently have little contribution to binding, but may have a significant role with D-glycosides. (c) 6′-CH_3 hydrophobic binding (e.g., with the L-rhamnose of ouabain) may have a significant role with α-L-glycosides, but not with β-D-glycosides. (For example, “adding” a 6′-OH group to a digitoxigenin α-L-rhamnoside to “make” the corresponding mannoside reduces activity more than 10-fold).

However, it has not been known whether these structural determinants generally apply to other Na+,K+-ATPase receptors. The present study indicates that the sugar binding sites of four Na+,K+-ATPase preparations of differing origin and intrinsic cardenolide sensitivity are similar with regard to recognition of the major structural determinants for binding (e.g., 4′-OH equatorial better than 4′-OH axial). Heterogeneous binding site behavior occurs only in relation to the less critical determinants of sugar structure (e.g., presence or absence of a 6′-OH group). Our data also indicate that the intrinsic sensitivity of the sugar binding site to sugar potentiation may vary among different enzyme preparations, and that this variation may be independent of the intrinsic sensitivity of an enzyme to inhibition by digitoxigenin.

Methods

Detailed descriptions of the methods used in these studies have recently been reported (From et al., 1990). Highlights are summarized below.

Digitalis Analogues

All the glycosides reported in this study were synthesized in our laboratories as previously reported (Fullerton et al., 1984; Rathore et al., 1986).

Preparation of ATPases

Hog kidney, rat brain, cat brain, and rabbit kidney enzymes were prepared according to the procedures outlined previously (Mayrand et al., 1982; Ahmed et al., 1983). The activity ranges of the hog, rat, cat, and rabbit Na+,K+-ATPase preparations were, respectively 1,000–1,300,450–550, 400–550, and 75–125 µmol Pi/mg of protein per h. The rat brain preparation as isolated by us exhibited the presence of only the more sensitive (α2) isoform of the enzyme (From et al., 1990).

Assay of ATPase

The Na+,K+-ATPase assay procedure has been described in detail previously (Ahmed et al., 1983). The preincubation period of 120 min under type I binding conditions was chosen because we have previously shown (Fullerton et al., 1984; Rathore et al., 1986) that I_50 (concentration of glycoside resulting in 50% inhibition of enzyme activity) values declined only modestly when preincubation times for monoglycosides exceeded 120 min. Although there is little doubt that true equilibrium binding may take many hours, a 120-min period of incubation at 37°C appears to induce near-equilibrium binding and from a practical standpoint avoids deterioration of the enzyme observed after prolonged periods of incubation (From et al., 1990).

To avoid significant dissociation of bound glycoside subsequent to initiating ATPase activity by the addition of KCl, the assay time after KCl addition was 10 min or less as previously described in studies with a variety of cardenolide genins (Ahmed et al., 1983). As digitalis monoglycosides are generally more tightly bound to the enzyme than genins (Table I; From et al., 1990), it seems unlikely that, upon addition of KCl, significant dissociation of the glycosides occurred during the subsequent brief assay period. Further, there is evidence that when glycoside is bound to the Na+,K+-ATPase under type I binding conditions, addition of KCl does not increase the dissociation rate (Yoda and Yoda, 1974). Each assay was performed in triplicate at least twice and there was ±5% or less variation in the ATPase activities of the replicates studied at each drug concentration.

Sensitivity of ATPases to Digitoxigenin and Monoglycoside Inhibition

Table I shows the I_50 values obtained for all compounds with regard to the four enzyme preparations. The relative sensitivities of the enzymes to digitoxigenin and the monoglycosides were in the following order: hog kidney > cat brain > rat brain > rabbit kidney. (Glycosides are numbered in order of hog kidney ATPase inhibition.) For any given sugar moiety the degree of potentiation varies considerably from enzyme to enzyme and is not well related to the intrinsic sensitivity of the enzyme to digitoxigenin. For example, inhibition by I and II was comparable in the hog and rabbit enzymes and greater than that observed in the other enzymes; however, the sensitivity to digitoxigenin was most disparate between these enzymes and closest between the hog and cat enzymes. The variation of the magnitude of inhibition was greatest for the most active compounds (I–V) and moderate for compounds of intermediate activity (VI–IX). For compounds with minimal activity (X–XII) such variation was minimal.

Figure 1. Example of digitoxigenin monoglycosides used in study.

Discussion

Our observations (Table I) suggest that the sugar binding sites of all the enzyme preparations are sensitive to the major structural features that determine the degree to which a sugar may potentiate genin activity. For example, all the β-D-galactosides (with 4′-OH axial) are much less active than the β-D-glucosides (with 4′-OH equatorial). In contrast, some of the less critical structural determinants seem to be affected by minor variations in the sugar binding site. For example, adding a 6′-OH to digitoxigenin β-D-digitoxoside to “make” the 6′-OH digitoxoside slightly reduces hog kidney ATPase inhibition, modestly reduces rabbit kidney ATPase inhibition, and slightly increases cat brain and rat brain ATPase inhibition. These kinds of changes suggest that the sugar binding site may not be entirely homogenous among ATPases.

In contrast, the genin binding site may be quite homogenous. Although the study of Schonfeld et al. suggests that the genin recognition site on ATPase may differ in low and high sensitivity enzymes (Schonfeld et al., 1986), our own work on the common dependence of genin activity on C17 lactone position (Ahmed et al., 1983; From et al., 1984) and recent amino acid sequence data (Price and Lingrel, 1988) support the concept of genin site homogeneity. Variation in overall genin sensitivity between enzymes may depend on amino acid substitutions in regions not directly involved in genin binding.

TABLE I
I_50 Values for Inhibition of Four Na,K-ATPases by Digitoxigenin and its C3 Monoglycosides

The data also suggest that there may be variation in overall sensitivity to sugar-induced potentiation of ATPase inhibition. For example, sugar potentiation is comparable and greatest in the hog and rabbit enzymes, but these enzymes show the greatest disparity in sensitivity to digitoxigenin.

The present data suggest a binding model in which extracellular domains may be present that recognize sugar structural characteristics favorable for binding, and these domains may show some structural diversity. Similarly, other independent structural features of the sugar binding site may determine overall sensitivity to sugar potentiation of genin activity. Thus, the inhibitory properties of a cardiac glycoside may be dependent not only on the structural features of both the genin and the sugar, but also the intrinsic sensitivities of both the genin and sugar binding sites in the Na+,K+-ATPase.

References

Ahmed, K., D. C. Rohrer, D. S. Fullerton, E. Kitatsuji, T. Deffo, and A. H. L. From. 1983. Interaction of Na+,K+-ATPase and digitalis genins: a general model for inhibitory activity. Journal of Biological Chemistry. 258:8092–8097.

From, A. H. L., D. S. Fullerton, and K. Ahmed. 1990. Digitals receptor sugar binding site characteristics: a model based upon studies of Na+,K+-ATPase preparations with differing digitalis sensitivities. Molecular and Cellular Biochemistry. 94:157–165.

From, A. H. L., D. S. Fullerton, T. Deffo, E. Kitatsuji, D. C. Rohrer, and K. Ahmed. 1984. The inotropic activity of digitalis genins is dependent upon C(17) side group carbonyl oxygen position. Journal of Molecular and Cellular Cardiology. 16:835–842.

Fullerton, D. S., M. Kihara, T. Deffo, E. Kitatsuji, K. Ahmed, B. M. Simat, A. H. L. From, and D. C. Rohrer. 1984. Cardiac glycosides. 2. A systematic study of digitoxigenin β-D glycosides. Journal of Medicinal Chemistry. 27:256–261.

Mayrand, R., D. S. Fullerton, and K. Ahmed. 1982. A simple method for the purification of rat brain Na+,K+-adenosine triphosphatase (ATPase). Journal of Pharmacological Methods. 7:279–288.

Price, E. M, and J. B. Lingrel. 1988. Structure-function relationships in the Na+,K+-ATPase α subunit: site directed mutagenesis of glutamine-111 to arginine and asparagine-122 to aspartic acid generates ouabain insensitive enzyme. Biochemistry. 27:8400–8408.

Rathore, H., A. H. L. From, K. Ahmed, and D. S. Fullerton. 1986. Cardiac glycosides. 7. Sugar stereochemistry and cardiac glycoside activity. Journal of Medicinal Chemistry. 29:1945–1952.

Schonfeld, W., R. Schonfeld, K-H. Menke, J. Weiland, and K. R. H. Repke. 1986. Origins of differences of inhibitory potency of cardiac glycosides in Na+/K+ transporting ATPase from human cardiac muscle, human brain cortex and guinea-pig cardiac muscle. Biochemical Pharmacology. 35:3221–3232.

Yoda, A. 1974. Association and dissociation rate constants of the complexes between various cardiac monoglycosides and Na+,K+-ATPase. Annals of the New York Academy of Science. 242:598–616.

Yoda, A., and S. Yoda, 1974. Influence of certain ligands on the dissociation rate constants of cardiac glycoside complexes with sodium- and potassium-dependent adenosine triphosphatase. Molecular Pharmacology. 10:810–819.

Studies of Digitalis Binding Site(s) in Na,K-ATPase by Covalent Labeling with a Photoactive Probe Located in the Genin Moiety

Reginald A. McParland, Dwight S. Fullerton, Robert R. Becker, Arthur H. L. From, and Khalil Ahmed

Department of Biochemistry and Biophysics, Oregon State University, Corvallis, Oregon 97331; College of Pharmacy, University of Utah, Salt Lake City, Utah 84112; U. S. Department of Veterans Affairs Medical Center and University of Minnesota, Minneapolis, Minnesota 55417

Introduction

Studies using a variety of chemical probes have suggested that the receptor site(s) for digitalis glycosides may be localized in the α-subunit of Na,K-ATPase (for references, see Fullerton et al., 1986). Distinct binding sites for the steroid and sugar moieties of the cardiac glycosides have been suggested (Yoda and Yoda, 1974). Studies on the primary structure of the α-subunit have also yielded much insight into the possible candidate sites for ouabain binding (Shull et al., 1985; Price and Lingrel, 1988; Lingrel et al., 1990). However, it is also well known that cardenolide interaction with Na,K-ATPase is influenced by a variety of factors, including both the structural determinants in the digitalis molecule and the conditions that influence the conformations of the ATPase molecule (e.g., Fullerton et al., 1986). Thus, it would seem reasonable that chemical approaches should also be developed in concert with the aforementioned molecular biological studies to investigate these sites. To that end, our strategy is to utilize photoactive probes specific for the digitalis binding site(s).

Our previous studies on the structural determinants of the cardiac glycosides for receptor binding have suggested that the C17 carbonyl oxygen geometry plays a major role in binding site compatibility for the genin portion of the digitalis glycoside molecule (Fullerton et al., 1986). To take advantage of this property we designed a 3H-labeled analogue of digitoxigenin 3β-D-digitoxoside with an acyclic C17 side group containing an azido group in the genin moiety. This analogue, [C20-3H]-(3β,5β,14β,20E)-24-azido-3-[(2,6-dideoxy-β-D-ribo-hexapyranosyl)oxy]-14-hydroxyl-21-norchol-20(22)-en-23-one or [3H]24-azido 3β-digitoxoside (3H-probe), was found to bind specifically to the ouabain binding site(s) in the α-subunit of hog kidney Na,K-ATPase (Deffo et al., 1983). We reasoned that this 3H-probe might serve to delineate the genin binding site(s) in Na,K-ATPase. Here we present our preliminary studies on the site(s) of attachment of the 3H-probe in the α-subunit of hog kidney Na,K-ATPase. At least two peptides carrying the covalently linked 3H-probe were initially identified in the proteolytic digests of the α-subunit (Ahmed et al., 1986). We decided to attempt further purification of the peptide with apparently higher 3H incorporation, and to determine its amino acid sequence; fractionation and identification of other labeled peptide material has not yet been undertaken. The initial results presented here hint that 3H-probe binding in the peptide we have studied was largely in the extracellular domain between the M3 and M4 domains.

Methods

Covalent Binding of 3H-Probe to Na,K-ATPase

Hog kidney outer medulla Na,K-ATPase (activity of 1,200–1,500 µmol Pi/mg of protein per h) was prepared as described previously (Ahmed et al., 1983). About 20 mg of enzyme protein, diluted to 0.3 mg/ml in 5 mM MgCl_2, 3 mM Pi, 2 mM EDTA, and 30 mM Tris-HCl, pH 7.2 (to promote the type II binding), was mixed with an equal amount (10−6 M, based on estimated 10−6 M ouabain binding sites in the enzyme) of 3H-probe (specific radioactivity, 1.89 Ci/mmol). All other details to promote covalent binding of the probe and isolation of the 3H-probe-labeled enzyme were as before (Deffo et al., 1983). The final pellet of 3H-probe-labeled ATPase was dissolved in a 5% SDS buffer (5% wt/vol SDS, 40 mM Tris-HCl, and 1.0 mM EDTA, pH 7.5) for further processing (see Results and Discussion).

Amino Acid Sequence Determination

A model 470A gas phase sequencer (Applied Biosystems, Inc., Foster City, CA) equipped with a trifluoroacetic conversion system was employed using standard procedures. PTH amino acids were analyzed on a Micro-Flow HPLC system (Beckman Instruments, Inc., Fullerton, CA) equipped with an IBM cyano column. 3H-Peptide (10 µg or 1 nM of protein) was dissolved in 30 µl of 1% SDS, heated for 5 min at 100°C, and applied to the glass fiber membrane containing 3 mg Polybrene (Sigma Chemical Co., St. Louis, MO). The sequencer was programmed to run the standard ABI 02CPTH cycle (cartridge temperature, 45°C).

Results and Discussion

3H-Probe-labeled α-subunit of Na,K-ATPase was isolated from Na,K-ATPase labeled with 3H-probe by Sepharose 6B column chromatography (Fig. 1). The peak corresponding to the β-subunit (tubes 104–112) also appeared to contain radioactivity. However, SDS/polyacrylamide gel electrophoresis of the labeled ATPase demonstrated that the covalently bound radioactivity was present only in the α-subunit, and there was none in the β-subunit. This suggests that the radioactivity in the β-subunit in the column profile represented noncovalent association of the photolyzed 3H-probe. Also, we did not detect any intermolecular cross-linking of the α-subunit. Tubes (84–92) corresponding to the α-subunit were pooled and dialyzed exhaustively against 0.1 M NH_4HCO_3/0.02% NaN_3. The nondiffusible material was collected, diluted 10-fold with dialysis buffer, and subjected to tryptic digestion (1:50 trypsin/α-subunit) at 37°C for 24 h. The digestion was terminated by the addition of SDS to a concentration of 0.1% and the mixture was lyophilized. The material was dissolved in SDS buffer followed by fractionation on a Sephadex G-100 column (not shown) which yielded a peak of radioactivity. This pooled 3H-peptide material (~ 3 mg) was dissolved in 2.5 ml of a solution containing 8 M urea, 2% SDS, 0.5 M Tris-HCl (pH 6.8), and 2% EDTA. It was covered with Argon followed by the addition of 50 M excess of dithiothreitol, and kept for 4 h at room temperature. Then 0.33 ml of 1.0 N NaOH followed by 1.2 M excess of iodoacetic acid were added. After another 15 min it was desalted on a column of P4 resin equilibrated with 0.1 M NH_4Ac (pH 5.0). Fractions containing carboxymethylated peptide were pooled, lyophilized, and dissolved in H_2O. The latter steps were repeated twice more to remove all of the NH_4Ac. The material was then dissolved in 0.5 ml of 3% SDS followed by 3.5 ml of 83% formic acid plus 5% SDS. To this, 10 mg of CNBr in 70% formic acid was added, and the mixture was incubated at room temperature for 20 h. It was diluted 10–20-fold with H_2O and lyophilized. The latter step was repeated to remove all of the formic acid. The material was then chromatographed on a Sephadex G-100 column and the radioactive peak was collected and subjected to another tryptic digestion followed by Sephadex column chromatography (not shown). Finally, the pooled 3H-labeled peptide was chromatographed through a SDS-hydroxylapatite column (Fig. 2). As shown, most of the radioactivity was recovered in tubes 20–27, which were slightly shifted from the absorbance peak. A possible explanation of the shift in the radioactivity peak (observed consistently) is that the presence of the covalently bound probe would probably alter the affinity of the peptide toward hydroxylapatite and delay its release from the column. Sequence analysis of the pooled peptide and radioactive peak (tubes 15–27) is shown in Fig. 3. The amino acids 263 (I) through 289 (G) were identified directly in the sequence analysis. Sequence reproducibility was excellent, and sequencer repetitive yield through this region was determined to be 95%. The remaining PTH amino acid derivatives were collected and radioactivity in each fraction was determined. Since the sequence of the first 29 amino acids corresponded precisely to the segment starting with amino acid 263 in the derived sequence of the α-subunit of Na,K-ATPase, we assigned the subsequent labeled amino acid fractions the positions in this segment of the α-subunit, leading to the radioactivity profile of amino acids labeled with the 3H-probe shown in Fig. 3. These results hinted that, under type II binding conditions, the labeled probe was able to bind to the extracellular domain (amino acids 307–312) between the M3 and M4 hydrophobic domains. It is interesting to note that a small amount of radioactivity was also detected in two amino acids (V and A) located in the M3 domain.

Figure 1. Sepharose 6B chromatography of dissociated Na,K-ATPase labeled with 3H-probe. Na,K-ATPase labeled with the 3H-probe was taken up in a SDS buffer as described in the text. It was chromatographed through a Sepharose 6B column equilibrated with 50 mM Tris-HCl (pH 7.5), 0.2% SDS, and 1 mM EDTA. The column was eluted at a rate of 15 ml/h and 2-ml fractions were collected. A278nm () and cpm/25 µl () were determined for each fraction.

Figure 2. SDS-hydroxylapatite chromatography of partially purified 3H-peptide. The carboxymethylated, CNBr cleaved peptides were dissolved in 2 ml of 10 mM sodium phosphate (pH 6.4) and 2% SDS, and heated at 100°C for 2 min. The mixture was diluted 10-fold and applied to a hydroxylapatite column (0.8 × 6.5 cm) previously equilibrated with 10 mM sodium phosphate (pH 6.4) containing 0.1% SDS. Peptide material was eluted with a linear gradient of 0.2–0.5 M sodium phosphate containing 0.1% SDS in a total volume of 60 ml. 1-ml fractions were collected and the A226nm () and cpm/100 µl () were determined.

An advantage of the experimental probe used in the present work is that the photoactive group would be relatively rigid, and unlike bulky probes used previously, would tend to be more specific (Fullerton et al., 1986). However, we also observed that the labeling efficiency of this probe was ~ 10%, and considering the losses during procedures to isolate a labeled peptide, it was estimated that the final recovery was ~0.01%. On the other hand, the peptide material containing the labeled fraction did not suggest significant sequence heterogeneity. Based on this, it can be tentatively postulated that the region that appears to contain the labeled amino acids (as designated here) may play a role in ouabain binding. Because of the experimental limitations, at present it is not possible to assign, with any degree of certainty, a specific binding site(s) for the 3H-probe. In addition, as mentioned earlier, it is quite possible that other labeled peptide materials were present in the initial peptic digestion of the labeled α-subunit. These various observations need to be expanded in further studies.

Figure 3. Sequence analysis and release of 3H during sequencing of the 3H-peptide. Fractions containing PTH amino acids were dried in a Speed-Vac, dissolved in 100 µl of 50% acetonitrile/H_2O, and transferred to 2 ml of Aquasol, and radioactivity was determined in Beckman LS6800 scintillation spectrometer. Only cpm's > 50 were considered significant. Raw data (solid bars); data normalized to 95% repetitive yield over the 55 cycles sequenced (open bars). All other details are given in the text.

It is noteworthy that based on molecular biological studies of the digitalis binding sites, it was proposed that the domains between M1 and M2 and between M3 and M4 may both participate in digitalis binding and in determining digitalis sensitivity of the enzyme (Price and Lingrel, 1988). Additional site-directed mutagenesis studies have further emphasized the importance of certain amino acid residues in the extracellular domain between M1 and M2 for influencing ouabain sensitivity in cells (Lingrel et al., 1990). The experimental approach adopted by us, despite its difficulties, has the advantage that it may allow us to determine whether binding conditions influence the site(s) of attachment. We are currently synthesizing probes that should be useful in determining the nature of binding site(s) in the Na,K-ATPase for the sugar moiety of the cardiac glycosides. The combination of the various approaches may lead to a detailed definition of the various digitalis binding sites in Na,K-ATPase.

Acknowledgments

This work was supported in part by research grant HL-21457 awarded by the Heart, Lung, and Blood Institute, NIH, and by the Medical Research Fund of the U.S. Department of Veterans Affairs.

References

Ahmed, K., R. McParland, R. Becker, A. H. L. From, M. Schimerlik, and D. S. Fullerton. 1986. Studies on the digitalis binding site in Na,K-ATPase. Federation Proceedings. 45:1488. (Abstr.)

Ahmed, K., D. C. Rohrer, D. S. Fullerton, T. Deffo, E. Kitatsuji, and A. H. L. From. 1983. Interaction of Na+,K+-ATPase and digitalis genins: a general model for inhibitory activity. Journal of Biological Chemistry. 258:8092–8097.

Deffo, T., D. S. Fullerton, M. Kihara, R. H. McParland, R. R. Becker, B. M. Simat, A. H. L. From, K. Ahmed, and M. I. Schimerlik. 1983. Photoaffinity labeling of the sodium- and potassium-activated adenosinetriphosphatase with a cardiac glycoside containing the photoactive group on the C-17 side chain. Biochemistry. 22:6303–6309.

Fullerton, D. S., K. Ahmed, A. H. L. From, R. H. McParland, D. C. Rohrer, and J. F. Griffin. 1986. Modelling the cardiac steroid receptor. Topics in Molecular Pharmacology. 3:257–284.

Lingrel, J. B., J. Orlowski, E. Price, E. Jewell, O. Szamraj, B. Pathak, and P. Tyson. 1990. Structure function studies of Na,K-ATPase and regulation of the α-subunit genes. Journal of General Physiology. 96:1a. (Abstr.)

Price, E. M., and J. B. Lingrel. 1988. Structure-function relationships in the Na+,K+-ATPase α subunit: site directed mutagenesis of glutamine-111 to arginine and asparagine-122 to aspartic acid generates ouabain insensitive enzyme. Biochemistry. 27:8400–8408.

Shull, G. E., A. Schwartz, and J. B. Lingrel. 1985. Amino acid sequence of the catalytic subunit of the (Na+ + K+)ATPase deduced from complementary DNA. Nature. 316:691–695.

Yoda, A., and S. Yoda. 1974. Influence of certain ligands on the dissociation rate constants of cardiac glycoside complexes with sodium- and potassium-dependent adenosine triphosphatase. Molecular Pharmacology. 9:810–819.

Characterization of Carbohydrate in Na+,K+-ATPase β-Subunit from Electrophorus

W. Timothy Link, Paul M. Rowe, and R. Wayne Albers

Laboratory of Neurochemistry, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892

Introduction

A role for the β-subunit of the Na+,K+-ATPase in enzymatic or ion transport function has not been defined; however, there is increasing evidence that it is involved in membrane insertion of the α-subunit and/or transport to the plasma membrane (for review see McDonough et al., 1990). It has also been suggested that glycosylation of the β-subunit may play a role in stabilizing the αβ complex once it is formed (Zamofing et al., 1988). Experiments with rat brain (Sweadner and Gilkeson, 1985), chick neuron (Tamkun and Fambrough, 1986), and dog kidney (Miller and Farley, 1988) all present evidence for three sites of N-glycosylation in the β-subunit (β1 form). In this report we present evidence for two sites of glycosylation of the β-subunit in Electrophorus electric organ and preliminary results on the type of carbohydrate moieties and their locations on the peptide chain.

Methods

HPLC Preparation of β-Subunit

Crude membrane preparation of Electrophorus electric organ were solubilized in dodecyl-β-D-maltoside (DDM; 2 mg/mg protein) containing 100 mM NaCl, 0.1 mM EDTA, and 1.0 mM DTT, and centrifuged at 100,000 g for 10 min (Beckman Airfuge; Beckman Instruments, Inc., Palo Alto, CA). The resulting soluble fraction was chromatographed by size exclusion on Zorbax GF-450 and GF-250 columns (DuPont Co., Wilmington, DE) in tandem, equilibrated with 0.2 M sodium phosphate, pH 7.0, 0.1 mM DTT, and 0.03% DDM. The collected fractions were rechromatographed on the same system, with 0.1% SDS replacing the DDM, to obtain the dissociated α- and β-subunits. For experiments involving digestion with glycosidases, the intact β-subunit was used in this form. In experiments involving chemical or proteolytic cleavage of the protein, the β-subunit was first reduced with 40 mM DTT and 7 M guanidine HCl and then alkylated with 100 mM iodoacetamide.

Glycosidase Treatment

Sialic acid residues were removed by treatment with neuraminidase (from C. perfringens; Sigma Chemical Co., St. Louis, MO), 5.0 mU/µg protein, in 20 mM Tris-maleate buffer, pH 6.0.

N-Glycanase (Genzyme Corp., Boston, MA) was used to remove N-linked glycosyl chains. Digestion was carried out with 40 mU enzyme per µg β-subunit in 0.1 M Na+-Bicine buffer at pH 8.6. After enzyme treatment, samples were run on 10% PAGE/SDS gels and electroblotted to Immobilon-P membrane (Millipore Corp., Bedford, MA). Blocker and diluent for the blots consisted of 2% BSA and 0.05% Tween 20. Blots were stained using peroxidase-labeled lectins from Triticum vulgaris or Ricinus communis (25 µg/ml in PBS; Sigma Chemical Co.). After blocking, blots were lectin-stained for 1 h at 37°C and then washed in 0.1 M Tris. 0.05% Tween 20, and 0.5 M NaCl. Blots were developed using 4-chloronaphthol.

Protein Digestion and Isolation of Fragments

Enzymatic digestions of reduced and alkylated β-subunit were carried out using V8 protease or endoproteinase ARG-C (Endo ARG-C; Genzyme Corp.) in 1% ammonium bicarbonate. Enzyme concentration was 1% of total protein. Other samples of the protein were treated with 70% formic acid at 37°C for 72 h. After digestion, the samples were chromatographed on a C8 column with 300 Å pore size (Rainin Instrument Co. Inc., Woburn, MA). The column was pre-equilibrated with 0.1% TFA and 10% acetonitrile in water. Peptide fragments were eluted with a linear gradient of 10–80% acetonitrile (0.1% TFA) over 30 min at a flow rate of 0.6 ml/min.

Sequencing of Peptide Fragments

Peaks from reverse-phase chromatography were further purified on a C4 column (Aquapore; Applied Biosystems, Inc., Foster City, CA) at 0.3 ml/min. Peptides were eluted with a 20–80% linear gradient of acetonitrile (0.1% TFA) and sequenced on a pulse/liquid peptide sequencer (model 477A; Applied Biosystems, Inc.).

Probing of Peptide Fragments with Lectins

HPLC fractions corresponding to detected peaks were dot-blotted to PVDF paper strips by multiple application. The strips were then wetted with 30% acetonitrile and blocked in 2% BSA/0.05% Tween 20 for 1 h. Incubation with lectins and development were as above.

Results

Purification by size exclusion resulted in a single, diffuse band of 50 K apparent mol wt on 10% gels (Fig. 1 A). Treatment with neuraminidase for 1 h resulted in a lower molecular weight band corresponding to ~ 45 K (Fig. 1 B, lane 1). No further observable decrease in molecular weight was apparent after 12 h of treatment (Fig. 1 B, lane 2) compared with 1 h. The increase in electrophoretic mobility is consistent with the removal of terminal sialic acid residues.

Digestion with N-glycanase resulted in the appearance of two lower molecular weight bands of ~ 40 K and 35 K, indicating the loss of at least two glycosyl chains, of ~ 5 K each, as shown in Fig. 1 C. A similar, two-step deglycosylation was observed after digestion with endoglycosidase F (not illustrated). Residual, intact β is apparent in lane 1 (4 h of treatment) but disappears after extended incubation (12 or 24 h; lanes 2 and 3, respectively). The middle (40 K) band is still sensitive to neuraminidase treatment as shown in Fig. 1 D, demonstrating that carbohydrate is still present. This band is highly resistant to treatment with N-glycanase, as indicated by its persistence after 24 h of treatment. Treatment periods > 36 h are required for most of the 40 K form to be degraded to the 35 K form (data not shown). The lower band (35 K) is insensitive to either enzymatic treatment, suggesting that it is the most deglycosylated form of the β-subunit.

Staining with carbohydrate-specific lectins revealed two important features: that treatment with neuraminidase exposed sites of high affinity for T. vulgaris and R. communis lectins, and that these sites are confined to separate carbohydrate chains. As shown in Fig. 2, lectin staining of the intact, untreated β-subunit (lane 1 of B and C) or subunit treated with N-glycanase alone (lane 2 of B and C) is absent; however, both lectins stain the neuraminidase-treated subunit (lane 3 of both B and C). After treatment with N-glycanase and neuraminidase, the staining by T. vulgaris (lane 4 of B) is lost, while staining by R. communis (lane 4 of C) is still present on the middle (40 K) band. This demonstrates that the carbohydrate chain with high affinity for T. vulgaris (specificity for N-acetylglucosamine) is lost in the first deglycosylation step. The staining for R. communis (specificity for β-D-galactose) is lost in the subsequent deglycosylation step as indicated by the absence of its staining of the lowest (35 K) band.

Figure 1. All lanes contain 2 µg of HPLC-purified β-subunit, run on 10% PAGE/SDS gels and stained with Commassie. (A) Untreated subunit. (B) Lanes 1 and 2 are treated with neuraminidase for 1 or 12 h, respectively. (C) Lanes 1–3 are after 4, 12, or 24 h of digestion with N-glycanase, respectively. (D) Lanes 1–3 are 4, 12, and 24 h of N-glycanase followed by 1 h of neuraminidase treatment.

Based on the above observations of differential lectin staining of glycosyl chains on the protein, we are currently attempting to determine the positions of these chains on the three potential N-glycosylation sites present on the subunit, as deduced from the Torpedo β sequence. We have obtained reproducibly generated fragments of the protein after proteolytic cleavage with V8 protease and Endo ARG-C, and also by mild acid cleavage. As indicated by our initial dot blots of HPLC peaks, the major fragments generated by both V8 and mild acid treatment contain both lectin-specific glycosyl chains. We are currently evaluating the fragmentation pattern generated by Endo ARG-C. The large fragment generated by mild acid cleavage has been sequenced twice and corresponds with high homology to the Torpedo sequence beginning with glycine (No. 172 in Torpedo). This fragment is of unknown length because the sequencing yield falls off abruptly at asparagine residue 193 (second potential N-glycosylation site). These preliminary data suggest that the lectin-specific carbohydrate chains are present on asparagine residues 193 and 266. We cannot rule out the first site (158) until we have obtained a fragment containing this residue. By using the major fragments generated in the above fashion as starting material, we should be able to cleave between residues 193 and 266 and separately probe these peptides to resolve this question.

Figure 2. A, B, and C are PVDF blots of 5 µg of β-subunit protein run on 10% PAGE/SDS gels after treatment as follows: untreated (lane 1); treated with N-glycanase for 36 h (lane 2); neuraminidase-treated for 1 h (lane 3); N-glycanase for 36 h followed by neuraminidase for 1 h (lane 4). Group A was stained with mouse anti-β serum (1:50,000) and shows all the intermediate forms. Group B was stained with peroxidase-labeled lectin from T. vulgaris (25 µg/ml). Group C was stained with peroxidase-labeled lectin from R. communis (25 µg/ml).

Summary

In this report we have demonstrated through lectin mapping that the β-subunit of Electrophorus may contain only two carbohydrate sidechains, in contrast to the pattern of three reported in other species. These chains can be readily distinguished from one another by differences in lectin binding and are cleaved at different rates by glycosidases. Using similar characterization techniques of other β-subunits, a meaningful pattern of glycosylation may be detected that could be useful in clarifying the role of this process and its contribution toward expression and/or function of the Na+,K+-ATPase.

References

McDonough, A. A., K. Geering, and R. A. Farley. 1990. The sodium pump needs its β subunit. Journal of the Federation of Applied Science and Experimental Biology. 4:1598–1605.

Miller, R. P., and R. A. Farley. 1988. All three potential N-glycosylation sites of the dog kidney (Na+ + K+)-ATPase β subunit contain oligosaccharide. Biochimica et Biophysica Acta. 954:50–57.

Sweadner, K. J., and R. C. Gilkeson. 1985. Two isozymes of the Na,K-ATPase have distinct antigenic determinants. Journal of Biological Chemistry. 260:9016–9022.

Tamkun, M. M., and D. M. Fambrough. 1986. The (Na+ + K+)-ATPase of chick sensory neurons. Journal of Biological Chemistry. 261:1009–1019.

Zamofing, D., B. C. Rossier, and K. Geering. 1988. Role of the Na,K-ATPase beta subunit in the cellular accumulation and maturation of the enzyme as assessed by glycosylation inhibitors. Journal of Membrane Biology. 104:69–79.

The Na-Pump α-Subunit Has N-linked Glycans Facing the Cytosol

Carlos H. Pedemonte and Jack H. Kaplan

Department of Physiology, University of Pennsylvania, Philadelphia, Pennsylvania 19104

Posttranslational processing of the Na-pump polypeptides is currently an area of great interest, and several investigations have been performed with the implicit assumption that the α-subunit has no N-linked oligosaccharides or no glycosides at all (Tamkun and Fambrough, 1986; Zamofing et al., 1988). The effects of inhibitors of glycosylation on the protein synthesis and membrane insertion have been interpreted solely in terms of effects on the β-subunit processing (Zamofing et al., 1988). The glycoprotein nature of the Na-pump β-subunit has been widely recognized; however, the glycosylation status of the α-subunit is controversial. The presence of carbohydrates in the α-subunit of the Na-pump from various species has been described; however, negative results have also been reported (for references, see Pedemonte et al., 1990). These differences may be due to the sensitivity of the methods used to detect the presence of the oligosaccharides. Therefore, it was interesting and timely to reinvestigate the glycosylation status of the Na-pump α-subunit.

We have used a very sensitive method to label the sugar moieties bound to the Na-pump subunits with radioactive galactose. This involves the use of UDP-[3H]Gal and bovine milk galactosyltransferase. This enzyme specifically catalyzes the addition of Gal, in a β1-4 linkage, to terminal N-acetyl glucosamine residues of protein and lipids (Magee et al., 1974). Separation of the protein in Polyacrylamide gel electrophoresis (PAGE) showed that both subunits of the Na-pump were labeled. Therefore, both subunits have attached oligosaccharides with terminal GlcNAc units. That the α-subunit contains bound oligosaccharides was proven by other independent experiments: the enzyme subunits were separated in PAGE and electroblotted to a nitrocellulose membrane, and the nitrocellulose membrane was treated with Concanavalin A (Con A). The α-subunit showed a positive reaction to Con A, indicating the presence of oligosaccharides. The reaction was prevented by a high concentration of glucose in the medium. Curiously, the β-subunit barely reacted with Con A. This lectin reacts specifically with α-mannosyl and α-glucosyl, and much less with GlcNAc, at the nonreducing ends of carbohydrate chains. Therefore, one or more of these groups are more exposed in the α-subunit than in the β-subunit after electroblotting. Omori et al. (1983) observed that both the α- and β-subunits of dog kidney Na-pump were not bound by a Con A–Sepharose affinity column. In the same way, we were unable to precipitate any of either subunit with Con A–Sepharose. Thus, the Con A–sensitive carbohydrates of the α-subunit are more exposed in the electroblotted nitrocellulose membrane than in the soluble condition used during the affinity precipitation.

We used right-side-out vesicles (RSOV), prepared from dog kidney outer medulla microsomes (Forbush, 1982), to determine the orientation of the galactose acceptor sites with respect to the cell membrane. The RSOV were at least 85% sealed as determined from their latent Na,K-ATPase activity. The RSOV were treated with UDP-[3H]Gal in the presence or absence of exogenous galactosyltransferase. This enzyme does not cross the cell membrane; therefore, unless the RSOV were permeabilized, only the extracellular-facing oligosaccharides would be labeled. This treatment resulted in a four- to eightfold increased incorporation of radioactivity in vesicles that were permeabilized by saponin before radiolabeling compared with that observed in intact vesicles. The observation that upon permeabilization many additional acceptor sites in the α-subunit became available for galactosylation indicates that most of the galactose acceptor sites in the Na-pump α-subunit are facing the cytosolic side of the cell membrane. The only difference between the Na-pump in intact vesicles and in vesicles that have been exposed to saponin is the access of impermeant reagents to their cytoplasmic surface (Kyte et al., 1987). To demonstrate that the higher labeling is due to the opening of the RSOV and not to the direct action of saponin on the Na-pump, other physical methods were used to permeabilize the vesicles. These included sonication, freezing and thawing, and passage though a French press. The vesicles were very resistant, and under optimum treatment conditions with the French press and sonication, we were only able to increase permeabilization (measured as Na,K-ATPase activity) twofold. In both cases, a close correlation of 1.8–2-fold increased [3H]Gal labeling was observed. As an alternative to saponin we used SDS, an anionic detergent. Even though both saponin and SDS act by a detergent action, they have very different structures and the sensitivity of the Na,K-ATPase to activation and inhibition by each of the reagents is very different (Kyte et al., 1987). As previously observed with saponin, treatment of the vesicles with SDS increased sixfold the labeling of the sugar moieties bound to the α-subunit of the Na-pump. Thus, these experiments confirm our conclusion that the sugar moieties bound to the α-subunit of the Na-pump are oriented toward the cytosol.

The possibility that O-linked sugars are bound to the Na-pump α-subunit was analyzed by β-elimination (Holt et al., 1987; Capasso et al., 1988). The Na-pump protein was radiolabeled using UDP-[3H]Gal and galactosyltransferase. The [3H]galactose-labeled α-subunit was separated by PAGE and excised from the gel, and the pieces of gel containing the α-subunit were treated with 1 M NaBH_4 in 50 mM NaOH, which under the conditions used preferentially release O-linked carbohydrates (Torres and Hart, 1984). More than 90% of the radioactivity was solubilized with this treatment. The soluble material was passed through a Sephadex-G50 column. The radioactive sugars were collected from the column with elution volumes that correspond to molecular weights higher than those of free carbohydrate chains. This means that most of the radiolabeled carbohydrates remain bound to soluble polypeptides produced by alkaline degradation of the α-subunit. Therefore, at least 90% of the radiolabeled oligosaccharide chain(s) bound to the Na-pump α-subunit are not accessible to mild alkaline degradation or, alternatively, are N-linked to the polypeptide.

The possibility that the radiolabeled sugars are N-linked to the α-subunit was examined by treating the Na pump polypeptides with glycopeptidase F (PNGase), which specifically hydrolyzes this kind of oligosaccharide–protein linkage. Increasing concentrations of PNGase produced a progressive reduction of the β-subunit apparent molecular weight in PAGE without affecting the mobility of the α-subunit at all. The fact that the mobility of the α-subunit in PAGE was not affected by the PNGase treatment might suggest that the radiolabeled oligosaccharides are not N-linked to the polypeptide. However, a fluorogram of the gel indicated that the radioactivity bound to the α-subunit was lost after treatment with PNGase. This clearly indicates the N-linkage of the sugars to the α-subunit.

In conclusion, our results indicate that the Na-pump α-subunit is a glycoprotein with the carbohydrates facing the cytosolic medium. This unusual topographic arrangement of the oligosaccharides has also recently been detected in some integral membrane proteins of endoplasmic reticulum (Abeijon and Hirschberg, 1988) and Golgi (Capasso et al., 1988). However, contrary to what has been reported for these membrane proteins, the Na-pump α-subunit presents N-bound oligosaccharides as a novel kind of linkage of the cytosolic-facing carbohydrates. This finding is in agreement with the orientation with respect to the membrane of the asparagines in the α-subunit that are candidates for N-glycosylation. N-linked oligosaccharides bind to asparagine residues that are always in the sequence Asn-X-Ser (or Thr), where X may be any amino acid, except perhaps aspartic or proline (Hart et al., 1979). The Na-pump α-subunit has five consensus sequences for glycosylation at asparagines 208, 398, 479, 631, and 713 in the primary structure of the sheep kidney Na,K-ATPase (Shull et al., 1985). The protein from dog kidney, with which our experiments were performed, has an additional consensus sequence at asparagine 493 (Tran et al., 1988). All six are located in what are considered to be cytoplasmic loops of the α-subunit polypeptide.

Another interesting observation that emerges from our results relates to the size of the carbohydrate moieties N-linked to the α-subunit. It is very well known that the Na-pump β-subunit has three large N-bound oligosaccharide moieties, which accounts for the difference in molecular weight of the polypeptide calculated from its cDNA and that observed in gel electrophoresis (Tamkun and Fambrough, 1986). In PAGE the β-subunit shows an apparent molecular mass ~ 20 kD higher than that corresponding to its amino acid content. Removal of each of its oligosaccharide moieties produces a reduction of ~7 kD in the apparent molecular mass (Pedemonte et al., 1990). On the contrary, the same treatment does not produce any noticeable change in the α-subunit apparent molecular mass. Since a change in molecular mass of as little as 2 kD would be observed under the present experimental conditions, it must be concluded that the N-linked oligosaccharides bound to the α-subunit are small. This is in agreement with the observation of Omori et al. (1983) that the carbohydrate content of the dog kidney Na-pump α-subunit is < 1% (wt/wt). This also conforms with the observation that in PAGE the α-subunit polypeptide presents a molecular mass that is similar to that predicted from its amino acid composition; and, contrary to what is observed with the β-subunit, the α-subunit did not change its apparent molecular mass after PNGase treatment. Therefore, the size of the oligosaccharide chains bound to Na-pump α-subunit must be smaller than those previously described for N-linked carbohydrate chains. Preliminary results appear to indicate that a single carbohydrate molecule is bound to the polypeptide.

The Na-pump α-subunit is the first glycoprotein described that has N-linked carbohydrates facing the cytoplasmic medium. This kind of protein glycosylation cannot be explained by the current hypothesis of glycoprotein biosynthesis, and a new model will be necessary to account for our observations.

Acknowledgments

This work was supported by NIH grants HL-30315 and GM-39500.

References

Abeijon, C., and C. B. Hischberg. 1988. Intrinsic membrane glycoproteins with cytosol-oriented sugars in the endoplasmic reticulum. Proceedings of the National Academy of Sciences, USA. 85:1010–1014.

Capasso, M. J., C. Abeijon, and C. B. Hirschberg. 1988. An intrinsic membrane glycoprotein of the Golgi apparatus with O-linked N-acetylglucosamine facing the cytosol. Journal of Biological Chemistry. 263:19782–19788.

Forbush, B. 1982. Characterization of right-side-out membrane vesicles rich in (Na,K)-ATPase and isolated from dog kidney outer medulla. Journal of Biological Chemistry. 257:12678–12684.

Hart, G. W., K. Brew, G. A. Grant, R. A. Bradshaw, and W. J. Lennarz. 1979. Primary structural requirements for the enzymatic formation of the N-glycosidic bond in glycoproteins. Journal of Biological Chemistry. 254:9747–9753.

Holt, G. D., R. S. Haltiwanger, C. R. Torres, and G. W. Hart. 1987. Erythrocytes contain cytoplasmic glycoproteins. Journal of Biological Chemistry. 262:14847–14850.

Kyte, J., K. Xu, and R. Bayer. 1987. Demonstration that lysine-501 of the a polypeptide of native sodium and potassium ion activated adenosine triphosphatase is located on its cytoplasmic surface. Biochemistry. 26:8350–8360.

Magee, S. C., R. Mawal, and K. E. Ebner. 1974. Biochemistry. 13:99–102.

Omori, K., S. Takemura, K. Omori, T. Mega, and Y. Tahiro. 1983. Isolation of the α and β subunits of canine (Na,K)ATPase by using SDS-PAGE and lectin-Sepharose. Journal of Biochemistry. 94:1857–1866.

Pedemonte, C. H., G. Sachs, and J. H. Kaplan. 1990. An intrinsic membrane glycoprotein with cytosolic oriented N-linked sugars. Proceedings of the National Academy of Sciences, USA. 87:9789–9793.

Shull, G. E., A. Schwartz, and J. B. Lingrel. 1985. Amino-acid sequence of the catalytic subunit of the (Na + K)ATPase deduced from a cDNA. Nature. 316:691–695.

Tamkun, M. M., and D. M. Fambrough. 1986. The (Na + K)-ATPase of chick sensory neurons. Journal of Biological Chemistry. 261:1009–1019.

Torres, C. R., and G. W. Hart. 1984. Polypeptide distribution of terminal acetylglucosamine residues on the surfaces of intact lymphocytes. Journal of Biological Chemistry. 259:3308–3317.

Tran, C. M., G. Scheiner-Bobis, W. Schoner, and R. A. Farley. 1988. The amino acid sequence of a peptide from the ATP-binding site of Na-K-ATPase labeled by 8-N_3-ATP. Biophysical Journal. 53:343a. (Abstr.)

Zamofing, D., B. C. Rossier, and K. Geering. 1988. Inhibition of N-glycosylation affects transepithelial Na but not Na-K-ATPase transport. Journal of Membrane Biology. 104:69–79.

Reaction Kinetics and Transport Modes

Steady-State Phosphorylation and Dephosphorylation of Na,K-ATPase in the Presence of Na+ and Na+ + K+

Jens Chr. Skou

Institute of Biophysics, Århus University, 8000 Århus C, Denmark

Introduction

In experiments on ligand effects on the rate of dephosphorylation of preformed phosphoenzyme of the Na,K-ATPase, the phosphorylation from AT^32P is usually stopped by addition of an excess of cold ATP. A problem is that in this procedure the reaction may be perturbed. In a discussion of the acid-stable phosphoenzyme as an intermediate in the normal reaction with Na+ + K+, I. M. Glynn at the 1987 conference on the Na-K-pump suggested that a way to circumvent the problem of perturbation is to let the enzyme react with cold ATP in the presence of the ligands, and at steady state add tracer amounts of AT^32P. Provided the phosphoenzyme is an intermediate, the initial rate of formation of E-32P should equal the rate of the overall hydrolysis (Glynn, 1988).

There is, however, a requirement to be fulfilled in order for this to be valid. Either the steady-state concentration of EATP must be negligible compared with the steady-state concentration of E-P, or the dissociation rate for ATP from EATP must be high compared with the rate of phosphorylation in order for the added AT^32P to give an immediate stop of formation of E-P from the cold ATP. As seen from the following, neither is the case in the presence of Na+ + K+, while in the presence of Na+ the steady-state concentration of EATP is negligible. Unfortunately, a comparison between the rate of formation of E-32P and the overall rate of hydrolysis in the presence of Na+ + K+ can therefore not give a clear-cut answer to the question of the relationship between phosphorylation and hydrolysis, only in the presence of Na+.

Methods

The enzyme was from rectal gland from shark (Squalus acanthias). The specific activity at 37°C with optimum ligand concentrations was 30 U/mg protein. The experiments were performed at 0°C in 30 mM histidine HCl, pH 7.4. In the experiments with Na+ but no K+, enzyme was reacted with 0.025 mM ATP in the presence of 150 mM Na+ and 1 mM Mg2+. At steady state (after 10 s) tracer amounts of AT^32P were added and the rate of formation of E-32P and release of 32P was followed. In the experiments with Na+ + K+ the enzyme was reacted with 0.025 mM ATP, 130 mM Na+, 20 mM K+, and 1 mM Mg2+ in a rapid mixing apparatus and at steady state (after 183 ms) mixed with a solution containing the same concentrations of ATP and ligands but with tracer amounts of AT^32P. The reaction was stopped at different time intervals by mixing with 5% trichloroacetic acid containing 1 mM pyrophosphate. The precipitated enzyme was washed and E-32P was measured as described by Klodos et al. (1981). 32P was measured by the method of Lindberg and Ernster (1956).

Results

Effect of Na+

Fig. 1 shows the results of an experiment in which tracer amounts of AT^32P were added after the reaction of enzyme with 0.025 mM ATP, 150 mM Na+, and 1 mM Mg2+ had come to steady state at 0°C.

After a delay the release of 32P became rectilinear with time and the slope gave the steady-state rate of hydrolysis, 0.77 nmol/mg protein per s. This must be equal to the steady-state release of Pi before the AT^32P is added, and this is given by a line through zero parallel with the rectilinear part of the 32P release. The ordinate value of the difference between these two lines represents the amount of Pi released after addition of AT^32P and amounts to 2.4 nmol/mg protein. The steady-state level of E-32P is 2.7 nmol/mg protein. The difference between these two values is inside the error of determination (mean of three determinations of the difference is 2.68 ± 0.25 (SEM) and of E-32P 2.67 ± 0.03). The amount of Pi released after the addition of the tracer is thus equal to the steady-state value of the phosphoenzyme. When the Pi release is plotted on the same scale as the E-32P (the left curve for the Pi release) it is seen that the initial rate of E-32P formation is equal to the rate of Pi release. As the rate of formation of E-32P in steady state is equal to the rate of dephosphorylation, this indicates that the dephosphorylation rate can account for the overall rate of hydrolysis, and that the phosphoenzyme is an intermediate in the hydrolysis reaction. The E-32P formation follows a hyperbolic curve and the observed rate constant is 0.27 s–1. With a steady-state concentration of E-P of 2.67 nmol/mg protein this gives a dephosphorylation rate of 0.72 nmol/mg protein per s, which with the error of determination is equal to the 0.77 nmol/mg protein per s measured from the slope of the 32P release.

Figure 1. Rate of formation of E-32P and release of 32P after tracer amounts of AT^32P in steady state (after 10 s) is added to enzyme in the presence of 150 mM Na+, 1 mM Mg2+, 0.025 mM ATP, and 30 mM histidine HCl, pH 7.4, 0°C. See text for explanation.

Effect of Na+ + K+

The results of an experiment with 130 mM Na+, 20 mM K+, 1 mM Mg2+, and 0.025 mM ATP at 0°C, pH 7.4, are given in Fig. 2. The slope of the linear part of the 32P release gives a hydrolysis rate of 15.8 nmol/mg protein per s. It is seen (cf. explanation to Fig. 1) that the amount of Pi released after addition of the tracer is 1.1 nmol/mg protein (1.0 and 1.2 nmol in other experiments) compared with the steady-state level of E-32P of 0.35 nmol/mg protein (E-Pmax of 2.7 nmol/mg protein). Furthermore, when the Pi release is plotted on the same scale as the E-32P (the left curve for the Pi release) it is seen that the initial rate of formation of E-32P is much lower than the overall rate of Pi release. When the enzyme under the same ionic conditions is reacted with ATP^32 and the phosphorylation is stopped by addition of cold ATP, the 32P release is 1.3 nmol/mg protein with a steady-state concentration of E-32P of 0.35 nmol/mg protein (not shown). In a cold to hot experiment with 0.005 mM ATP the Pi release is 0.37 nmol/mg with a steady-state E-P concentration of 0.14 nmol/mg, which shows that the amount of extra released Pi decreases when the ATP concentration is decreased.

Figure 2. Rate of formation of E-32P and release of 32P after tracer amounts of AT^32P in steady state (after 183 ms) is added to enzyme in the presence of 130 mM Na+, 20 mM K+, 0.025 mM ATP, and 30 mM histidine HCl, pH 7.4,0°C. See text for explanation.

A Pi release that is higher than the steady-state level of phosphoenzyme must mean either that there is a source for Pi release outside the phosphoenzyme or that there is a precursor for formation of the phosphoenzyme, and that the procedures used do not stop the continued formation of phosphoenzyme from the precursor. A precursor could be EATP with an off rate for ATP, which is too low compared with the rate of phosphorylation in order for the added AT^32P in the cold to hot experiment, and the added cold ATP in the hot to cold experiment to give an immediate stop of phosphorylation from the cold respectively hot ATP.

That this is the case is seen from Fig. 3. The enzyme was phosphorylated (in a rapid mixing apparatus) in the presence of 150 mM Na+, 1 mM Mg2+, and 0.025 mM AT^32P at 10°C. At the time where about half of the enzyme was phosphorylated (14 ms), 5 mM cold ATP was added. As seen, this did not result in an immediate stop of phosphorylation (addition of trichloroacetic acid instead of ATP gives an immediate stop).

Figure 3. Effect of addition of 5 mM ATP on phosphorylation of enzyme from 0.025 mM AT^32P with 150 mM Na+, 1 mM Mg2+ at 10°C. The cold ATP was added (in a rapid mixing apparatus) after the enzyme had reacted for 14 ms with AT^32P (zero time). The reaction was stopped after different time intervals and the E-32P was determined. E-Pmax was 2.6 nmol/mg protein.

Discussion

As pointed out by Glynn, there are a number of experiments that suggest that the acid-stable phosphoenzyme is involved in the normal cycle of the Na+,K+-ATPase in the presence of Na+ + K+. A problem, however, is that none of the more direct experiments on the effect of K+ on dephosphorylation has been able to give a clear-cut answer (Glynn, 1988). This may be due to perturbation of the reaction by the procedures used to stop phosphorylation, or to the fact that the experiments as they are normally performed with the cations added consecutively cannot give an answer to the hydrolysis reaction in which both cations are present simultaneously. However, as seen from the present experiments, even when these two potential errors are eliminated it is not possible to get an answer. The explanation seems to be that a relatively large fraction of the enzyme in steady state in the presence of Na+ plus K+ is in the EATP form and that the off rate for ATP is too low for the added hot ATP to stop a continued phosphorylation from the cold ATP. This also explains why the cold to hot method can be used with Na+ but not K+. With Na+ practically all the enzyme is in the phosphoform in steady state, with very little left for EATP.

In the experiment shown in Fig. 2, the Pi that comes from the precursor is 1.1 – 0.35 = 0.75 nmol/mg. As not all of the EATP is converted to phosphoenzyme, since a part must exchange cold ATP for hot, the amount of EATP in steady state must be higher than the 0.75 nmol/mg (E-Pmax is 2.7 nmol/mg). With 0.005 mM ATP the extra release is 0.37 –0.14 = 0.22 nmol/mg protein. The increase in the steady-state amount of EATP with the ATP concentration suggests that the rate-limiting step shifts with an increase in the ATP concentration from deocclusion of K+ to phosphorylation, which probably means occlusion of Na+.

References

Glynn, I. M. 1988. The coupling of enzymatic steps to the translocation of sodium and potassium. In The Na+,K+-pump. Part A: Molecular Aspects. J. C. Skou, J. G. Nørby, A. B. Maunsbach, and M. Esmann, editors. Alan R. Liss, Inc., New York. 435–460.

Klodos, I., J. G. Nørby, and I. G. Plesner. 1981. The steady-state kinetic mechanism of ATP hydrolysis catalyzed by membrane-bound (Na+ + K+)-ATPase from ox brain. II. Kinetic characterization of phosphointermediates. Biochimica et Biophysica Acta. 643:463–482.

Lindberg, O., and L. Ernster. 1956. Determination of organic phosphorus compounds by phosphate analysis. Methods in Biochemical Analysis. 3:1–22.

Effect of Oligomycin on the Rate of Phosphorylation of Na,K-ATPase in the Presence of Na+

Jens Chr. Skou

Institute of Biophysics, University of Århus, 8000 Århus C, Denmark

Introduction

Phosphorylation of the Na,K-ATPase leads to occlusion of Na+ on the ADP-sensitive phosphoenzyme (Glynn et al., 1984). By “occlusion” it is meant that bound Na+ has a low rate of exchange with Na+ in the medium. It probably means that Na+ has been transferred to the membrane phase. It seems likely that the transition of phosphate from ATP to the enzyme is a fast reaction, while occlusion, which must involve a conformational transition, is a slow reaction. A comparison between the rate of phosphorylation with and without Na+ preoccluded may give information on the step at which occlusion occurs in the phosphorylation reaction. Is occlusion rate limiting for formation of the ADP-sensitive phosphoenzyme, or does occlusion follow from formation of the phosphoenzyme?

Methods

The enzyme is from rectal glands from shark (Squalus acanthias). The specific activity is 35 U/mg protein at 37°C with optimum ligand concentrations. The Na-ATPase activity at 0°C is 1.1 nmol/mg protein per s. Na+ is preoccluded on the nonphosphorylated enzyme by preincubation with oligomycin (Esmann and Skou, 1985), 20 μg/ml at room temperature for 2 min. Thereafter the temperature is decreased to 0°C and the phosphorylation is started by mixing enzyme and Na+ in one syringe with Mg2+ and AT^32P in another in a fast mixing apparatus. Phosphorylation is stopped by mixing with 5% trichloroacetic acid with 1 mM pyrophosphate. The precipitated enzyme is washed, and E-32P is determined as described by Klodos et al. (1981). Parallel experiments are performed without oligomycin.

Results

With 150 mM Na+ at 0°C without oligomycin the rate of formation of the phosphoenzyme increases with the ATP concentration up to 100 μM and the observed maximum rate is ~23 s-1. This is shown in Fig. 1 in a semilogarithmic plot as the rate of disappearance of nonphosphorylated enzyme. When enzyme is preincubated with Na+ in the presence of oligomycin, the rate of phosphorylation also increases with the ATP concentration up to 100 μM (Fig. 2). But at each of the ATP concentrations the rate is higher and the maximum rate is ~ 81 s-1. K0.5 for ATP for the increase in the observed rate of phosphorylation is ~ 8 μM without oligomycin, while it is higher, ~ 14 μM, in the presence of oligomycin. Oligomycin increases the affinity for Na+ for the effect on the rate of phosphorylation. With 25 μM ATP without oligomycin the observed rate is 12 s-1 with 5 mM Na+, 28 s-1 with 30 mM Na+, and 20 s-1 with 150 mM Na+. With oligomycin the values are the same, 77 s-1, with 5 mM Na+ and with 30 mM Na+, while with 150 mM Na+ it is 53 s-1. 6 mM Na+ thus gives about a half-maximal effect without oligomycin, while with oligomycin the half-maximal value must be obtained with no more than half of the 5 mM Na+ (the lowest tested).

Figure 1. Rate of formation of phosphoenzyme as a function of the ATP concentration in the presence of 150 mM Na+, 1 mM Mg2+, and 30 mM histidine HCl, pH 7.4,0°C. This is shown in a semilogarithmic plot as the disappearance of nonphosphorylated enzyme. The ordinate is percent of maximum of E-P (Et, in the figure), which is 3.1 nmol mg protein-1; the abscissa is milliseconds.

Discussion

In the present experiments we measured the sum of all acid-stable phosphoenzymes, ADP-as well as K+-sensitive. The rate of dephosphorylation is very low in the presence of Na+. With the enzyme used in the present experiments it is 0.35 s-1 at 0°C, while the rate of phosphorylation with saturating ATP and 150 mM Na+ is 23 s-1. Oligomycin decreases the rate of dephosphorylation, but neither this, nor a decreased rate of transition from the ADP-sensitive to the K+-sensitive phosphoenzyme, can explain the observed higher rate of phosphorylation in the presence of oligomycin. It must be due to an increased rate of formation of the ADP-sensitive phosphoenzyme.

An increased rate of phosphorylation could be due to an effect on the phosphotransfer reaction as such, an increased rate of transition of phosphate from ATP to the enzyme. There are no known indications that oligomycin has such an effect. However, it is known, as mentioned above, that phosphorylation leads to occlusion of Na+ and that oligomycin occludes Na+ on the nonphosphorylated enzyme. It seems likely, therefore, that the increased rate of phosphorylation in the presence of oligomycin is due to the preocclusion of Na+. This means that occlusion of Na+ is rate limiting in formation of the phosphoenzyme and that occlusion does not follow formation of the phosphoenzyme but must at the latest be simultaneous with the formation.

Figure 2. As in Fig. 1, but the enzyme has been preincubated with 150 mM Na+ in the presence of 20 μg oligomycin/ml.

An alternative is that occlusion precedes formation of the phosphoenzyme; i.e., it is occlusion of Na+ which confers phosphotransferase activity to the enzyme.

References

Esmann, M., and J. C. Skou. 1985. Occlusion of Na+ by the Na,K-ATPase in the presence of oligomycin. Biochemical and Biophysical Research Communications. 127:857–863.

Glynn, I. M., Y. Hara, and D. E. Richards. 1984. The occlusion of sodium ions within the mammalian sodium-potassium pump: its role in sodium transport. Journal of Physiology. 351:531–547.

Klodos, I., J. G. Nørby, and I. G. Plesner. 1981. The steady-state kinetic mechanism of ATP hydrolysis catalyzed by membrane-bound (Na+ + K+)-ATPase from ox brain. II. Kinetic characterization of phosphointermediates. Biochimica et Biophysica Acta. 643:463–482.

Anion Effects on the Steady-State Ratio of the Phosphoenzymes of Na,K-ATPase as Measured by Dephosphorylation and Oligomycin Inhibition

Irena Klodos and Liselotte Plesner

Institute of Biophysics, University of Aarhus, DK-8000 Aarhus C, Denmark

Introduction

The phosphoenzymes of the Na,K-ATPase exist in two major forms: the ADP-sensitive E_1P and the K-sensitive E_2P. It has been generally accepted that the steady-state ratio of E_1P/E_2P is a function of the Na+-concentration, but recently evidence was presented that the anions are also of importance (Post, 1988; Post and Suzuki, 1991). According to Post, more lyotropic (chaotropic) anions like Br- or SCN- increase the E_1P/E_2P ratio, whereas less lyotropic (kosmotropic) anions like SO2-4 and acetate decrease the ratio. The ratio was estimated from ADP- or K-chase experiments, and the salt composition during the chase was the same as in the steady state. Further studies of the anion effects have recently revealed (Klodos, 1991) that the salt composition of the chase medium affects the observed dephosphorylation pattern; i.e., the measured K-sensitivity of EP was not only a function of the steady-state conditions, but was also affected by the chase conditions. This raises the question of whether the anion effect observed by Post reflected an anion-induced change in the steady-state ratio of E_1P/E_2P, or, instead, was a function of the anions present in the dephosphorylation medium. In this paper we applied two methods of evaluation of EP composition. In the first, the dephosphorylation kinetics were studied in experiments where the anion composition of the phosphorylation and the dephosphorylation media were varied independently. In the second, oligomycin inhibition of ATP hydrolysis was used as a measure of the steady-state amount of E_1P present in media differing with respect to salt composition.

Materials and Methods

The experiments were performed with ox brain Na,K-ATPase (Klodos et al., 1975) with a specific activity of 4.7 U/mg protein.

Na,K-ATPase was phosphorylated for 60 s at 0°C in 3 ml of medium containing 0.1 mM MgCl_2, 25 μM AT^32P, 30 mM imidazole buffer at pH 7.4, and 600 mM Na+. The anion was either Br- or SO2-4. At time zero the dephosphorylation was started by the addition of 3 ml of a chase containing 1 mM unlabeled ATP and 20 mM KCl. The concentration of Na+ was kept constant (600 mM) throughout the experiment, and the anion composition of the chasing solution is given in the legend. Dephosphorylation was stopped by the addition of TCA, and the amount of E^32P was measured (Klodos et al., 1981).

The rate of AT^32P hydrolysis was measured in a histidine buffer, 30 mM, pH 7.4 at 37°C and in the presence different sodium salts, but at constant [Na+] equal to 450 mM. [ATP] = [Mg2+] = 3 mM and KCl, when present, was 20 mM. The reaction was started with ATP after preincubation of the enzyme for 10 min at room temperature with oligomycin at assay conditions. 32Pi was determined according to Lindberg and Ernster (1956).

Results and Discussion

Dephosphorylation Kinetics

In the experiments described below, two sodium salts, NaBr and Na_2SO_4, were used, keeping the Na+ concentration both during the phosphorylation and in the chase equal to 600 mM.

It has been previously shown, in experiments where the effect of the sodium salts was compared at constant [Na+] that: (a) Br-, a chaotropic anion, when present in the phosphorylation medium, increased the steady-state ratio of E_1P/E_2P (Post, 1988) and when added with the K-chase decreased the K-reactivity of the preformed EP (Klodos, 1991), both in comparison with the effect of Cl-. (b) SO_42-, a kosmotropic anion, elicited a decrease in the steady-state ratio and an increase in the K-reactivity, again in comparison with the effect of Cl-.

Figure 1. K-sensitivity of phosphointermediates formed in the presence of either NaBr or Na_2SO_4. The enzyme was phosphorylated in 3 ml of reaction medium containing either 600 mM NaBr (circles) or 300 mM Na_2SO_4 (squares). At time zero dephosphorylation was started by the addition of 3 ml chasing solution containing ATP and KCl (final concentrations 1 and 20 mM, respectively) and either 600 mM NaBr or 300 mM Na_2SO_4. The dephosphorylation medium contained 600 mM NaBr (open circles), 300 mM Na_2SO_4 (open squares), or 300 mM NaBr + 150 mM Na_2SO_4 (filled symbols).

The results of a dephosphorylation experiment shown in Fig. 1 support both previous findings. The EPs formed in the presence of NaBr and Na_2SO_4 displayed a very significant difference in both K-reactivity (Fig. 1) and ADP-reactivity (not shown) when the chase solution had the same salt composition as the respective phosphorylation media. However, when the EPs formed in either NaBr or Na_2SO_4 were both dephosphorylated in the medium consisting of 300 mM NaBr + 150 mM Na_2SO_4, the difference in their reactivity to K+ (Fig. 1) and ADP (not shown) decreased substantially.

Steady-State Hydrolysis of ATP

Oligomycin inhibition of ATP hydrolysis at steady state was measured at constant Na+ concentration equal to 450 mM, using either NaBr, NaCl, Na_2SO_4, or sodium acetate. The ATP hydrolysis rates were measured at saturating concentration of substrate [ATP] = [Mg2+] = 3 mM in the absence (Fig. 2 A) and in the presence (Fig. 2 B) of 20 mM KCl. The results are presented in the form of Dixon plots, where the ordinate is the ratio of the rate of hydrolysis measured in the absence (v0 olig) and in the presence of oligomycin (volig), and where the abcissa is the free oligomycin concentration, determined according to Plesner and Plesner (1991). In this plot the slope is a measure of the oligomycin inhibition.

The oligomycin inhibition, both in the absence and in the presence of K+, was found to depend on the anions present in the reaction medium. The inhibition was increased by chaotropic anions and decreased by kosmotropic anions in the sequence Br-> Cl- > SO2-4 > acetate (Fig. 2). Na+ in concentrations > 450 mM was found to inhibit the rate of hydrolysis at saturating [ATP] and [Mg2+], in the absence and in the presence of K+. Oligomycin was found to increase the Na+ affinity, explaining the inhibition caused by oligomycin under the conditions of these experiments (Plesner and Plesner, 1991). The inhibition caused by Na+ at concentrations > 450 mM was dependent on the anion present in the reaction medium. The reduction in the rate of hydrolysis for a specific increase in Na+ concentration was found to increase when the anion was more chaotropic; i.e., the inhibition was more pronounced in the presence of Br-> Cl- > SO2-4 > acetate (Plesner, L., and I. Klodos, unpublished observations).

Figure 2. Influence of anions on oligomycin inhibition of ATP hydrolysis in the absence (A) and in the presence of 20 mM KCl (B). Ordinate: V0 olig/Volig (i.e., the hydrolysis rate in the absence of oligomycin divided by the hydrolysis rate in its presence. Abcissa: free oligomycin concentration. Assay concentrations: [ATP] = [Mg2+]= 3 mM, [Na+] = 450 mM. The anions are shown.

This result indicates that the apparent affinity for the inhibitory effect of high Na+ concentrations depends on the anions in the medium and explains why oligomycin, by increasing Na+ affinity, caused a stronger inhibition in the presence of chaotropic anions than in the presence of kosmotropic anions. The inhibition at a given oligomycin concentration could be taken as an expression of the Na+ affinity, for the inhibitory effect of high Na+ concentrations, of the enzyme species at a given steady state. A specific oligomycin concentration will give rise to a weak inhibition under conditions where the affinity is low; that is, Na+ dissociates more readily from the enzyme, and the same oligomycin concentration will cause strong inhibition under conditions where Na+ affinity is high (i.e., where Na+ binds firmly, preventing its dissociation from the enzyme that is necessary for the reaction cycle to proceed).

The transition of E_1P to E_2P is associated with the deocclusion and release of Na+ from the enzyme. As shown by Post (1988) and in the dephosphorylation experiments above, this transition seems to be affected by the anions present in the medium. It therefore seems likely that the degree of oligomycin inhibition is also an expression of the transition and thus a measure of the E_1P/E_2P ratio.

Conclusions

At constant [Na+] = 600 mM, dephosphorylation patterns of preformed phosphoenzymes were obtained in a series of experiments where the anion composition of the steady-state and the chase media were varied independently. The measured K- and ADP-sensitivity of the preformed phosphointermediates, although affected by the dephosphorylation conditions, also reflected changes at steady state in the E_1P/E_2P ratio.

The oligomycin inhibition of steady-state ATP hydrolysis, at constant [Na+] and in the presence and absence of K+, was found to increase in the presence of chaotropic anions (Br-) and to decrease in the presence of kosmotropic anions (SO_42- and acetate-), both in comparison with the inhibition observed in the presence of Cl-.

Both observations seem to be consistent with the observation reported by Post, that the chaotropic anions increase the E_1P/E_2P ratio, whereas kosmotropic anions decrease the ratio.

Acknowledgments

This work was supported by the Danish Medical Research Council and The Biomembrane Research Center, University of Aarhus, Aarhus, Denmark.

References

Klodos, I. 1991. Effect of lyotropic anions on the dephosphorylation of Na,K-ATPase phosphointermediates. In The Sodium Pump: Recent Developments. J. H. Kaplan and P. De Weer, editors. The Rockefeller University Press, New York. 333–337.

Klodos, I., J. G. Nørby, and I. W. Plesner. 1981. The steady-state kinetic mechanism of ATP hydrolysis catalyzed by membrane-bound (Na+ + K+)-ATPase from ox brain. II. Kinetic characterization of phosphointermediates. Biochimica et Biophysica Acta. 643:463–482.

Klodos, I., P. Ottolenghi, and A. A. Boldyrev. 1975. Large-scale preparation of Na,K-ATPase from ox brain. Analytical Biochemistry. 67:397–403.

Lindberg, O., and L. Ernster. 1956. Determination of organic phosphorous compounds by phosphate analysis. Methods of Biochemical Analysis. 3:1–22.

Plesner, L., and I. W. Plesner. 1991. Kinetics of oligomycin inhibition and activation of Na+/K+-ATPase. Biochimica et Biophysica Acta. 1076:421–426.

Post, R. L. 1988. Lyotropic ions on reactivity of phosphoenzyme of sodium, potassium-ATPase. Biophysical Journal. 53:138a. (Abstr.)

Post, R. L., and K. Suzuki. 1991. A Hofmeister effect on the phosphoenzyme of Na,K-ATPase. In The Sodium Pump: Structure, Mechanism, and Regulation. J. H. Kaplan and P. De Weer, editors. The Rockefeller University Press, New York. 201–209.

Transient Kinetics of Dephosphorylation of Na,K-ATPase after Dilution of NaCl

Irena Klodos and Bliss Forbush III

Institute of Biophysics, University of Aarhus, DK-8000 Aarhus C, Denmark; and Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06510

Introduction

The phosphorylation of Na,K-ATPase is described as a sequential process in which formation of ADP-sensitive E_1P (from enzyme-ATP complex in the presence of Na+ and Mg2+) is followed by transition to K-sensitive E_2P (Albers, 1967; Post et al., 1969).

NaCl in the medium is not only required for the phosphorylation to occur, but also affects the ratio of E_1P to E_2P. It has been previously shown that at very high concentrations of NaCl, EP consists almost exclusively of E_1P, while at low [NaCl] the predominant EP form is K-sensitive E_2P (Taniguchi and Post, 1975; Nørby et al., 1983).

There has been long-standing disrepancy among different measurements of the rate of transition of E_1P to E_2P. One of the methods used to determine the rate was K-chase experiments, where the dephosphorylation was studied after the addition of high concentrations of KCl to the preformed E^32P. The chase results in an “instantaneous” removal of K-sensitive E_2P followed by a relatively slow decay of K-insensitive EP. The rate of decay of the slow component should be at least equal to the rate of transition of E_1P to E_2P. However, the rate has generally been found to be too low for the transition to be a step in the overall hydrolytic reaction (Nørby and Klodos, 1988). Moreover, the rate obtained from K-chase experiments was several orders of magnitude lower than the rate deduced from other experiments, where the formation of EP and the pre–steady-state ratio of E_1P/E_2P was measured (Hobbs et al., 1985). It has therefore been suggested that K+, added to the phosphoenzyme formed in the presence of Na+ alone, inhibits the transition of E_1P to E_2P (Nørby et al., 1983).

The aim of present study was an investigation of effects of NaCl dilution in the dephosphorylation medium on K-reactivity of EPs preformed from [32P]ATP in the presence of high [NaCl], We find that on dilution of NaCl during a K-chase, E_1P breaks down rapidly.

Materials and Methods

Experiments were performed with dog kidney and ox brain Na,K-ATPase prepared according to Forbush (1982) and Klodos et al. (1975), respectively. Phosphorylation of both enzymes was performed at 0°C. The dephosphorylation experiments were performed either at 0°C (both kidney and brain enzymes) or at 20°C (kidney enzyme).

Measurements of 32P Release from Na,K-ATPase

The dog kidney enzyme was phosphorylated in the presence of 1 M NaCl, 1 mM MgCl_2, and 10 μM [32P]ATP for 5 s. It was diluted, filtered, and rinsed in a medium of the same composition as the phosphorylation medium except that [32P]ATP was replaced with unlabeled ATP (total time 15–20 s at 0°C).

The dephosphorylation of the EP was performed at 0 or 20°C in a rapid filtration apparatus to measure the rate of 32P release from E^32P (Forbush, 1984). The “first solution” in the rapid filtration apparatus had the same composition as the phosphorylation medium. The dephosphorylation was started simultaneously with the dilution of NaCl by the addition of chase (“second solution”) containing K+ and concentrations of NaCl shown in the figures.

Measurements of Acid-stable E^32P

The ox brain enzyme was phosphorylated for 60 s in 1 ml of medium containing 600 mM NaCl, 0.1 mM MgCl_2, 25 μM [32P]ATP, and 30 mM imidazole buffer at pH 7.4.

Figure 1. Fuglsø experiment. Effect of NaCl dilution on K-dependent dephosphorylation. The dog kidney enzyme was phosphorylated in the presence of 1 M NaCl. The K-chase accompanied by dilution of NaCl to 100 mM is shown by the upper curve. The lower control curve shows that there is almost no K-dependent dephosphorylation at 1 M NaCl.

Two types of dephosphorylation experiments were performed. In the first the K-chase was concomitant with the dilution of NaCl in the medium. In the second type of experiments K-chase preceded the dilution of NaCl. The phosphorylation of the enzyme was stopped by the addition of chasing solution of the same composition as the phosphorylation medium. 3 s later NaCl in the medium was diluted by addition of the second chasing solution. In all chases the concentration of unlabeled ATP and KCl was 1 and 20 mM, respectively. Final concentrations of NaCl are shown in the figures.

The dephosphorylation was stopped by the addition of TCA, and the amount of E^32P was measured (Klodos et al., 1981).

Results and Discussion

The results of the original experiment performed during, and discussed at, the 5th International Conference on Na,K-ATPase is shown in Fig. 1. The enzyme was phosphorylated at 1 M NaCl to form E_1P and the K-chase at 0°C was accompanied by dilution of NaCl to 100 mM. The rationale of the experiment was that the dilution of NaCl during the chase would lead to formation of E_2P, and the newly formed K-sensitive EP would be instantaneously dephosphorylated; i.e., the rate of dephosphorylation should be equal to the rate of transition of E_1P to E_2P. As seen from the control curve (1 M NaCl during the dephosphorylation) EP formed at 1 M NaCl is almost K-insensitive. The dilution of NaCl in the dephosphorylation medium leads to a significant increase in the K-sensitivity, indicating an increase in the rate of transition of E_1P to E_2P.

The dependence of the transition rate on [NaCl] was studied in experiments performed both with kidney enzyme, where the release of 32P from EP was measured, and with ox brain enzyme, where the amount of acid-stable E^32P remaining after the chase was measured. Fig. 2A shows the result of such an experiment with ox brain enzyme phosphorylated in the presence of 600 mM NaCl. The K-chase at 0°C was accompanied by the dilution of NaCl in the dephosphorylation medium to 300 or 100 mM. It is clearly seen from the figure that: (a) K-dependent dephosphorylation is biphasic, (b) the ratio of rapidly and slowly decaying EP depends on the concentration of NaCl in the dephosphorylation medium, but (c) the rate of the slow phase is almost independent of [NaCl]. The comparison of the K-dependent dephosphorylation at 20 or 50 mM KCl shows clearly that neither the magnitude of the rapid phase nor the rate of decay in the slow phase is affected by an increase in K+ concentration (not shown).

Figure 2. [NaCl] in the dephosphorylation medium. The ox brain enzyme was phosphorylated in the presence of 600 mM NaCl. (A) The K-chase was accompanied by the dilution of NaCl. (B) At time zero K was added. NaCl was diluted by the addition of the second chasing solution 3 s after the addition of the K-chase. The final concentrations of NaCl are shown.

In experiments with kidney enzyme the dilution of NaCl during the K-chase results in a similar biphasic dephosphorylation pattern, where the ratio between rapidly and slowly decaying components was found to be determined by [NaCl] in the dephosphorylation medium (not shown). The similarity of the results of experiments, where dephosphorylation was measured as 32P release and by the determination of acid-stable phosphointermediates, shows that the slow component is not caused by a formation of an acid-lable “buffer” pool of E^32P.

It has been recently shown by Post (1988) and Post and Suzuki (1991) that the steady-state distribution of EPs is not only determined by [Na+], but also by lyotropic ions. In our experiments the changes in K-sensitivity, previously ascribed to a specific effect of [Na+], clearly result from lyotropic effects of a decrease in Cl- concentration, as illustrated by the following observation. The enzyme was phosphorylated in the presence of 300 mM TrisCl and 100 mM NaCl. Although the K-chase was accompanied only by the dilution of TrisCl, the K-sensitivity of EP changed in exactly the same way as after the dilution of NaCl, except that the magnitude of slow component is determined by [TrisCl] in the dephosphorylation medium (not shown).

The appearance of K-sensitive EP upon the dilution of salt in the medium is due to the transition of E_1P to E_2P, as indicated by the observation shown in Fig. 2 B. In this experiment the K-sensitive EP was “removed” by K-chase. 3 s after the chase the remaining EP consists exclusively of K-insensitive E_1P, as indicated both by an apparently monoexponential dephosphorylation of the remaining EP in the presence of K+, and by a rapid and complete dephosphorylation of the remaining EP by ADP (not shown). The dilution of NaCl at this point of time causes a rapid dephosphorylation of some of EP followed by a reappearance of the slow component, decaying at the same [NaCl] independent rate. The fraction of the slow component is clearly determined by the concentration of NaCl. Moreover, successive “fast phase” bursts of dephosphorylation and successive reappearances of the slow component were observed when the first dilution was followed 4 s later by the subsequent decrease in [NaCl] (not shown).

The experiments described above were performed at 0°C. In experiments where the dephosphorylation was performed at 20°C, the rate of decay after K-chase was also found to depend on the salt concentration in the dephosphorylation medium. Experiments were carried out in the presence of 1 M NaCl alone or 100 mM NaCl + 1 M N-methyl-D-glucamine chloride (NMGCl), and a K-chase was accompanied by dilution of NaCl or by the dilution of NMGCl. In both types of experiment the decay of EP was clearly accelerated by a decrease in the salt concentration in the dephosphorylation medium (not shown). However, the complex biphasic dephosphorylation pattern, characteristic of experiments at 0°C, was not observed (not shown), but instead the dephosphorylation of EP was found to be nearly monoexponential at 20°C.

Conclusions

These data demonstrate that the E_1P → E_2P transition is greatly slowed at high NaCl concentrations. The effect of NaCl is not specific for Na+, but is a salt effect (after Post), (a) At 20°C the salt effect on the dephosphorylation is consistent with relatively simple kinetic schemes. (b) At 0°C, however, an anomalous slow component of dephosphorylation is observed, which cannot be fit with simple kinetic models. The magnitude of this K-insensitive component, but not its rate of dephosphorylation, depends on the salt concentration.

Acknowledgments

This work was supported by The Danish Medical Research Council, The Biomembrane Research Center, University of Aarhus, and NIH grant GM-31782.

References

Albers, R. W. 1967. Biochemical aspects of active transport. Annual Review of Biochemisty. 36:727–756.

Forbush, B., III. 1982. Characterization of right-side-out membrane vesicles rich in (Na,K)-ATPase and isolated from dog kidney outer medulla. Journal of Biological Chemistry. 257:12678–12684.

Forbush, B., III. 1984. An apparatus for rapid kinetic analysis of isotopic efflux from membrane vesicles and of ligand dissociation from membrane proteins. Analytical Biochemistry. 140:495–505.

Hobbs, A. S., R. W. Albers, and J. F. Froehlich. 1985. In The Sodium Pump. I. M. Glynn and J. C. Ellory, editors. The Company of Biologists, Ltd., Cambridge, UK. 355–361.

Klodos, I., J. G. Nørby, and I. W. Plesner. 1981. The steady-state kinetic mechanism of ATP hydrolysis catalyzed by membrane-bound (Na+ + K+)-ATPase from ox brain. II. Kinetic characterization of phosphointermediates. Biochimica et Biophysica Acta. 643:463–482.

Klodos, I., P. Ottolenghi, and A. A. Boldyrev. 1975. Large-scale preparation of Na,K-ATPase from ox brain. Analytical Biochemistry. 67:397–403.

Nørby, J. G., and I. Klodos. 1988. The phosphointermediates of Na,K-ATPase. Progress in Clinical and Biological Research. 268A:249–270.

Nørby, J. G., I. Klodos, and N. O. Christiansen. 1983. Kinetics of Na-ATPase activity by the Na,K pump. Interactions of the phosphorylated intermediates with Na+, Tris+, and K+. Journal of General Physiology. 82:725–759.

Post, R. L. 1988. Lyotropic ions on reactivity of phosphoenzyme of sodium, potassium-ATPase. Biophysical Journal. 53:138a. (Abstr.)

Post, R. L., S. Kume, T. Tobin, B. Orcutt, and A. K. Sen. 1969. Flexibility of an active center in sodium-plus-potassium adenosine triphosphatase. Journal of General Physiology. 54:306s–326s.

Post, R. L., and K. Suzuki. 1991. A Hofmeister effect on the phosphoenzyme of Na,K-ATPase. In The Sodium Pump: Structure, Mechanism, and Regulation. J. H. Kaplan and P. De Weer, editors. The Rockefeller University Press, New York. 201–209.

Taniguchi, K., and R. L. Post. 1975. Synthesis of adenosine triphosphate and exchange between inorganic phosphate and adenosine triphosphate in sodium and potassium ion transport adenosine triphosphatase. Journal of Biological Chemistry. 250:3010–3018.

Effect of Lyotropic Anions on the Dephosphorylation of Na,K-ATPase Phosphointermediates

Irena Klodos

Institute of Biophysics, University of Aarhus, DK-8000 Aarhus C, Denmark

Introduction

The phosphointermediates formed by Na,K-ATPase in the presence of Na+, Mg2+, and ATP consist of ADP- and K-sensitive phosphoforms (cf. Nørby and Klodos, 1988). Recently, Post (1988) and Post and Suzuki (1991) have shown that the steady-state ratio of K- and ADP-sensitive EPs is not only determined by the concentration of Na+ in the medium, as previously believed, but is also affected by unspecific interactions with lyotropic anions present during phosphorylation. The evaluation of the steady-state distribution of EPs has been based on the use of a rapid quench technique, in which the phosphorylation of the enzyme by AT^32P is stopped by addition of unlabeled ATP and high concentrations of either K+ (K-chase) or ADP (ADP-chase). In both types of experiments biphasic dephosphorylation of the preformed E^32P is observed. The rapidly decaying phosphoenzyme is taken to represent steady-state K- and ADP-sensitive phosphoforms, respectively. However, the degree of K-sensitivity is not only a function of the steady-state conditions, but, as shown by Klodos and Forbush (1991), is also influenced by whether a high or low concentration of salt is present in the dephosphorylation medium. A necessary step in the description of steady-state distribution of phosphoenzymes is therefore an evaluation of the influence of dephosphorylation conditions on K- and ADP-sensitivity. The present study is an attempt to distinguish between specific effects of [Na+], and effects of ionic strength and lyotropic anions on the K-sensitivity of preformed EP.

Materials and Methods

The experiments were performed with ox brain Na,K-ATPase, prepared according to Klodos et al. (1975). The enzyme was phosphorylated for 60 s at 0°C in 1 ml of incubation medium containing 0.1 mM MgCl_2, 25 μM [γ-32P]ATP, 30 mM imidazole buffer (pH 7.4 at 0°C), and 600 mM NaCl. This was followed by the addition of 5 ml chasing solution, which contained 1 mM unlabeled ATP (spontaneous dephosphorylation) or 1 mM unlabeled ATP + 20 mM KCl (K-chase) and the ligands described below and in figure legends. [MgCl_2] and [imidazole] were kept constant throughout the experiment.

The reaction was stopped by the addition of TCA, and the amount of E^32P was measured (Klodos et al., 1981).

Results and Discussion

Experiments at Constant [Cl-], but at Varying Concentrations of Na+ in the Dephosphorylation Medium

In these experiments the K-chase contained N-methyl-D-glucamine chloride (NMGCl) and NaCl so that [Cl–] remained unchanged, while the concentration of [Na+] decreased from 600 to 100 mM. Surprisingly, as seen from Fig. 1, the decrease in [Na+] has only little effect on the K-sensitivity of the preformed EP. This result is in clear contradiction to previously observed effects of the dilution of NaCl in the dephosphorylation medium (Fig. 2 in Klodos and Forbush, 1991). The next series of experiments was therefore performed at constant [Na+], but with varying anion composition of the dephosphorylation medium.

Experiments at Constant [Na+], but with Varying Anion Composition of the Dephosphorylation Medium

The replacement of Cl- by other anions has a pronounced effect on the K-reactivity of the preformed EP, as shown by the experiment in Fig. 2, where Cl- was replaced by either I- or citrate^3-.

Figure 1. The effect of [Na+] on K-sensitivity of phosphointermediates at constant [Cl-]. Na,K-ATPase from ox brain was phosphorylated at 600 mM NaCl in 1 ml of reaction medium. Concomitant with the K-chase (volume = 5 ml) NaCl was replaced by NMGCl, keeping the sum of [NaCl] and [NMGCl] equal to 600 mM. The upper curve represents the control experiment, where the chase contained the same [NaCl] as the phosphorylation medium and no NMGCl. Final concentrations of NaCl in the dephosphorylation medium are shown.

The ranking of anions with respect to their ability to increase the K-reactivity of the preformed EP was citrate3- > SO2-4 > Cl- > Br- > SCN- > I-. The spontaneous dephosphorylation was also affected by anions (not shown) and the sequence was exactly the same. The removal of K-sensitive EP before the replacement of Cl-, by chasing with K+ 3 s before the replacement of salts, leads to the same anion-dependent changes in the stability of the remaining EP (not shown). And again the sequence of anions is identical with the above ranking.

One of the reasons for the destabilizing effect of citrate^3- and SO2-4 in comparison with that of monovalent anions could be a change in the ionic strength of the dephosphorylation medium. However, the identity of dephosphorylation behavior observed in experiments with Na_3citrate (ionic strength = 1.1 M) and Na_2Hcitrate adjusted to pH 7.4 with NMG (ionic strength = 1.6 M) indicates that in the range of salt concentrations used, the K-sensitivity of EP is not affected by changes in ionic strength (not shown).

Figure 2. K-stimulated dephosphorylation and anions present in dephosphorylation medium. Na,K-ATPase was phosphorylated at 600 mM NaCl in 1 ml of reaction medium. The K-chase (volume = 5 ml) contained 600 mM Nal or 167 mM Na_3citrate, adjusted to pH 7.4 at 0°C with a small amount of NaOH. In the control experiment (open circles) the chase contained 600 mM NaCl. Final concentrations of salts are shown.

The sequence of anions in promoting a decreased stability of EP in the absence and prescence of K+ corresponds to the Hofmeister series of lyotropic anions and is identical with that reported by Post (1988) and Post and Suzuki (1991) for the increase in the relative amount of K-sensitive EP in steady-state experiments.

Experiments at Constant [NaCl], but at Varying [H_2O] in the Dephosphorylation Medium

One of the textbook explanations for the differences in protein precipitating potency of dissolved salts, as ranked in the Hofmeister series, is that they differ in their effects on the water activity in the medium. Therefore, in the third series of experiments the chasing solution contained NaCl and either dimethylsulfoxide (DMSO), polyethyleneglycol (PEG), ethyleneglycol (EG), or glycerol, keeping the concentration of NaCl constant.

Replacement of up to 40% of the medium water with EG or glycerol did not affect the stability of EP, either in the absence or in the presence of K+ (not shown). However, when DMSO or PEG was used to replace the medium water an increased K-reactivity of the preformed EP was observed (Fig. 3). A DMSO concentration around 15% was optimal (not shown).

The similarities between DMSO and PEG are limited to their effect on the dephosphorylation in the presence of K+, while the two substances have opposite effects on the spontaneous dephosphorylation. Whereas the replacement of the medium water with PEG decreased the stability, DMSO caused a concentration-dependent increase in the stability of the preformed EP (not shown).

Figure 3. The effect of DMSO and PEG on K-stimulated dephosphorylation. Na,K-ATPase was phosphorylated at 600 mM NaCl in 1 ml of reaction medium that did not contain any DMSO or PEG. The K-chase (volume = 5 ml) contained 18% DMSO or PEG and NaCl in concentrations required to keep [NaCl] equal to that of the phosphorylation medium.

Since only PEG elicits the dephosphorylation behavior (both spontaneous and K-induced) similar to that observed in the presence of anions like SO2-4 or citrate3-, it seems safe to conclude that the effect of these anions on the dephosphorylation pattern is not connected with their ability to bind the medium water.

Summary and Conclusions

K-stimulated dephosphorylation of the phosphointermediates of Na,K-ATPase, formed in the presence of Na+ alone, depends on the composition of dephosphorylation medium: (a) [Na+] seems to be of minor importance for K-sensitivity of preformed EP as long as the concentration of Cl- is kept constant. (b) K-sensitivity of the preformed phosphoenzymes is affected by anions. (c) The difference in the effect of Cl-as compared with SO2-4 or citrate3- cannot be explained by changes in ionic strength of the medium.

Anions in the dephosphorylation medium affect the spontaneous decay of EP (no K+ added with the chase) in parallel with their effect on K-stimulated decay.

The effect of a decrease in the water concentration in the medium on the stability of the preformed EP depends on the compound replacing H_2O: (a) DMSO and PEG both have an “anion-like” effect on K-reactivity of preformed phosphoenzymes, while EG and glycerol have no effect. (b) Only PEG seems to mimic the effect of less lyotropic anions on both spontaneous and K-stimulated dephosphorylation.

In conclusion, the properties of the phosphointermediates of Na,K-ATPase are not only a function of specific interactions of the enzyme with specific ligands, as previously believed, but are also affected by protein-anion and protein-water interactions.

Acknowledgments

This work was supported by Danish Medical Research Council and The Biomembrane Research Center, University of Aarhus, Aarhus, Denmark.

References

Klodos I., and B. Forbush III. 1991. Transient kinetics of dephosphorylation of Na,K-ATPase after dilution of NaCl. In The Sodium Pump: Recent Developments. J. H. Kaplan and P. De Weer, editors. The Rockefeller University Press, New York. 327–331.

Klodos, I., J. G. Nørby, and I. W. Plesner. 1981. The steady-state kinetic mechanism of ATP hydrolysis catalyzed by membrane-bound (Na+ + K+)-ATPase from ox brain. II. Kinetic characterization of phosphointermediates. Biochimica et Biophysica Acta. 643:463–482.

Klodos, I., P. Ottolenghi, and A. A. Boldyrev. 1975. Large-scale preparation of Na,K-ATPase from ox brain. Analytical Biochemistry. 67:397–403.

Nørby, J. G., and I. Klodos. 1988. The phosphointermediates of Na,K-ATPase. Progress in Clinical and Biological Research. 268A:249–270.

Post, R. L. 1988. Lyotropic ions on reactivity of phosphoenzyme of sodium, potassium-ATPase. Biophysical Journal. 53:138a. (Abstr.)

Post, R. L., and K. Suzuki. 1991. A Hofmeister effect on the phosphoenzyme of Na,K-ATPase. In The Sodium Pump: Structure, Mechanism, and Regulation J. H. Kaplan and P. De Weer, editors. The Rockefeller University Press, New York. 201–209.

Oligomycin Activation of Na,K-ATPase Hydrolysis

Liselotte Plesner and Igor W. Plesner

Danish Biomembrane Research Center; and Institutes of Biophysics and Chemistry, University of Aarhus, DK-8000 Aarhus C, Denmark

Introduction

Oligomycin is generally thought to induce the E_1 conformation of Na,K-ATPase and to exert inhibition of ATP hydrolysis by blocking the conversion of E_1P to E_2P. On the other hand, it has been shown in equilibrium binding experiments that oligomycin causes increased Na+ binding (Matsui and Homareda, 1982) and promotes Na+ occlusion (Esmann and Skou, 1985). Oligomycin also decreases the concentration of Na+ necessary for a maximal Na+ effect on the rate of phosphorylation (Skou, 1991), and decreases the apparent affinities for Na+ in hydrolysis experiments (Robinson et al., 1984). Under conditions where Na+ activation is predominant, activation by oligomycin has been reported (Blostein and Whittington, 1973).

The experiments to be reported were performed (a) to test the hypothesis that perhaps the sole effect of oligomycin is to increase the specific Na+ concentration under all conditions (i.e., to increase the affinity for Na+, and thus to enhance the effect of Na+ on the hydrolysis rate whether it is inhibitory or activating); and (b) to elucidate whether this view of the effect of oligomycin is equivalent to the opening statement of this article.

We first measured hydrolysis rates for Na-ATPase and Na,K-ATPase at saturating ATP concentrations as a function of the Na+ concentration. From the effect of oligomycin on these rates it was indeed possible to propose that oligomycin acts (inhibits) by enhancing the Na+ inhibition of hydrolysis, and that this is the only property necessary for an understanding of the inhibition obtained under these conditions. If this is true, one should expect oligomycin activation of the hydrolysis under conditions where Na+ activation of the enzyme predominates. We present evidence for such an activation, an effect which, as mentioned above, has previously been observed in the red blood cell by Blostein and Whittington (1973).

Materials and Methods

Enzyme

Na,K-ATPase was prepared from ox brain according to Klodos et al. (1975). The specific activity was 4.4 U/mg protein.

Assay

The assays were performed in 30 mM histidine, pH 7.4, at 37°C. Assay concentrations of ATP, NaCl, KCl, and oligomycin are given in the legends to the figures. 32Pi liberation was determined according to the method of Lindberg and Ernster (1956). Ionic strength varied with the salt concentration. The reaction was started with ATP after preincubating the enzyme for 10 min at room temperature with oligomycin under the assay conditions. The concentrations of free oligomycin were determined as detailed in Plesner and Plesner (1991).

Results

When the ATP hydrolysis rate was measured as a function of [NaCl] at [ATP] = [Mg2+] = 3 mM (Fig. 1), bell-shaped curves were obtained with maximum at [NaCl] = 450 mM and 150 mM in the absence (Fig. 1 a) and the presence (Fig. 1 b) of K+, respectively.

Figure 1. Activity vs. [Na+] in the absence (a) and the presence (b) of 20 mM KCl, and with free oligomycin at the (micromolar) concentrations indicated. The ordinate units (micromolar per minute) correspond to nanomoles per milliliter of undiluted enzyme per minute. [ATP] = [Mg2+] = 3 mM.

Addition of oligomycin in increasing concentrations resulted in a shift of the curves to the left; i.e., the K0.5 for Na+ inhibition seemed to decrease with increasing oligomycin concentration. Likewise, the degree of oligomycin inhibition increased with the Na+ concentration (see Fig. 2), as one would expect because of the principle of detailed balance at equilibrium.

From the curves in Fig. 1 it is seen that Na+ inhibition is more pronounced in the presence of K+ (Fig. 1 b) than in its absence (Fig. 1 a). Accordingly, oligomycin inhibition is more pronounced in the presence of K+ (Fig. 2 b) than in its absence (Fig. 2 a). The most economical way to explain the data presented in Figs. 1 and 2 is to assume that the only effect of oligomycin is to increase the binding of Na+, and thus Na+ will inhibit more effectively the higher the concentration of oligomycin. If this is the case, we would predict that under conditions where Na+ activation predominates (i.e., at low Na+ concentrations, so that the low-affinity inhibitory sites are not occupied) and low, nonsaturating ATP concentrations (because the binding of ATP is activated by Na+ acting at high-affinity sites), oligomycin enhancement of the Na+ effect should lead to activation by oligomycin. Experiments to test this prediction are shown in Fig. 3. In the absence of K+, at [ATP] = [Mg2+] = 0.3 μM, oligomycin caused a concentration-dependent activation at Na+ concentrations in the range of 1–3 mM (Fig. 3 a). In the presence of K+, at [ATP] = [Mg2+] = 3 μM, a similar effect of oligomycin is observed with Na+ concentrations in the range of 5–20 mM (Fig. 3 b).

Figure 2. Dixon plots of (v(0)/v(oli) vs. the free oligomycin concentration) in the absence (a) and the presence (b) of 20 mM KCl at the indicated concentrations of NaCl. v(0)/v(oli) is the hydrolysis rate in the absence of oligomycin divided by the rate in its presence. The lines are the least-squares regression lines fitted to each set of points. [ATP] = [Mg2+] = 3 mM.

We conclude that whether Na+ causes inhibition (Figs. 1 and 2) or activation (Fig. 3), the effect of oligomycin is to enhance the Na+ effect. E_1 forms of the enzyme are generally thought to be responsible for the occlusion and transport of Na+ ions, and E_1 forms are often defined as the Na forms. Thus, the proposal expressed above for the action of oligomycin is phenomenologically equivalent to the first part of the opening statement in the Introduction: oligomycin increases, in a concentration-dependent way, the Na+ affinity of the form of the enzyme that is sensitive to Na+.

It is difficult, however, to reconcile the concentration-dependent oligomycin activation seen from the data in Fig. 3 with the idea that oligomycin per se blocks the conversion of E_1P to E_2P. Inhibition of this transition is seen only under conditions where the (specific) Na+ concentration is high enough to prevent or inhibit the dissociation of Na+ from the low-affinity, external unloading sites, which is necessary for the reaction cycle to proceed, and oligomycin “blocks” this step only by increasing the specific Na+ concentration.

Figure 3. Dixon plots of (v(0)/v(oli) vs. [oligomycin]) from experiments at low Na+ concentrations (millimolar), as indicated in the figures, and nonsaturating ATP concentrations. (a) [ATP] = [Mg2+] = 0.3 μM; [K+] = 0. (b) [ATP] = [Mg2+] = 3 μM; [K+] = 20 mM. The lines are the least-squares regression lines. In this plot a negative slope corresponds to activation.

References

Blostein, R., and E. S. Whittington. 1973. Studies of high potassium and low potassium sheep erythrocyte membrane sodium-adenosine triphosphatase: interactions with oligomycin, adenosine triphosphate, sodium, and potassium. Journal of Biological Chemistry. 248:1772–1777.

Esmann, M., and J. C. Skou. 1985. Occlusion of Na+ by the Na,K-ATPase in the presence of oligomycin. Biochemical and Biophysical Research Communications. 127:857–863.

Klodos, I., P. Ottolenghi, and A. A. Boldyrev. 1975. Large scale preparation of Na,K-ATPase from ox brain. Analytical Biochemistry. 67:397–403.

Lindberg, O., and L. Ernster. 1956. Determination of organic phosphorous compounds by phosphate analysis. In Methods of Biochemical Analysis. Vol. 3. D. Gick, editor. Interscience, New York. 1–22.

Matsui, H., and H. Homareda. 1982. Interaction of sodium and potassium ions with Na,K-ATPase. I. Ouabain-sensitive alternative binding of three Na+ and two K+ to the enzyme. Journal of Biochemistry. 92:193–217.

Plesner, L., and I. W. Plesner. 1991. Kinetics of oligomycin inhibition and activation of Na+/K+-ATPase. Biochimica et Biophysica Acta. 1076:421–426.

Robinson, J. D., L. J. Robinson, and N. J. Martin. 1984. Effects of oligomycin and quercitin on the hydrolytic activities of the (Na+ + K+)-dependent ATPase. Biochimica et Biophysica Acta. 772:295–306.

Skou, J. C. 1991. Effect of oligomycin on the rate of phosphorylation of Na,K-ATPase in the presence of Na+. In The Sodium Pump: Recent Developments. J. H. Kaplan and P. De Weer, editors. The Rockefeller University Press, New York. 317–319.

Order-of-Magnitude Estimation of Rate Constants for the Interconversion of the Phosphoenzymes of Na,K-ATPase, Based on Oligomycin Inhibition Experiments

Igor W. Plesner

Department of Chemistry, University of Aarhus, Aarhus, Denmark

Introduction

During the last few years there has been some debate in the literature, as well as at sodium pump conferences, as to whether the phosphoenzyme forms of Na,K-ATPase, specifically the ADP-sensitive form E_1P, is kinetically competent, so that the Post-Albers model can account for the large activation of the enzyme by K+. This issue is addressed here by attempting to determine the values that the phosphoenzyme interconversion rate constants must have in order for the Post-Albers model to account for the observed activities. In this volume (Plesner and Plesner, 1991) data for oligomycin inhibition and activation are presented, and it is argued that these may be understood by assuming that oligomycin exerts its effect on the enzyme merely by enhancing the binding of Na+, and therefore Na+ must also affect the binding of oligomycin. However, to arrive at numerical values of the rate constants, we adopt here, as an approximation, the assumption that the binding affinity of oligomycin is independent of Na+. As a result, because of this approximation, the best we can hope for is probably to obtain order-of-magnitude estimates of the rate constants for the interconversion and dephosphorylation of the phosphoenzyme, if the Post-Albers model is assumed to hold. Using the kinetic results from oligomycin inhibition it is shown that an “iterative” procedure can be used to obtain the rate constants at 37°C as functions of the Na+ concentration in the range 50 mM < [Na+] < 1,650 mM. The procedure also yields the fractional concentrations of E_1P and E_2P. It is found that with these assumptions the interconversion rate constants are quite large at physiological conditions, and that the transition E_1P → E_2P is not rate limiting. Whether or not these findings are true must of course be decided by experiments.

Procedure

We consider the simple version of the Post-Albers model shown in Fig. 1. The kinetic states shown may consist of several species differing in the number of Na+ ions bound. We assume that the equilibrium (dissociation) constants for oligomycin binding are independent of the Na+ concentration. For this model it has been shown (Plesner, 1987) that the ratio of Vm(0), the maximum velocity in the absence of oligomycin, to Vm(oli) is given by

where f_1 is the fractional concentration of E_1P at steady state and Ki is the dissociation constant of oligomyein from E_1P. Thus, from the slope of such a Dixon plot of the data (the linear part; see Plesner, 1987) f_1 can be obtained if Ki is known, and vice versa.

Kinetic data were obtained in the absence and presence of 20 mM KCl, and with [Na+] varying from 50 to 1,650 mM (examples of data are exhibited in Plesner and Plesner, 1991). The slopes from the Dixon plots increased in all cases as [Na+] increased. It was also found that for [Na+] < 750 mM, the slopes were larger in the presence than in the absence of K+, whereas the reverse was true for larger Na+ concentrations. With the model used here, referring to Eq. 1, this means that the fractional E_1P concentration, f_1, increased with the Na+ concentration. Calculation of the rate constants now proceeds as follows.

Step 1. We assume that with 1,650 mM NaCl and no K+, corresponding to the largest slope found, f_1 = 1. Since this slope by assumption is just 1/Ki, we can find f_1 in all the other runs from the slopes found in each case, using Eq. 1.

Figure 1. The Post-Albers model for Na,K-ATPase showing the presumed action of oligomycin. The numbers indicate rate constants, and the K’s are oligomycin dissociation constants in the corresponding equilibrium pools.

Step 2. In the absence of K+ and oligomycin, all the enzyme is phosphorylated. Assuming that addition of KCl results in an essentially zero steady-state concentration of E_2P, we therefore have, at a particular Na+ concentration a, for the activities v_0(a) and vK(a) measured in the absence and presence, respectively, of K+, but in the absence of oligomycin,

Since the set f_1 is known, Eq. 2a yields a value of k_3E_0, and since by assumption k_3 is independent of K+ (K+ does not bind to E_1P in the Post-Albers model), this value can be used in Eqs. 2b and 2c to calculate values of k-3E_0 and k_4E_0, the spontaneous dephosphorylation rate constant for E_2P. Clearly, because of the assumption that f_1Na (NaCl = 1,650) = 1, Eq. 2c cannot be used at this concentration. However, a look at the values obtained for k_4E_0 as a function of [NaCl] shows that k_4E_0 becomes independent of [NaCl] at large concentrations. We therefore use this limiting value and Eq. 2c to calculate a new value for f_1, at 1,650 mM NaCl, which in turn yields a new value for 1/Ki to be used for the calculation of a new set of f_1's for all NaCl concentrations. The value of Ki obtained in this way was

Step 3. The calculation of rate constants as in step 2 is now repeated, using the new set of f_1 values. The values so obtained constitute the self-consistent set of rate constants to be assigned to the appropriate steps in Fig. 1. The results for the rate constants are shown in Table I.

It is seen that as [Na+] increases in the range investigated, k_3 decreases, while k-3 appears to be independent of NaCl. k_4 is best described by a function that saturates at small Na+ concentrations (Kd for Na+ in the E_2P pool ~9 mM; see legend to Fig. 2). A good fit of the results for k_3 is obtained if it is assumed that (a) only the E_1P pool contains Na-bound phosphoenzyme, at three independent and equivalent sites, and (b) that only the E_1P species devoid of Na+ can be converted to E_2P. The resulting equation describing the Na+ dependence of k_3 is given in the legend to Fig. 2. The parameters are

where KNa is the dissociation constant for Na+ from a site, and k°3 is the intrinsic rate constant for the conversion of E_1P, devoid of Na+, to E_2P. The fitted curves for k_3 and k_4 are shown in Fig. 2.

TABLE I
Phosphoenzyme Rate Constants (cf. Fig. 1)

The scheme implied in the above fitting of k_3 is not compatible with the property of k_4 being a saturable function of [Na+], which in turn implies that Na+ can also bind to E_2P (with high affinity; see above). If so, one should also expect the rate constant k-3 to be a function of [Na+]. This inconsistency may be a result of the basic assumption that the Na+ dependence of the oligomycin binding affinity can be ignored. Also, to see the Na+ dependence of k_4 we would need data at < 50 mM Na+ concentrations.

The results for the distribution of the phosphoenzyme species as a function of the salt concentration is shown in Fig. 3. Also shown is the fractional E_1P concentration in the presence of 20 mM KCl. It is seen from the figure that even at 1,650 mM NaCl the fractional E_1P concentration is only ~74%. It is also noteworthy that according to the results presented here the fractional E_1P concentration at 1,650 mM Na+ and 20 mM K+ is still ~25%.

In conclusion, it is shown here that if the Post-Albers model is to account for the data, and if the oligomycin binding affinity is assumed to be independent of Na+, then the inhibition kinetics of oligomycin can be used to find a self-consistent set of rate constants for the phosphoenzyme interconversion. The Na+ dependence of these rate constants, however, does not yield a consistent picture for the interaction of Na+ with the phosphoenzymes.

Figure 2. The rate constants k_3 and k_4 (inset) as functions of [Na+] as obtained by the procedure described in the text. The continuous lines are the fitted equations:

k_3 = k_3o/ (1+ [Na+]/452)3; k_4 = 5/ (1+ 8.8/[Na+])
with [Na+] and associated Kd's in units of millimolar.

Figure 3. The fractional concentrations of the phosphoenzymes, calculated as described in the text.

It is of interest that k-3 is found to be ~10 times as large as k_4 at physiological conditions, and therefore the phosphoenzymes may be presumed to be in rapid equilibrium in the absence of K+. The step E_1P → E_2P is therefore not rate limiting under physiological conditions.

The magnitude of the rate constant k_3 at physiological conditions is somewhat surprising (~3,000 s-1), since this is the step presumed to be associated with deocclusion of Na+ and the conformational transition to E_2P. Whether this is the correct order of magnitude awaits an experimental determination.

References

Plesner, I. W. 1987. Oligomycin inhibition of Na,K-ATPase. Cell Biophysics. 11:279–307.

Plesner, L., and I. W. Plesner. 1991. Oligomycin activation of Na,K-ATPase hydrolysis. In The Sodium Pump: Recent Developments. J. H. Kaplan and P. De Weer, editors. The Rockefeller University Press, New York. 339–343.

A Procedure for the Simulation of Unidirectional (Exchange) Rates in Enzyme Catalyzed Reactions in the Presence of Both Substrates and Products

Igor W. Plesner

Department of Chemistry, University of Aarhus, Aarhus, Denmark

Introduction

Radioactively labeled compounds are frequently used in the measurement of steady-state rates in enzyme catalyzed reactions. Such rates therefore represent the unidirectional exchange rates which, in the presence of both substrates and products, differ from the net “chemical” rates. For quantification purposes one needs a rate equation for the exchange, but even for relatively simple models such equations, which can be derived using the diagram techniques of Cleland (1967) or Chen (1990), are far too complex to be useful in detailed studies. For example, for the Post-Albers model considered below it can be calculated that the denominator in the exchange rate for Pi/ATP exchange contains 315 terms, each a product of a number of rate constants.

A procedure for simulation of unidirectional rates at steady state in an enzyme kinetic mechanism is best described by consideration of a specific model. The method, however, is quite general and can easily be implemented for any model for enzyme action, and can be used for the calculation of isotope exchange rates at equilibrium, pre-steady state rates for isotope exchange, and isotope exchange fluxes in membrane transport models.

Procedure

The model shown in Fig. 1 (without the states marked with an asterisk) is a simplified version of the Post-Albers scheme for the Na,K-ATPase reaction. In broken membrane preparations the only reaction that can be monitored is the hydrolysis of ATP to ADP and inorganic phosphate:

The activity of the enzyme is strongly influenced by Na+ and K+, but in Fig. 1 only the action of the latter is explicitly included.

The usual simulation procedure is to solve the steady-state equations for the model, having the (matrix) form

where A is the coefficient matrix, consisting of linear combinations of rate constants, x is the vector of steady-state concentrations of the kinetic intermediates, and B is a vector of constants, whose elements are all zero except one, which is 1. The solutions are the (fractional) steady-state concentrations of the intermediates, from which the net rate in any step may be calculated. For example, for the model in Fig. 1 the net hydrolysis rate is obtained from

and similar expressions. It should be noted, however, that in the presence of both substrates and products, neither of the individual terms in Eq. 3 represents the unidirectional flux in the model. For that calculation one needs to know the fraction of each steady-state concentration stemming from that point in the model at which the labeled species adds. This is achieved as follows:

1. We first expand the model such that those enzyme states that will become labeled during measurement of a particular exchange rate are explicitly present in the model and distinguishable from the corresponding unlabeled states. For the rates for ATP-Pi and for Pi-ATP exchange, the expansion of the model results in the scheme shown in Fig. 1, where the states becoming labeled during the exchange have been marked with an asterisk. (Note that for graphical reasons the state E_1 is represented twice in the diagram). The rate constants connecting the labeled species are the same as those connecting the corresponding unlabeled ones.

Figure 1. The expanded model used for Pi/ATP or ATP/Pi exchange. The states becoming labeled are indicated by an asterisk. The Post-Albers model for Na,K-ATPase is obtained when the states with an asterisk are ignored. T ≡ ATP.

2. The second order rate constants corresponding to ligand addition have to be multiplied by the appropriate ligand concentrations. For Pi/ATP exchange in the “labeled cycle” we take for the phosphate concentration to multiply the rate constant connecting E_2 and E_2P*

where α is an arbitrary, small number, and P is the total concentration of inorganic phosphate. The Pi concentration to be used for the corresponding unlabeled addition is then (1 – α)P. In addition, for this experiment the concentration of labeled ATP is taken as zero.

3. The steady-state equations, having the same form as Eq. 2, but with enlarged coefficient matrix A and vectors x and B corresponding to the enlarged model, are now solved by computer. It may be shown that a range of (small) α values (typically 10-6–10-3) may be found such that the resulting steady-state concentrations of the unlabeled species are not noticeably perturbed relative to the values obtained for the original model.

4. From the solutions the fractional concentration of a particular enzyme species deriving from the addition of labeled Pi, [Ei]P, is then

and for the Pi/ATP exchange rate we then obtain

where [E_2T]P stands for the sum [E_2T]P + [E_2KT]P. With α in the proper range, the concentrations (Ei)P are independent of the particular choice for α. An analogous procedure is used for the calculation of the exchange rate in the opposite direction (ATP/Pi exchange).

It is noteworthy that the fraction of the steady-state concentration of a species stemming from the addition of the labeled compound varies from species to species. Accordingly, it can be shown that experimental determination of the amount of label in an enzyme form, and using this to represent the actual steady-state concentration of that enzyme form, will in general lead to quantitatively and qualitatively erroneous results. The simultaneous solution for the nonlabeled steady-state concentrations provides a check on the calculations, since we must have

where the rates on the right hand side are the unidirectional rates, and vnet is the net rate of hydrolysis, calculated from Eq. 3.

Simulation

The procedure described here has been applied to a preliminary simulation of recent experiments on [32P]ATP synthesis from 32Pi and ADP by Na,K-ATPase (Plesner et al., 1990). Several features not explicitly shown in the model in Fig. 1 were included in the computations. These include the competitive inhibition of the enzyme by ADP and several small cycles: E_2-E_2P-E_2KP-E_2K-E_2 and E_2-E_2K-E_2KATP-E_2ATP-E_2. The species in boxes in Fig. 1 are presumed to be in rapid equilibrium. With these features the total number of parameters in the model (rate constants and dissociation constants) is 24. Some of these were extracted from the literature, some were estimated using recent experiments on oligomycin inhibition (Plesner, 1991), and some were adjusted by trial and error. The rate constants are not all independent, but are subject to restraints of detailed balance at equilibrium, including that for the overall hydrolysis cycle:

where Πbc and Πbcc are the clockwise and counterclockwise products of rate constants in cycle b (Fig. 1), respectively, and KATP is the equilibrium constant for the hydrolysis reaction (Eq. 1). The parameters used here are shown in Table I.

TABLE I
Parameters Used in the Simulations T ≡ ATP

*Calculated from detailed balance relation
‡Valid for [Na+] = 0.6 M. (From Plesner, 1991)

A preliminary simulation of two data sets is shown in Fig. 2. Simulations were carried out also for the dependence of the synthesis and hydrolysis (exchange) rates, in the presence and absence of K+, on the concentrations of Na+, ADP, and inorganic phosphate, but are not shown due to space limitations. Although the fits are not satisfactory in a quantitative sense, the qualitative features of the experiments are reproduced by the model. It may be concluded that the Post-Albers model shown in Fig. 1 can, at least qualitatively, explain the observed results. Of course, only a refinement of the simulations can decide whether or not quantitative agreement with the experiments can be obtained. A more detailed account will be published elsewhere.

Figure 2. Simulation of the Pi/ATP exchange (vsyn) and ATP/Pi exchange (vhyd) for Na,K-ATPase as a function of [K+]. [ATP] = 1 mM; [ADP] = 3 mM; [Pi] = 10 mM; [NaCl] = 600 mM. The experimental points are from Plesner et al. (1990).

References

Chen, Y. 1990. The diagram methods for the evaluation of exchange fluxes in membrane transport systems. Biophysical Chemistry. 35:55–63.

Cleland, W. W. 1967. Enzyme Kinetics. Annual Review of Biochemistry. 36:77–112.

Plesner, I. W. 1991. Order-of-magnitude estimation of rate constants for the interconversion of the phosphoenzymes of Na,K-ATPase, based on oligomycin inhibition experiments. In The Sodium Pump: Recent Developments. J. H. Kaplan and P. De Weer, editors. The Rockefeller University Press, New York. 345–349.

Plesner, L., B. Karlsmose, and M. E. Lüscher. 1990.32P-ATP synthesis in steady state from 32Pi and ADP by Na,K-ATPase from ox brain and pig kidney: activation by K+. Biochimica et Biophysica Acta. 1040:167–174.

Chemical Relaxation Studies of Fluorescein 5′-Isothiocyanate Modified Sodium Pump

Larry D. Faller, Georgios Scheiner-Bobis, and Robert A. Farley

CURE, Department of Medicine, UCLA School of Medicine and Department of Veterans Affairs Medical Center, West Los Angeles, California 90073; and the Department of Physiology and Biophysics, University of Southern California School of Medicine, Los Angeles, California 90033

Introduction

The working hypothesis in most models of mammalian active transport is that the carrier can assume different conformations, designated E_1 and E_2, in which the ion binding sites are alternately exposed to the cytosol and to the lumen. Experimental evidence for a conformational change has been obtained by covalent modification of the Na+-pump with fluorescein 5′-isothiocyanate. K+ quenches the fluorescence of fluorescein-labeled Na,K-ATPase. Na+ prevents or reverses the K+-quench. Karlish (1980) has explained the effects of the transported ions by a mechanism

in which the conformational change between E_1K and E_2K is rate limiting, and Na+ reverses the conformational change by competing with K+ for the same ion binding site(s) on the E_1 conformer. A study comparing the mechanisms of ion interaction with the sodium and proton pumps and investigating the mechanism of the E_1E_2 conformational change by varying the temperature is being published elsewhere (Faller).[1] In this report we focus on chemical relaxation analysis of the rates and extraction of equilibrium data from the kinetic curves.

Methods

Chemical Relaxation Treatment of Rates

Stopped-flow fluorimetric measurements in which enzyme is mixed with monovalent cation can be thought of as concentration-jump, chemical relaxation experiments. For a step perturbation, the reactants approach their new equilibrium values exponentially. The reciprocal of the time constant is the relaxation time (τ).

The functional form of the relaxation expression depends on the mechanism of the reaction. For the mechanism in Eq. 1,

where KK = k–1/k_1 and KNa are the dissociation constants of K+ and Na+, respectively, from the E_1 conformer of the enzyme. Kc = k_2/k–2 is the equilibrium constant between E_2K and E_1K. Eq. 2 predicts hyperbolic dependence of 1/τ on [K+] ([Na+] = 0) and an inverse relationship between 1/τ and [Na+] ([K+] fixed).

Analysis of Amplitudes

The maximum fluorescence change observed in a mixing experiment depends on the equilibrium distribution of fluorophore-labeled enzyme between the E_1 and E_2 conformers at the final rectant concentrations, so the amplitudes (∆Fo) deduced from kinetic experiments contain the same information obtainable from static, fluorimetric titrations.

Figure 1. Relaxation effect observed when equal volumes of 300 μg ml–1 Na,K-ATPase, preincubated in 10 mM KCl, and NaCl were mixed at 15°C. The reaction was buffered at pH 7.4 with 50 mM Tris-HCl and the ionic strength was kept constant with choline chloride. Lower curves, 200 mM NaCl, 1/τ = 0.199 and 0.197 s–1, ∆Fmax = 0.423 au. Upper curves, 40 mM NaCl, 1/τ = 0.298 and 0.291 s–1, ∆Fmax = 0.275 and 0.288 au.

The mechanism in Eq. 1 predicts hyperbolic dependence of ∆F_0 on ion concentration. For the K+-quench:

where

For Na+ reversal of the K+-quench (assuming Na+ and K+ compete):

where

Figure 2. Sigmoidal dependence of ∆Fo for K+-quench on log[K+] at 15°C. The theoretical curve was calculated for binding to a single class of sites with K0.5K =1.4 mM and ∆Fmax = −0.67 au.

Results

Fig. 1 shows that Na+ exponentially reverses the quench of fluorescein fluorescence caused by K+ and that 1/τ for Na+ reversal of the K+-quench is inversely related to [Na+]. KNa, k–2, k_2, and KK were estimated from nonlinear, least-square fits of the experimental data to Eq. 2.

Fig. 2 is a “kinetic” titration of fluorescein-labeled Na,K-ATPase with K+. ∆Fo depends sigmoidally on log[K+] as predicted by Eq. 3. K0.5K and K0.5Na were estimated from nonlinear, least-square fits of the experimental data to Eq. 3 or Eq. 5.

Table I compares the equilibrium estimates of K0.5K and K0.5Na from amplitude data with the corresponding values calculated from kinetic constants with Eq. 4 or Eq. 6.

Discussion

Hyperbolic dependence of 1/τ on [K+] and unequivocal evidence for a maximum rate are observed for K+ quench ([Na+] = 0) of the Na,K-ATPase. The nonzero ordinate intercept ([K+] = 0) predicted by the relaxation expression (Eq. 2) cannot be demonstrated for the Na+-pump because the back reaction is slow (k–2 = 0.44 s–1 at 22°C). However, a nonzero ordinate intercept (k–2 = 120 s–1 at 22°C) and maximum 1/τ = k_2 + k–2 = 363 s–1 are observed with the gastric H+-pump.[1]

TABLE I
Comparison of Measured and Calculated Parameters

The inverse relationship between 1/τ and [Na+] predicted by Eq. 2 ([K+] fixed) is observed with the Na,K-ATPase (Fig. 1). KNa estimated from the shape of a 1/τ versus [Na+] plot at 22°C (0.19 mM) and KK estimated from K+-quench data (10 mM) are in good agreement with the Na+ (0.19 mM) and K+ (9 mM) dissociation constants from intracellular sites that Garay and Garrahan (1973) deduced from studies of Na+ efflux from red blood cells at 37°C.

The reproducibility of the kinetic data, illustrated in Fig. 1, makes it feasible to analyze amplitudes. The hyperbolic dependence of ∆Fo on ion concentration predicted by Eq. 3 is observed when the fluorescence of fluorescein-modified Na+-pump is quenched by K+ (Fig. 2). With another monovalent cation that dramatically shifts Kc, the smaller maximum fluorescence change predicted by Eq. 4 has been demonstrated (unpublished results).

Since experimentally KNa (app) < [Na+], the second term in the numerator of Eq. 5 can be neglected, and the expected hyperbolic dependence of ∆F_0 on [Na+] has been demonstrated for Na+ reversal of the K+-quench of the Na,K-ATPase.[1] The nonzero ordinate intercept predicted by Eq. 5 is too small to observe experimentally because K0.5Na is numerically at least an order of magnitude bigger than KNa (app).

The value of K0.5Na estimated from amplitude measurements is in satisfactory agreement with the value calculated from kinetic constants (Table I). Qualitatively, K0.5K is smaller than KK as predicted by Eq. 4, but quantitatively the equilibrium and kinetic estimates of K0.5K differ by more than an order of magnitude. Karlish (1980) reported a comparable discrepancy (3–15-fold) between static fluorimetric and kinetic estimates of K0.5K. The inconsistency may be exaggerated in the case of the Na+-pump by the difficulty inherent in measuring apparently very tight binding, because satisfactory agreement between equilibrium and kinetic values of K0.5K was obtained with the H,K-ATPase, for which the measured K0.5K = 4 mM.[1]

In general, the study of fluorescence changes in fluorescein-modified sodium pump supports the mechanism of ion interaction with the enzyme in Eq. 1. The predictions of a kinetic equation derived by considering the measurements as relaxation experiments (Eq. 2) have been confirmed. Expressions for the amplitude of the fluorescence change (Eqs. 3 and 5) have been derived, and, with the exception of K0.5K in the case of the Na,K-ATPase, the kinetic and equilibrium data are quantitatively consistent. The demonstration that amplitudes measured in stopped-flow experiments can be successfully analyzed obviates the need for independent, static fluorescence measurements.

Acknowledgments

This work was supported by NIH grants DK-36873 (to L. D. Faller) and GM-28673 (to R. A. Farley), NSF grant DMB8704525 (to L. D. Faller), a Boehringer Ingelheim Fonds fellowship (to G. Scheiner-Bobis), and a VA Merit Review award (to L. D. Faller).

References

Garay, R. P., and P. J. Garrahan. 1973. The interaction of sodium and potassium with the sodium pump in red cells. Journal of Physiology. 231:297–325.

Karlish, S. J. D. 1980. Characterization of conformational changes in (Na,K) ATPase labeled with fluorescein at the active site. Journal of Bioenergetics and Biomembranes. 328:295–316.

[1] Faller, L. D., R. A. Diaz, G. Scheiner-Bobis, and R. A. Farley, manuscript submitted for publication.

Fluorescence Energy Transfer between N-[P-(2-benzimidazolyl)-phenyl] maleimide, N-(1-anilinonaphthyl-4)maleimide, Fluorescein 5′-isothiocyanate, and Anthroylouabain in Na+,K+-ATPase

Eiji Shinoguchi, Eiichi Ito, Akinobu Kudo, Shinji Nakamura, and Kazuya Taniguchi

Department of Chemistry, Faculty of Science, and Department of Orthodontics, School of Dentistry, Hokkaido University, Sapporo 060, Japan

Conformational differences between reaction intermediates in Na+,K+-ATPase have been clearly shown by various methods, including proteolytic digestion (Jorgensen, 1982) and intrinsic and extrinsic fluorescence probes (Karlish and Yates, 1978; Karlish, 1980; Taniguchi et al., 1980; Glynn, 1985). The enzyme preparations modified with N-[P-(2-benzimidazolyl)-phenyl]maleimide (BIPM) have been especially useful in following the conformational changes accompanying the sequential appearance of reaction intermediates in real time (Taniguchi et al., 1980,1982,1984, 1986, 1988, 1990; Suzuki et al., 1987): the relative fluorescence intensities of Na+ bound enzyme (NaE_1), ADP-sensitive phosphoenzyme (E_1P), K+-sensitive phosphoenzyme (E_2P), and K+ bound enzyme (KE_2) can be clearly distinguished. The transition from E_1P to E_2P induced the largest fluorescence change. However, quantitative aspects of conformational changes remain to be elucidated. To measure the change in the distance between covalently bound fluorescence probes in various enzyme states (Post et al., 1969; Albers, 1976; Glynn, 1985) would be very valuable in helping to elucidate the mechanism of energy transduction in Na+,K+-ATPase. In this paper, we have estimated the distance between covalently bound fluorescence probes such as BIPM (Cys-964) and fluorescein 5′-isothiocyanate (FITC; Lys-501) and the distance between these probes and the anthroylouabain (AO) binding site which is assumed to be the first extracellular domain of the α-chain (Price et al., 1989).

Results and Discussion

Distance between BIPM and ANM Probes in α-Chain of Na+,K+-ATPase

The BIPM treated enzyme (Nagai et al., 1986) was further treated with N-(1-anilinonaphthyl-4)maleimide (ANM). The resulting preparations retained their original Na+,K+-ATPase activity and contained ~0.9 mol of ANM/BIPM/α-chain. The emission spectrum showed the maximum values at ~360 nm (BIPM fluorescence) and 425 nm (ANM fluorescence). The addition of sodium dodecylsulfate (SDS) almost completely abolished the fluorescence at 425 nm, with a significant increase in the fluorescence at 360 nm. The data suggested that fluorescence energy transfer between these probes was present, and that SDS destroyed the tertiary structure of the enzyme which increased the distance between the probes to reduce the energy transfer.

To estimate the distance between these probes, the energy transfer efficiencies in NaE_1, E_1P, E_2P, KE_2, and ouabain E_2P were calculated from the decrease of the BIPM fluorescence due to ANM binding to be 53.8 ± 1.2, 54.7 ± 1.2, 53.7 ± 1.2, 54.3 ± 1.2, and 53.7 ± 1.2% (n = 10). The quantum yields of BIPM probe in these enzyme species were 0.103 ± 0.005, 0.101 ± 0.005, 0.112 ± 0.005, 0.110 ± 0.005, and 0.112 ± 0.005 (n = 10). The overlap integral between the BIPM and the ANM absorption spectrum was 1221 × 10–17 ± 3 M–1cm^3 (n = 10). The distances between BIPM and ANM were calculated assuming the orientation factor to be ⅔ (random orientation between BIPM and ANM probes) and the refractive index of the medium to be 1.4 (Forster, 1960). These values in NaE_1, E_1P, E_2P, KE_2, and ouabain E_2P were 23.5, 23.3, 23.9, 23.7, and 23.9 Å, respectively. The means of the differences estimated from 10 experiments were 0.6 (E_2P – E_1P), 0.4 (KE_2 – E_1P), and 0.6 Å (ouabain E_2P – E_1P), respectively (P < 0.01). The data suggest that the change in the distance is around one-hundredth of the assumed net distance of Na+ and K+ movements across the membrane (~50 Å).

The Distance between ANM and FITC

The BIPM-ANM-treated enzyme was further treated with FITC (Karlish, 1980). The resulting preparation showed only 2% of original Na+,K+-ATPase activity. The ANM fluorescence (the maximum at 430 nm) was reduced but the FITC fluorescence (520 nm) increased. The energy transfer efficiency in NaE_1 was calculated from the decrease of the ANM fluorescence to be 4.4 ± 0.8% (n = 16). The quantum yields of ANM probe in NaE_1 were 0.0548 ± 0.0015 (n = 16). The overlap integral between ANM emission spectrum and FITC absorption spectrum was 9,332 × 1O–17 ± 3 M–1 cm^3. Thus the distance was calculated to be 50.6 ± 1.7 Å (n = 10). The change in the distance in various conformational states was too small to be detected under present experimental conditions.

The distance between BIPM and FITC was not calculated because the decrease of the BIPM fluorescence due to FITC binding was too small to detect accurately. However, it is possible to estimate the distance between 58 and 74.5 Å. The former value was obtained assuming that the energy transfer efficiency is 3%, which should be detected in our experimental conditions. The latter was the summation of the distance of ANM-BIPM and ANM-FITC.

The Distance between BIPM–AO and FITC–AO

The energy transfer efficiency of anthroylouabain E_2P was calculated from the decrease of BIPM fluorescence by binding of AO to be 4.35%. The overlap integral between BIPM emission spectra and AO absorption spectrum was found to be 965.2 × 10–17 M–1cm^3. Thus the distance was calculated to be 39 Å using the quantum yield of BIPM probe as 0.112.

The distance between FITC and AO has already been reported to be 66–83 Å (Carilli et al., 1982). As a control experiment, this distance was calculated. The energy transfer efficiency in AOE_2P was calculated from the decrease of the fluorescence intensity of AO probe due to FITC binding to be 5.6%. The overlap integral between AO emission spectrum and FITC absorption spectrum was 9,944 × 10 –17 M–1 cm^3. Thus the distance was calculated to be 72.5 Å using the quantum yield of AO as 0.57.

These data are summarized in Fig. 1 using the model of Modyanov and co-workers (Arzamazova et al., 1988).

The distance between BIPM and AO (39 Å) is smaller than the distance of 72.5 Å between AO and FITC or trinitrophenyl ATP (64–78 Å; Carrilli et al., 1982; Fortes and Aguilar, 1988) or the distance of 58–74.5 Å between BIPM and FITC. These data suggest that the BIPM reactive site, Cys-964, is located in the transmembrane segment near the extracellular region as suggested (Arzamazova et al., 1988), rather than near the cytoplasmic region of the membrane. The estimated distance between BIPM and AO is consistent with a model of mutual disposition of the α-chain (Lutsenko et al., 1990). The BIPM probe at Cys-964 senses the change in Na+ binding states (Taniguchi et al., 1990). The same transmembrane segment (ILeu-946 to Leu-971) contains two Glu-954, -955 residues which may be related to Na+ binding.

Figure 1. Scheme of distribution and distance of fluorescence probes in the model of α-chain (Arzamazova et al., 1988). Closed and open circles show the BIPM (Cys-964) and FITC (Lys-501) binding sites, respectively. Open triangles in the first and fifth transmembrane segments are Cys-104 and 802. The fifth segment is nearer to the seventh segment containing Cys-964 (Lutsenko et al., 1990), while the distance between the first extracellular segment and the BIPM probe at Cys-964 is 39 Å.

The distance between ANM and BIPM probes is ~23.9 Å, which is much smaller than the thickness of the bilayer (~50 A). This suggests that the ANM probe is also located near the extracellular region in the membrane. There are two such Cys residues, Cys 104 and Cys 802. However, the model of Lutsenko et al. (1990) and the present data suggest that the latter is the ANM reactive site.

Rather small differences in the distance between ANM and BIPM probes accompanying sequential appearance of reaction intermediates were obtained in steady-state fluorescence measurements. Data presented here suggest the importance of measurement of transient fluorescence changes of both donor and acceptor probes accompanying formation and breakdown of the intermediates and of validation of the orientation factor in each intermediate.

Acknowledgments

This work was supported in part by a Grant-in-Aid for Scientific Research on Priority Areas of “Bioenergetics” to K. Taniguchi from the Ministry of Education, Science and Culture, Japan and the Naito Foundation.

References

Albers, W. 1976. The (sodium plus potassium)-transport ATPase. In The Enzymes of Biological Membranes. Vol. 3. A. Martonossi, editor. Plenum Publishing Corp., New York. 283–301.

Arzamazova, N. M., E. A. Arystarkhova, N. M. Gevondyan, N. M. Luneva, R. G. Efremov, N. A. Aldanova, V.-A. Nesmeyanov, and N. N. Modyanov. 1988. Sequence analysis of exposed domains of membrane-bound Na+,K+-ATPase: model of transmembrane arrangement. Progress in Clinical and Biological Research. 268A:57–64.

Carilli, C. T., A. F. Farley, D. M. Perlman, and L. C. Cantley. 1982. The active site structure of Na+- and K+-stimulated ATPase. Journal of Biological Chemistry. 257:5601–5606.

Forster, T. 1960. Transfer mechanisms of electronic excitation energy. Radiation Research Supplement. 2:326–339.

Fortes, P. A., and R. Aguilar. 1988. Distance between 5- iodoacetamido-fluorescein and the ATP and ouabain sites of (Na,K)-ATPase determined by fluorescence energy transfer. Progress in Clinical and Biological Research. 268A:197–204.

Glynn, I. M. 1985. The Na+,K+-transporting adenosine triphosphatase. In The Enzymes of Biological Membranes. Vol. 3. A. Martonossi, editor. Plenum Publishing Corp., New York. 35–114.

Jorgensen, P. L. 1982. Mechanism of the Na,K-pump: protein structure and conformation of the pure Na+,K+-ATPase. Biochimica et Biophysica Acta. 694:27–68.

Karlish, S. J. D. 1980. Characterization of conformational changes in Na+,K+-ATPase labeled with fluorescein of the active site. Journal of Bioenergetics and Biomembranes. 12:111–136.

Karlish, S. J. D., and D. W. Yates. 1978. Tryptophan fluorescence of (Na+,K+)-ATPase as a tool for study of the enzyme mechanism. Biochimica et Biophysica Acta. 527:115–130.

Lutsenko, S., E. Chertova, and N. Modyanov. 1990. Hydrophobic photochemical labeling of Na+,K+-ATPase. The Journal of General Physiology. 96:29a. (Abstr.)

Nagai, M., K. Taniguchi, K. Kangawa, S. Matsuo, S. Nakamura, and S. Iida. 1986. Identification of N-[p-(2- benzimidazolyl)phenyl]maleimide-modified residue participating in dynamic fluorescence changes accompanying Na+,K+-dependent ATP hydrolysis. Journal of Biological Chemistry. 261:13197–13202.

Post, R. L., S. Kume, T. Tobin, B. Orcutt, and A. K. Sen. 1969. Flexibility of an active center in sodium plus potassium adenosine triphosphatase. Journal of General Physiology. 54:306S-326S.

Price, E. M., D. A. Rice, and B. Lingrel. 1989. Site-directed mutagenesis of a conserved extracellular aspartic acid residue affects the ouabain sensitivity of sheep Na+,K+-ATPase. Journal of Biological Chemistry. 264:21902–21906.

Suzuki, K., K. Taniguchi, and S. Iida. 1987. The acceleration of Na+,K+-ATPase activity by ATP and ATP analogues. Journal of Biological Chemistry. 262:1175–1177.

Taniguchi, K., T. Sasaki, E. Shinoguchi, Y. Kamo, and E. Ito. 1990. The change in Na+ binding states before and after accumulation of ADP-sensitive phosphoenzyme in Na+,K+-ATPase. The Journal of General Physiology. 96:58a–59a. (Abstr.)

Taniguchi, K., H. Tosa, K. Suzuki, and Y. Kamo. 1988. Microenvironment of two different extrinsic fluorescence probes in Na+,K+-ATPase changes out of phase during sequential appearance of reaction intermediates. Journal of Biological Chemistry. 263:12943–12947.

Taniguchi, K., K. Suzuki, T. Sasaki, H. Shimokobe, and S. Iida. 1986. Reversible change in light scattering following formation of ADP-sensitive phosphoenzyme in Na+,K+-ATPase modified with N-[p-(2- benzimidazolyl)phenyl]maleimide. Journal of Biological Chemistry. 261:3272–3281.

Taniguchi, K., K. Suzuki, D. Kai, I. Matsuoka, K. Tomita, and S. Iida. 1984. Conformation change of sodium-and potassium-dependent adenosinetriphosphatase. Journal of Biological Chemistry. 259:15228–15233.

Taniguchi, K., K. Suzuki, and S. Iida. 1982. Conformational change accompanying transition of ADP-sensitive phosphoenzyme to potassium-sensitive phosphoenzyme of (Na+,K+)-ATPase modified with N-[p-(2-benzimidazolyl)phenyl]maleimide. Journal of Biological Chemistry. 257:10659–10667.

Taniguchi, K., K. Suzuki, J. Shimizu, and S. Iida. 1980. ATP dependent reversible conformational change of Na+,K+-ATPase modified with N-[P-2-(benzimidazolyl)phenyl]maleimide. Journal of Biochemistry. 88:609–612.

Effect of Na+ and Amino Compounds on the E_2 → E_1 Transition, Studied by Eosin Fluorescence

T. J. F. Van Uem, F. M. A. H. Schuurmans Stekhoven, G. F. Borm, A. G. M. Theeuwes, H. G. P. Swarts, and J. J. H. H. M. De Pont

Departments of Biochemistry and Mathematical Statistical Analysis, University of Nijmegen, Nijmegen, The Netherlands

Introduction

In a previous article from our laboratory (Schuurmans Stekhoven et al., 1988) we reported on the stimulatory and inhibitory effects of substituted and unsubstituted amines on the steady-state phosphorylation level of Na,K-ATPase.

As the steady-state phosphorylation level is determined by partial reactions preceding and following the phosphorylation step, we decided to investigate the effect of these amino compounds on the E_2 → E_1 transition. This conformational transition has been studied with the stopped-flow technique, monitoring the increase of eosin fluorescence (Skou and Esmann, 1983).

Experimental Procedures

The rate of the transition between the E_2 and E_1 forms of Na,K-ATPase was measured at room temperature with a stopped-flow apparatus using in each experiment 1 μM eosin and 0.1 mg/ml Na,K-ATPase in 1 mM imidazole, pH 7.0. The effect of every concentration of Na+ or an amino compound on the E_2 → E_1 transition is the average of five tracings. The excitation wavelength was 530 nm and emission was recorded at 560 nm after passage through a 535-nm cut-off filter. Slits were 10 nm and 2,100 data points were collected per tracing. Control experiments of the E_2 → E_1 transition were carried out with Na+ and varying concentrations of K+. The influence of amino compounds on the rate of this transition was studied without addition of K+. This can be done since the equilibrium between the E_1 and E_2 forms of Na,K-ATPase in the presence of 1 mM imidazole, pH 7.0, and in the absence of cations is poised toward E_2. The effect of K+ was not studied here since the amino compounds cannot drive the E_2 to E_1 transition in the presence of low K+ concentrations.

Results

Effect of Na+

Fig. 1 shows a typical stopped-flow tracing of the E_2 → E_1 transition when 200 mM imidazole, pH 7.0, is mixed with 0.2 mg/ml Na,K-ATPase. Not only in this example but also in the other experiments with Na+ or amino compounds all tracings appear to consist of a fast and a slow phase. Therefore, they have been fitted by an expression of the form:

where F(t) is the fluorescence in arbitrary units, λ_1 and λ_2 (λ1,2 ≥ 0) are the observed rate constants for the fast and slow phases, respectively, and a_1 and a_2 are the amplitudes (al + a_2 = 100%) of the fast and slow phases. Figs. 2 and 3 show the dependency of λ_1 and λ_2 on the Na+ concentration after preincubation of the enzyme with varying concentrations of K+. As can be seen, there is an inhibitory effect of K+ and a stimulatory effect of Na+ on both rate constants.

Figure 1. Effect of 100 mM imidazole, pH 7.0, on the rate of transition from E_2 to E_1. Na,K-ATPase (0.2 mg/ml) in 1 μM eosin and 1 mM imidazole, pH 7.0, was mixed (1:1) at 20°C with 200 mM imidazole, pH 7.0, with 1 μM eosin.

The relation between the amplitude a_1 of the fast phase and the K+ concentration is shown in Fig. 4. This relation indicates that the enzyme form with the fast transition rate can be driven by K+ to a form with a slow transition rate (Esmann and Skou, 1983). The amplitudes of the fast and slow phases do not change during the Na+-driven E_2→ E_1 transition, as can be seen in Fig. 5. These observations can be interpreted with the following scheme:

There are two low-fluorescence enzyme forms, E_2 and E_2K+ (occluded K+), which are in equilibrium before Na+ is added but which cannot interchange during the transition to the high-fluorescence form E_1, as has been shown in Fig. 5. The transition from E_2 to E_1 represents the fast fluorescence change, while the transition from E_2K+ to E_1 + K+ represents the slow change. The relation between the observed rate constants λ1,2 and the intrinsic rate constants kl,2 and k–1, –2 of the given scheme can be given by the empirical equation:

For the fast transition we then get:

and for the slow phase:

These results show that both rate constants are dependent on Na+ and K+ with different affinities.

Figure 2. Relation between Na+ and the rate constant λ_1 (s–1) of the fast phase at different K+ concentrations. The concentrations are given as final concentrations after mixing. () 0 μM K+ K+, (∆) 20 μM K+, () 50 μM K+, and () 75 μM K+.

Figure 3. Relation between Na+ and the rate constant λ_2 (s–1) of the slow phase at different K+ concentrations. The concentrations are given as final concentrations after mixing. () 0 μM K+, (∆) 20 μM K+, () 50 μM K+, and () 75 μM K+.

Figure 4. The effect of K+ on the amplitude al (%) of the fast phase. The relation follows the equation al = 52 – 0.45·[K+].

Figure 5. Relation between Na+ and the amplitude a_1 (%) of the fast phase at 20 (∆) and 50 μM K+ ().

Effect of Amino Compounds

Five amino compounds, having a very diverse effect on the steady-state phosphorylation level of Na,K-ATPase, have been tested on the E_2 → E_1 transition without added K+. Of the five compounds tested, four show a stimulatory effect on the rate constants λ_1 and λ_2 with increasing concentration. As the K+ concentration in these experiments is about zero, we can reduce the empirical equation to:

where L stands for ligand. The results for the amino compounds are represented in Table I. In this table only the values for the forward rate constants are given, since the values for the backward rate constants are about zero.

TABLE I
Effect of Amino Compounds on the Rate Constants k_1 and k_2 of the E_2 → E_1 Transition

n, Number of experiments; m, number of concentrations per experiment; C, concentration of triallylamine (millimolar).
*Instead of kl,2, λ1,2 is represented, which is in this case apparently independent of the concentration of triallylamine.

Conclusions

(a) The ratio of the fast/slow pool is linearly dependent on K+ and is stable during the time course of the experiment (except in case of triallylamine). (b) The velocity of each transition depends on K+ concentration with different affinities. (c) The kinetics of the transitions (k_1 = 0.07–0.20 s–1·mM–1, k_2 = 0.005–0.010 s–1·mM–1) display little dependence on the type of ligand except for triallylamine, as the most effective buffer component (k_1 = 18 s–1·mM–1, (k_1 = 0.56 s–1·mM–1), and for the (k_2 = 0.022 s–1·mM–1) of Na+, indicating an easier displacement of high-affinity K+.

References

Esmann, M., and J. C. Skou. 1983. The effect of K+ on the equilibrium between the E_2 and the K+-occluded E_2 conformation of the (Na+ + K+)-ATPase. Biochimica et Biophysica Acta. 748:413–417.

Schuurmans Stekhoven, F. M. A. H., H. G. P. Swarts, G. K. 't Lam, Y. S. Zou, and J. J. H. H. M. De Pont. 1988. Phosphorylation of (Na+ + K+)-ATPase: stimulation and inhibition by substituted and unsubstituted amines. Biochimica et Biophysica Acta. 937:161–176.

Skou, J. C., and M. Esmann. 1983. The effects of Na+ and K+ on the conformational transitions of (Na+ + K+)-ATPase. Biochimica et Biophysica Acta. 746:101–113.

Slow and Rapid Components of Dephosphorylation Kinetics of Na,K-ATPase

Kuniaki Suzuki and Robert L. Post

Department of Molecular Physiology and Biophysics, Vanderbilt University Medical School, Nashville, Tennessee 37232-0615

Introduction

To better understand the mechanism of Na,K-ATPase, we studied dephosphorylation kinetics of its phosphoenzyme intermediate, including components sensitive to dephosphorylation upon addition of ADP or K+ (E_1P or E_2P, respectively). For analysis of this kinetics it is important to know whether phosphoenzyme decay is single exponential or not; this question has not been resolved (Glynn, 1988). We improved the chase method over those of previous investigators and found non-single exponential decay kinetics. We isolated rapid and slow components and analyzed non–single exponential decay as a combination of two components.

Materials and Methods

Membrane-bound Na,K-ATPase from dog kidney outer medulla was prepared by Jørgensen's zonal method (Jørgensen, 1974). The specific activity was 30–35 U/mg at 37°C and the phosphoenzyme level was 2.1–2.8 nmol/mg protein. Phosphorylation and dephosphorylation of the enzyme were done at 2°C as follows unless otherwise stated. In 0.9 ml the reaction system contained 57–79 μg of membrane protein, 40 μmol of imidazole/MOPS buffer at pH 7.1,0.1–0.5 μmol of MgCl_2, and 30–512 μmol of NaCl with or without 0.1 mg of bovine serum albumin. The enzyme was labeled by adding 10 nmol of [γ-32P]ATP in 0.1 ml. After 10, 20, or 30 s phosphorylation was stopped either by an acid quench or by 0.1 ml of a chase solution containing 0.11–1.84 μmol unlabeled ATP and/or 6–6.7 μmol 1,2-diaminocyclohexane-N,N,N′,N′-tetraacetic acid (CDTA) without or with 4.4 nmol–1.43 μmol of ADP or with 6.6 μmol of KCl. After various times the chase was terminated with 10 ml of 0.35 M trichloroacetic acid containing 0.6 mM ATP and 13 mM H_3PO_4. The samples were filtered, washed, and counted. Concentrations given in the legends are final concentrations of reaction mixtures.

Results

Search for the Best Chase Solution

To terminate formation of [32P]phosphoenzyme and observe its decay, a chase by unlabeled ATP or by a chelator of magnesium ion is usually used. By the former chase labeled ATP is diluted by cold ATP and new formation of [32P]phosphoenzyme is terminated. By the latter chase phosphoenzyme formation is terminated by chelation of Mg2+, which is essential for phosphorylation. Both chase methods had problems. At low concentrations of NaCl (Fukushima et al., 1984) an ATP chase slowed dephosphorylation compared with a CDTA chase (not shown). At high concentrations of NaCl the fraction of ADP-sensitive and K+-insensitive phosphoenzyme, E_1P, increases (Taniguchi and Post, 1975). In this case contamination of ATP with ADP reduced the amount of [32P]phosphoenzyme. In the presence of 512 mM NaCl we phosphorylated the enzyme from [32P]ATP and chased with a 10-fold molar ratio of CDTA to Mg2+. After this plain CDTA chase 28% of initial phosphoenzyme remained after 20 s. Addition to 100 μM cold ATP to the chase solution decreased the remaining phosphoenzyme to 9%. Further addition of 4 μM ADP decreased the phosphoenzyme to 3% (not shown).

For a plain chase with CDTA we varied the ratio of CDTA to Mg2+. Dephosphorylation was almost maximal at a 20-fold molar ratio of CDTA to Mg2+. However, 4% of phosphoenzyme remained at 80 s after the start of the chase. So we tested the effects of adding CDTA to the phosphorylation mixture before [32P]ATP. Phosphorylation reached 4% of maximal phosphoenzyme level at 110 s after addition of [32P]ATP even in the presence of a 30-fold ratio of CDTA to Mg2+ (not shown). Finally, we chose a combination of CDTA (CDTA/Mg2+ = 20:1) and cold ATP (cold ATP/hot ATP = 10:1) as the best chase solution. When this chase solution was added to the phosphorylation mixture before [32P]ATP, <0.25% of maximal phosphoenzyme appeared at 90 s after the addition of [32P]ATP (not shown).

Figure 1. Non–single exponential decay of phosphoenzyme (EP). [NaCl] was 30 mM and [MgCl_2] was 0.1 mM. As described in Materials and Methods, phosphorylation proceeded for 30 s. EP was chased with the best chase solution (see text). The solid line fits this equation: EP = (70 × e–0105t) + (30 × e–0.021t).

Isolation of Rapid and Slow Components of Decay Kinetics

Phosphoenzyme formed with 30 mM NaCl and 0.1 mM MgCl_2 showed non–single exponential decay with the best chase solution (Fig. 1). We separated rapid and slow components of the non–single exponential decay. The rapid component was detected as follows. The enzyme was first phosphorylated with unlabeled ATP and then chased with CDTA for 3 s. Rapidly dephosphorylated enzyme was rephosphorylated with [32P]ATP and excess MgCl_2 for 2 s and then chased with unlabeled ATP. This technique revealed rapid single exponential decay (K = –0.105/s) (Fig. 2). When the enzyme was phosphorylated in the presence of 0.1 mM Mg2+ plus 0.4 mM CDTA, only 20% of maximum phosphoenzyme appeared. After a chase this phosphoenzyme showed slow and slightly curvilinear decay (Fig. 2). In the presence of a low free Mg2+ concentration the rate of phosphoenzyme formation was probably slower than that of dephosphorylation of the rapid component. This curved kinetics was analyzed as a combination of rapid and slow components by using K = –0.105/s for the rapid component. A best fit showed that this 22% of maximal phosphoenzyme was composed of 7.4% of a rapid component and 14.8% of a slow component (K = –0.021/s) (Fig. 2).

Analysis of Non–Single Exponential Decay as a Combination of Rapid and Slow Components

Non–single exponential decay was analyzed using the rate constants of the rapid and slow components. A best fit showed that 70% of phosphoenzyme was the rapid component and 30% was the slow component (Fig. 1). We tested the sensitivity of the slow component to ADP, to K+, or to both. With 30 mM NaCl the slow component was sensitive to K+ and insensitive to ADP (not shown). With 512 or 860 mM NaCl ~45% or 10–20%, respectively, of the slow component was sensitive to K+. In both cases almost all phosphoenzyme was sensitive to ADP (not shown). The slow component seems to have a normal sensitivity to K+ or ADP. We also found that both ADP- and K+-resistant components of the phosphoenzyme showed non–single exponential kinetics (Fig. 3). Thus, both E_1P and E_2P contained rapid and slow components.

Figure 2. Isolation of rapid and slow components of decay kinetics. [NaCl] was 30 mM and [MgCl_2] was 0.1 mM. To isolate the rapid component, phosphorylation was initiated by addition of 10 nmol of unlabeled ATP instead of [32P]ATP as in Materials and Methods. At 10 s after ATP, EP was chased with 2 μmol of CDTA (CDTA/Mg = 20:1). 3 s later, rapidly dephosphorylated enzyme was rephosphorylated with 0.1 ml of [32P]ATP and excess MgCl_2. 2 s later, [32P]EP was chased with 1 mM cold ATP (+). By this method 14.7% of the maximum EP level of this enzyme was phosphorylated by [32P]ATP. To isolate the slow component, phosphorylation was done as in Materials and Methods except that 0.4 mM CDTA (CDTA/Mg = 4:1) was added before [32P]ATP. At 30 s after [32P]ATP, EP was chased with best chase solution ().

In other experiments ADP or K+ sensitivity of the phosphoenzyme or the rate constant of dephosphorylation varied with the time of storage after enzyme purification or with the enzyme fraction prepared by the zonal method (not shown). Thus, different enzyme preparations can have different proportions of rapid and slow components.

Discussion

In models of the enzyme where rapid and slow components are interconvertible more slowly than the turnover time, rapid and slow molecules of the enzyme react with ATP separately and in parallel. Perhaps kinetic separation of rapid and slow components is related to lateral phase separation of phospholipids in the bilayer of the fragments of membrane in which the enzyme was embedded. Alternatively, a model of protomer interactions may prove useful: a molecule of phosphoenzyme might react more rapidly if adjacent molecules in the bilayer are also phosphorylated.

Figure 3. Non–single exponential dephosphorylation of ADP-or K-resistant EP. [NaCl] was 512 mM and [MgCl_2] was 0.1 mM. At 20 s after phosphorylation, EP was chased with the best solution without (+) or with 0.5 mM free ADP (∆) or 6 mMK().

Acknowledgments

This work was supported by grant HL-01974 from NHLBI, NIH.

References

Fukushima, Y., S. Yamada, and M. Nakao. 1984. ATP inactivates hydrolysis of K+-sensitive phosphoenzyme of kidney Na+,K+-transport ATPase and activates that of muscle sarcoplasmic reticulum Ca2+-transport ATPase. Journal of Biochemistry. 95:359–368.

Glynn, I. M. 1988. The coupling of enzymatic steps to the translocation of sodium and potassium. In The Na+,K+-Pump. Part A: Molecular Aspects. J. C. Skou, J. G. Nørby, A. B. Maunsbach, and M. Esmann, editors. Alan R. Liss, Inc., New York. 435–460.

Jørgensen, P. L. 1974. Purification and characterization of (Na,K)-ATPase. III. Purification from the outer medulla of mammalian kidney after selective removal of membrane components by sodium dodecylsulfate. Biochimica et Biophysica Acta. 356:36–52.

Taniguchi, K., and R. L. Post. 1975. Synthesis of ATP and exchange between inorganic phosphate and ATP in sodium and potassium transport ATPase. Journal of Biological Chemistry. 250:3010–3018.

High Concentrations of ATP Induce a Transient Extra Phosphorylation of the Na,K-ATPase

Ruben D. Peluffo, Patricio J. Garrahan, and Alcides F. Rega

Instituto de Química y Fisicoquímica Biológicas (UBA-CONICET), Junín 956, 1113 Buenos Aires, Argentina

It is generally accepted that ATP plays two different roles in the Na,K-ATPase reaction: (a) it acts as the substrate with high affinity (Km ≈ 0.2 μM) and (b) it accelerates the release of occluded K+, which is the rate-limiting step of the ATPase reaction, acting with low affinity (Km ≈ 150 μM) and through a mechanism that does not involve its hydrolysis (see Glynn, 1985). In agreement with this, when assayed in media without K+, the low affinity effect of ATP during the steady-state hydrolysis of ATP by the ATPase is not apparent. In addition, in the absence of K+ phosphorylation of the ATPase is ~100-fold faster than dephosphorylation (Mårdh and Lindhal, 1977), so that in the steady state most of the enzyme is phosphorylated. It seems reasonable, therefore, to expect the phosphorylation reaction of the Na,K-ATPase in media without K+ to depend solely on the binding of ATP with high affinity. The experiments reported here yielded results that seem to conflict with this expectation.

The Na,K-ATPase used was partially purified from the red outer medulla of pig kidney following either the simpler of the two procedures described by Jörgensen (1974) or the procedure of Jensen et al. (1984). Phosphorylation was carried out in a rapid mixing apparatus adapted for chemical quenching (Intermekron, Uppsala, Sweden). Incubations were performed in media with (in mM): 40 imidazole-HCl or Tris-HCl, 150 NaCl or choline chloride, 2 EDTA, and enough MgCl_2 to give a final free Mg2+ concentration of 0.5 mM. The pH was 7.5 and the temperature was 25°C. The reaction was started by mixing the enzyme in the incubation buffer with enough [γ-32P]ATP to give a final concentration of either 10 or 500 μM ATP. Phosphoprotein levels were calculated from the difference between 32P incorporation in the above media and in media without MgCl_2. Control experiments performed under conditions identical to those used during the phosphorylation reaction showed that ATPase activity in media with Na+ and without K+ remained constant between 10 and 500 μM ATP.

Fig. 1 shows the time course of phosphorylation in media with either 10 or 500 μM ATP. At 10 μM ATP, a concentration ≈ 50 times higher than the Km in media without K+ (Rossi and Garrahan, 1989), phosphorylation followed simple exponential kinetics with a t1/2 of 9 ms and reached a steady-state value of 2 nmol/mg protein. This confirms previous findings from other authors (Mårdh and Post, 1977). However, when the concentration of ATP was raised to 500 μM the time course of phosphorylation became biphasic and the reaction became so fast that it was not possible to sample points in the rising part of the curve. At the shortest time interval (2.5 ms) phosphorylation reached a value three times higher than that obtained with 10 μM ATP. After this early maximum the level of phosophoprotein decayed, reaching in ~15 ms the same steady-state level as with 10 μM ATP.

The extra and transient phosphorylation observed at 500 μM ATP during the experiment in Fig. 1 persisted in media with 40 mM imidazole when choline chloride replaced all the NaCl. The phenomenon, however, proved to be fully dependent on Na+ when the buffer was Tris-HCl. This probably reflects the known ability of imidazole, but not of Tris, to replace Na+ as cofactor for phosphorylation (Schuurmans et al., 1985). Phosphorylation at 500 μM ATP was inhibited by ouabain: a 10-min preincubation at 25°C in media with 1 mM ouabain led to complete inhibition of the phenomenon when phosphorylation was performed in media with Na+ and to 85% inhibition when the media contained imidazole and choline chloride.

LDS/PAGE electrophoresis showed that phosphoprotein made with 500 μM ATP could be quantitatively recovered in a band with the mobility of the phosphoprotein made with 10 μM ATP. At pH 6, 150 mM hydroxylamine accelerated more than 17-fold the nonenzymatic release of 95% of the phosphate from the phosphoprotein made with 500 μM ATP. These results strongly suggest that the phosphoprotein formed with 500 μM ATP is a phosphoenzyme of the Na,K-ATPase with the phosphate linked to the enzyme through an acyl-phosphate bond.

Figure 1. The time course of formation of phosphoprotein in media with 10 () or 500 () μM ATP. The incubation media contained NaCl and imidazole. See text for other details.

The transient phosphorylation at 500 μM ATP completely disappeared when the ATPase was preincubated for 25 min at 25°C in media with 0.200 mM fluorescein isothiocyanate (FITC). The effect of FITC was partially prevented by 5 mM ATP. Since FITC seems to be a specific inhibitor of the binding of ATP to E_1E_2 ATPases (Karlish, 1980), this result suggests that the sites involved in phosphorylation at 500 μM ATP are either the same as or closely related to the sites for the nucleotide that participate in the hydrolysis cycle of th Na,K-ATPase.

The results indicate that the Na,K-ATPase has considerably more sites than those that are phosphorylated with 10 μM ATP in media without K+, conditions under which the catalytic site of the enzyme should be almost fully occupied. This finding is not in keeping with the current reaction schemes of the Na,K-ATPase (Glynn, 1985). Although we do not yet have an explanation for our finding, it could be that different sites are phosphorylated at 10 than at 500 μM ATP, or that as a consequence of a noncatalytic effect of ATP at 500 μM, more of the sites that are phosphorylated at 10 μM ATP are exposed and transiently phosphorylated.

Acknowledgments

This work was supported by grants from CONICET and Universidad de Buenos Aires. P. J. Garrahan is grateful to Fundación Antorchas for a travel grant to attend the meeting.

References

Glynn, I. M. 1985. The Na+,K+-transporting adenosine triphosphatase. In The Enzymes of Biological Membranes. 2nd ed. A. Martonosi, editor. Plenum Publishing Corp., New York. 33–114.

Jensen, J., J. C. Nørby, and P. Ottolenghi. 1984. Sodium and potassium binding to the sodium pump of pig kidney: stoicheiometry and affinities evaluated from nucleotide-binding behaviour. The Journal of Physiology. 346:219–241.

Jörgensen, P. L. 1974. Purification and characterization of (Na+ + K+)-ATPase. III. Purification from the outer medulla of mammalian kidney after selective removal of membrane components by SDS. Biochimica et Biophysica Acta. 356:36–52.

Karlish, S. J. D. 1980. Characterization of conformational changes in (Na,K)ATPase labelled with fluorescein at the active site. Journal of Bioenergetics and Biomembranes. 12:111–136.

Mårdh, S., and S. Lindahl. 1977. On the mechanism of sodium and potassium-activated adenosine triphosphatase. Time course of intermediary steps examined by computer simulation of transient kinetics. The Journal of Biological Chemistry. 252:8058–8061.

Mårdh, S., and R. L. Post. 1977. Phosphorylation from adenosine triphosphate of sodium and potassium-activated adenosine triphosphatase. Comparison of enzyme-ligand complexes as precursors to the phosphoenzyme. The Journal of Biological Chemistry. 252:633–638.

Rossi, R. C., and P. J. Garrahan. 1989. Steady-state kinetic analysis of the Na+/K+-ATPase: the inhibition by potassium and magnesium. Biochimica et Biophysica Acta. 981:105–114.

Schuurmans Stekhoven, F. M. A. H., H. G. P. Swarts, J. J. H. H. M. de Pont, and S. L. Bonting. 1985. Na +-like effect of imidazole on the phosphorylation of (Na+ + K+)-ATPase. Biochimica et Biophysica Acta. 815:16–24.

High Affinity Inhibitory Effect of Cytoplasmic K+

Flemming Cornelius and Jens Chr. Skou

Institute of Biophysics, University of Aarhus, Aarhus, Denmark

Introduction

Using unsided, membrane-bound Na,K-ATPase it has been shown that the high affinity activating effect of K+ in the presence of Na+ disappears at low ATP concentration and is replaced by a high affinity inhibition that is dependent on the ATP concentration and the temperature (Skou, 1979). The high affinity effect of K+ has been explained as being due to extracellular K+, which with a high affinity promotes dephosphorylation, becomes occluded, and deoccludes very slowly at low ATP concentration, causing the overall rate of hydrolysis to become lower than in the presence of Na+ alone. The low affinity inhibitory effect of K+ is due to a competition for Na+ at the cytoplasmic aspect of the pump.

The present experiments were performed to investigate directly the sidedness of the K+ inhibition by using reconstituted Na,K-ATPase from salt glands of the spiny dogfish and analyzing the effect of cytoplasmic K+ on the ATP hydrolysis-dependent Na+:Na+ exchange. This exchange, from the point of view of stoichiometry and electrogenicity, behaves like a Na+:K+ exchange, suggesting that the reaction follows the same pathway as the Na+:K+ exchange, but with a lower rate (Cornelius and Skou, 1985).

Methods

The methods for preparing the membrane-bound enzyme, solubilized enzyme, and its reconstitution into liposomes have been described in previous papers (Cornelius and Skou, 1984; Cornelius, 1988) as has the method for the determination of the fraction of molecules oriented inside-out (10%), right-side out (65%), and nonoriented (25%).

The rate of Na+:Na+ exchange was assessed by its accompanying hydrolytic activity as previously described (Cornelius and Skou, 1985). Identical results were obtained if Na+ tracer-flux into the proteoliposomes was measured instead of ATPase activity. The membrane-bound and the reconstituted enzyme preparations differ not only in their sidedness but also in that the sided enzyme has been dissolved in C_12E_8, and even if C_12E_8 is removed later with bio-beads the preparation still contains traces of C_12E_8. Huang et al. (1985) have demonstrated effects of nonsolubilizing concentrations of C_12E_8 on K0.5 for ATP and K+, indicating that the detergent has effects resembling low affinity ATP binding. Similar observations were found in the present study; therefore, to compare results from the sided and the unsided preparations we used both membrane-bound enzyme and reconstituted, nonoriented enzyme as controls for the unsided preparation. The reconstituted, nonoriented fraction of enzyme has been through the same procedures as the fraction of inside-out orientated enzyme, and is tested in the presence of the same traces of C_12E_8. The difference in ATP sensitivity of the Kcyt effect of the two unsided preparations is noted by comparing Figs. 1 and 2.

Results and Discussion

Temperature and ATP

In Fig. 1 the combined effect of inversely varied Na+ and K+ (their sum kept constant at 110 mM) on the hydrolytic activity of unsided, membrane-bound Na,K-ATPase is compared with the reconstituted, inside-out oriented Na,K-ATPase, where the extracellular Na+ concentration is kept constant and only the cytoplasmic Na+ and K+ concentrations are varied. The ATP concentration is 1 μM and the temperature is 0°C. Under such conditions the high affinity activation by K+ in unsided preparations disappears; instead, the activity decreases steadily with the increase in the K+/Na+ ratio as previously shown (Skou, 1979). As seen from Fig. 1, with the membrane-bound Na,K-ATPase (lower curve) both high and low affinity inhibitory effects of K+ are present. The same picture is seen at 23°C but at a lower ATP concentration of 0.1 μM (not shown). The high and low affinity inhibitory effects of K+ are also present with the sided preparation (upper curve) with only K+ on the cytoplasmic side of the enzyme. However, the affinity for the inhibitory effect of cytoplasmic K+ is apparently lower.

Figure 1. Comparison of the activation of unsided, membrane-bound Na,K-ATPase (lower curves, ∆) and sided, reconstituted Na,K-ATPase (upper curve, ○) at 0°C. Na+ and K+ are varied inversely, their sum kept constant at 110 mM. The temperature in both cases is 0°C and the ATP concentration is 1 μM.

When the same type of experiments are performed at room temperature (Fig. 2) the deactivating effect of K+ is also seen at the lower ATP concentrations (1 μM) for both sided and unsided (nonoriented enzyme) preparations (panels A and B). However, by increasing the ATP concentration (≥ 25 μM) the well-known activation of K+ is observed in the unsided preparation (panel D), whereas cytoplasmic K+ still produces inhibition in the sided preparation (panel C). Since the high affinity inhibitory effect of cytoplasmic K+ in sided preparations could not be counteracted by high ATP concentrations at 23°C or 0°C (on the contrary, it persists even with a concentration of ATP [3 mM] that is saturating for hydrolysis), it is presumably not connected with the disappearance of the K+ inhibition by increased ATP concentration in the unsided preparations. The apparent affinity for the high affinity inhibitory effect of K+ relative to the affinity for Na+ decreases with the increase in the ATP concentration.

Figure 2. Activation of hydrolytic activity accompanied by Na+:Na+ exchange of reconstituted i:o-oriented (left panels) and n:o-oriented (right panels) Na,K-ATPase by inversely varied cytoplasmic Na+ and K+ at 23°C. Extracellular (intraliposomal) Na+ is kept at 115 mM. The ATP concentrations are 1 μM (A and B) and 25 μM (C and D).

Model Simulations

The explanation given for the high affinity inhibition seen in membrane-bound, unsided Na,K-ATPase during conditions of low ATP and temperature, ascribing it to extracellular K+ and a slow deocclusion of the E_2(K_2)-form, cannot account for the observed high affinity inhibition of K+ using sided preparations (e.g., by a backward occlusion of cytoplasmic K+), since the effect could not be relieved by high ATP concentrations.

In light of the characteristics described above, especially the pronounced inhibition of 1 mM Kcyt, in the presence of 130 mM Nacyt, we found it necessary to include in the model for Na+:Na+ exchange a hybrid pool formed by K+-binding to an enzyme species with bound Na+, and which contains enzyme species that cannot bind ATP. The outline of the model is shown in Fig. 3 and will be described in more detail elsewhere (Cornelius, F., and J. C. Skou, manuscript in preparation). In the model it is assumed that in addition to the spontaneous deocclusion of E_2(Na_2) (via k′5), high affinity cytoplasmic binding of K+ to this enzyme species can occur before Na+ release (via K'7), forming a hybrid complex E_2Na_2K contained in the hybrid pool. Inside the hybrid pool a rapid equilibrium between E_2-enzyme species exists, so that at increasing Kcyt, and decreasing Nacyt the two Na+ will discharge and binding of another cytoplasmic K+ ion takes place. In the forward direction the two K+ are then occluded and this is followed by a return to the E_1A pool in the main scheme by low affinity ATP binding. The high affinity inhibition of cytoplasmic K+ is a result of the trapping of enzyme species in the hybrid pool, which only slowly, governed by k′8, are released into the main scheme. Although no actual fitting of experimental data to the model is attempted, the experimental observations on both sided and unsided Na,K-ATPase can be simulated by the hybrid binding model depicted in Fig. 3.

Figure 3. The hybrid model. In the model each box signifies a pool of enzyme species in rapid equilibrium. k′ denotes apparent rate constants. Cytoplasmic K+ can act at two places: on the E_1A pool where low affinity competition between cytoplasmic Na+ and K+ can take place, and through high affinity binding to the occluded E_2-form, which contains two occluded (extracellular) Na+. The hybrid pool contains the following enzyme species in rapid equilibrium: E_2Na_2K = E_2NaK = E_2K = E_2K_2. The interaction of E_2(Na_2) with Kcyt causes the high affinity inhibition in the sided preparations, due to the slow returning into the main scheme governed by the rate constant k′8 and through low affinity ATP binding (k′9).

Acknowledgments

The technical assistance of H. Zakarias and the financial support from The Danish Medical Council, Novo Foundation, and The Danish Biotechnology Centre for Biomembranes is gratefully acknowledged.

References

Cornelius, F. 1988. Incorporation of C_12E_8-solubilized Na+,K+-ATPase into liposomes: determination of sidedness and orientation. Methods in Enzymology. 156:156–167.

Cornelius, F., and J. C. Skou. 1984. Reconstitution of (Na+ + K+)-ATPase into phospholipid vesicles with full recovery of its specific activity. Biochimica et Biophysica Acta. 772:357–373.

Cornelius, F., and J. C. Skou. 1985. Na+-Na+ exchange mediated by (Na+ + K+)-ATPase reconstituted into liposomes: evaluation of pump stoichiometry and response to ATP and ADP. Biochimica et Biophysica Acta. 818:211–221.

Huang, W.-H., S. S. Kakar, and A. Askari. 1985. Mechanism of detergent effects on membrane-bound (Na+ + K+)-ATPase. Journal of Biological Chemistry. 260:7356–7361.

Skou, J. C. 1979. Effects of ATP on the intermediary steps of the reaction of the (Na+ + K+)-ATPase. IV. Effect of ATP on K0.5 for Na+ and on hydrolysis at different pH and temperature. Biochimica et Biophysica Acta. 567:421–435.

Sidedness of Na and K Effects on Phosphorylation and Conformations of Pig Kidney Na,K-ATPase Interacting with Inorganic Phosphate or Acetyl Phosphate

G. Berberián and L. Beaugé

Instituto de Investigación Médica “Mercedes y Martin Ferreyra,” 5000 Córdoba, Argentina

Introduction

All preparations that show Na,K-ATPase activity consistently display phosphatase activity (Glynn, 1985). The real meaning of the phosphatase activity is still under debate and its reaction cycle is far from established. Recent studies on ligand interactions support the idea that the phosphatase activity has similarities with the reversible binding of inorganic phosphate (see Beaugé et al., 1990, for references). The aim of this work was to examine this notion by studying the sidedness of the effects of Na and K on the steady-state phosphorylation levels acquired with acetyl phosphate and inorganic phosphate, and to determine the most abundant phosphoenzyme conformation induced by these two phosphoryl group donors.

Results

Partially purified pig kidney Na,K-ATPase (Jorgensen, 1974) was used throughout this work. In the first group of experiments, we studied, in a broken membrane preparation, the effects of 50 mM Na and 10 mM K, alone and in combination, on the steady-state levels of phosphoenzyme obtained with ATP, Pi, and AcP. As expected (Glynn, 1985), in the absence of both Na and K, phosphorylation was negligible with ATP; under the same conditions, 1 mM Pi gave values close to 70% of the maximal [E-P], whereas with 5 mM AcP [E-P] reached 63% of that maximal. In the presence of Na alone, ATP reached the maximal value, while with AcP the level was even higher than that with no monovalent cations (83% of that seen with ATP); on the other hand, with Pi there was no E-P formation. With K alone the picture was qualitatively similar to that seen with no Na or K (only Pi and AcP resulted in E-P formation) but the absolute values were about halved. Finally, in the presence of Na plus K, E-P formation was only noticeable with AcP. We also found that in the presence of 10 mM K the addition of 1 mM ADP reduced the Pi- and AcP-dependent 32P incorporation by 50%.

The sidedness of ligand effects on E-P from different phosphoryl donors was studied by using liposomes with incorporated Na,K-ATPase (Karlish and Pick, 1981). The basal vanadate-sensitive K-K exchange fluxes in these liposomes were low and not affected by 0.5 mM Pi alone. However, in the presence of 0.5 mM Pi, 0.1 mM ATP increased the exchange 5.7-fold (Karlish et al., 1982); in addition, under these conditions the levels of E-P were reduced by only 30%. Thus, a large ATP stimulation of Pi-dependent K-K exchange fluxes coexisted with a small reduction in the steady-state levels of enzyme phosphorylation. The results on sidedness can be summarized as follows (Figs. 1 and 2): (a) The sites at which Na inhibited Pi-dependent phosphorylation were required for phosphorylation from ATP, and stimulated E-P formation from AcP, were all cytoplasmic. (b) Cytoplasmic K (Kcyt) had no effect on the levels of E-P formed from Pi, whereas it increased those obtained with AcP by 30%. (c) E-P formed from Pi, with or without Kcyt, was reduced ~50% in the presence of external K (Kcxt). At 10 mM Kcyt, Naext (100 mM), with or without 10 mM Kext, had no effect. (d) When E-P was formed from AcP, a reduction due to Kext (~50%) occurred only if there was K or Na in the cytoplasmic side. (e) The maximal levels of [E-P] from Pi came about in the absence of any monovalent cation on both membrane phases. (f) The Nacyt-dependent phosphorylation from ATP decreased to 10% in the presence of Kext. (g) In the absence of Kext, the maximal levels of E-P formation obtained from ATP in the presence of Naeyt, matched those seen with AcP in the presence of either Kcyt or Nacyt. (h) The [E-P] formed from AcP in the absence of Na and K at both sides of the membrane resembles that observed in broken membranes incubated in the absence of both monovalent cations. (i) Finally, the addition of intracellular ATP or ADP resulted in a 30–40% fall in [E-P] formed from inorganic phosphate or acetyl phosphate in the presence of Kcyt and Kext.

Figure 1. Effects of cytoplasmic and extracellular Na and K on the steady-state levels of phosphorylation from 0.5 mM [32P]Pi of Na,K-ATPase incorporated into liposomes. The solution compositions were: (a) intravesicular (extracellular): 140-([Na + K]) mM Tris-HCl (pH 7.0 at 20°C); (b) extravesicular (cytoplasmic): 135- ([Na + K]) mM Tris-HCl (pH 7.0 at 20°C), 3 mM MgCl_2, and 0.5 mM [32P]Pi. Temperature was 20 ± 2°C.

In the experiments with FITC-labeled Na,K-ATPase, broken membranes were used. In this case, there were ligands that induced quenching unrelated to the enzyme conformation. The quenching was 20% with 3 mM MgCl_2 and 6% with 5 mM AcP; therefore, the initial baselines were established in the presence of AcP (without or with Mg and with no monovalent cations) or Mg (without and with Pi). The changes observed after the addition of Na and K were related to the signals obtained under those initial conditions. In the presence of acetyl phosphate under Mg-free conditions, 2 mM K reduced the relative fluorescence by 5.5 ± 0.3% (n = 3) and this effect was fully counteracted by 20 mM Na. On the other hand, 10 mM Na alone had no effect on the basal fluorescence, while the inclusion of 5 mM K in addition to Na resulted in a 5.6% fluorescence decrease. In the presence of 5 mM AcP and 3 mM MgCl_2, the addition of 2 mM K led to a reduction in fluorescence similar to that seen in Mg-free conditions (5.6 ± 0.3% [n = 3]; here, 20 mM Na not only did not reverse the action of K, but when added alone resulted in an even larger reduction (8.6 ± 0.5% [n = 3]). The fact that when either Na or K was added to an enzyme in the presence of AcP and Mg the signal was systematically deflected downward, is compatible with the notions that (a) the initial state of the enzyme was of an E_1-P type, and (b) both Na and K shifted that state toward an E_2-P form. In the presence Pi and Mg the enzyme behaved as if it were in an E_2-P state (Karlish, 1980; Beaugé et al., 1990), and was not modified by the addition of K ions.

Figure 2. Effects of cytoplasmic and extracellular Na and K on the steady-state levels of phosphorylation from 5 mM [32P]AcP of Na,K-ATPase incorporated into liposomes. The solution compositions were: (a) intravesicular (extracellular): 140-([Na + K]) mM Tris-HCl (pH 7.0 at 20°C); (b) extravesicular (cytoplasmic): 135-([Na + K]) mM Tris-HCl (pH 7.0 at 20°C), 3 mM MgCl_2, and 5 mM [32P]AcP. Temperature was 20 ± 2°C. All experiments were carried out in duplicate; the differences between those duplicates never exceeded 5%.

Discussion

One rule that seems to emerge from these results is that regardless of the phosphoryl group donor and the reaction taking place, the levels of phosphoenzyme in the E_2 conformation are lower when phosphorylation occurs in the presence of Kext than when it occurs in the absence of Kext.

Phosphorylation from Pi takes the enzyme into an E_2 state (Karlish, 1980; this work) and is maximal in the absence of any monovalent cation on both membrane surfaces or in the presence of intracellular K (Fig. 1). This notion is compatible with the fact that it is intracellular Na, taking the enzyme toward the E_1 state, that inhibits Pi phosphorylation. In proteoliposomes, the steady-state levels of [E-P] obtained from Pi were reduced when there was Kext in both the absence and presence of Kcyt. This suggests that the similar reduction observed in broken membranes, either at 0°C (Post et al., 1975) or 20°C (this work), is a consequence of K acting on extracellular sites. In addition, also in broken membranes and at 0°C, Post et al. (1975) observed that K ions were ineffective if added after E-P was formed. Our results indicate that E-P formed from Pi and Mg is sensitive only to Kext, provided that cation is attached to extracellular sites during phosphorylation.

At first sight, one would be tempted to identify the E-P formation from AcP in the absence of any monovalent cations with that obtained from Pi under similar conditions. However, there are four major differences with AcP: (a) the E-P levels are not maximal; (b) the addition of intracellular K increases [E-P]; (c) the phosphoenzyme is in the E_1 conformation; and (d) the levels of Pi incorporation are not modified by Kext unless intracellular K is simultaneously present. A second rule to be extracted then is that Kext does not affect the levels of phosphoenzyme in the E_1-P state. Similarly, it is internal K which is responsible for bringing the enzyme into E_2.

Intracellular K increases the levels of phosphorylation from AcP, changes the conformation toward an E_2-P state, and stimulates phosphatase activity (Drapeau and Blostein, 1980; Beaugé et al., 1990). In addition, the reduction in the [E_2-P] from AcP due to ADP is accompanied by an inhibition of the phosphatase activity (not shown). Therefore, at least with acetyl phosphate and with beta-(2-furyl)acryloyl phosphate (Beaugé et al., 1990; this work) the rate of phosphatase activity runs parallel with the levels of E_2-P.

Acknowledgments

This work was supported by CONICET, CONICOR, Fundación Antorchas, and Fundación Pérez Companc).

References

Beaugé, L., G. Berberán, and M. Campos. 1990. Potassium occlusion in relation to cation translocation and catalytic properties of Na,K-ATPase. In Regulation of Potassium Transport across Biological Membranes. L. Reuss, J. M. Russell, and G. Szabo, editors. University of Texas Press, Austin. 29–63.

Drapeau, P., and R. Blostein. 1980. Interactions of K+ with (Na,K)-ATPase. Orientation of K+-phosphatase sites studied with inside-out red cell membrane vesicles. Journal of Biological Chemistry. 258:7827–7834.

Glynn, I. M. 1985. The Na,K-transporting adenosine triphosphatase. In The Enzyme of Biological Membranes. Vol. 3.2nd ed. A. N. Martonosi, editor. Plenum Publishing Corp., New York. 35–114.

Jorgensen, P. L. 1974. Purification and characterization of Na,K-ATPase. III. Purification from the outer medulla of mammalian kidney after selective removal of membrane components by sodium dodecyl sulfate. Biochimica et Biophysica Acta. 356:36–52.

Karlish, S. J. D., 1980. Characterization of conformational changes in Na,K-ATPase labeled with fluorescein at the active site. Journal of Bioenergetics and Biomembranes. 12:111–136.

Karlish, S. J. D., W. R. Lieb, and W. D. Stein. 1982. Combined effects of ATP and phosphate on rubidium exchange mediated by Na,K-ATPase reconstituted into phospholipid vesicles. Journal of Physiology. 328:333–350.

Karlish, S. J. D., and U. Pick. 1981. Sidedness of the effects of sodium and potassium ions on the conformational state of the sodium-potassium pump. Journal of Physiology. 312:505–520.

Post, R. L., G. Toda, and G. N. Rogers. 1975. Phosphorylation by inorganic phosphate of sodium plus potassium ion transport adenosine triphosphatase. Four reactive states. Journal of Biological Chemistry. 250:691–701.

Phosphatase Activity and Potassium Transport

G. Berberián and L. Beaugé

Instituto de Investigación Médica “Mercedes y Martin Ferreyra,” 5000 Córdoba, Argentina

Introduction

The different transport modes of the Na pump are associated with specific biochemical reactions. Thus, forward Na-K exchange, ATP-dependent Na-Na exchange, and uncoupled Na efflux are associated with the overall cycle of ATP hydrolysis stimulated by external K or Na, or in the absence of external monovalent cations; ATP plus ADP-dependent Na-Na exchange has its counterpart in the ATP-ADP exchanges; the K-K exchange is supported by the reversible binding of phosphate to the enzyme in the presence of intracellular Mg and ATP (or its nonhydrolyzable analogues), while the Na-K reverse exchange goes along with the biochemical reversal or ATP synthesis (see Glynn, 1985). In the presence of Mg and K the enzyme can show a phosphatase activity that has some similarities, or at least seems to share some intermediate, with the reversible phosphorylation from phosphate (see Beaugé et al., 1990). The present experiments were performed to see if, under conditions of phosphatase activity, it was possible to induce K translocation.

Results

Vanadate-sensitive K-K exchange and net K uptake into liposomes with incorporated pig kidney Na,K-ATPase (Karlish and Stein, 1982a) were measured at 20°C and pH 7.0. The cytoplasmic (extravesicular) solutions contained no phosphate compounds (basal fluxes) or 5–10 mM phosphate, AcP, or pNPP in the absence and presence of 3 mM MgCl2. In addition, they had 140 mM Tris-HCl, 5 mM [86Rb]KCl, and 1 mM ouabain with and without 0.5 mM vanadate. 1 mM ADP or ATP was subsequently added. Mg-free solutions contained 2 mM CDTA. The intravesicular (extracellular) media had 0 or 100 mM KCl and 130 – [KCl] mM of Tris-HCl. All solutions were Na free.

In the first group of experiments (Fig. 1) intracellular MgCl_2 was omitted, preventing phosphorylation and phosphatase activity. As expected (see Glynn, 1985), ADP alone increased the basal exchange by ~1.8-fold; 5 and 10 mM pNPP also increased the basal exchange by 1.3- and 1.7-fold, respectively, indicating that this effect was exerted with low apparent affinity. On the other hand, under these conditions the addition of AcP only (either 5 or 10 mM) showed no effect on the K-K exchange fluxes.

The effects of phosphate, pNPP, and AcP on K-K exchange fluxes in the presence of enzyme phosphorylation and/or phosphatase activity (depending on the ligand used) were followed in proteoliposomes incubated with 3 mM cytoplasmic MgCl_2. A crucial problem we had to deal with first was phosphate contamination, which was not only due to the hydrolysis of pNPP and AcP, but also to the phosphate present in these compounds as a contaminant. This was because of the need to clearly distinguish a possible action due to pNPP or AcP from that of Pi simultaneously present. This problem was minimized by using a phosphate-trapping system (PiTS) based on glycogen degradation into glucose-6-phosphate via an enzymatic pathway catalyzed by phosphorylase a, glucose-6-phosphate dehydrogenase, and phosphoglucomutase. 1-h preincubation of the solutions at 20°C (without the proteoliposomes) with PiTS resulted in an almost complete disappearance of up to 200 μM phosphate in the media. As a further precaution against phosphate accumulation from hydrolysis, the working hematocrit was reduced from the usual 50% to 5%.

The K-K exchange experiments in the presence of MgCl_2 are illustrated in Fig. 2. Under conditions of high phosphatase activity, 5 mM AcP (4.6 nmol Pi / min per 10 μM liposomes) did not alter the K-K exchange. On the other hand, with 5 mM pNPP (phosphatase activity of 1.4 nmol / min per 10 μl liposomes) there was a small increase in the exchange fluxes; the increase was twice as large as that seen in Mg-free solutions. The simultaneous presence of 1 mM ADP and 5 mM pNPP or 5 mM AcP raised the basal K-K exchange to values comparable to those observed with 10 mM phosphate and 1 mM ATP (see Karlish et al., 1982), while the phosphatase activity was reduced by 30%.

Figure 1. Effects of ADP, pNPP, and AcP, alone or in combination, on K-K exchange in liposomes with Na,K-ATPase incorporated, in the absence of cytoplasmic Mg2+. Potassium fluxes were estimated from the uptake of 86Rb after 2 min incubation at 20°C. The compositions were as follows (in mM): extracellular (intravesicular), 100 KCl, 30 Tris-HCl (pH 7.0 at 20°C); cytoplasmic (extravesicular), the ligands indicated in this figure plus 2 CDTA, 5 [86Rb]KCl, and 140 Tris-HCl (pH 7.0 at 20°C). Each entry is the mean ± SEM of quadruplicate determinations. To each value, the average basal flux of 0.118 ± 0.006 nmol/10 μl liposomes per min was subtracted.

When we studied net potassium movements, we found that net [86Rb]K uptake (into K-free vesicles) in the presence of 3 mM MgCl_2 was not modified by 5 mM AcP or pNPP. This occurred despite the fact that the usual values of pNPPase and AcPase activities were observed. On the other hand, 5 mM Pi produced the anticipated considerable inhibition of this net K uptake (see Karlish and Stein, 1982b).

Discussion

Large K-K exchange fluxes observed when proper [ATP], [Pi], and [Mg2+] are present are thought to be a consequence of a vectorial effect of Pi and ATP (see Kenney and Kaplan, 1988). The Pi-induced K release to the outside seems ordered (Glynn et al., 1985; Forbush, 1987), while that due to ATP to the inside appears random (Forbush, 1987). In addition, there is a lack of stoichiometry between the K fluxes and water-phosphate oxygen exchange (Dahms and Boyer, 1973). The phosphatase activity of Na,K-ATPase is stimulated by intracellular K (Drapeau and Blostein, 1980); on the other hand, although occluded E_2(K) might be a phosphatase intermediate, the most abundant conformation at maximal rates of that activity is an E_2 state where K is not occluded. Furthermore, in contrast to what happens with Pi, the degree of K deocclusion during phosphatase activity goes well above 50% in the absence of K in the media (a random release?) (see Beaugé et al., 1990). On that basis, if phosphatase activity includes a succession of transitions between E_2(K) formed from the inside and E_2K with the K sites looking outward, one would expect that K transport could occur without the need for ATP or ADP. But that was not the case (Figs. 1 and 2). Actually, the only condition under which phosphatase activity coexisted with K-K exchange was when ATP or ADP were also present (Fig. 2). In broken membranes (see Beaugé et al., 1990), as well as in proteoliposomes (Berberián and Beaugé, 1991), we found an acid-stable E-P formation from AcP acting as phosphatase substrate. It is not inconceivable, then, that the stimulation of K-K exchange by phosphatase substrates in the presence of Mg2+ and ADP can utilize the E-P formed in that reaction without necessarily being coupled to the overall biochemical events leading to phosphatase activity. This possibility is illustrated in Fig. 3, which shows (upper part) the overlapping of reaction steps corresponding to K-K (and reversible enzyme phosphorylation from Pi) in the Na,K-ATPase cycle and the phosphatase activity. In addition, the whole K deocclusion during the phosphatase reaction may be related to a different E-P intermediate that might have the second product (acetate or pNP) still bound to the enzyme (lower part of the figure). Finally, these results confirm the ATP-like effects of pNPP on the low affinity ATP site (see Beaugé et al., 1990).

Figure 2. Effects of ATP, Pi, AcP, pNPP, and ADP, alone or in combination, on vanadate-sensitive K-K exchange in liposomes with Na,K-ATPase incorporated, in the presence of 3 mM cytoplasmic MgCl_2. The general experimental procedure, carried out in triplicate, was identical to that described in the legend to Fig. 1. Note that (a) the K-K observed in the presence of 0.5 mM vanadate was not affected by the tested ligands; (b) with AcP and pNPP the fluxes were measured, in the presence of 3.5 mg/ml glycogen, 2 IU/ml Phosphorylase a, 2 IU/ml glucose-6-phosphate dehydrogenase, 2 IU/ml phosphoglucomutase, 1 mM NADP, and 0.016 mM glucose-l,6-diphosphate (PiTS); and (c) with pNPP the solutions were also preincubated for 1 h at 20°C with PiTS.

Figure 3. Scheme showing the possible overlapping between (a) reaction steps of the K-K exchange and reversible Pi phosphorylation in the Na,K-ATPase cycle and (b) phosphatase activity supporting K-K in the presence of ADP or ATP.

Acknowledgments

We were aided by grants from CONICET, CONICOR, Fundación Antorchas, and Fundación Pérez Companc.

References

Beaugé, L. A., G. Berberián, and M. Compos. 1990. Potassium occlusion in relation to cation translocation and catalytic properties of Na,K-ATPase. In Regulation of Potassium Transport across Biological Membranes. L. Reuss, J. M. Russell, and G. Szabo, editors. University of Texas Press, Austin. 29–63.

Berberián, G., and L. Beaugé. 1991. Sidedness of Na and K effects on phosphorylation and conformations of pig kidney Na,K-ATPase interacting with inorganic phosphate or acetyl phosphate. In The Sodium Pump: Recent Developments. J. H. Kaplan and P. De Weer, editors. The Rockefeller University Press, New York. 389–393.

Dahms, A. S., and P. D. Boyer. 1973. Occurrence and characteristics of 18–0 exchange reaction catalyzed by sodium- and potassium-dependent adenosine triphosphatase. Journal of Biological Chemistry. 248:3155–3162.

Drapeau, P., and R. Blostein. 1980. Interactions of K+ with (Na,K)-ATPase. Orientation of K+-phosphatase sites studied with inside-out red cell membrane vesicles. Journal of Biological Chemistry. 258:7827–7834.

Forbush, B. 1987. Rapid release of 42K or 86Rb from two distinct transport sites on the Na,K-pump in the presence of Pi or vanadate. Journal of Biological Chemistry. 262:11116–11127.

Glynn, I. M. 1985. The Na,K-transporting adenosine triphosphatase. In The Enzymes of Biological Membranes. Vol. 3. 2nd ed. A. N. Martonosi, editor. Plenum Publishing Corp., New York. 35–114.

Glynn, I. M., J. L. Howland, and D. E. Richards. 1985. Evidence for the ordered release of rubidium ions occluded within the Na,K-ATPase of mammalian kidney. Journal of Physiology. 368:453–469.

Karlish, S. J.D., and W. D. Stein. 1982a. Passive rubidium fluxes mediated by Na,K-ATPase reconstituted into phospholipid vesicles when ATP- and phosphate-free. Journal of Physiology. 328:295–316.

Karlish, S. J. D., and W. D. Stein. 1982b. Effects of ATP or phosphate on passive rubidium fluxes mediated by Na,K-ATPase reconstituted into phospholipid vesicles. Journal of Physiology. 328:317–331.

Karlish, S. J. D., W. R. Lieb, and W. D. Stein. 1982. Combined effects of ATP and phosphate on rubidium exchange mediated by Na,K-ATPase reconstituted into phospholipid vesicles. Journal of Physiology. 328:333–350.

Kenney, L. J., and J. H. Kaplan. 1988. The vectorial effects of ligands on occluded intermediate in red cell sodium pump transport. Progress in Clinical and Biological Research. 268A: 525–530.

The Order of ADP and Pi Release in a Single Cycle of Na,K-ATPase

Tim J. H. Walton and José D. Cavieres

Department of Physiology, Leicester University, Leicester LE1 9HN, United Kingdom

Introduction

Although the general properties of the acid-stable phosphoenzymes broadly agree with the Post-Albers scheme and with the results of flux studies, there are indications that, under some conditions, the acid-stable phosphoenzyme of Na,K-ATPase might not be kinetically competent as an intermediate in the overall reaction.

Whereas in K+ -free conditions the acid-stable phosphoenzyme turns over fast enough to account for the steady-state Na-ATPase activity, this does not seem to be the case for K+ -containing solutions. In the latter case, the dephosphorylation is at least biphasic, the fastest component being attributed to E_2P breakdown and the slowest to E_1P decay via interconversion to E_2P. However, the product of the slow phase rate constant and the steady-state concentration of E_1P has been found in some cases (Plesner et al., 1981; Nørby et al., 1983) to fall short by a factor of between 2 and 10 of the observed steady-state Na,K-ATPase rate. One possibility is that E_1P is not an intermediate in the ATP hydrolysis catalyzed by Na+ plus K+ (Plesner et al., 1981); another is that under these conditions the measured steady-state level of intermediate is too low due to partial acid-induced breakdown.

In an attempt to investigate these possibilities, we decided to study the presteady state kinetics of ADP and Pi release from purified Na,K-ATPase with a nondestructive method. Not only should ADP appear to be released ahead of Pi, but the enzyme-associated γ-phosphate should also meet the kinetic requirements of a phosphointermediate. To do this study, we designed a novel “flow-kinetics” technique.

Fast separation of the enzyme from its substrate and products should be achieved if the substrate flows past immobilized enzyme at a high speed. Since the enzyme, purified from pig kidneys (Jørgensen, 1974), is in membrane-bound form, we immobilized it by entrapment on cellulose filters. The substrate was injected with a Hamilton syringe driven at constant linear speed by the ram of a quenched-flow apparatus. The ram displacement was monitored from the output of a potentiometric wire along the ram path and the ram speed was calculated by an on-line Apple II microcomputer. The time resolution was obtained by collecting the effluent jet onto a disc of filter paper rotating on a Garrard turntable whose speed was calibrated with a stroboscope.

In several preliminary experiments, the syringe was filled with 1–10μ,M [γ^32P,3H]μATP, which was injected past immobilized enzyme at 0.8 ml s–1. After drying out, the jet track was cut up into 20-ms sectors and the eluted nucleotide mixture was resolved by thin-layer chromatography (Cavieres, 1987). The ATP, ADP, and AMP/Pi fractions were then counted for 3H and 32P and the molar masses of ATP, ADP, and Pi were calculated.

Results and Discussion

Fig. 1 shows the time interval profile of ADP and Pi released at 21°C when 6 μM [γ^32P,3H]ATP in a Na + plus K+ medium was injected through a filter supporting 100 μg Na,K-ATPase protein. Every fraction contained more released ADP than Pi for up to ~ 300 ms, but the predominance became random at longer times. The time summation of released ADP and Pi is shown in Fig. 2 and is referred to the mass of immobilized protein. The difference between the integrated ADP and Pi release curves, denoted as E(P), is also shown on an expanded vertical scale.

Figure 1. Interval product release by immobilized Na,K-ATPase. This preliminary experiment was conducted at 21°C and at a flow rate of 0.8 ml s–1. Turntable speed, 78 rev min–1. The [3H]ADP and [32P]Pi contents of each time fraction are shown as superimposed bars.

Control experiments show that the different initial rate of ADP and Pi release is not due to differential Pi and ADP binding by the filters or by unrelated components of the SDS-extracted microsomes. When injected together through unloaded filters, [3H]ADP and [14C]glucose appear with similar profiles. The same holds true for the [32P]Pi and [3H]glucose pair. When [3H]ADP and [32P]Pi at equimolar concentrations are injected simultaneously through an unloaded filter, the time profiles are practically superimposed on each other. Furthermore, an equimolar mixture of [3H]ADP and [32P]Pi (at the relevant concentration) flows unchanged when injected through a filter supporting ouabain-treated enzyme. Thus, one may conclude that the mass deficit of γ-Phosphate should be found on the active enzyme.

The time integrals of ADP and Pi release tend to become parallel at long times, implying identical steady-state rates of ADP and Pi release, as expected. The rates calculated from these slopes are about twice as high as the rate measured in similar conditions over 30 min with a [γ^32P]ATPase assay. This would be the situation if steady state has not quite been reached at 500 ms in this filtration experiment.

The level of E(P) reaches apparent steady state at 0.6 nmol (mg protein)–1. This represents ~60% of the level of acid-stable phosphorylation obtained at 10–20 s in a K+-free Na+-containing medium at 0°C with the same preparation. This fraction seems higher than the steady-state ratio of acid-stable phosphoenzymes in the presence and absence of K ions (Mårdh, 1975; Hobbs et al., 1985).

One drawback of the method as described is the time resolution for the onset of the reaction, which is limited by the time taken to fill up the aqueous dead space of the filters (~ 20 μM). In the conditions described here, tracer flow takes up to 120 ms to build up to steady state, and the initial rates of product release must be faster than they appear in the present results. We have recently succeeded in reducing the dead time, and this, together with a reduction of the reaction temperature, should minimize the limitation of the observed rate.

Figure 2. Time summation of the data of Fig. 1. The difference between the two integral curves, labeled E(P), has been plotted on an expanded scale (right).

The flow-kinetic experiments are now being performed at a flow rate of 5 ml s–1 at 1°C. To compare the two estimates of phosphoenzyme formation, we are now also measuring acid-stable phosphorylation within a comparable time scale using the quenched-flow machine.

Acknowledgments

We thank Dr. C. R. Bagshaw and Dr. A. Maxwell (Department of Biochemistry, Leicester University) for allowing us to use their quenched-flow apparatus.

We thank the Wellcome Trust for research and travel grants, the Medical Research Council for an equipment grant, and The Royal Society for a travel grant.

References

Cavieres, J. D. 1987. Fast reversal of the initial reaction steps of the plasma membrane (Ca2+ + Mg2+)-ATPase. Biochimica et Biophysica Acta. 899:83–92.

Hobbs, A. S., R. W. Albers, and J. P. Froehlich. 1985. Quenched-flow determination of the E_1P to E_2P transition rate constant in electric organ Na,K-ATPase. In The Sodium Pump. I. Glynn and C. Ellory, editors. The Company of Biologists, Ltd., Cambridge, UK. 355–361.

Jørgensen, P. L. 1974. Purification and characterization of (Na+ + K+)-ATPase. III. Purification from the outer medulla of mammalian kidney after selective removal of membrane components by sodium dodecylsulphate. Biochimica et Biophysica Acta. 694:27–68.

Mårdh, S. 1975. Bovine brain Na+,K+-stimulated ATP phosphohydrolase studied by a rapid-mixing technique: K+ -stimulated liberation of [32P]orthophosphate from [32P]phosphoenzyme and resolution of the dephosphorylation into two phases. Biochimica et Biophysica Acta. 391:448–463.

Nørby, J. G., I. Klodos, and N. O. Christiansen. 1983. Kinetics of Na-ATPase activity by the Na,K pump. Interactions of the phosphorylated intermediates with Na+, Tris+, and K+. Journal of General Physiology. 82:725–759.

Plesner, I. W., L. Plesner, J. G. Nørby, and I. Klodos. 1981. The steady-state kinetic mechanism of ATP hydrolysis catalyzed by membrane-bound (Na+ + K+)-ATPase from ox brain. III. A minimal model. Biochimica et Biophysica Acta. 643:483–494.

High and Low Affinity ATP Binding Sites as Well as Na+ and K+ Forms Coexist in Na+,K+-ATPase

E. Buxbaum, E. H. Serpersu, R. Antolovic, E. Hamer, M. Willeke, and W. Schoner

Institut für Biochemie und Endokrinologie, Justus-Liebig Universität, D-6300 Giessen, Germany; and Department of Biochemistry, University of Tennessee, Knoxville, Tennessee 37996-0840

Introduction

There is a long-standing debate over whether the sodium pump works according to the Albers-Post model as an α, β protomer or according to the Repke-Stein model as an α_2β_2 diprotomer of interacting subunits. We found recently that Co(NH_3)4PO_4 and CO(NH_3)4ATP inactivate Na+/ K+-ATPase (Scheiner-Bobis et al., 1987, 1988) by forming a stable complex with the low affinity ATP binding site. Since this inactivation did not affect the formation of E_1-P, we concluded that Na+/K+-ATPase presumably works as an α_2β_2 diprotomer of interacting subunits. Moreover, we found that inactivation by Co(NH_3)4ATP shifts the high affinity ATP binding site to the Na+ form (Scheiner-Bobis et al., 1989). To test this conclusion further, we were interested to learn (a) how inactivation by Co(NH_3)4PO_4 affects Na+/K+-transport and the partial reactions of the sodium pump; (b) whether the induction of a sodium form after fixation of the enzyme in the E_2 conformation by Co(NH_3)4ATP can also be demonstrated by the trypsin cleavage; and (c) whether in addition to Co(NH_3)4ATP further ATP analogues exist which specifically interfere with the low affinity ATP binding site.

Materials and Methods

8-Azido-2'-O-dansyl-ATP and other dansyl-ATP derivatives were synthesized by a modification of the procedure of Chuan et al. (1989). Trypsin digestion of Cr(H_2O)4ATP–, Co(NH_3)4ATP–, or 8-azido-Co(NH_3)4ATP-inactivated Na+/K+- ATPase was done in the absence of cations in an analogous way to the procedure described by Jørgensen (1975). Affinity of ATP analogues for the ATP binding sites was tested by their interference with the inactivation by Cr(H_2O)4ATP (high affinity site) and Co(NH_3)4ATP (low affinity ATP binding site).

Results and Discussion

When Na+/K+ -ATPase in the purified enzyme or in everted human red blood cells was inactivated by incubation with Co(NH_3)4PO_4, the overall reaction of the Na+/K+- pump and Na+/K+-ATPase activity decreased proportionally with the partial activities of the E_2 conformation, including Rb+/Rb+ exchange, Rb+ occlusion, and K+ -activated p-nitrophenylphosphatase. On the contrary, partial activities of the E_1 conformation were unchanged, such as Na+/Na+ exchange, uncoupled Na+ transport, ATP/ADP exchange activity, and Na+-ATPase and Na+-dependent formation of a phosphointermediate (Buxbaum and Schoner, 1990). Furthermore, we could observe that incorporation of 130 pmol of [γ–32P]Co(NH_3)4ATP or [32P]Co(NH_3)4PO_4 per unit of enzyme led to complete inactivation. Such an enzyme preparation was still able to incorporate the same amount of radioactivity from [γ–32P]Cr(H_2O)4ATP. This double incorporation of ligands was independent of the specific activity used (Buxbaum and Schoner, 1990). These observations are consistent with the RepkeStein postulate that during the Na+/K+ pumping process catalytic α-subunit interact in an α_2,β_2 diprotomer.

Figure 1. Time course of the formation of tryptic fragments from Na+/K+-ATPase, which had been inactivated by Co(NH_3)4-8-azido-ATP. The sample was digested with a trypsin/ATPase weight ratio of 4:100 and SDS-electrophoresis according to Laemmli was performed.

Limited tryptic digestion under controlled conditions reveals the existence of sodium and potassium forms of Na+/K+-ATPase (Jørgensen, 1975). Although Co(NH_3)4ATP and Co(NH_3)4PO_4 interfere with the low affinity ATP binding site of the E_2 conformation, inactivation of the enzyme by these MgATP and MgPO_4 analogues leads to the induction of a sodium form of the high affinity ATP binding site as detected by eosin fluorescence (Scheiner-Bobis et al., 1989; Buxbaum and Schoner, 1990). In fact, this observation can also be verified by limited tryptic digestion of the Co(NH_3)4ATP– and 8-azido-Co(NH_3)4ATP-inactivated and washed enzyme which is digested in the absence of Na+ or K+ (Fig. 1). Tryptic digestion of the enzyme inactivated by [γ–32P]-8-azido-Co(NH_3)4ATP shows the slow appearance of an 80-kD (Na+-like) tryptic fragment, but the immediate formation of 60- and 40-kD (K+ -like) tryptic fragments. Autoradiograms revealed labeling of the 60-kD fragment but not of the 80-kD fragment, which both interacted with antibodies against the sequence 991–1,006 of the catalytic α-subunit (not shown). Provided that two ATP binding sites would exist per α-subunit, labeling of the 80- and 60-kD tryptic fragments should be expected. The simultaneous formation of Na+ and K+-like tryptic patterns in the Co(NH_3)4ATP and 8-azido-Co(NH_3)4ATP-inactivated enzyme strongly favors the conclusion that Na+/K+-ATPase works as an α_2β_2 diprotomer of interacting catalytic subunits as predicted by the Repke-Stein model.

To further verify the conclusion that high and low affinity ATP binding sites interact, we looked for ATP analogues, which specifically bind with high affinity to the low affinity ATP binding site. In fact, 8-azido-2'-O-dansyl-ATP binds with a Kd value of 20 μM to the low affinity ATP binding site and not to the high affinity ATP binding site (Table I). Other 2'-O-dansyl-ATP derivatives behave similarly but bind with lower affinity. Since the substitution of the adenine ring by the 8-azido group favors the syn-conformation, we examined whether arabino-ATP behaved similarly. Indeed, specific interference with the low affinity ATP binding site was apparent with this compound (Table I), which also has a syn-conformation (Scheit, 1980). It appears that high and low affinity ATP binding sites of Na+/K+-ATPase prefer different conformations of the ATP molecule. Similar conclusions from other lines of evidence have been presented during this meeting (Stewart et al., 1991).

TABLE I
Study on the Nucleotide Triphosphate Specificity of High- and Low-Affinity ATP Binding Sites of Na+/K+-ATPase

The interaction with the high affinity ATP binding site was measured from the competition with the inactivation by Cr(H_2O)4ATP and the interaction with the low affinity ATP-binding site by competition with Co(NH_3)4ATP. All experiments were done at 37°C and pH 7.25.

Acknowledgments

This work has been supported by SFB 249 Giessen Pharmakologie Biologischer Makromoleküle and the Fonds der Chemischen Industrie Frankfurt/M.

References

Buxbaum, E., and W. Schoner. 1990. Blocking of Na+/K+ transport by the MgPO_4 complex analogue Co(NH_3)4PO_4 leaves the Na+/Na+-exchange reaction of the sodium pump unaltered and shifts its high affinity ATP-binding site to a Na+-like form. European Journal of Biochemistry. 195:355–360.

Chuan, H., J. Lin, and J. H. Wang. 1989. 8-Azido-2-O'-dansyl-ATP. A fluorescent photoaffinity reagent for ATP binding proteins and its application to adenylate kinase. Journal of Biological Chemistry. 264:7981–7988.

Jørgensen, P. L. 1975. Purification and characterization of Na+,K+ -ATPase. V. Conformational changes in the enzymes: transitions between the Na+ -form and K+ -form studied with tryptic digestion as a tool. Biochimica et Biophysica Acta. 410:399–415.

Scheiner-Bobis, G., E. Buxbaum, and W. Schoner. 1988. The catalytic subunits cooperate in (Na+ + K+)-ATPase: demonstration with MgPO_4 and MgATP complex analogues. Progress in Clinical and Biological Research. 268A:219–226.

Scheiner-Bobis, G., M. Esmann, and W. Schoner. 1989. Shift to the Na+ form of Na+/K+-transporting ATPase due to modification of the low affinity ATP binding site by Co(NH_3)4ATP. European Journal of Biochemistry. 183:173–178.

Scheiner-Bobis, G., K. Fahlbusch, and W. Schoner. 1987. Demonstration of cooperating α subunits in working (Na+ + K+)-ATPase by the use of the MgATP complex analogue cobalt tetrammine ATP. European Journal of Biochemistry. 168:123–131.

Scheit, K. H. 1980. Nucleotide Analogs. John Wiley & Sons, Inc., New York. 145–149.

Stewart, J. M. McD., T. A. Kuntzweiler, C. Klevickis, and C. M. Grisham. 1991. Structural features of intermediate states of sheep kidney Na+,K+-ATPase. In The Sodium Pump: Structure, Mechanism, and Regulation. J. H. Kaplan and P. De Weer, editors. The Rockefeller University Press, New York. 143–158.

Kinetics of Magnesium Interaction with Na,K-ATPase

Yolanda G.-M. Covarrubias and Paul De Weer

Department of Physiology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6085

It is known that Mg2+ at micromolar concentrations stimulates several reactions of the Na,K-ATPase, including ATP hydrolysis and ADP/ATP exchange in the presence of Na+ or Na+ + K+, but at millimolar concentrations it inhibits these reactions (Fahn et al., 1966; Plesner and Plesner, 1988). Although it has been suggested (Robinson, 1976) that two Mg2+ binding sites are required to account for this dual effect, a single site suffices (De Weer et al., 1983).

Figure 1. Na,K-ATPase reaction schemes. Various possible reaction mechanisms of Na,K-ATPase with binding of either MgATP complex (models 1–4) or ATP and Mg2+ sequentially (models 5–9). E.N.P. represents the unresolvable equilibrium E.ATP E-P.ADP. The low-affinity Mg2+ release step is also shown. Steady-state kinetic equations derived for these models with King-Altman procedures were fit by least-squares techniques to the hydrolysis and exchange data of Figs. 2 and 3.

Previous attempts have been made to resolve the question of whether a MgATP complex rather than ATP and Mg2+ sequentially binds to the enzyme (Skou, 1974; Plesner and Plesner, 1981). To reexamine this question we have fitted ATP hydrolysis and ADP/ATP exchange data, obtained over a 4,000- to 6,000-fold free [Mg2+] range, with steady-state rate equations derived for nine different “minimal” models. Four such models assume MgATP complex to be the substrate; five assume sequential ATP and Mg2+ binding (Fig. 1). Na,K-ATPase, purified from dog kidney outer medulla according to Jørgensen (1979), was incubated (1.7–2.7 μg/ml) at 20°C in chloride media containing 30 mM imidazole, pH 7.5, 5 mM each of CDTA and HEDTA, and Mg2+, Na+, K+, and labeled and unlabeled nucleotides as specified in the figure legends. Activity was defined as that sensitive to 1 mM ouabain (only ~ 2% was insensitive). Incubation times were adjusted to allow only 10–15% hydrolysis or exchange. Reaction products (ATP, ADP, Pi) were separated by thin-layer chromatography, and their radioactivity was measured in a liquid scintillation counter. Data shown represent means ± SEM of three to six determinations.

Figure 2. Effects of free [Mg2+] on rate of ATP hydrolysis at various [ATP]. The medium contained 150 mM Na+, 20 mM K+, and 100, 500, or 3,000 μM [32P]ATP. The curves are simultaneous least-squares fits (appropriate for the three ATP concentrations) of the equation derived for model 6. Equally good fits were obtained with all other models of Fig. 1 except model 1, which gave a poor fit.

We report that steady-state hydrolysis experiments alone cannot resolve the question regarding the enzyme's “true” substrate since, by the least-squares criterion, only one of the models (model 1 in Fig. 1) can be excluded and all eight others fit the data equally well (Fig. 2). Note that the involvement of a single Mg2+ ion allows for both stimulation and inhibition of the reaction.

On the other hand, analysis of steady-state ADP/ATP exchange kinetics allows a distinction between “MgATP complex” models and “sequential” models. None of the “MgATP complex” models fit our exchange data well, and only the separate-binding models 6 and 9 yielded a good fit (Fig. 3). We conclude that models with separate addition of ATP and Mg are more likely than those in which MgATP complex is the “true” substrate, and that a second binding site for Mg2+ is not needed to account for the observed kinetics of ATP hydrolysis and ADP/ATP exchange.

Simultaneous least-squares fitting of model 6 or 9 (each containing nine rate constants) to both hydrolysis and exchange data yields five phenomenological (adjustable) parameters at fixed [ATP], and seven when [ATP] is varied systematically. Variation of [ADP], and the determination of steady-state phosphorylation levels, should yield additional phenomenological reaction parameters. Resolution of all nine rate constants may require pre–steady state kinetic analysis.

Figure 3. Effects of free [Mg2+] on rates of ATP hydrolysis and ADP/ATP exchange at various [ATP]. The medium contained 150 mM Na+, 50 μM ADP, and 100, 500, or 3,000 μM ATP. Free [Mg2+] was varied from 0.5 to 3,000 μM. Hydrolysis (squares) and exchange (circles) rates were measured under identical conditions except for the nature of the radiolabel ([γ–32P]ATP and [3H]ADP, respectively). The curves are simultaneous least-squares fits (appropriate for the three [ATP]) of the hydrolysis and exchange equations derived for model 6 of Fig. 1. (For the sake of clarity, only the hydrolysis data and fit at 100 μM ATP are shown.) Equally good fits were obtained with model 9. None of the “MgATP complex” models could fit the data well, nor could the “sequential” models 5, 7, or 8.

Acknowledgments

We thank Dr. G. E. Breitwieser for carrying out some of the initial experiments, and R. W. Ratzlaff for programming expertise.

This work was supported by the American Heart Association.

References

De Weer, P., G. E. Breitwieser, H. G. Smith, and B. G. Kennedy. 1983. ADP-ATP exchange in internally dialyzed squid axons. Current Topics in Membranes and Transport. 19:665–669.

Fahn, S., G. J. Koval, and R. W. Albers. 1966. Sodium-potassium-activated adenosine triphosphatase of Electrophorus electric organ. I. An associated sodium-activated transphosphorylation. Journal of Biological Chemistry. 241:1882–1889.

Jørgensen, P. L. 1979. Purification and characterization of (Na+ + K+)-ATPase. III. Purification from the outer medulla of mammalian kidney after selective removal of membrane components by sodium dodecylsulphate. Biochimica et Biophysica Acta. 356:36–52.

Plesner, L., and I. W. Plesner. 1981. The steady-state kinetic mechanism of ATP hydrolysis catalyzed by membrane-bound (Na+ + K+)-ATPase from ox brain. I. Substrate identity. Biochimica et Biophysica Acta. 643:449–462.

Plesner, L., and I. W. Plesner. 1988. Distinction between the intermediates in Na+ – K+-ATPase reactions. II. Exchange and hydrolysis kinetics at micromolar nucleotide concentrations. Biochimica et Biophysica Acta. 937:63–72.

Robinson, J. D. 1976. The (Na+ + K+)-dependent ATPase: mode of inhibition of ADP/ATP exchange activity by MgCl_2. Biochimica et Biophysica Acta. 440:711–722.

Skou, J. C. 1974. Effect of ATP on the intermediary steps of the reaction of the (Na+ + K+)-dependent enzyme system. II. Effect of a variation in the ATP/Mg++ ratio. Biochimica et Biophysica Acta. 339:246–257.

The Change in Na+ Binding States before and after Accumulation of ADP-sensitive Phosphoenzyme in Na+,K+-ATPase

Toshio Sasaki, Eiji Shinoguchi, Yoshiko Kamo, Eiichi Ito, and Kazuya Taniguchi

Department of Chemistry, Faculty of Science, and Department of Pharmacology, School of Dentistry, Hokkaido University, Sapporo 060, Japan

To better understand the mechanism of energy transduction in Na+,K+-ATPase, it is important to study its conformational changes. The N-[p-(2-benzimidazolyl) phenyl] maleimide(BIPM)-modified enzyme makes it possible to clearly show the sequential appearance (Taniguchi et al., 1984) of reaction intermediates accompanied by conformational changes (Taniguchi et al., 1982, 1986; Suzuki et al., 1987) through the Post-Albers mechanism (Post et al., 1969; Glynn and Karlish, 1975; Mardh, 1975; Albers, 1976).

Dynamic light-scattering changes (Taniguchi et al., 1986) have shown the sequential appearance of three different states of ADP-sensitive phosphoenzyme (E_1P), while double-labeled enzyme preparations with BIPM at Cys-964 (Nagai et al., 1986) and fluorescein-5'-isothiocyanate at Lys-501 of the α-chain (Schull et al., 1985) have shown the presence of at least two different conformational states of E_1P that are sensitive to acetate but not to ADP (Taniguchi et al., 1988). However, the reason for the conformational difference in E_1P's has not been made clear. The presence of a tight Na+ binding state has been reported as the occlusion of Na+ in E_1P by using chymotrypsin- or N-ethylmaleimide-treated enzymes to stabilize E_1P, in the phosphorylated complex from the β,γ-bidentate complex of chromium with ATP (Glynn and Karlish, 1990), and in an enzyme-Na+-oligomycin complex, (Na)E_1, in the absence of phosphorylation (Esmann and Skou, 1985). However, the relationship between these occluded Na+ forms of the enzyme and their conformational states remain to be elucidated. This paper suggests that the dynamic decrease in the fluorescence of the BIPM probe at Cys-964 mainly reflects various states of Na+ in the enzyme before and after the formation of E_1P.

Results and Discussion

The Decrease of the Fluorescence Intensity Induced by the Addition of Oligomycin to Na+-bound Enzymes

To compare the conformations of Na+-bound enzyme (NaE_1), (Na)E_1,and E_1P, the change in the fluorescence intensity of BIPM-treated enzyme induced by oligomycin was measured in the presence of 2 M NaCl and 0.43 mM MgCl_2. The intensity decreased with a Hill coefficient, nh, of 1.88 with Kh 0.016 μg/ml. The effects of oligomycin under various concentrations of Na+ were also measured. These data show that the decrease caused by oligomycin did not saturate in the presence of 16 mM Na+, a concentration that seems to saturate Na+ occlusion (Esmann and Skou, 1985).

The relative fluorescence intensity was measured in the presence of 2 M NaCl and 0.43 mM MgCl_2 while changing the order of addition of ATP and oligomycin, each of which presumably induces Na+ occlusion with or without phosphorylation. Oligomycin induced ~ 80% of the fluorescence decrease observed after accumulation of E_1P. To compare the ability of NaE_1 with that of (Na)E_1 as a precursor to E_1P, BIPM-treated enzymes in the presence of 2 M Na+ with or without oligomycin were phosphorylated by [32P]ATP. The enzymes preincubated with Na+ + oligomycin with or without Mg2+ were phosphorylated more rapidly than the enzymes preincubated without oligomycin.

Oligomycin induces Na+ occlusion in the absence of phosphorylation with simultaneous reduction of sensitivity to K+ (Esmann and Skou, 1985) and acceleration of phosphorylation (Hobbs et al., 1983). Oligomycin accelerated the ATP + Mg2+-induced conformation change. It reduced the amplitudes of fluorescence changes dependent on the initial Na+ concentration and accelerated the phosphorylation. These data suggest that Na+'s in (Na)E_1 formed with Na+ and oligomycin are better substrates for ATP + Mg2+-induced E_1P formation than those in NaE_1.

Comparison of the Rate of Fluorescence Change with That of Phosphorylation Induced by ATP + Mg2+ or Ca2+

Fluorescence changes of species of (Na)E_1 formed with oligomycin in the presence of 16 mM NaCl + 1984 mM choline chloride or 2 M NaCl were followed after the addition of Mg2+ + ATP with NaCl or choline chloride, respectively, to give the same final ligand concentration. The apparent first-order rate constant and extent of fluorescence decrease were 12.1 ± 1.2/s and 0.9 ± 0.1% or 10.1 ± 3.2/s and 0.4 ± 0.1%, respectively. The apparent rate constants for phosphorylation estimated were ~ 20–21/s. Control experiments using NaE_1 formed with 16 mM and 2 M Na+ showed that the apparent rate constants of fluorescence change were 3.4 ± 0.3/s and 8.6 ± 0.7/s, respectively, with the same amplitude, 1.7 ± 0.1%, and the rate of phosphorylation, 13/s, irrespective of the concentration of Na+ preincubated. These data show that Mg2+ + ATP induced much faster phosphorylation of species of NaE_1 and (Na)E_1 before fluorescence changes. The turnover of Na+,K+-dependent ATP hydrolysis of BIPM enzymes used in the present experiment was in the same range to that of various preparations (Jorgensen, 1982). The rate constant of fluorescence changes was 3.4–12.1/s at 25°C, pH 7.4, and the rate of ATP hydrolysis was ~0.2/s. Thus, the ATP-dependent fluorescence changes are rapid enough to be candidates for the reflection of conformational changes accompanying ATP hydrolysis.

It has been shown that Ca2+ + ATP induces a rapid fluorescence decrease of the NaE_1 before phosphorylation (Taniguchi et al., 1984). To investigate the effect of oligomycin, the fluorescence changes and the amount of phosphoenzyme of the species of (Na)E_1 formed with 16 mM Na+ + 1,984 mM choline chloride and 2 M Na+ were measured after the addition of Ca2+ + ATP to give the same final ligand concentration. The rate constant and the extent of the fluorescence change were 20.6 ± 2.7/s and 0.6 ± 0.1% and 16.6 ± 3.7/s and 0.3 ± 0.1%, respectively. The corresponding rate constants of phosphorylation were 1.5 and 1.4/s, respectively.

The values of the conformational changes of NaE_1 formed with low and high Na+ concentrations were 10.7 ± 0.4/s and 1.4 ± 0.1% and 19.8 ± 2.7/s and 1.4 ± 0.1%, respectively. The corresponding rate constants of phosphorylation were both estimated to be 1.3/s, irrespective of initial Na+ concentrations.

These data show that Ca2+ + ATP induced much faster fluorescence changes of species of NaE_1 and (Na)E_1 before phosphorylation. The rate of phosphorylation was one order of magnitude smaller than those caused by Mg2+ + ATP.

The fluorescence intensity of the BIPM enzyme in the presence of oligomycin decreased with increases in the concentrations of Na+. The amplitude of ATP-induced fluorescence change in (Na)E_1 with 16 mM Na+ was larger than that with 2 M Na+ whatever divalent cation was used. The gross fluorescence change induced by ATP + Ca2+ was completed before the phosphorylation. The apparent Kd for Na+ binding in the presence of oligomycin was reported to be 0.06 mM (Matsui and Homareda, 1982). These data suggested the presence of different conformational states in (Na)E_1 rather than the presence of enzyme-oligomycin complexes that did not bind enough Na+ to saturate the binding. The phosphorylation of species of NaE_1 and (Na)E_1 by ATP + Mg2+ was completed before completion of the fluorescence change. The data are consistent with previous results showing multiple conformational states of E_1P (Taniguchi et al., 1986, 1988).

Figure 1. A model for change in the Na+ binding state or Na+ migration detected by BIPM fluorescence at Cys-964 preceding formation of K+-sensitive phosphoenzyme (E_2P). Ellipses designate Na+,K+-ATPase embedded in membranes. In this model the number of Na+ ions binding is omitted for simplicity. A shows a possible Na+ binding state in NaE_1. B shows the change in Na+ binding states to form (Na)E_1 by oligomycin or the Na+ migration (dotted line) induced by ATP with Ca2+ or Mg2+ to form divalent cation-Na-ATP-enzyme complexes with or without oligomycin (E_1S*) and E_1P; thus, these changes occur before and after formation of E_1P (see text). C shows a state of E_2P that has lower affinity for Na+.

The fluorescence decreases after the transition from NaE_1 to (Na)E_1 and E_1P were not additive. The former was ~80% of that of E_1P in the presence of oligomycin, which suggests the presence of a conformational difference between them. The fluorescence decrease also occurred irrespective of the completion of phosphorylation: rapid fluorescence decrease occurred before phosphorylation induced by ATP + Ca2+ as already reported (Taniguchi et al., 1984) and after that induced by ATP + Mg2+. From these results we propose that the ATP-dependent BIPM fluorescence changes reported in this paper reflect conformational events accompanying the changes in Na+ binding states presumably related to Na+ migration in the enzyme (Fig. 1). The present data also indicate that changes in the Na+ binding state and the formation of E_1P are not tightly coupled.

The role of ATP and Mg2+ may be to change the Na+ binding state in NaE_1 with a rather small decrease of the fluorescence intensity, which is enough to accelerate the formation of E_1P as observed in the presence of oligomycin with rather lower concentrations of Na+. The delay of phosphorylation in the absence of oligomycin may reflect the time required to induce such a change by ATP + Mg2+ (see also Skou, 1990).

Acknowledgments

This work was supported in part by a Grant-in-Aid for Scientific Research on Priority Areas of “Bioenergetics” to K. Taniguchi from the Ministry of Education, Science and Culture, Japan, and The Naito Foundation.

References

Albers, W. 1976. The (sodium plus potassium)-transport ATPase. In The Enzymes of Biological Membranes. Vol. 3. A. Martonossi, editor. Plenum Publishing Corp., New York. 283–301.

Esmann, M., and J. C. Skou. 1985. Occlusion of Na+ by Na,K-ATPase in the presence of oligomycin. Biochemical and Biophysical Research Communications. 127:857–863.

Glynn, I. M., and S. J. D. Karlish. 1990. Occluded cations in active transport. Annual Review of Biochemistry. 59:171–205.

Glynn, I. M., and S. J. D. Karlish. 1975. The sodium pump. Annual Review of Physiology. 37:13–55.

Hobbs, A. S., R. W. Albers, and J. P. Froehlich. 1983. Effects of oligomycin on partial reactions of the sodium plus potassium stimulated adenosine triphosphatase. Journal of Biological Chemistry. 258:8163–8168.

Jorgensen, P. L. 1982. Mechanism of the Na,K-pump: protein structure and conformation of the pure Na,K-ATPase. Biochimica et Biophysica Acta. 694:27–68.

Karlish, S. J. D., and D. W. Yates. 1978. Tryptophan fluorescence of (Na+,K+)-ATPase as a tool for study of the enzyme mechanism. Biochimica et Biophysica Acta. 527:115–130.

Mardh, S. 1975. Bovine brain Na+,K+-ATP-stimulated ATP hydrolase studied by a rapid-mixing technique. Biochimica et Biophysica Acta. 391:448–463.

Matsui, H., and H. Homareda. 1982. Interaction of sodium and potassium ion with Na+,K+-ATPase. Journal of Biochemistry. 92:193–217.

Nagai, M., K. Taniguchi, K. Kangawa, S. Matsuo, S. Nakamura, and S. Iida. 1986. Identification of N-[p-(2-benzimidazolyl) phenyl] maleimide-modified residue participating in dynamic fluorescence changes accompanying Na+,K+-dependent ATP hydrolysis. Journal of Biological Chemistry. 261:13197–13202.

Post, R. L., S. Kume, T. Tobin, B. Orcutt, and A. K. Sen. 1969. Flexibility of an active center in sodium plus potassium adenosine triphosphatase. Journal of General Physiology. 54:306S–326S.

Schull, G. E., A. Schwarts, and J. B. Lingrel. 1985. Amino acid sequence of the catalytic subunit of (Na+,K+)-ATPase deduced from a complementary DNA. Nature. 316:691–695.

Skou, J. C. 1990. Effect of oligomycin on rate of phosphorylation of Na,K-ATPase in the presence of Na+. Journal of General Physiology. 96:51a-52a. (Abstr.)

Suzuki, K., K. Taniguchi, and S. Iida. 1987. The acceleration of Na+,K+-ATPase activity by ATP and ATP analogues. Journal of Biological Chemistry. 262:11752–11757.

Taniguchi, K., K. Suzuki, and S. Iida. 1982. Conformational change accompanying transition of ADP-sensitive phosphoenzyme to potassium-sensitive phosphoenzyme of (Na+,K+)-ATPase modified with N-[p-(2-benzimidazolyl)phenyl]maleimide. Journal of Biological Chemistry. 257:10659–10667.

Taniguchi, K., K. Suzuki, D. Kai, I. Matsuoka, K. Tomita, and S. Iida. 1984. Conformation change of sodium- and potassium-dependent adenosinetriphosphatase. Journal of Biological Chemistry. 259:15228–15233.

Taniguchi, K., K. Suzuki, T. Sasaki, H. Shimokobe, and S. Iida. 1986. Reversible change in light scattering following formation of ADP-sensitive phosphoenzyme in Na+,K+-ATPase modified with N-[p-(2-benzimidazolyl) phenyl] maleimide. Journal of Biological Chemistry. 261:3272–3281.

Taniguchi, K., H. Tosa, K. Suzuki, and Y. Kamo. 1988. Microenvironment of two different extrinsic fluorescence probes in Na+,K+-ATPase changes out of phase during sequential appearance of reaction intermediates. Journal of Biological Chemistry. 263:12943–12947.

Occlusion of the K+-Congener TI+ through the Phosphointermediates of Na,K-ATPase

Rolando C. Rossi and Jens G. Nørby

Institute of Biophysics, University of Aarhus, DK-8000 Aarhus C, Denmark

Introduction

The current model for the reaction mechanism of Na,K-ATPase proposes a cycle with a Na+- and Mg2+-dependent phosphorylation by ATP and a K+-dependent dephosphorylation, accompanied by conformational transitions. Post et al. (1972) found evidence that K+ and its congeners, after promoting dephosphorylation, could remain tightly bound to the pump. This phenomenon, called occlusion, was interpreted as the expression of a transport intermediate in which the cations are “traveling” across the membrane and therefore are temporarily inaccessible to the aqueous solutions. Cation-occluding species (presumably involved in K+-transport) have been subsequently isolated in experiments using radioactive K+, Rb+, and TI+, where the free ions were removed either by ion exchange or by filtration techniques (Beaugé and Glynn, 1979; Forbush, 1984; Nørby and Jensen, 1989). The release of cations from the occluded state was found to be activated by the presence of ATP in the millimolar range, the nucleotide acting through a mechanism distinct from that as phosphate donor. Thus, transport of K+ was proposed to occur through this sequence: binding of external K+ to the phosphoenzyme → K+-dependent dephosphorylation → occlusion → ATP-stimulated release of K+ to the intracellular medium.

The present work aimed at characterizing quantitatively the K+-transport steps at 20°C using 204TI+ as a probe for K+. With this goal we measured, in parallel experiments, the time courses for both phosphoenzyme formation and breakdown, and TI+-occlusion via the dephosphorylation pathway.

Materials and Methods

All experiments were carried out at 20°C in media containing 150 mM NaNO_3, 0.5 mM MgSO_4, and 20 mM imidazole-HNO_3, pH 7.4, using Na,K-ATPase partially purified from pig kidney (Jensen et al., 1984). To follow the time courses (in the millisecond scale) of phosphorylation, dephosphorylation, and TI+-occlusion, a rapid-mixing apparatus was used (Mårdh and Zetterqvist, 1974). The level of phosphoenzyme (EP) was measured through the amount of acid-stable 32P incorporated by the enzyme from [γ^32P]ATP after stopping the reaction with an ice-cold solution containing 10% trichloroacetic acid (TCA) and 2 mM Na_4P_2O_7. Aliquots of the suspension were transferred to Millipore filters (type GS, 0.22 μm pore size), where the phosphoenzyme was washed five times with 4 ml of the same ice-cold solution. For the blanks, similar experiments were done in media where Na+ was replaced by K+.

To measure TI+-occlusion (TIOCCL) we used 204TI+. After the appropriate time of reaction (Fig. 1), the enzyme was collected in a filtration chamber on a Millipore filter (type AA, 0.8 μm pore size), through which an ice-cold solution of 30 mM KNO_3,20 mM imidazole-HNO_3, pH 7.4 at 0°C, was running with a flow of 10–14 ml/s. This system was devised to instantaneously slow the reactions and quench a further binding of TI+ as well as to remove the unbound TI+. Usually, 50 ml of washing solution was used per filter. The blanks were estimated from the amount of radioactive TI+ remaining on the filters in experiments where ATP was omitted during the reaction; i.e., where there was no phosphorylation of the enzyme. These blanks showed no significant difference with blanks where the enzyme was absent. Membrane protein was determined for each case by the Lowry method, after dissolving the filter in ice-cold acetone, which precipitates the enzyme, washing two times with 2 ml of ice-cold acetone and finally evaporating the remaining solvent.

Figure 1. Parallel measurements of the time courses of TI+-dependent dephosphorylation (●) and TI+-occlusion , after addition of 0.7 ml of TI+ 300 μM to 1.4 ml of medium containing the phosphoenzyme. EP was formed in the absence of TI+ by mixing equal volumes of enzyme (0.15 mg of protein/ml) and ATP (20 μM). All the media contained 150 mM Na+, 0.5 mM Mg2+, and 20 mM imidazole-HNO_3, pH 7.4 at 20°C. Addition of TI+ (time 0 in the figure) took place after 120–160 ms of phosphorylation, when steady-state [EP] was reached. The level of phosphoenzyme in the absence of TI+ (EP_0) was 1.048 ± 0.065 nmol/mg, independent of time from 120 to 240 ms. The lines are the graphical representations of Eq. 1 and Eq. 2, using the best-fitting values of the parameters.

Results and Discussion

In preliminary experiments carried out at 20°C (not shown), it was observed that TI+ behaves qualitatively like K+ in its effects on the level of phosphoenzyme, in agreement with previous results obtained at 0°C (e.g., Post et al., 1972). TI+ was found to decrease the steady-state level of phosphoenzyme with a K0.5 of ~ 30 μM in media with 10 μM ATP, 150 mM NaNO_3, 0.5 mM MgSO_4, and 20 mM imidazole-HNO_3, pH 7.4. When, under the same conditions, the time course of dephosphorylation was followed after the addition of TI+ to the phosphoenzyme formed in the absence of the cation, the initial rate of dephosphorylation varied with [TI+] with a K0.5 much higher than the value obtained for the effect on the steady-state level of phosphoenzyme. This feature allowed us to use concentrations of TI+ that are high enough to substantially reduce the level of EP when compared with the level obtained in the absence of the cation (90% or more), and low enough as to measure accurately the time course of TI+-dependent dephosphorylation. The difference in K0.5 could be explained assuming that the product of the dephosphorylation by TI+ is a species (presumably the one containing TI+ occluded) that converts slowly to the form that can be phosphorylated.

The formation of a TI+-occluded species was detected in media with 100 μM TI+ and 150 mM NaNO_3 only when the enzyme had been in contact with micromolar concentrations of ATP, under conditions where phosphorylation takes place. In an experiment performed using the rapid-mixing apparatus coupled to a filtration chamber (results not shown), the enzyme was first mixed with a medium containing either 0 or 20 μM of ATP in the absence of TI+ and after 150 ms TI+ was added (composition of the media are the same as described in the legend to Fig. 1). The reaction with TI+ continued for a fixed time (120 ms) before quenching by instantaneous exposure to the washing solution, as described above. Different washing times were assayed by varying the volume of washing solution. When ATP was absent during the reaction, the radioactive TI+ remaining on the filters decayed exponentially to 0 within the washing time, while when the nucleotide was present, an extra amount of enzyme-bound TI+ could be measured which, under the conditions of the washing procedure, remained constant with the washing time from 2 to 10 s. These results are in agreement with previous findings for Rb+-occlusion (Glynn and Richards, 1982). This procedure was applied to follow the time course of TI+-occlusion after addition of TI+ (100 μM, final concentration) to the phosphoenzyme formed in the absence of the cation (Fig. 1). The results are compared with those obtained in a parallel experiment for the time course of dephosphorylation. Both sets of data were analyzed by nonlinear regression. For the time course of dephosphorylation, the following equation was used:

where EP_0 and EP∞ are the phosphoenzyme levels at t = 0 and t → ∞ respectively, and k1 is a rate coefficient. The best-fit values were: EP∞ = 0.11 ± 0.07 nmol/mg and k1 = 21.5 ± 3.2 s–1. The value of EP_0 was determined separately (see legend to Fig. 1). For the time course of TI+-occlusion, the following equation was used:

where TIOCCL∞ is the amount of occluded TI+ when the time tends to infinity and k2 is a rate coefficient. The best-fit values were TIOCCL∞ = 1.93 ± 0.08 nmol/mg and k2 = 19.2 ± 1.5 s–1. Comparing the values found for k1 and k2, it is apparent that dephosphorylation by TI+ is mirrored by occlusion of the cation. The ratio between the amount of occluded TI+ and the corresponding amount of phosphoenzyme that was dephosphorylated at any given time after the addition of TI+ was calculated for the data in Fig. 1. The values obtained were independent of the time and agreed very closely with the stoichiometry of 2 already known for the transport of K+. It should be noted that the small delay observed after the addition of TI+ is better simulated by a model that takes the rate of binding of TI+ into account. However, the application of such a model (unpublished) does not invalidate the above conclusions on stoichiometry and rate.

To investigate whether these results could be influenced by the fact that TI+ was added to the phosphoenzyme formed in its absence, the time courses of phosphorylation and TI+-occlusion at 150 mM Na+ were followed after mixing the enzyme and the ATP (10 μM final concentration) when TI+ (100 μM) was present in the media (the rest of the composition was as described in the legend to Fig. 1). A control of the time course of phosphorylation in the absence of TI+ was also carried out. Results showed that during the initial phase the time course of phosphorylation in the presence of TI+ follows the same curve as the one obtained in the absence of the cation, suggesting that the enzyme becomes sensitive to TI+ only after the appearance of a phosphorylated species. This conclusion is consistent with a lag period observed for the time course of TI+ occlusion, which follows a “precursor–product” pattern when compared with the time course of phosphorylation.

Conclusion

Taking due account of the experimental conditions used, the results would indicate that: (a) in the presence of Na+ and ATP, the Na,K/Pump becomes reactive to TI+ only after the appearance of a phosphorylated intermediate; (b) the TI+-dependent dephosphorylation requires the binding of two TI+ per phosphoenzyme unit; (c) if any intermediate exists between the reactive phosphoenzyme with two TI+ bound and the TI+-occluded dephosphoform, its concentration is so small that it cannot be detected. This means that, in a sequence involving two reactions (namely, occlusion and dephosphorylation), it is not possible to define whether the occlusion of two TI+ is the first step and dephosphorylation the second or vice versa, but in any case the second reaction must be much faster than the first one in order that dephosphorylation and occlusion can be considered as simultaneous processes.

Acknowledgments

This work was supported by the Consejo Nacional de Investigaciones Científicas y Técnicas de la República Argentina, the Biomembrane Research Center, University of Aarhus, and the Danish Natural Science Research Council.

References

Beaugé, L., and I. M. Glynn. 1979. Occlusion of K+ ions in the unphosphorylated sodium pump. Nature. 280:510–512.

Forbush, B., III. 1984. An apparatus for rapid kinetic analysis of isotopic efflux from membrane vesicles and ligand dissociation from membrane proteins. Analytical Biochemistry. 140:495–505.

Glynn, I. M., and D. E. Richards. 1982. Occlusion of rubidium ions by the sodium-potassium pump: its implications for the mechanism of the potassium transport. Journal of Physiology. 330:17–43.

Jensen, J., J. G. Nørby, and P. Ottolenghi. 1984. Sodium and potassium binding to the sodium pump of pig kidney: stoichiometry and affinities evaluated from nucleotide-binding behaviour. Journal of Physiology. 346:219–241.

Mårdh, S., and Ö. Zetterqvist. 1974. Phosphorylation and dephosphorylation reactions of bovine brain (Na+-K+)-stimulated ATP phosphohydrolase studied by a rapid-mixing technique. Biochimica et Biophysica Acta. 350:473–483.

Nørby, J. G., and J. Jensen. 1989. A model for the stepwise radiation inactivation of the α_2-dimer of Na,K-ATPase. The Journal of Biological Chemistry. 264:19548–19558.

Post, R. L., C. Hegyvary, and S. Kume. 1972. Activation by adenosine triphosphate in the phosphorylation kinetics of sodium and potassium ion transport adenosine triphosphatase. The Journal of Biological Chemistry. 247:6530–6540.

Competitive Relationship between K+ and (Z)-5-Methyl-2- [2-(1-naphthyl)ethenyl] -4- piperidinopyridine for Access to the K+-translocating Channel of (Na,K)-ATPase

Yoshihiro Fukushima, Junji Takada, Hideaki Nagamune, and Mitsuo Yano

National Children's Medical Research Center, Tokyo 154, Japan; Banyu Pharmaceutical Co. Ltd., Tokyo 153, Japan; and Tokushima University, Tokushima 770, Japan

Introduction

A Novel Hydrophobic Fluorescent Amine as a Probe for the K+ Site

To identify the location of the K+-translocating channel in the peptide region of the (Na,K)-ATPase molecule, and to monitor the microenvironment around it, we have obtained a new fluorescent compound (Z)-5-methyl-2-[2-(1-naphthyl)ethenyl]-4-piperidinopyridine (AU-1421; Takada et al., 1990). Labeling of (Na,K)-ATPase with AU-1421 was expected to provide a guide for future point-mutation experiments. The peak wavelengths of excitation and emission of AU-1421 in water, in 25 mM histidine containing 0.1 mM EDTA, and in methanol were 340 and 490 nm, 330 and 415 nm, and 325 and 460 nm, respectively. These peak wavelengths are very different from those of tryptophan residues. If (Na,K)-ATPase can be labeled with AU-1421, it should be possible to estimate the distance between the K+ site and the ATP site from the energy transfer with FITC attached to the Lys-501 residue.

AU-1421 was found to inactivate (Na,K)-ATPase when they were preincubated together in the absence of monovalent inorganic cations followed by determination of ATP-hydrolyzing activity or phosphorylation. Inactivation experiments (Takada et al., 1990) revealed: (a) Protection of the enzyme by K+ or its congeners followed simple competitive kinetics, giving a binding constant for K+ of 30 μM, consistent with the reported value obtained from conformation change experiments. (b) Na+ at a concentration of several tens of millimolar also protected the enzyme, probably acting as a weak substitute for K+, thus showing that the protection did not depend on the enzyme conformation E_1 or E_2, but only on the occupation of the K+ site. (c) Saturation of the low affinity site of ATP impaired the K+ protection of the enzyme from AU-1421. (d) Saturation of only the high-affinity site for Na+ stimulated the inactivation, and a high concentration of Tris, imidazole, or choline, which all act similarly to Na+ in stabilizing the E_1 conformation of the enzyme, also accelerated the inactivation induced by AU-1421. (e) Phosphorylation of the enzyme with ATP was suppressed under similar conditions and was maintained by the presence of K+ during preincubation. (f) The inactivation proceeded slowly at 37°C, but there was little inactivation at 0°C.

These results indicated that AU-1421 interacts with the K+ occlusion center through the E_1 conformation (Fig. 1 A). This mode of interaction is similar to that reported for Ca2+ upon interaction with the K+ occlusion center (Vasallo and Post, 1986). As the E_1 state facilitates inactivation, the AU-1421 access pathway is presumably the K+-releasing site of the occlusion center in the E_1 conformation opening toward the cytoplasmic side.

Results and Discussion

Competitive Inhibition by AU-1421 of K+-sensitive Dephosphorylation

If these assumptions are valid, it would be interesting to investigate whether AU-1421 binds to the K+ uptake site of the K+-translocating channel. According to the current reaction scheme for (Na,K)-ATPase, K+ uptake occurs at the extracellular surface of E_2P. Therefore, we examined competition between AU-1421 and K+ with respect to the reactivity of E_2P (i.e., the hydrolyzing activity of the phosphoester bond of E_2P [Fig. 1 B]).

Figure 1. Sidedness of the reaction for AU-1421 and the K+-translocating channel opening to the cytoplasmic face (A) or to the external face (B) of the cell. E_1 and E_2 indicate the two principal conformations of (Na,K)-TPase, and –P indicates phosphorylation of the enzyme.

Membrane-bound (Na,K)-ATPase was prepared from crude microsomes of sheep kidney outer medulla by treatment with sodium dodecyl sulfate. E_2P was made by adding 10 μM [32P]ATP to the enzyme (0.1 mg/ml) in the presence of 10 or 100 mM NaCl, 0.5 mM MgCl_2, and 25 mM histidine (pH 7.0) at 0°C. The reactive state of the E_2P was observed in terms of dephosphorylation after stopping the phosphorylation by addition of CDTA to chelate free Mg2+. The effect of AU-1421 on the reactive E_2P was tested by adding a solution of AU-1421 in distilled water. AU-1421 dissolved in water up to a concentration of 2 mM, and this solution was stored shielded from light in a refrigerator.

When various concentrations of AU-1421 were added to E_2P, the dephosphorylation rate was immediately slowed (Fig. 2). The IC_50 value was ~10 μM. This rapid inhibition at 0°C was remarkable; inactivation of the nonphosphorylated enzyme required a preincubation time in the order of minutes at 37°C, as described in (f) and discussed above. This is because the affinity of the nonphosphoenzyme for AU-1421 is lower than that of the phosphoenzyme (Fig. 1). Moreover, the inhibition was readily abolished by simple addition of K+; AU-1421 appeared to dissociate from E_2P by saturation with K+,

and the E_2P∙K showed rapid splitting.

A similar contrast between the phospho-form and non-phospho-form with regard to accessibility from the aqueous phase and competition with K+ in the reactivity of E_2P were also observed in the inactivation or inhibition by several other amine compounds, such as arginine (Fukushima, 1987, 1990). Similarly to the AU-1421 experiment, monovalent cations afforded protection, which was prevented by a high concentration of ATP, suggesting that the amine binding site is related to the cation site. E_2P of Ca-ATPase from the sarcoplasmic reticulum was not sensitive to amines. Thus, amine compounds seem to be general inhibitors of (Na,K)-ATPase, although the effect appears only at concentrations of several tens of millimolar in the case of hydrophilic substances such as arginine. The hydrophobicity of AU-1421 (log∙ P = 5.5) was probably the reason for its high affinity for the enzyme.

Figure 2. Rapid inhibition of E_2P by AU-1421 and its prevention by K+. (Na,K)-ATPase was phosphorylated by adding 10 μM [32P]ATP in the presence of 100 mM NaCl, 0.5 mM MgCl_2, and 25 mM histidine at 0°C. At 15 s after the start of phosphorylation, 9 mM CDTA was added to impair the phosphorylation reaction (○,□). This is indicated as zero time in the figure. At 5 s after CDTA addition, 4, 8, 16, or 83 μM AU-1421 was added to the phosphoenzyme (●). After a further 5 s, 3.8 mM KCl was added (□,■).

To investigate the relationship between the binding sites for AU-1421 and K+, various concentrations of K+ were added to the AU-1421–inhibited E_2P. The splitting of E_2P was stimulated by increased concentrations of K+, and the apparent affinity (50% protective concentration) for K+ decreased with an increase in AU-1421 concentration, although there was no effect on the maximum rate of dephosphorylation (Fig. 3). Therefore, AU-1421 appears to compete with K+ at the site on E_2P. The Ki value for AU-1421 was estimated to be 7.2 μM under these conditions.

Figure 3. Competition between AU-1421 and K+ on E_2P. Almost the same experiments as those in Fig. 2 were carried out in the presence of 10 mM NaCl. AU-1421 was added at a concentration of 0 (●), 8 , 16 (□), 24 (∆), or 41 (○) μM, then various concentrations of KCl were added. The K+ sensitivity at each concentration of KCl was taken as the difference between the phosphoenzyme level after no addition of KCl and that after addition of KCl. The percentage of K+-sensitive phosphoenzyme is indicated as a function of KCl concentration.

Although AU-1421 inhibited E_2P, unlike K+, it did not stimulate dephosphorylation in spite of the apparent interaction with the K+ site. Thus, stimulation may require the occupation of the K+ occlusion center. K+ can reach the occlusion center, but AU-1421 may not be able to pass into the K+-translocating channel of E_2P (Fig. 1 B). The same idea may be applied to the action of Ca2+ (cf. Vasallo and Post, 1986). Simple addition of Ca2+ to E_2P inhibited by AU-1421 or other amines (Fukushima, 1987) restored the normal rate of dephosphorylation, but did not bring about rapid splitting. Thus Ca2+ seems to compete with AU-1421 only at the surface of the E_2P.

References

Fukushima, Y. 1987. Inhibition by amine bases or by sodium ions and protection by divalent cations in the hydrolysis of (Na,K)-ATPase. Journal of Biological Chemistry. 262:11000–11005.

Fukushima, Y. 1990. Destabilization of (Na,K)-ATPase by nitrogen basic compounds. Biomedical Research. 11:337–344.

Takada, J., Y. Hioki, M. Yano, and Y. Fukushima. 1990. A novel hydrophobic amine, (Z)-5-methyl-2-[2-(1-naphthyl)ethenyl]-4-piperidinopyridine, as a probe of the K+ occlusion center of Na+ /K+-ATPase. Biochimica et Biophysica Acta. 1037:373–379.

Vasallo, P. M., and R. L. Post. 1986. Calcium ion as a probe of the monovalent cation center of sodium, potassium ATPase. Journal of Biological Chemistry. 261:16957–16962.

Characterization of Lanthanides as Competitors of Na+ and K+ in Occlusion Sites of Renal (Na+,K+)-ATPase

Peer David and Steven J. D. Karlish

Department of Biochemistry, Weizmann Institute of Science, Rehovot, 76100, Israel

Introduction

Trivalent lanthanides have been used as competitors of Ca2+ in the sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPase (Highsmith and Head, 1983; Scott, 1984, 1985; Jona and Martonosi, 1986; Hanel and Jencks, 1990). The ionic radii of the lanthanides (0.98–1.16 Å) are similar to Ca2+ ionic radii (1.00 Å) (Martin and Richardson, 1979; Martin, 1984), but their affinity to the two Ca2+ binding sites is much higher due to their higher charge density (Scott, 1984). Lanthanides, like Ca2+, stabilize E_1 (Jona and Martonosi, 1986). The luminescence properties of Tb3+ have been used in energy transfer experiments to estimate the distances between the two Ca2+ binding sites, and the distance between the Ca2+ and the FITC binding sites (Scott, 1985).

Lanthanides have also been able to substitute for Na+ (1.02-Å ionic radii) on the brush border Na+ proline cotransport carrier (Birnir and Wright, 1987). It is therefore of interest to examine the possibility that lanthanides are also competitors of Na+ and/or K+ on the (Na+,K+)-ATPase.

Results and Discussion

Is Lanthanum a Competitor of Rb+ and Na+ in an Occlusion Assay?

The 86Rb+ and 22Na+ occlusion assays are as previously described (Shani et al., 1987; Shani-Sekler et al., 1988). Na+ occlusion is that induced by oligomycin (Esmann and Skou, 1985). In brief, purified (Na+,K+)-ATPase membranes at a concentration of 0.8 mg/ml were incubated in a reaction mixture containing 150 mM Tris, pH 7.0, and 0.1–2 mM RbCl + 86Rb+. After incubation for 3 min at room temperature, we diluted the mixture 10-fold with ice-cold sucrose and passed the suspension through Dowex-50 columns. The bound Rb+ in the effluent is considered to be “occluded.” In a typical Rb+ dose–response curve with different La3+ concentrations (0, 25, 37.5, 50 μM), pseudo-hyperbolic curves were obtained, with La3+ drastically affecting the apparent affinity of the enzyme for Rb+ (466, 910, 1,120, 2,580 μM, respectively), but with no change in the maximal Rb+ binding capacity (4.25 ± 0.28 nmol Rb+/mg protein). The binding affinity of La3+, calculated from a simple competitive model, was found to be 26.5 μM. Therefore, La3+ is a Rb+ competitor.

In a typical Na+ occlusion experiment, the medium contains 100 mM final salt concentration of (Tris + Na+), pH 7.0, and 125 μg/ml of oligomycin is added for 10 min after incubation of the enzyme with (22Na+ + Na+) at the desired concentration. In experiments examining Na+ occlusion at various doses of Na+, sigmoid curves are observed. It has been observed here and elsewhere that La3+ (up to 40 μM) behaves competitively with Na+, Na+ affinity being reduced in the presence of La3+ (K = 5.12 mM increased to 48.07 mM), while maximal binding (5.16 ± 0.8 nmol Na+/mg protein) and the cooperativity index (nH = 1.39 ± 0.27) are not changed.

Comparison of Inhibition Potency between Different Lanthanides

All lanthanides are potent inhibitors of the Rb+ occlusion at 60 μM. The maximal inhibition was seen with Pr3+ (66%) and the minimal inhibition with Er3+ (17%). The order of potency was Pr > Nd > La > Eu > Tb > Ho > Er.

Involvement of the Divalent Cation Binding Site?

It might be thought that La3+ does not act directly on the monovalent cation binding site, but rather indirectly via the divalent cation binding sites. To test this we looked for a possible effect of Mg2+ on La3+ inhibition of Rb+ occlusion. 10 mM Mg2+ does not affect occlusion at 1 mM Rb+ (1.33 ± 0.1 and 1.30 ± 0.09 nmol Rb+/mg protein, respectively), or the inhibitory potency of La3+ (0.47 ± 0.07 and 0.44 ± 0.05 nmol Rb+/mg protein, respectively, at 1 mM Rb+ + 60 μM La3+). The results suggest a direct La3+ effect on the monovalent cation binding site.

Further evidence comes from the use of (Na+,K+)-ATPase, extensively trypsinized in the presence of Rb+ and absence of Ca+, to produce a 19-kD major fragment and smaller fragments derived from the α-subunit of the (Na+,K+)-ATPase. The β-subunit under these conditions remains intact. The digestion abolishes all ATPase- and ATP-dependent activities, but Rb+ and Na+ occlusion remain intact (Karlish et al., 1990). In experiments that measure Rb+ occlusion to trypsinized enzyme in the presence and absence of 40 μM La3+, the maximal specific Rb+ binding of the trypsinized enzyme is double that of control enzyme (Bmax = 8.40 ± 0.38 and 4.56 ± 0.28 nmol Rb+/mg protein, respectively), but the pseudo-hyperbolic binding curve of Rb+ is not altered. La3+ again appears competitive since there is the same maximal Rb+ binding but a reduction in the apparent affinity of Rb+. K_1 calculated for La3+ is 26 μM. Thus, in an enzyme that has lost about half of its protein content and lacks the (Mg2+)-ATP catalytic domain, La3+ still competes with Rb+, again suggesting a direct La3+ interaction with the monovalent cation binding site.

Effects of Lanthanides on Conformation

In principle, a competitor for both Rb+ and Na+ in the occlusion assay could act like Rb+ in stabilizing the E_2 state or like Na+ in stabilizing the E_1 state. These possibilities were distinguished using FITC-labeled (Na+,K+)-ATPase (see Karlish, 1980, and Rephaeli et al., 1986 for labeling procedure). It turns out that La3+ acts like Na+ ions. In a high ionic strength medium (E_1), La3+ ions do not affect fluorescence. However, if Rb+ (K+) ions are added first to induce the quenched (E_2(K)) state, La3+, like Na+ ions, reverses the fluorescence change. Does La3+ compete with Rb+ ions? In experiments where different Rb+ concentration were used to quench the fluorescence and then different La3+ concentrations were added in a dose-dependent manner to reverse the fluorescence quench, it is clear that as the Rb+ concentration is increased, the amount of La3+ needed to reverse half of the fluorescence increases as well. A plot of (K1 app) as a function of Rb+ concentration is linear, K1 app = 0.95·[Rb+] + 0.583, suggesting that La3+ is indeed a competitor of Rb+. The intrinsic affinity in the absence of Rb+ is only ~ 0.583 μM La3+.

Rate of Conformational Transitions

Since La3+, like Na+, stabilized the E_1 form of the pump, it was of interest to look at La3+ effects on the rates of the E_1 ↔ E_2 conformational transitions, by stopped-flow measurements (at 25°C). In the first type of experiment, enzyme with bound K+ was mixed with a high concentration of La3+ (80 μM) to induce the E_2(K) → E_1La transition, similar to the E_2(K) → E_1Na transition with a high concentration of Na+ ions (Karlish, 1980). The rate of the transition is about sixfold slower than that with Na+ (0.09 and 0.523 s–1, respectively). Even in the presence of a saturating Na+, La3+ (80 μM) still inhibited three- to fourfold (0.153 and 0.523 s–1 respectively). This is unexpected, for it suggested a real if moderate inhibition by La3+ of the conformational transition, in addition to its Na-like effect.

In a high ionic strength, the FITC-labeled enzyme is stabilized in the E_1 form of the pump and addition of K+ induces the E_1 + K+ → E_2(K) transition. The rate of this transition shows saturation behavior with respect to K+ concentration (K0.5 ~ 75 mM) and a maximal rate of ~300 s–1. At 75 mM K+, La3+ appears to slightly accelerate this rate (210 and 328 s–1, respectively).

It was conceivable that the unexpected effects of La3+ on the rate of the conformational transition occurs at Mg2+ sites. Previously, Mg2+ ions have been shown to partially reverse K+-induced fluorescence quenching (Karlish, 1980). The rate of this process is ~ 10-fold faster than E_2(K) → E_1(Na) (4.426 and 0.523 s–1, respectively), and is not affected by Na+ or La3+ (5.22 and 3.75 s–1, respectively). Therefore, the La3+ effects are different from Mg2+ effects. One possibility is that the La3+ effects on the conformational rates of the E_2(K) → E_1(X) and the E_1 + K+ → E_2(K) transitions occur from nonspecific La3+ binding sites (discussed below).

Dissociation of La3+ Ions

FITC-enzyme prebound with La3+ ions (8 μM) was mixed with a high concentration of K+ ions (75 mM) in the stopped-flow fluorimeter. The rate of fluorescence quenching upon conversion to the E_2(K) form was slow, k = 1.74 s–1. Since the rate of E_1 + K+ → E_2(K) at 75 mM in the absence of La3+ is 200–300 s–1, the observed rate is limited by and reflects the dissociation rate of prebound La3+. The calculated second order binding rate for the equilibrium E_1 + La ↔ E_1(La) using kd = 1.741 s–1 and Kd = 4.45 × 10–7 M, is kf = 3.9 × 10^6 s–1 · M–1. This rate is at the lower range of values expected for the kf of a simple binding process. Thus, La3+ is probably not tightly occluded.

Nonspecific Binding of La3+ Ions

A striking discrepancy appears between the La3+ intrinsic affinity found in the Rb+ occlusion assay (26.5 μM) and the La3+ intrinsic affinity found from its effect on the conformational change (0.58 μM). The conditions of the two different assays are similar (150 mM Tris, pH 7.0), but there is a large difference in protein concentration in the two assays, 0.01 mg/ml in the fluorescence assay compared with 0.8 mg/ml in the Rb+ occlusion assay. It has now been found that the concentration of La3+ needed to reverse half of the fluorescence quench, with 2 mM Rb+, is strongly affected by protein concentration, with a linear correlation (Kl app = 32.4·[protein concentration] + 1.82 μM La3+). Two extrapolations should be made. First, K1 app at zero protein is 1.82 μM La3+ in the presence of 2 mM Rb+. Considering a simple competitive model between Rb+ and La3+ so that K1 app = K_1·(1 + [Rb+]/K0.5), the intrinsic affinity calculated (K0.5 = 650 μM Rb+; not shown) is 0.445 μM La3+. Extrapolating the intrinsic affinity found (0.445 μM La3+) to the protein concentration in the Rb+ occlusion assay (0.8 mg/ml), the estimated K1 app is 26.36 μM La3+ (i.e., indistinguishable from the K_1 found directly in the Rb+ occlusion assay [26.5 μM La3+]). These results suggest strongly nonspecific binding of La3+ to the enzyme preparation.

Tb3+ binding to the (Na+,K+)-ATPase as measured with dipicolinic acid (Barela and Sherry, 1976) proved the existence of nonspecific binding. 2 μM of Tb3+ were added to an enzyme (0.014 mg/ml) in 150 mM, Tris-HCl, pH 7.0, in the presence or absence of 4 mM Rb+, at 4°C for 10 min. The samples were transferred through ice-cold Dowex-50 columns. 1 mM of dipicolinic acid was added to the eluate. Upon excitation at 280 nm and emission at 543 nm, the Tb-dipicolinic complex fluorescence can be detected (Barela and Sherry, 1976). Tb3+ binding in the presence or absence of Rb+ was found to be 20.5 ± 0.7 and 20.2 ± 0.4 nmol Tb3+/mg protein, respectively. It is clear that Rb+-displacable binding was not detected under these conditions. All the Tb3+ binding was nonspecific. It can be suggested that most if not all of the nonspecific binding of the lanthanides is bound to the membrane phospholipids rather than to the protein. This interpretation is supported by the fact that the La3+ affinity on the 19 kD-containing membranes, which lost half of their protein content, remained constant as compared with control preparation.

Conclusion

This work presents evidence that the trivalent cations, lanthanides, are Na+ analogues at the monovalent cation binding site of the renal (Na+,K+)-ATPase with the highest binding affinity reported to date. However, the lanthanides do not appear to be occluded in the monovalent cation binding sites. Additional nonspecific lanthanide binding is detected, probably to the membrane phospholipids. The paramagnetic and/or luminescence properties of several members of this family may serve, in the future, as tools for studying the geometry of the monovalent cation binding domain and/or the environment of the cation binding site.

References

Barela, T. D., and A. D. Sherry. 1976. A simple, one step fluorometric method for determination of nanomolar concentrations of terbium. Analytical Biochemistry. 71:351–357.

Birnir, B., and E. M. Wright. 1987. Lanthanide/proline cotransport across rabbit renal brush borders. Journal of Membrane Biology. 100:221–227.

Esmann, M., and J. C. Skou. 1985. Occlusion of Na+ by (Na+,K+)-ATPase in the presence of oligomycin. Biochemical and Biophysical Research Communications. 127:857–863.

Hanel, A. M., and W. P. Jencks. 1990. Phosphorylation of the calcium-transporting adenosinetriphosphatase by lanthanum ATP: rapid phosphoryl transfer following a rate-limiting conformational change. Biochemistry. 29:5210–5220.

Highsmith, S. R., and M. R. Head. 1983. Tb3+-binding to Ca2+ binding sites on sarcoplasmic reticulum ATPase. Journal of Biological Chemistry. 258:6858–6862.

Jona, I., and A. Martonosi. 1986. The effects of membrane potential and lanthanides on the conformation of the Ca2+-transport ATPase in sarcoplasmic reticulum. Biochemical Journal. 234:363–371.

Karlish, S. J. D. 1980. Characterization of conformational changes in (Na+,K+)-ATPase labeled with fluorescein at the active site. Journal of Bioenergetics and Biomembranes. 12:111–135.

Karlish, S. J. D., R. Goldshleger, and W. D. Stein. 1990. A 19 KDa C-terminal tryptic fragment of a-chain of (Na+,K+)-ATPase is essential for occlusion and transport of cations. Proceedings of the National Academy of Sciences, USA. 87:4566–4570.

Martin, R. B. 1984. Bioinorganic chemistry of calcium. Metal Ions in Biology. 17:2–51.

Martin, R. B., and F. S. Richardson. 1979. Lanthanides as probes for calcium in biological systems. Quarterly Reviews of Biophysics. 12:181–209.

Rephaeli, A., D. E. Richards, and S. J. D. Karlish. 1986. Conformational transitions in fluorescein-labeled (Na+,K+)-ATPase reconstituted into phospholipid vesicles. Journal of Biological Chemistry. 261:6248–6254.

Scott, T. L. 1984. Luminescence studies of Tb3+ bound to the high affinity sites of the Ca2+-ATPase of sarcoplasmic reticulum. Journal of Biological Chemistry. 259:4035–4037.

Scott, T. L. 1985. Distances between the functional sites of the (Ca2+ + Mg2+)-ATPase of sarcoplasmic reticulum. Journal of Biological Chemistry. 260:14421–14423.

Shani, M., R. Goldshleger, and S. J. D. Karlish. 1987. Rb+ occlusion in renal (Na+ + K+)- ATPase characterized with a simple manual assay. Biochimica et Biophysica Acta. 904:13–21.

Shani-Sekler, M., R. Goldshleger, D. Tal, and S. J. D. Karlish. 1988. Inactivation of Rb+ and Na+ occlusion on (Na+,K+)-ATPase by modification of carboxyl groups. Journal of Biological Chemistry. 263:19331–19342.

Kinetics of Na+,K+-ATPase Conformational Change with Alternate Substrates

P. R. Pratap, J. D. Robinson, and M. I. Steinberg

State University of New York Health Science Center, Syracuse, New York 13210

Introduction

Several substrates energize transport mediated by the dog kidney Na pump. In addition to ATP, GTP, and CTP, alternative substrates include carbamyl phosphate (carbP), acetyl phosphate, and 3-O-methylfluorescein phosphate (Guerra et al., 1990). In the absence of K, these substrates are also hydrolyzed by the enzyme in broken membrane preparations (equivalent to the Na-ATPase activity of the enzyme).

In this study we compare ATP, CTP, and carbP as indicators of substrate site requirements, using measurements of steady-state hydrolysis of these substrates and fluorescence stopped-flow techniques to examine conformational changes associated with enzyme activity. Enzyme conjugated with 5-iodoacetamidofluorescein (IAF), which has been used to study kinetics of conformational changes of dog kidney enzyme (Steinberg and Karlish, 1989), is used here to compare conformational changes energized by ATP with those energized by CTP and carbP as indicators of (a) the rate of conversion of E_1 to E_2P, expressed as a rate constant k, and (b) the steady-state catalytic rate, represented by the steady-state fraction of enzyme in E_2 conformations and expressed as %ΔF.

Materials and Methods

Enzyme purified from the red outer medulla of frozen dog kidneys (Jorgensen, 1974a) was labeled with IAF (Steinberg and Karlish, 1989). Na+,K+-ATPase activity of unlabeled enzyme was estimated to be 10–12 μmol Pi·mg protein–1·min–1 at 37°C (Jorgensen, 1974b). Except for this measurement, all experiments were performed at 24°C and in the absence of K+. A fluorescent assay (Banik and Roy, 1990) was used to measure enzyme activity with different substrates with or without oligomycin (5 mg/ml stock in ethanol). Protein content was determined by a Lowry assay (Jorgensen, 19746). Rapid kinetics were measured with a stopped-flow fluorometer (λex = 492 nm; emission cutoff filter λ1/2max = 529 nm).

Results

Steady-state hydrolysis of ATP was approximately the same as that of CTP and three times that of carbP. Na+-ATPase activity was 0.14 μmol Pi·mg protein–1·min–1 ≈ 1 mol Pi·mol enzyme–1·s–1. This rate is comparable to rates measured by Guerra et al. (1990). Oligomycin reduces enzyme activity by 40–60%.

Fig. 1 shows the rate (K) and magnitude (%∆F) of the fluorescence change under stopped-flow conditions as a function of substrate. Nonlinear least-squares fits of a Michaelis-Menten equation to the data yielded estimates of maximum rate (kmax) for fluorescence changes energized by ATP, CTP, and carbP of 73, 40, and 8 s–1, respectively. The corresponding half-maximum substrate concentrations (K1/2) were 12, 103, and 1,400 μM. Kmax for fluorescence change energized by ATP is ~ 100 times larger than the rate of steady-state hydrolysis of ATP.

Oligomycin reduced the rate of fluorescence change energized by 1 mM ATP and CTP by 98–99%, while this rate with 1 mM carbP was reduced by only 40%. Oligomycin reduced %ΔF by 20–25% with all three substrates.

Figure 1. Rate (k; A) and magnitude (%ΔF; B) of fluorescence change as functions of substrate concentration. IAF-enzyme (50–70 μg/ml protein) in buffer (155 mM NaCl, 4 mM MgCl_2, 1 mM Et_3N-EDTA, and 25 mM imidazole, pH 7.0, 24°C) was rapidly mixed with an equal volume of varying concentrations of three substrates (ATP, CTP, and carbP) in the same buffer (one “shot”). Final concentration of enzyme in the reaction chamber was 25–35 μg/ml. The fluorescence of eight shots was recorded and the average and standard error of the mean were calculated. A single exponential was fitted by a nonlinear leastsquares algorithm to this average to give parameter estimates with errors. (ATP, three experiments; CTP, three experiments; carbP, one experiment).

Fig. 2 shows k and %ΔF as a function of [Na] with or without oligomycin. %ΔF does not vary significantly over the range of [Na] tested (1–155 mM). k varies with [Na], with [Na]1/2 ≈ 7 mM. Oligomycin reduces k by 50% at 1 mM [Na] and by 98–99% at [Na] > 10 mM, but did not significantly affect %ΔF.

Discussion

Under stopped-flow conditions, the fluorescence change of the IAF enzyme may be attributed to the transition from E_1 to E_2P. Since the step from E_1P to E_2P is the same for all substrates, the variations in the rate of fluorescence change are attributable to variations in the rates of substrate binding, enzyme phosphorylation, and/or product release. When the transition E_1 to E_2P is energized by ATP or CTP, oligomycin reduces this rate by 98–99%. With carbP, oligomycin reduces this rate only by half, probably because carbP itself slows this transition by a factor of 10.

Enzyme activity under steady-state conditions reflects the cycling of the enzyme from E_1 to E_2P and back to E_1 The rate of hydrolysis is two orders of magnitude slower than the fluorescence response seen under stopped-flow conditions, and reflects the rate of some step that occurs after the E_1 to E_2P transition. Under these conditions oligomycin still reduces the rate of E_1 to E_2P transition by 98–99%, but this effect is not seen because of the slow overall rate.

Figure 2. Rate (k; A) and magnitude (%ΔF; B) of fluorescence change as functions of Na concentration with or without oligomycin. IAF-enzyme (50–70 μg/ml protein) in buffer (x mM NaCl, (155 - x) mM choline chloride, 4 mM MgCl_2, 1 mM Et_3N- EDTA, and 25 mM imidazole, pH 7.0, where x was varied from 1 to 155) was rapidly mixed with the same buffer containing ATP (final [ATP] = 1 mM for experiments shown as open squares and 0.5 mM for the others). In experiments with oligomycin (two experiments), 4 μM of 5 mg/ml oligomycin stock solution in ethanol was added to 2 ml of enzyme suspension (final [oligomycin] = 10 μg/ml in the syringe and 5 μg/ml in the reaction chamber). In parallel experiments without oligomycin, an equal volume of ethanol was added as control. Data were analyzed as described in Fig. 1.

References

Banik, U., and S. Roy. 1990. A continuous fluorimetric assay for ATPase activity. Biochemical Journal. 266:611–614.

Guerra, M., J. D. Robinson, and M. Steinberg. 1990. Differential effects of substrates on three transport modes of the Na+/K+-ATPase. Biochimica et Biophysica Acta. 1023:73–80.

Jorgensen, P. L. 1974a. Purification and characterization of (Na+ + K+)-ATPase. III. Purification from the outer medulla of mammalian kidney after selective removal of membrane components by sodium dodecyl-sulphate. Biochimica et Biophysica Acta. 356:36–52.

Jorgensen, P. L. 1974b. Isolation of (Na+ + K+)-ATPase. Methods in Enzymology. 32B:277–290.

Steinberg, M., and S. J. D. Karlish. 1989. Studies on conformational changes in Na,K-ATPase labeled with 5-iodoacetamidofluorescein. Journal of Biological Chemistry. 264:2726–2734.

Photo-cleavable Cryptands as a New Tool to Perform Na+ and K+ Concentration Jumps for Binding and Transport Studies

R. Warmuth, E. Grell, and J. M. Lehn

Max-Planck-Institute for Biophysics, Frankfurt, Germany; and Université Louis Pasteur, Institut Le Bel, Strasbourg, France

Introduction

In many cases it is difficult to carry out kinetic or mechanistic studies concerning alkali ion binding to membrane bound enzymes as well as alkali ion transport across membranes. This is especially so when the application of fast mixing techniques for the investigation of certain biological systems is not appropriate.

These principal problems can be avoided by using photoinduced cation concentration changes. The initial free concentration of the selected cation is reduced upon complexation by a suitable light-sensitive ligand. As a result of the UV light–induced photochemical reaction, the affinity of the photoproduct to the cation is decreased (e.g., as a consequence of bond cleavage or rearrangement) and the cation is released into solution within a millisecond time range. Depending on the prechosen ligand to cation ratio it is possible to obtain either cation concentration jumps (equimolar amounts of ligand and cation) or cation concentration pulses (ligand is present in large excess) as shown in Fig. 1.

This new principle has been successfully applied to generate Ca2+ and Mg2+ concentration jumps by Tsien and Zucker (1986), Kaplan and Ellis-Davies (1988), and Adams et al. (1988).

To carry out cation-selective, photo-induced alkali ion concentration jumps we have synthesized light-sensitive cryptands bearing a photo-cleavable o-nitrobenzylether bond (Fig. 2). These new compounds, which are described here, can be used for performing binding studies in solution as well as for transport investigations on black lipid membranes or cellular systems.

Experimental Methods

Four new, light-sensitive cryptands with different cavity sizes and hence different alkali ion selectivities have been synthesized according to the general procedure described by Dietrich et al. (1973): a Li+-selective (NC211) with n = m = 0, R = H, a Na+-selective (NC221) with n = 1, m = 0, R = H, and two K+-selective ligands with n = m = 1, R = H (NC222) or R = OCH_3 (DMNC222) (cf. Fig. 2). The photo-sensitive o-nitrobenzylether bond was introduced via a 1-(o-nitrophenyl)-ethane-1,2-diol prepared according to the procedure of Ellis-Davies and Kaplan (1988). The structures of the new compounds are confirmed by 1H- and 13C-NMR spectroscopy, elementary CHN analysis, FAB mass spectroscopy, and partially by x-ray structure analysis.

Figure 1. Schematic generation of a cation concentration jump (A) and concentration pulse (B) upon photolysis. Men+, cation; L, ligand; L*, photo-product of the ligand.

Results and Discussion

The alkali ion binding properties of the light-sensitive cryptands have been determined in aqueous solution (Fig. 3). In view of competition with protonation the apparent stability constants (Kapp) increase with increasing pH and are similar to those of the unsubstituted cryptands without the o-nitrophenyl substituent (Lehn and Sauvage, 1975).

Figure 2. Model illustration of the photolytically induced K+ release due to UV illumination of caged K+ (ligand NC222) (n = m = 1, R = H).

Figure 3. pH dependence of the apparent K+ binding constant for the cryptands DMNC222 (○), NC222 (□), and [222] (—), and Na+ binding constant for the cryptands NC221 (Δ) and [221] (— —). Stability constants (Kapp) have been determined using alkali ion-selective glass electrodes at 25°C. Values for the unsubstituted cryptands [221] and [222] have been calculated according to the data of Lehn and Sauvage (1975).

The selective release of K+ in the presence of 1 equiv. of Na+ upon photolysis of the K+-selective cryptand DMNC222 and its time dependence is demonstrated in Fig. 4. While the K+ concentration increases during the UV light–induced decomposition of the cryptand, the Na+ concentration remains nearly constant. After complete photo-cleavage of the ligand all of the bound K+ was released into solution, indicating that the photoproduct exhibits no significant affinity to either K+ or Na+. These results show that the light-sensitive cryptands are indeed suitable to induce highly selective alkali ion concentration changes upon UV photolysis.

Summary

Alkali ion-selective and photo-cleavable cryptands exhibiting high affinity for a cation of given size have been synthesized. Upon a short UV light pulse the coordinated cation is liberated, leading to a concentration jump, which allows us to carry out physiological studies in cellular systems and kinetic investigations.

Figure 4. Selective release of K+ in dependence of duration of UV irradiation performed in a spectrofluorimeter using a 140-W xenon lamp under continuous stirring in a standard 2-cm cuvette containing 5 ml 0.75 mM cryptand DMNC222 in the presence of 1 equiv. KCl (0.75 mM) and 1 equiv. NaCl (0.75 mM) at pH 9.2 (100 mM (CH_3)4NOH/H_3BO_3 at 25°C). Curves are not calculated.

Acknowledgments

We thank Mr. E. Lewitzki (Max-Planck-Institute for Biophysics, Frankfurt, Germany) and Dr. B. Dietrich (Université Louis Pasteur, Strasbourg, France) for helpful assistance and stimulating discussions. We also wish to thank Dr. J. W. Bats and Prof. G. Quinkert (University of Frankfurt) for performing and discussing the x-ray structure analysis.

References

Adams, S. R., J. P. Y. Kao, G. Grynkiewicz, A. Minta, and R. Y. Tsien. 1988. Biological useful chelators that release Ca2+ upon illumination. Journal of the American Chemical Society. 110:3212–3220.

Dietrich, B., J.-M. Lehn, J.-P. Sauvage, and J. Blanzat. 1973. Cryptates - X. Tetrahedron. 29:1629–1645.

Ellis-Davies, G. C. R., and J. H. Kaplan. 1988. A new class of photolabile chelators for the rapid release of divalent cations: generation of caged Ca and caged Mg. Journal of Organic Chemistry. 53:1966–1969.

Kaplan, J. H., and G. C. R. Ellis-Davies. 1988. Photolabile chelators for the rapid photorelease of divalent cations. Proceedings of the National Academy of Science, USA. 85:6571–6575.

Lehn, J.-M., and J.-P. Sauvage. 1975. [2]-Cryptates: stability and selectivity of alkali and alkaline-earth macrobicyclic complexes. Journal of the American Chemical Society. 97:6700–6707.

Tsien, R. Y., and R. S. Zucker. 1986. Control of cytoplasmic calcium with photo-labile tetracarboxylate 2-nitrobenzhydryl chelators. Biophysical Journal. 50:843–853.

Precision Titrations to Determine Affinity and Stoichiometry of Alkali, Alkaline Earth, and Buffer Cation Binding to Na,K-ATPase

E. Grell, R. Warmuth, E. Lewitzki, and H. Ruf

Max-Planck-Institute for Biophysics, Frankfurt, Germany

Introduction

Na,K-ATPase actively transports Na+ and K+ across many biological membranes. Besides the selective binding and occlusion of these alkali ions as well as of Mg2+, it is assumed that several other ions, such as buffer cations and amines (Skou and Esmann, 1981; Robinson and Davies, 1987; Schuurmans Stekhoven et al., 1988), are also bound to this enzyme. The binding of these additional cations may shift the equilibrium between the main conformational states E_1 and E_2, and may also affect certain enzymatic reaction steps. To investigate the interaction of Na,K-ATPase with cations, binding studies were performed on its fluorescent FITC derivative, initially introduced and investigated by Karlish (1979), Hegyvary and Jørgensen (1981), Post et al. (1987), and Mezele et al. (1988). Fluorescence emission intensity of the covalently bound FITC group was used to differentiate between E_1 and E_2: the high intensity E_1 state (found in the presence of Na+) and the low intensity E_2 state (observed with K+). Apart from such assignments, the results of the binding studies provide information about the energetic stability of the cation complexes formed in solution and the corresponding stoichiometries. Except for T1+ (Jensen and Norby, 1989), up to now only equilibrium constants related to 1:1 stoichiometries have been reported for alkali ion binding.

Experimental Methods

Membrane-bound Na,K-ATPase was prepared and purified from pig kidney as specified in Mezele et al. (1988). Fluorescein isothiocyanate (isomer I) coupling was performed according to a modified procedure of Karlish (1979). Fluorescence measurements at 25°C under conditions of minimum filter effects caused by light scattering were carried out using a specially designed titration system (Mezele et al., 1988).

Results and Discussion

Fig. 1 shows some selected spectrofluorometric titrations with mono- and divalent cations in two different buffer systems. In 10 mM histidine-HCl, pH 7.5, surprising results are observed. Similar to the Na+ titration, the relative FITC fluorescence intensity increases with increasing concentrations of many different monovalent cations, as shown for the protonated form of imidazole. This fluorescence intensity change, caused by cation binding, is attributed to the E_2 → E_1transition. Based on an evaluation related to a 1:1 complex formation model, a mean log K value around 2.3 is found for all monovalent cations given in Fig. 2. Thus, the log K values are essentially independent of the size and nature of the cation, which is indicative of unselective, electrostatic binding to a negatively charged site of the enzyme. According to this interpretation, it is easy to understand why the conformational properties of the enzyme can, for example, depend on buffer concentration. Na+ binding, however, is much better described by a 2:1 stoichiometry (two binding sites per enzyme unit).

Figure 1. Spectrofluorometric titrations of different preparations of FITC Na,K-ATPase (~0.17 µM) with cations in 10 mM histidine-HCl, pH 7.5 (ImH+, Na+, Mg2+, EnH_22+), and 25 mM imidazole-HCl, pH 7.5, containing 0.1 M (K+) resp. 80 mM choline chloride (NH_4+): plots of relative fluorescence (R.F.) emission intensity versus logarithm of cation (resp. total amine) concentration. In the case of evaluations including 2:1 stoichiometry (solid line), the broken line represents the best fit concerning 1:1 stoichiometry. Affinity is expressed as stability constant K; for abbreviations, see Fig. 2.

Fig. 1 also shows a titration with Mg2+ and one with an organic divalent cation (protonated ethylene diamine, EnH_22+) in the same buffer system. As referred to above, the typical E_2 → E_1fluorescence change is observed for both divalent cations. Similar results are obtained for numerous other divalent cations. Data evaluation, based again on 1:1 stoichiometry, yields log K values around 4.4, which are remarkably similar for all divalent cations (Fig. 2). However, this value is significantly larger than that found for monovalent cations, which is also consistent with electrostatic binding.

Selective cation binding is found for the larger alkali ions (e.g., K+) and for NH_4+ in 25 mM imidazole-HCl, pH 7.5, containing choline chloride (Fig. 1). Here, clear evidence for 2:1 stoichiometry is found. The fact that both log K values are very similar in the case of the K+ titrations suggests cooperative binding of this cation. The fluorescence change found here is typical for a conformational equilibrium shift from E_1 to E_2.

Figure 2. Log K values related to 1:1 stoichiometry for complexes between FITC Na,K-ATPase and different cations (resp. protonated amines) in 10 mM histidine-HCl, pH 7.5, with chloride as the related anion. Im, imidazole; Ch, choline; Gua+, guanidinium; TMA+, tetramethylammonium; NMG, N-methyl-D-glucamine; En, ethylene diamine; Pn, 1,3-propane diamine.

According to Fuoss (1958), the stability constant for electrostatic ion or ion pair binding based on 1:1 stoichiometry can be readily calculated. Assuming a binding distance of 0.45 nm and a dielectric constant of 41, a log K value of 2.0 results for the interaction of a monovalent cation with a divalent anion, and a value of 4.6 results for the binding between a divalent cation and a divalent anion. From the good correspondence of the calculated values with the experimentally determined ones (in the histidine buffer and assuming 1:1 stoichiometry), it is concluded that the electrostatic model is a realistic one and that the binding site of the enzyme is doubly negatively charged. Electrostatic binding of cations, and consequently the subsequent conformational rearrangement, is assumed to be sensitive to the electric field, which is one conclusion from this study.

The type of unselective binding discussed here is illustrated in the molecular model shown in Fig. 3. Two closely situated carboxylate side chain groups are thought to act as the site of interaction. Binding, strongest in case of protonated ethylene diamine, leads to charge neutralization and hence may create a distance decrease between the carboxylate groups, thus inducing the subsequent rearrangement from E_2 to E_1. No differentiation between amines which exhibit a stimulating or inhibiting effect as concluded from phosphorylation studies by Schuurmans Stekhoven et al. (1988) was observed here.

Figure 3. Molecular model of unselective, electrostatic cation binding to a doubly negatively charged site of Na,K-ATPase inducing the subsequent conformational main transition from E_2 to E_1.

Summary

Unselective, electrostatic binding of numerous mono- and divalent cations including buffer ions to a probable doubly negatively charged site within the enzyme induces the E_2 → E_1 conformational transition. Evidence for selective binding exhibiting 2:1 stoichiometry has been found for the larger alkali ions and for NH_4+ in a buffer medium, preferentially populating E_1.

Acknowledgments

The authors are grateful to Mrs. A. Ifftner for expert technical assistance and to the Germdan Research Foundation (SFB 169) for support.

References

Fuoss, R. M. 1958. The equilibrium between ion pairs and free ions. Journal of the American Chemical Society. 80:5059–5061.

Hegyvary, C., and P. L. Jørgensen. 1981. Conformational changes of renal sodium plus potassium iontransport adenosine triphosphatase labeled with fluorescein. The Journal of Biological Chemistry. 256:6296–6303.

Jensen, J., and J. G. Nørby. 1989. Thallium binding to native and radiation-inactivated Na+/K+-ATPase. Biochimica et Biophysica Acta. 985:248–254.

Karlish, S. J. D. 1979. Cation induced conformational states of Na,K-ATPase studied with fluorescent probes. In Na,K-ATPase Structure and Kinetics. J. C. Skou and J. G. Norby, editors. Academic Press, New York and London. 115–128.

Mezele, M., E. Lewitzki, H. Ruf, and E. Grell. 1988. Cation selectivity of membrane proteins. Berichte der Bunsen-Gesellschaft für Physikalische Chemie. 92:998–1004.

Post, R. L., S. M. Seiler, and P. M. Vasallo. 1987. Action of hydrogen ions on sodium, potassium adenosine triphosphatase labelled with fluorescein. In Perspectives of Biological Energy Transduction. Mukohata, Morales, and Fleischer, editors. Academic Press Japan, Tokyo. 273–283.

Robinson, J. D., and R. L. Davies. 1987. Buffer, pH, and ionic strength effects on the (Na+ + K+)-ATPase. Biochimica et Biophysica Acta. 912:343–347.

Schuurmans Stekhoven, F. M. A. H., H. G. P. Swarts, G. K. ’t Lam, Y. S. Zou, and J. J. H. H. M. De Pont. 1988. Phosphorylation of (Na+ + K+)-ATPase: stimulation and inhibition by substituted and unsubstituted amines. Biochimica et Biophysica Acta. 937:161–176.

Skou, J. C., and M. Esmann. 1981. Eosin, a fluorescent probe of ATP binding to the (Na+ + K+)-ATPase. Biochimica et Biophysica Acta. 647:232–240.

Ca-dependent Inhibition of the Na,K-ATPase: Estimation of the Molecular Weight of Calnaktin

Jing Ye-Hu, Virginia Barrett, Guangsheng Wang, and Douglas R. Yingst

Department of Physiology, Wayne State School of Medicine, Detroit, Michigan 48201

Introduction

Hemolysate and membrane extracts of human red cells contain a heat- and trypsin-sensitive factor that increases the sensitivity of the Na,K-ATPase to inhibition by Ca (Yingst and Marcovitz, 1983), suggesting a protein (calnaktin) that increases the sensitivity of the Na,K-ATPase to inhibition by Ca (see Yingst, 1988, for review). To date calnaktin has not been conclusively identified.

The present study had two objectives. The first was to determine the apparent molecular weight (Mr) of active samples of calnaktin from hemolysate and membrane extract by means of size-exclusion chromatography. The second was to determine if active fractions from the size-exclusion column contain a unique protein that correlates with the observed activity as visualized by means of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).

Materials and Methods

Hemolysate was made from packed human red blood cells in P20 phosphate buffer using a modification of the Dodge procedure (Dodge et al., 1963) as previously described (Yingst and Marcovitz, 1983), except that the hemolyzing solution also contained 0.1 mM phenylmethylsulfonyl fluoride. Membranes were separated from the soluble proteins by filtration using a Pellicon apparatus (HVLP cassette, 0.45 µM; Millipore Corp., Bedford, MA). Membrane extract was made by the method of Mauldin and Roufogalis (1980). Hemolysate (in P20) and membrane extract (in 1 mM Tris and 0.1 mM ethylenediamine tetraacetic acid [EDTA], pH 8.0) were first purified by anion exchange and Ca-dependent hydrophobic-interaction chromatography (Ca-dep HIC) using batch procedures. These samples were then concentrated and washed by ultrafiltration, and applied to a size-exclusion column. The anion exchange step was carried out by adding diethylaminoethyl (DEAE) cellulose (Sigma Chemical Co., St. Louis, MO) to the membrane-free solutions of hemolysate and membrane extract. These solutions were stirred for 60 min at room temperature and the DEAE was collected on a Buchner funnel. The DEAE from the hemolysate was washed with P20; the DEAE from the membrane extract was washed with 1 mM Tris, 0.1 mM EDTA, pH 8.0. The DEAE from each was then washed with 200 mM NaCl and 5 mM HEPES, pH 7.4, and the sample was eluted with 500 mM NaCl and 5 mM Hepes, pH 7.4. Ca-dep HIC was conducted by adding phenylsepharose (PS) (Sigma Chemical Co.) and Ca to a final concentration of 1 mM to the DEAE-eluted samples. The resulting solutions were stirred for 60 min at room temperature. The PS was collected on a filter, washed first with 500 mM NaCl, 5 mM HEPES, and 1 mM Ca at pH 7.4 and then with 5 mM HEPES and 1 mM Ca, pH 7.4. The sample was eluted from the PS with 5 mM HEPES and 1 mM EGTA, pH 7.4, and then concentrated and washed on a PM10 filter (Amicon Corp., Danvers, MA), with 5 mM HEPES and 0.1 mM EGTA, pH 7.4.

TABLE I
Membrane Extract and Hemolysate Purified by Anion Exchange and Ca-dep HIC

*A unit of activity is defined as a 10% increase in the percent inhibition of the Na,K-ATPase at 2 µM free Ca/µg of protein under the standard assay conditions described in Materials and Methods.
‡The data are for a starting volume of 140 ml packed cells.

Figure 1. Absorbance at 280 nm as a function of time on a size-exclusion column (Bio-Sil TSK-125; Bio-Rad Laboratories, Richmond, CA; run on a Waters HPLC system [Millipore Corp., Bedford, MA]) loaded with 46 µg of membrane extract, previously purified to the stage shown in Table I by anion exchange, Ca-dep HIC, and ultrafiltration. The column was equilibrated and run in 100 mM NH_4HCO_3 and 0.1 mM EGTA, pH 6.8. The arrow shows the time that samples containing calnaktin, as shown by the assay in Fig. 3, were eluted. (Inset) Calibration curve of Mr standards run under the same conditions as the membrane extract. The arrow shows the calculated Mr of the active fractions.

Figure 2. Absorbance at 280 nm as a function of time on a size-exclusion column loaded with 15 mg of hemolysate previously purified to the stage shown in Table I by anion exchange, Ca-dep HIC, and ultrafiltration. The column conditions were similar to those described for the membrane extract in the legend to Fig. 1. Fractions that eluted between 7.5 and 10 min were pooled and then concentrated by ultrafiltration on a PM10 membrane. From this pooled sample 30% (1.8 mg) was reapplied to the same column and rerun as before. Samples from this second column were assayed as shown in Fig. 4. The arrow shows the time that the active fractions containing calnaktin were eluted from the column. (Inset) Calibration curve of the Mr standards run under the same conditions as the hemolysate. The arrow shows the calculated Mr of the active fractions.

Na,K-ATPase assays were carried out using human red cell membranes as previously described (Yingst and Marcovitz, 1983). The assay solution contained 18 mM NaCl, 30 mM KCl, 112 mM choline chloride, 20 mM HEPES, 0.2% bovine serum albumin, 1 mM ATP, and either 5.5 mM MgCl_2 (zero free Ca) or 1.84 mM CaCl_2 and 3.68 mM MgCl_2 (2 µM free Ca). The Na,K-ATPase is equal to the difference in activity with and without 0.5 mM ouabain. The percent inhibition represents the inhibition of the Na,K-ATPase at 2 µM free Ca compared with the activity in the absence of Ca. SDS-PAGE was run by the method of Laemmli (1970). Gels were stained with silver (Eschenbruch and Bürk, 1982).

Figure 3. Effect of fractions off the size-exclusion column shown in Fig. 1 on Ca-dependent inhibition of the Na,K-ATPase. Equal volumes of each fraction were added to the assay. Fractions were labeled according to the time at which they were eluted from the column. The vertical bars are standard deviations. The value labeled C is the control representing the inhibition at 2 µM free Ca in the absence of added fractions.

Results and Discussion

Anion exchange, Ca-dependent hydrophobic interaction chromatography (Ca-dep HIC) and ultrafiltration were used to partially purify calnaktin from membrane extract and hemolysate from human red blood cells (Table I). Size-exclusion chromatography was then used to further purify membrane extract (Fig. 1) and hemolysate (Fig. 2). Fractions from these columns were tested for their effect on Ca-induced inhibition of the Na,K-ATPase (Figs. 3 and 4). Active fractions of both extract and hemolysate gel filtered at an apparent Mr of 43,000–45,000 were compared to proteins of known Mr (Figs. 1 and 2, insets).

Active fractions of hemolysate that gel filtered at 45,000 Mr contained small amounts of a light yellow–staining protein of 12,000 MI, which appears white in Fig. 5 (see arrow). Inactive fractions did not contain this protein. A very similar band was visible in active fractions from columns loaded with membrane extract (data not shown). In general the activity recovered from the size-exclusion columns was 10–20% of the applied amount. The specific activity of the active fractions was increased 3–10-fold over the levels given in Table I. In these experiments the rate of Na,K-ATPase activity at zero free Ca ranged from 6 to 9 nmol P_1/mg membrane protein per min.

Figure 4. Effect of hemolysate fractions from the size-exclusion column described in Fig. 2 on Ca-dependent inhibition of the Na,K-ATPase. Equal volumes of each fraction were added to the assay. Fractions were labeled according to the time at which they were eluted from the column. The vertical bars are standard deviations. The value labeled C is the control as defined in the legend to Fig. 3.

The above data show that the two objectives of this study were met. First, the Mr of calnaktin was determined by size-exclusion chromatography. Second, a unique protein that correlated with calnaktin activity was identified. The results indicate that calnaktin activity is associated with a 12,000 Mr protein which can form higher order complexes.

Figure 5. SDS-PAGE of hemolysate fractions purified by size-exclusion chromatography as described in Fig. 2. The light-colored protein at the arrow had a Mr of 12,000, compared with the Mr standards (see below). Equal volumes of each fraction were lyophilized and loaded onto the gel. In this experiment 50% of each fraction was put on the gel. The amount of protein loaded on the gel was 10 times that added to each of the assay tubes to determine Na,K-ATPase activity. Fractions were labeled according to the time (minutes) at which they were eluted from the column in Fig. 2. 4 µg of protein (0.63 µg/band) was applied as Mr markers in the first lane (S). The proteins used as Mr standards were: (a) phosphorylase B (92,500), (b) bovine serum albumin (66,200), (c) ovalbumin (45,000), (d) carbonic anhydrase (31,000), (e) soybean trypsin inhibitor (21,500), and (f) lysozyme (14,400).

Acknowledgments

We thank Dr. Michael Buck for running the gel filtration columns.

This work was supported by the NIH (RCDA AM-01253), the Cystic Fibrosis Foundation, and the American Heart Association of Michigan.

References

Dodge, J. T., C. Mitchell, and D. T. Hanahan. 1963. The preparation and chemical characteristics of hemoglobin-free ghosts of human erythrocytes. Archives of Biochemistry and Biophysics. 100:119–150.

Eschenbruch, M., and R. R. Bürk. 1982. Experimentally improved reliability of ultrasensitive silver staining of proteins in polyacrylamide gels. Analytical Biochemistry. 125:96–99.

Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 227:680–685.

Mauldin, D., and B. D. Roufogalis. 1980. A protein activator of Mg2+-dependent, Ca2+-stimulated ATPase in human erythrocyte membranes distinct from calmodulin. Biochemical Journal. 187:507–513.

Yingst, D. R. 1988. Modulation of the Na,K-ATPase by Ca and intracellular proteins. Annual Review of Physiology. 50:291–303.

Yingst, D. R., and M. Marcovitz. 1983. Effect of hemolysate on calcium inhibition of the (Na+ + K+)-ATPase of human red blood cells. Biochemical Biophysical Research Communications. 111:970–979.

Na/K-Pump Activity and Cell Volume Changes during Maturation of Rat Reticulocytes

Heimo Mairbäurl and Joseph F. Hoffman

Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06510

Introduction

Stimulation of erythropoiesis by application of the hormone erythropoietin (EPO) became possible after the introduction of recombinant human EPO for the treatment of renal anemia (Winearls et al., 1986; Eschbach et al., 1987). This technique thus represents a tool for obtaining premature red cells without having to stress animals by treatment, for instance with phenylhydrazine, thiamphenicol, or repeated bleeding (for review see Rapoport, 1986).

The purpose of this work was to evaluate changes in cell volume, composition, and cation transport during the course of maturation of reticulocytes and aging of mature cells. This paper describes a technique suitable for stimulating reticulocyte production in rats by injection of EPO, isolation of these cells by density gradient centrifugation, and subsequent characterization with regard to various parameters of interest.

Methods

Male Sprague-Dawley rats (average weight 450 g) were injected intraperitoneally with a single dose of 150 U/kg of recombinant human EPO (Epogen®; Amgen Inc., Thousand Oaks, CA). Control rats were injected with saline. 3 d after treatment, when in EPO-treated animals the peak of the reticulocytosis occurs (Siri et al., 1966), the rats were anesthetized and then exsanguinated by cardiac puncture. Whole blood (5 ml) was layered on density gradients (25 ml) prepared from Percoll (Pharmacia, Piscataway, NJ) by adjusting the density (1.095 g/ml) and the osmolarity (285 mosmol/liter) with a balanced salt solution (Mairbäurl et al., 1983). The gradients were centrifuged at 20,000 g, 10°C for 30 min (Sorval RC-5 centrifuge, SS-34 rotor; DuPont, Wilmington, DE). Four fractions (F1 = top, F2, F3, F4 = bottom) were obtained by sampling from the top of the gradient after removal of supernatant plasma and buffy coat. The cells from all four fractions were filtered through a mixture of alpha-cellulose and micro-crystalline cellulose (to remove contaminant leukocytes), and then washed three times with saline.

In vitro maturation of reticulocytes was carried out by washing and incubating cells from the top fraction F1 at a hematocrit (Hct) of ~ 0.3% with culture medium. The medium (RPMI 1640; Gibco BRL, Grand Island, NY) was substituted with 5% fetal calf serum, 2 mM glutamine, 10 mM HEPES, pH 7.3, and antibiotics). Incubation was carried out in a shaking water bath. During the incubation, which lasted several days, the samples were gassed with humidified 5% CO_2 in oxygen after passage through a 0.22-µm filter. All equipment used was autoclaved.

Hct was determined after microcentrifugation and hemoglobin was measured after conversion to cyanmethemoglobin. Reticulocytes were counted on air-dried smears of cells after staining with new methylene blue (Sigma Chemical Co., St. Louis, MO). Intracellular Nai and Ki were determined on cells packed (Hct > 97%) in special narrow bore lucite tubes and analyzed by flame photometry. The ratio between Cli and Cl_0 (rcl) was determined after incubating the cells with 36Cl.

Na efflux was measured after altering Nai and Ki (each to usually ~ 50 mmol/liter cells) at the same time as loading the cells with trace amounts of 22Na by means of the nystatin technique. The flux medium was composed of (in mM): 140 choline Cl, 2.5 KCl, 9 glucose, and 20 HEPES-Tris, pH 7.4, at 37°C. The Hct was ~0.5%. Fluxes were measured between zero and 5 min (in the presence of 5 mM ouabain) or 90 s when no ouabain was present. Diethyl-phthalate was used to separate cells and medium during microcentrifugation. Radioactivity was measured by gamma counting. The linearity of the fluxes over the indicated periods of time was tested in separate experiments.

Results and Discussion

In contrast to treatment with phenylhydrazine or thiamphenicol, which both increase the reticulocyte count in blood to > 90% over a period of several days, the application of a single dose of EPO caused an increase in the percentage of reticulocytes to about four to five times the normal value in our experiments. However, by subsequent centrifugation on density gradients reticulocytes could be enriched to a purity > 95% (i.e., the F1 fraction). It should be noted that the reticulocytes obtained after EPO treatment were lighter than the red cells with the lowest buoyant density of control rats. Furthermore, the density of RNA precipitated by new methylene blue was higher, indicating that this group of cells represents reticulocytes that are on average younger than normal (Rapoport, 1986).

Reticulocyte volume, indicated by the decrease in the mean cellular hemoglobin concentration (MCHC) was found to be ~30% larger than that of the whole or unfractionated cell population. Also, Nai content was lower and Ki was elevated. Similar results were observed in pig reticulocytes (Kim et al., 1976), but are in contrast to the results of Furukawa et al. (1981), who found Nai and Ki in rat reticulocytes, obtained after phenylhydrazine treatment, to be essentially the same as those in mature erythrocytes. Our results show that, as MCHC increases in red cells of increased buoyant density, Nai increased and Ki decreased to values similar to those found in unfractionated red cells. A slightly higher value of rCl was found in F4 cells than in reticulocytes (F1).

When reticulocyte maturation was followed in vitro, changes similar to those observed during in vivo maturation (assessed by density gradient centrifugation) were observed: MCHC and Nai increased, whereas Ki decreased as the percentage of reticulocytes decreased during the course of the 3-d incubation to reach values similar to those found in unfractionated red cells. A similar time course for the maturation of rat reticulocytes was seen by Noble et al. (1989) after induction of reticulocytosis using thiamphenicol. These results, taken together, indicate that changes in volume and ion content of red cells of different densities are taking place during the maturation of reticulocytes.

The sensitivity of the Na/K-pump to inhibition by ouabain appears to be similar in reticulocytes and mature red cells. The Na/K-pump, with 2.5 mM Ko, was essentially completely inhibited by 5 mM ouabain since no further inhibition was found after addition of scillaren. Thus all experiments on the Na/K-pump were carried out in the presence or absence of 5 mM ouabain and 2.5 mM Ko.

At Vmax for Nai (Nai > 30 mmol/liter cells when Nai was varied reciprocally with Ki) the Na/K-pump activity in reticulocytes was about three to five times higher than that of mature or unfractionated red cells. Similar observations were made on the Na,K-ATPase in red cell membranes prepared from high K and low K sheep (Blostein et al., 1983), rat (Furukawa et al., 1981), pig (Kim et al., 1976), and human (Mairbäurl and Hoffman, 1989). There was no difference in pump fluxes between unfractionated red cells and cells from fraction F4, which represent the oldest cells in the population (Chalfin, 1956). When reticulocytes were kept in culture for several days, the pump flux at Vmax decreased in proportion to the disappearance of reticulocytes, reaching values of unfractionated red cells on day 2 in culture. Since the pump flux at Vmax is a measure of the number of active pumps or copies of the Na/K-pump in the red cell membrane, it can be concluded from these results that the decrease in pumps during red cell aging is almost fully accounted for by the reduction in the number of active pumps during the maturation of reticulocytes. It is not clear whether the decrease in active pumps is caused by the loss of membrane protein or by inactivation. An energy-dependent step appears to be involved in the loss of pumps, since ATP depletion during the culturing of reticulocytes lowers the rate of pump loss in sheep reticulocytes (Blostein et al., 1974). Evidently no further decrease in the number of pump sites appears to occur during the aging of mature red cells.

In fractionated red cells the ouabain-insensitive Na efflux was highest in reticulocytes and decreased with increasing cell density and age. Similar results were reported in human red cells from patients with reticulocytosis (Wiley, 1981). A portion of the ouabain-insensitive Na efflux can be attributed to Na/K/2Cl cotransport, since this flux was inhibited by bumetanide. Na/K/2Cl cotransport at Vmax for Nai and Ki (but not for Mgi) appeared to be higher in red cells with high density (F4) than in reticulocytes (F1).

During reticulocyte maturation in culture, ouabain-insensitive Na efflux increased during the incubation and reached values similar to or exceeding the relative pump flux. It is not yet known whether this is due to an activation of a transport system or to an increase in passive diffusion.

Our results indicate that, at least in vitro, reticulocytes rapidly adjust their volume, ionic composition, and cation transport capacity to that characteristic of the whole cell population. The changes that subsequently occur as the mature population ages are slow in comparison (Suzuki and Dale, 1988; Zirman et al., 1988). It is not yet known what the transport basis is for the decrease in reticulocyte steady-state volume that takes place during maturation. Whether these changes are related to a loss in membrane material (Johnstone et al., 1989) or in the activity of certain glycolytic enzymes (e.g., Rapoport, 1986) also remains to be clarified.

References

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Furukawa, H., J. P. Bilezikian, and J. N. Loeb. 1981. Potassium fluxes in the rat reticulocyte: ouabain sensitivity and changes during maturation. Biochimica et Biophysica Acta. 649:625–632.

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Mairbäurl, H., and J. F. Hoffman. 1989. The number of Na/K/Cl-cotransport copies in the membrane determines its contribution to the regulation of steady-state red cell volume. FASEB Journal. 3:A579. (Abstr.)

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Suzuki, T., and G. L. Dale. 1988. Senescent erythrocytes: isolation of in vivo aged cells and their biochemical characteristics. Proceedings of the National Academy of Sciences, USA. 85:1647–1651.

Wiley, J. S. 1981. Increased erythrocyte cation permeability in thalassemia and conditions of marrow stress. Journal of Clinical Investigation. 67:917–922.

Winearls, C. G., D. O. Oliver, M. J. Pippard, C. Reid, M. R. Downing, and P. M. Cotes. 1986. Effect of human recombinant erythropoietin derived from recombinant DNA on the anemia of patients maintained by chronic haemodialysis. Lancet. ii: 1175–1178.

Zirman, A., S. Torem, and E. Beutler. 1988. The in vivo ageing of red cell enzymes: direct evidence of biphasic decay from polycythaemic rabbits with reticulocytosis. British Journal of Haematology. 69:67–70.

Is the Pump of Intact Red Blood Cells Saturated for ATP at Physiological Concentrations?

Marina Marjanovic and John S. Willis

Department of Physiology and Biophysics, University of Illinois, Urbana, Illinois 61801

Introduction

Reports of the affinity of the Na-K pump for ATP in intact cells are conflicting. Studies of Na-K ATPase in broken membranes (Dunham and Glynn, 1961; Sachs, 1988) show that the pump of human red blood cells should be saturated at concentrations between 1 and 2 mM, as confirmed in one study of ouabain-sensitive Na efflux in resealed ghosts of human red cells (Kennedy et al., 1986). However, with ATP enrichment in intact red blood cells and rabbit kidney cells, measures of pump activity appear to rise linearly with concentration to 3–4 mM (Brown and Lew, 1983; Soltoff and Mandel, 1984), and in perfused squid giant axon there was no saturation of ATP-dependent Na efflux up to 10 mM ATP (Brinley and Mullins, 1968).

A complication of such studies is the availability and role of Mg, both as part of the substrate and as modifier of pump activity. Using Na-K ATPase of broken membranes as our standard of comparison, we have sought to determine to what extent ATP and Mg2+ limit pump activity in intact red blood cells.

Methods

ATP concentration in freshly drawn red blood cells was lowered by incubation in 10 mM 2-deoxy-D-glucose and increased by incubation in 10 mM adenosine, 10 mM pyruvate, and 50 mM phosphate (Brown and Lew, 1983). Pump activity in intact cells was estimated as ouabain-sensitive K influx using 42K. Simultaneously, ATP concentrations were determined by luciferin-luciferase assay. When needed, free Mg2+ concentration in the cells was changed by incubating them in medium containing different concentrations of MgCl_2 in the presence of the ionophore, A23187 (Ellory et al., 1983). ATPase in broken membranes was determined by measuring 32Pi liberated from gamma-labeled ATP.

Results and Discussion

In our experiments, Na-K ATPase activity measured in broken membrane preparations of both human and guinea pig red blood cells followed a simple hyperbolic form at higher concentrations and showed saturation between 1.5 and 2 mM ATP. When we lowered ATP concentration with 2-deoxy-D-glucose, the dependence of ouabain-sensitive K influx on the concentration of ATP followed the lower part of a Michaelis-Menten curve for each species. At concentrations of ATP typical of fresh red cells of guinea pig (0.4–0.6 mM), ouabain-sensitive K influx did not show saturation; activity was only 60% of the estimated Vmax value. Although fresh human red cells have an ATP concentration of 1–1.2 mM, ouabain-sensitive K influx was only ~ 80% of estimated Vmax (Fig. 1).

Figure 1. Effect of ATP on the Na-K pump in intact red blood cells. Pump activity was measured as ouabain-sensitive K influx in both ATP-depleted (open circles, open triangles) and ATP-enriched (filled circles, filled triangles) intact red blood cells of guinea pig and human, with unaltered concentration of free Mg2+. The endogenous rates of untreated cells are marked by an asterisk.

When ATP concentration was elevated by the method of Brown and Lew (1983), pump activity increased by a maximum of 10% in human cells and 30% in guinea pig cells (Fig. 1). In both cases maximal values appeared at ~ 1.5 mM ATP. Further increase in ATP concentration above 1.5 mM resulted in lower pump rates, but usually not lower than the endogenous rates of untreated cells.

Stimulation of pump activity at concentrations above 1.5 mM ATP may be constrained by other factors, such as availability of free Mg2+. To check this possibility, we increased ionized Mg content in the cell by loading Mg2+ in the presence of A23187. This caused an increase in ouabain-sensitive K influx at every concentration of ATP tested and abolished the fall-off at high ATP concentrations. Stimulation of pump activity at high ATP concentrations was observed with all concentrations of MgCl_2 used for loading (1, 2, and 5 mM). Fig. 2 compares the results obtained with guinea pig red blood cells: ATPase activity measured in broken membranes and ouabain-sensitive K influx loaded with 2 mM Mg2+. It appears that even when ATP and Mg2+ are not limiting, pump activity reaches maximum levels between 1 and 2 mM.

Figure 2. Comparison of ouabain-sensitive K influx of Mg-loaded red cells with Na-K ATPase of broken membranes. Figure shows ATPase activity (open triangles) and ouabain-sensitive K influx in intact, ATP-depleted (open circles) and ATP-enriched (filled circles) red cells from guinea pig. Cells were loaded with 2 mM Mg2+. ATP/Mg2+ in broken membrane preparations was 1:1. The Michaelis-Menten curve (Km = 250 mM) is fitted only to ATPase values.

Conclusion

In red blood cells of guinea pig and human there is no disparity between the Na-K pump of intact cells and the Na-K ATPase of broken membranes in their dependence on higher concentrations of ATP.

However, because of the lower ATP concentration in fresh red blood cells of guinea pig, their pump is only ~ 70% saturated; in human fresh red blood cells the pump is ~ 90% saturated.

At higher ATP concentrations availability of Mg2+ is limiting.

References

Brinley, F. J., and L. J. Mullins. 1968. Sodium fluxes in internally dialyzed squid axons. Journal of General Physiology. 52:181–211.

Brown, A. M., and V. L. Lew. 1983. The effect of intracellular calcium on the sodium pump of human red cells. Journal of Physiology. 343:455–492.

Dunham, E. T., and I. M. Glynn. 1961. Adenosinetriphosphatase activity and the active movements of alkali ions. Journal of Physiology. 156:274–292.

Ellory, J. C., P. W. Flatman, and G. W. Stewart. 1983. Inhibition of human red cell sodium and potassium transport by divalent cations. Journal of Physiology. 340:1–17.

Kennedy, B. G., G. Lunn, and J. F. Hoffman. 1986. Effects of altering the ATP/ADP ratio on pump-mediated Na/K and Na/Na exchanges in resealed human red blood cell ghosts. Journal of General Physiology. 87:47–72.

Sachs, J. R. 1988. Interaction of magnesium with the sodium pump of the human red cell. Journal of Physiology. 400:575–591.

Soltoff, S. P., and L. J. Mandel. 1984. Active ion transport in the renal proximal tubule. Journal of General Physiology. 84:643–662.

Characterization of Purified Na,K-ATPase Isolated from Mink (Mustela vison) Kidney

Otto Hansen, Tove N. Clausen, and Søren Wamberg

Institute of Physiology, Aarhus University, DK-8000 Aarhus C, Denmark; Fur Research Farm West, Tvis, Holstebro, Denmark; and the Department of Physiology, Odense University, Odense M, Denmark

Introduction

Carnivores are usually able to concentrate their urine considerably. This feature is dependent on the structure of the medullary zone of the kidney. Its ability to create interstitial hyperosmolarity implies sufficient sodium pump capacity of this zone. Since the maximal urine osmolality in mink is ~ 3,000 mosmol/liter (Eriksson et al., 1984), mink kidney would seem a good source for purification of Na,K-ATPase with maximum capacity for ligands and agonists like ouabain. It is important to establish the maximum capacity per milligram protein of pure enzyme to determine whether the mode of action of the Na,K-ATPase is as an αβ-protomer or an α_2β_2-dimer.

In our study of a common metabolic disorder in nursing mink (nursing sickness) we noticed that severe electrolyte disturbances, hyperosmolarity, hyperaldosteronism, dehydration, and, in spite of these facts, an inability to concentrate urine, were prominent features of the disease (Clausen and Hansen, 1989; Clausen et al., 1990). For these reasons we thought it worthwhile to characterize the kidney Na,K-ATPase of healthy and sick mink.

Materials and Methods

The kidneys were removed from healthy mink and mink with nursing sickness immediately after sacrifice (overdose of sodium pentobarbital) and pelting. They were placed in ice-cold imidazole-sucrose-EDTA as prescribed by Jørgensen (1974, 1988). After removal of the capsule, the kidneys were cut in longitudinal sections. The slices were placed for 1 h in the isotonic buffer to make the red outer medulla more easily visible, after which time the zone was dissected out. Enzyme preparation by homogenization, preparation of crude membranes by differential centrifugation, SDS-activation in the presence of ATP and isopycnic zonal centrifugation were carried out as described (Jørgensen, 1974, 1988).

Part of the crude membrane fraction obtained by differential centrifugation was used for comparison of sodium pump capacity in healthy and sick animals. The SDS concentration necessary for optimum Na,K-ATPase activity was determined and the activated preparation was used in ouabain binding experiments. [3H]Ouabain was obtained from Amersham International (Amersham, UK) and purified by chromatography on Na,K-ATPase. Ouabain binding was determined at equilibrium as described elsewhere (Hansen, 1976).

SDS-polyacrylamide gel electrophoresis was performed with mini gel equipment and a 4–16% polyacrylamide gel. The gels were stained with Coomassie brilliant blue R-250 or transferred to Immobilon PVDF membranes in a semidry electroblotter. The blots were incubated with diluted culture supernatant from anti–α-subunit antibody secreting hybridoma clones. (Splenocytes were harvested from rats or mice immunized with pig or chicken kidney Na,K-ATPase, respectively.) Bound antibodies were detected with a horseradish peroxidase–conjugated anti–mouse or anti–rat IgG (Dakopatts, Copenhagen, Denmark or Zymed Lab., San Francisco, CA) and 3-amino-9-ethylcarbazole (Aldrich-Chemie, Steinheim, Germany) and H_2O_2 in acetate buffer.

Figure 1. (A) Activation of Na,K-ATPase activity of crude membrane fractions from mink and pig kidney at different SDS concentrations. After differential centrifugation of the homogenate, the crude membrane fraction (1.5 mg·ml-1) was incubated with SDS at the indicated concentrations for 30 min at 20°C in the presence of imidazole (pH 7.5, 20°C), EDTA, and Na_2ATP (Jørgensen, 1988). (B) Scatchard plots of ouabain binding isotherms with purified enzyme or membrane fractions from healthy and sick mink kidney. Binding took place with 0.015 mg protein·ml-1 (purified enzyme), 0.1 mg·ml-1 (healthy mink) or 0.097 mg·ml-1 (sick mink) in the presence of 3 mM Mg2+, 3 mM Pi, and 40 mM Tris-HCl (pH 7.25). Binding isotherms were determined at equilibrium as described (Hansen, 1976).

Results and Discussion

During the critical step of detergent treatment of the crude membrane fraction, extraneous protein is released from the membranes and latent Na,K-ATPase activity is unmasked, probably due to the opening of vesicles. In Fig. 1 A it is seen that unmasking of latent Na,K-ATPase activity in crude membranes from pig and mink kidney takes place at identical SDS concentrations. A three- to fourfold increase in specific activity is obtained in both cases at optimum concentration of SDS. Before zonal centrifugation, activation by an SDS concentration of 0.3 mg · ml-1 was selected at the indicated protein concentration.

The most active fractions of the mink kidney enzyme purified by zonal centrifugation were characterized with respect to ouabain binding affinity and capacity. An example is given in the upper part of Fig. 1 B, which is a Scatchard plot of binding isotherms at binding equilibrium. Only one ouabain binding component is seen. From the ordinate intercept and the slope of the straight line, respectively, the binding capacity is 3.0 nmol · (mg protein)-1 and the apparent dissociation constant is 1.0 nM (Hansen, 1984). The affinity for cardiac glycosides is thus very high, even though kidney enzymes usually are of the α_1-isozyme type (see below).

TABLE I
Hydrolytic Activity and Ouabain Binding Capacity of the Most Active Fractions from Two Productions of Purified Na,K-ATPase from Mink Kidney by Sucrose Gradient Centrifugation

The SDS-activated crude membrane fraction without further purification was used to see whether an adaptation took place in mink with nursing sickness, since this fraction, according to Jørgensen (1988), contains the major part of Na,K-ATPase of the homogate. The lower part of Fig. 1 B is a plot of ouabain binding isotherms to the activated crude membrane fraction isolated from kidney outer medulla of healthy mink and dams with nursing sickness (20 vs. 7 animals). It is seen that the apparent ouabain affinity is somewhat lower (Kd 1.7 nM) than in purified enzyme and the ouabain binding capacity per milligram protein is 18% higher in sick mink than in healthy ones. Sick dams thus seem to have a higher capacity for reabsorption of Na+, an observation that is consistent with the very high concentration of aldosterone measured in dams with nursing sickness (Clausen et al., 1990).

A number of observations on purified mink kidney enzyme are gathered in Table 1. The hydrolytic activity is high immediately after preparation of the enzyme but some 20% decrease in activity is seen after freezing. Still, since a careful activation procedure is used and since the enzyme, judging from SDS-gel electrophoresis (Fig. 2), is pure, the ouabain binding capacity seems remarkably low. In the absence of impurities and denatured protein, twice the observed ouabain binding capacity per milligram protein would be expected. This observation suggests the presence of inactive protein (e.g., the existence of a diprotomer containing one active and one inactive α-subunit).

Figure 2. SDS-polyacrylamide gel electrophoresis (A) and immunoblotting (B) of purified Na,K-ATPase from pig kidney and mink kidney. Lanes a, b, f, and g, 2.6 µg (mink); lanes c, h, and k, 1.6 µg (pig); lanes d, e, i, and j, 1.6 µg (mink). Electrophoretic blots on Immobilon membranes (B) were incubated with a 1:100 diluted solution of the culture supernatant from hybridoma clone 2F (immunogen chicken renal Na,K-ATPase).

Fig. 2 also contains immunoblots of mink kidney and, for comparison, pig kidney Na,K-ATPase. It is apparent that the mobility of the β-peptide from mink kidney is lower than that of pig kidney Na,K-ATPase. Immunostaining reveals that mink α-peptide is recognized by 2F-antibody from mice, which does not recognize pig kidney α-peptide. Canine renal Na,K-ATPase also reacts with 2F-antibody, and mink enzyme thus seems to be related to other carnivore α_1-types of the enzyme (personal communication by Dr. D. M. Fambrough, Johns Hopkins University, who kindly supplied us with the mouse hybridoma).

Acknowledgments

We would like to thank Mr. Gert Almind, Ms. Inga Edney, Ms. Lis Hygom, and Mr. Toke Nørby for technical assistance.

This work was supported by the Danish Biomembrane Research Centre and the Danish Agricultural and Veterinary Research Council grant 13–4338.

References

Clausen, T. N., and O. Hansen. 1989. Electrolytes in mink with nursing sickness. Dansk Veterinær Tidsskrift. 72:266–268.

Clausen, T. N., O. Hansen, and S. Wamberg. 1990. Electrolyte and acid/base changes in lactating female mink (Mustela vison) subject to nursing sickness. Scientifur. 14:117.

Eriksson, L., M. Valtonen, and J. Mäkelä. 1984. Water and electrolyte balance in male mink (Mustela vison) on varying dietary NaCl intake. Acta Physiologica Scandinavica Suppl. 537:59–64.

Hansen, O. 1976. Non-uniform populations of g-strophanthin binding sites of (Na+ + K+)-activated ATPase. Biochimica et Biophysica Acta. 433:383–392.

Hansen, O. 1984. Interaction of cardiac glycosides with (Na+ – K+)-activated ATPase: a biochemical link to digitalis-induced inotropy. Pharmacological Reviews. 36:143–163.

Jørgensen, P. L. 1974. Purification and characterization of (Na+ + K+)-ATPase. III. Purification from the outer medulla of mammalian kidney after selective removal of membrane components by sodium dodecyl sulphate. Biochimica et Biophysica Acta. 356:36–52.

Jørgensen, P. L. 1988. Purification of Na+,K+-ATPase: enzyme sources, preparative problems, and preparation from mammalian kidney. Methods in Enzymology. 156:29–43.

Immunocytochemical Evidence for Protein Kinase C–Induced Internalization of Sodium Pumps in Xenopus laevis Oocytes

Günther Schmalzing, Kristin Mädefessel, Winfried Haase, and Kathi Geering

Max-Planck-Institut für Biophysik, Frankfurt/Main, Germany; and Institut de Pharmacologie de l'Université de Lausanne, Lausanne, Switzerland

Introduction

Recent evidence suggests that the downregulation of the sodium pump activity of Xenopus oocytes during meiotic maturation is the result of an endocytotic removal of virtually all pump molecules from the plasma membrane (Schmalzing et al., 1990). Similar to progesterone-induced maturation, activators of protein kinase C like phorbol 12-myristate 13-acetate (PMA) and synthetic diacylglycerols like 1,2-dioctanoyl-sn-glycerol (diC_8) have been found to diminish the current generated by the sodium pump and the ouabain binding capacity of the cell surface (Vasilets et al., 1990, 1991). The parallel reduction of membrane capacitance suggested that protein kinase C–induced inhibition of sodium pump activity is also due to internalization of pump molecules. In this study we took advantage of an antibody that specifically recognizes functional αl-subunits of the sodium pump in Xenopus oocytes to provide direct immunocytochemical evidence for endocytosis as a mechanism of regulation of the sodium pump activity.

Results

To localize sodium pumps in oocytes, paraffin sections were incubated with antibody Aα_1N (Ackermann and Geering, 1990), which was raised against a fusion protein containing 98 amino acids of the sodium pump α1-subunit cloned recently from Xenopus laevis kidney cells (Verrey et al., 1989). Preliminary data, obtained by Western blotting, indicate that Aα_1N mainly detects the trypsin-resistant, functionally active α-subunit expressed at the plasma membrane (data not shown). Selective detection of functional sodium pumps is important since Xenopus oocytes contain a large amount of nonfunctional α-subunits that remain trypsin sensitive, presumably because they are in excess of β-subunits (Geering et al., 1989; Geering, 1991).

In control sections, Aα_1N intensely labels the plasma membrane of the vegetal hemisphere (Fig. 1 c). The less intense immunofluorescence of the plasma membrane of the animal hemisphere (Fig. 1 a) indicates that sodium pumps are more abundant at the vegetal than at the animal pole. Islets of immunofluorescence 1–2 µm underneath the cell surface of the vegetal hemisphere (Fig. 1 c) are supposed to represent functional intracellular sodium pumps disclosed previously by ouabain binding measurements in permeabilized cells (Schmalzing et al., 1989).

Figure 1. Localization of α1-subunits of sodium pumps in control and PMA-stimulated oocytes by immunofluorescence. Oocytes were incubated in the absence or presence of 50 nM PMA at 20°C. After 30 min the cells were fixed with Carnoy's solution, dehydrated, and embedded in paraffin at 46°C. Sections cut at 4 µm were dewaxed, rehydrated, and incubated with polyclonal antibody Aα_1N (IgG fraction) directed against the NH_2-terminus of the α1 isoform of the Xenopus sodium pump, followed by biotinylated goat anti–rabbit F(ab)˝ and rhodamine-conjugated streptavidin (Dianova, Hamburg, Germany). (a and b) Animal pole; (c and d) vegetal pole. Left panels, control sections; right panels, sections of PMA-treated oocytes. Upper and lower photographs, same fields as in the adjacent fluorescence images, but photographed with Nomarski optics. Arrows, location of the plasma membrane.

In sections from cells exposed to PMA, labeling of the plasma membrane is greatly reduced at both poles (Fig. 1, b and d). There is a parallel increase of intracellular immunofluorescence, especially at the vegetal pole (Fig. 1 d), indicating that surface sodium pumps were translocated to the cell interior. In addition, PMA treatment changes the intracellular distribution of internal sodium pumps, which are located deeper inside the cells. At the animal pole, dots of immunofluorescence that replace the labeling of the plasma membrane are located underneath the cell surface (compare Fig. 1 a and 1 b). Parallel ouabain binding experiments indicated that 30 min of exposure to 50 nM PMA led to a 70% decrease of the ouabain binding capacity of the outer cell surface. Similar changes in the distribution of immunofluorescence were observed in sections of oocytes exposed to 10–30 µM diC_8 for 60 min (results not shown).

Figure 2. Phorbol myristate acetate stimulates endocytosis as demonstrated by incorporation of Lucifer yellow. Oocytes were incubated in the presence of 0.5 mg/ml of Lucifer yellow CH with (left) and without (right) 50 nM PMA. After 30 min the cells were fixed with formaldehyde and embedded in Spurr's resin according to Bernardini et al. (1987). Sections cut at 1 µm were examined with a Zeiss epifluorescence microscope. Lower panels, same fields as in the adjacent fluorescence images, but photographed with Nomarski optics.

To visualize endocytosis of fluid phase which may accompany internalization of sodium pumps, oocytes were stimulated with PMA in the prescence of Lucifer yellow, a fluid phase marker (Swanson et al., 1985). After fixation and embedding of the cells, Lucifer yellow was detected in sections by its intense fluorescence. PMA-treated oocytes show numerous macropinosomes ~ 5 µm underneath the cell surface (Fig. 2 a). In sections of control oocytes fluorescent vesicles are virtually absent (Fig. 2 b).

A reduction of the cell surface area could directly be demonstrated by electron microscopy, as shown in Fig. 3. The left panel shows a cross-section through the cortical region of a control oocyte, the surface of which is enlarged by numerous microvilli. By contrast, oocytes exposed for 30 min to 50 nM PMA (middle) or for 60 min to 10 µM diC_8 (right) have a smooth plasma membrane. This smoothing is apparently due to a complete disappearance of the microvilli.

Discussion

Our findings provide direct evidence that activation of protein kinase C results in a translocation of sodium pumps from the plasma membran to intracellular membranes (Fig. 1). Stimulation of endocytosis by PMA (Fig. 2) has been observed previously in macrophages, yet without loss of cell surface area (Swanson et al., 1985, cf. Fig. 3). Decreases in the area of the plasma membrane (Kado et al., 1981) and activity of surface sodium pumps (Richter et al., 1984) occur also during progesterone-induced maturation of Xenopus oocytes. Moreover, a protein kinase C–dependent pathway has been implicated to be involved in the progesterone effect on meiosis (Varnold and Smith, 1990). Despite some similarities, however, there are differences in the details of the downregulation of the sodium pump. During meiotic maturation, the capacity of the plasma membrane to bind ouabain is completely abolished (Richter et al., 1984), suggesting that a specific sorting event precedes endocytosis such that all pump molecules can be internalized. By contrast, PMA induces an incomplete downregulation of surface sodium pumps only, the basis of which appears to be essentially the reduction of the cell surface area (Vasilets et al., 1990). Nonspecificity of the PMA effect is supported by the finding that internalized sodium pumps could not be recovered in an endosome fraction of low buoyant density by sucrose gradient centrifugation and ouabain binding or immunoblotting (results not shown), in contrast to the situation after meiotic maturation (Schmalzing et al., 1990; K. Geering, unpublished results).

Figure 3. Loss of cell surface area induced by activators of protein kinase C. Oocytes were treated with 0.1% ethanol (controls, left), 50 nM PMA (middle), or 10 µM diC_8. After 30–60 min cells were fixed with glutaraldehyde and processed for electron microscopy as described (Vasilets et al., 1990).

Alterations of cell shape induced by phorbol ester are microtubule dependent (Lyass et al., 1988) and certain components of the cytoskeleton are known to serve as direct substrates for protein kinase C (Inagaki et al., 1987). In addition, the sodium pump itself has been shown to be tightly associated with cytoskeletal elements such as ankyrin, fodrin, and cadherin (Nelson et al., 1991). Therefore, the cytoskeleton might be the primary target of action of protein kinase C described above. It should be mentioned, however, that of a variety of cytoskeletal modifiers tested, none was capable of blocking the PMA effect (results not shown).

Acknowledgments

We thank Silke Kröner for carrying out the immunofluorescence staining.

This work was supported by grants from the Deutsche Forschungsgemeinschaft (Sonderforschungsbereich 169) to G. Schmalzing and the Swiss National Fund for Scientific Research (31–26241.89) to K. Geering.

References

Ackermann, U., and K. Geering. 1990. Mutual dependence of Na,K-ATPase α- and β-subunits for correct posttranslational processing and intracellular transport. FEBS Letters. 269:105–108.

Bernardini, G., M. Ferraguti, and R. Stipani. 1987. Fertilization induces endocytosis in Xenopus eggs. Cell Differentiation. 21:255–260.

Geering, K. 1991. Posttranslational modifications and intracellular transport of sodium pumps: importance of subunit assembly. In The Sodium Pump: Structure, Mechanism, and Regulation. J. H. Kaplan and P. De Weer, editors. The Rockefeller University Press, New York. 31–43.

Geering, K., I. Theulaz, F. Verrey, M. T. Häuptle, and B. C. Rossier. 1989. A role for the β subunit in the expression of functional Na+-K+-ATPase in Xenopus oocytes. American Journal of Physiology. 257:C851–C858.

Inagaki, M., Y. Nishi, K. Nishizawa, M. Matsuyama, and C. Sato. 1987. Site-specific phosphorylation induces disassembly of vimentin filaments in vitro. Nature. 328:649–652.

Kado, R. T., K. Marcher, and R. Ozon. 1981. Electrical membrane properties of the Xenopus laevis oocyte during progesterone-induced meiotic maturation. Developmental Biology. 84:471–476.

Lyass, L. A., A. D. Bershadsky, J. M. Vasiliev, and I. M. Gelfand. 1988. Microtubule-dependent effect of phorbol ester on the contractility of cytoskeleton of cultured fibroblasts. Proceedings of the National Academy of Sciences, USA. 85:9538–9541.

Nelson, W. J., R. W. Hammerton, and H. McNeill. 1991. Role of the membrane-cytoskeleton in the spatial organization of the Na,K-ATPase in polarized epithelial cells. In The Sodium Pump: Structure, Mechanism, and Regulation. J. H. Kaplan and P. De Weer, editors. The Rockefeller University Press, New York. 77–87.

Richter, H.-P., D. Jung, and H. Passow. 1984. Regulatory changes of membrane transport and ouabain binding during progesterone-induced maturation of Xenopus oocytes. Journal of Membrane Biology. 79:203–210.

Schmalzing, G., P. Eckard, S. Kröner, and H. Passow. 1990. Downregulation of surface sodium pumps by endocytosis during meiotic maturation of Xenopus laevis oocytes. American Journal of Physiology. 258:C179–C184.

Schmalzing, G., S. Kröner, and H. Passow. 1989. Evidence for intracellular sodium pumps in permeabilized Xenopus laevis oocytes. Biochemical Journal. 260:395–399.

Swanson, J. A., B. D. Yirinec, and S. C. Silverstein. 1985. Phorbol esters and horeseradish peroxidase stimulate pinocytosis and redirect the flow of pinocytosed fluid in macrophages. Journal of Cell Biology. 100:851–859.

Varnold, R. L., and L. D. Smith. 1990. Protein kinase C and progesterone-induced maturation in Xenopus oocytes. Development. 109:597–604.

Vasilets, L. A., K. Mädefessel, G. Schmalzing, and W. Schwarz. 1991. Inhibition of the Na+/K+ pump in Xenopus oocytes by diacylglycerol analogues. In The Sodium Pump: Recent Developments. J. H. Kaplan and P. De Weer, editors. The Rockefeller University Press, New York. 89–93.

Vasilets, L. A., G. Schmalzing, K. Mädefessel, W. Haase, and W. Schwarz. 1990. Activation of protein kinase C by phorbol ester induces downregulation of the Na+,K+-ATPase in oocytes of Xenopus laevis. Journal of Membrane Biology. 118:131–142.

Verrey, F., P. Kairouz, E. Schaerer, P. Fuentes, K. Geering, B. C. Rossier, and J.-P. Kraehenbuhl. 1989. Primary sequence of Xenopus laevis Na+/K+-ATPase and its localization in A6 kidney cells. American Journal of Physiology. 256:F1034–F1043.

Effect of Phosphatidylcholine on the Na,K-ATPase Activity of Basolateral Plasma Membranes from Proximal Tubular Cells of Rat Kidney

Reinaldo Marín, Teresa Proverbio, and Antonio J. Rodríguez

Centro de Biofísica y Bioquímica, Instituto Venezolano de Investigaciones Científicas, Caracas, Venezuela

Introduction

It has been demonstrated that the Na,K-ATPase activity can be modified by the surrounding lipid microenvironment (Kimelberg and Papahadjopoulos, 1972, 1974; Hegner and Platt, 1975; Kimelberg, 1975; Proverbio et al., 1985). Thus, the Na,K-ATPase activity is partially inhibited by disturbances in the lipid bilayer such as an increase in the cholesterol/phospholipid ratio (Kroes and Ostwald, 1971; Proverbio et al., 1985), a decrease in the degree of unsaturation of the fatty acid residues of the plasma membrane (Kimelberg and Papahadjopoulos, 1974), and an increase in the lipid peroxidation (Kako et al., 1988; Thomas and Reed, 1990). Since it has been suggested that lipid peroxidation participates in the aging process (Sawada and Carlson, 1987), it is likely that an increase in lipid peroxidation can produce a partial inhibition of the Na,K-ATPase activity, leading to disturbances in the Na+ and K+ electrochemical gradients across cell membranes. A decrease in the level of lipid peroxidation could then return the activity of the Na,K-ATPase to normal values. Phosphatidylcholine (PC) has been used as antioxidant (Abramova and Oksengendler, 1985), and PC given orally has been shown to increase the rat liver plasma membrane fluidity (Hegner and Platt, 1975).

In this work we evaluated the effect of giving PC, rich in unsaturated fatty acid residues (66% dilinoleyl-phosphatidylcholine), to 30-d-old rats (weighing 100 g), on the Na,K-ATPase activity of basolateral plasma membranes of kidney proximal tubular cells. 25 mg of soybean PC, suspended in distilled water, was either given orally or injected intraperitoneally during two consecutive days. On the third day, basolateral plasma membrane preparations were obtained from homogenates of outermost kidney cortex slices, and their Na,K-ATPase activity was determined.

Results and Conclusions

Table I shows the effect of both routes of PC administration on the Na,K-ATPase activity of the rat kidney cortex homogenates. It can be seen that in both cases there was an increase in the ATPase activity of ~30%. Similar results were obtained for purified basolateral plasma membranes (data not shown). This increase in activity was not due to an increase in the number of molecules, since the ouabain binding was similar for control and experimental membranes. The ATPase activity was partially characterized and it was found that the Km values for Na+, K+, and Mg:ATP, the K0.5inh for ouabain, and the optimal pH and temperature values were similar for control and experimental membranes.

TABLE I
Effect of PC on the Na,K-ATPase Activity of Rat Kidney Cortex Homogenates

Condition

Na,K-ATPase activity

nmol Pi/mg protein · min

Control

85 ± 4

Orally given PC

107 ± 5

Intraperitoneal PC

105 ± 4

The values are expressed as means ± SE (n = 8).

The lipid peroxidation for both conditions was measured using the thiobarbituric (TBA) assay. As shown in Table II, the TBA-reactive material (malondialdehyde) is lower in the experimental membranes when compared with control membranes.

It has been demonstrated that a decrease in the lipid peroxidation of the plasma membranes produces an increase in their fluidity (Ohta et al., 1989). It is likely, then, that the treatment with PC reduces the lipid peroxidation by replacing peroxidized fatty acid residues with nonperoxidized ones, increasing in this way the membrane fluidity. This, in turn, increases the Na,K-ATPase activity. In keeping with this, Hegner and Platt (1975) showed that essential phospholipids given orally (containing 50% of dilinoleyl-PC) are incorporated in rat kidney plasma membranes.

Our results are in agreement with those of Hegner and Platt (1975), who showed that rat liver plasma membranes increase their Na,K-ATPase activity after 2 d of feeding with essential phospholids (rich in PC). In our work we used four times less PC (with higher levels of unsaturated fatty acid residues) than Hegner and Platt, demonstrating similar results on the lipid peroxidation and Na,K-ATPase activity. This work may have important applications for treating diseases or processes involving a decrease in the Na,K-ATPase activity due to an increase in lipid peroxidation.

TABLE II
TBA-reactive Material Formed from Rat Kidney Basolateral Plasma Membranes

Condition

TBA-reactive material

nmol MDA/mg protein

Control

10.60 ± 0.93

PC

6.89 ± 0.79

TBA, thiobarbituric acid; MDA, malondialdehyde.
The values are expressed as means ± SE (n = 4).

Acknowledgments

The authors wish to acknowledge Dr. F. Proverbio for his criticisms and fruitful discussions.

References

Abramova, Ch. I., and G. I. Oksengendler. 1985. Chelovek y Protivookyslytelnye Veshestva. N. V. Savateev, editor. Ed. Nauka, Akademia Nauk, Leningrad. 75–78.

Hegner, D., and D. Platt. 1975. Effect of essential phospholipids on the properties of ATPases of isolated rat liver plasma membranes of young and old animals. Mechanisms of Ageing and Development. 4:191–200.

Kako, K., M. Kato, T. Matsuoka, and A. Mustapha. 1988. Depression of membrane-bound Na+-K+-ATPase activity induced by free radicals and by ischemia of kidney. American Journal of Physiology. 254:C330–C337.

Kimelberg, H. K. 1975. Alterations in phospholipid-dependent (Na+ – K+)-ATPase activity due to lipid fluidity. Effects of cholesterol and Mg2+. Biochimica et Biophysica Acta. 413:143–156.

Kimelberg, H. K., and D. Papahadjopoulos. 1972. Phospholipid requirements for (Na+ + K+)-ATPase activity: head-group specificity and fatty acid fluidity. Biochimica et Biophysica Acta. 282:277–292.

Kimelberg, H. K., and D. Papahadjopoulos. 1974. Effects of phospholipid acyl chain fluidity, phase transitions, and cholesterol on (Na+ + K+)-stimulated adenosine triphosphatase. Journal of Biological Chemistry. 249:1071–1080.

Kroes, J., and R. Ostwald. 1971. Erythrocyte membranes-effect of increased cholesterol content on permeability. Biochimica et Biophysica Acta. 249:647–650.

Ohta, A., T. Mohri, and T. Ohyashiki. 1989. Effect of lipid peroxidation on membrane-bound Ca2+-ATPase activity of the intestinal brush-border membranes. Biochimica et Biophysica Acta. 984:151–157.

Proverbio, F., T. Proverbio, and R. Marín. 1985. The effect of age and cholesterol content on Na,K-ATPase activity of basolateral plasma membranes from rat kidney. In The Sodium Pump. I. Glynn and J. C. Ellory, editors. The Company of Biologists, Ltd., Cambridge, UK. 713–716.

Sawada, M., and J. C. Carlson. 1987. Changes in superoxide radical and lipid peroxide formation in the brain, heart and liver during the lifetime of the rat. Mechanisms of Ageing and Development. 41:125–137.

Thomas, C. E., and D. J. Reed. 1990. Radical-induced inactivation of kidney Na+,K+-ATPase: sensitivity to membrane lipid peroxidation and the protective effect of vitamin E. Archives of Biochemistry and Biophysics. 281:96–105.

Cellular Regulation of the [Na+, K+]-ATPase Activity

Haywood Blum and Robert G. Johnson, Jr.

Department of Medicine, University of Pennsylvania Medical Center, Philadelphia, Pennsylvania 19104

Introduction

Although the sodium pump is the best understood ion translocating ATPase, little is known about the molecular regulation of its activity within the cell, independent of its substrates, Na+, K+, and Mg-ATP. We have studied the activity of the [Na+, K+]-ATPase and the kinetics of its activation and deactivation within the electric organ of the live intact electric fish, Narcine brasiliensis. This system was chosen for a number of reasons: the electric organ tissue is quite homogeneous, there is a high density of sodium pumps within the electrocyte, and the sodium pump subunits are largely homologous with mammalian Na+ pump subunits. In addition, the Narcine electric organ is remarkable for the stringency of control of the pump activity.

Pump activity could be monitored noninvasively by 31P-nuclear magnetic resonance (NMR). As shown in Fig. 1, the activation of the electric organ and the accompanying utilization of ATP may be monitored by depletion of phosphocreatine (PCr) through the creatine kinase reaction. This is the only known biochemical pathway for PCr. The electric organ has an abundant quantity of creatine kinase, and PCr comprises almost 50% of the phosphorylated compounds found in the resting organ. Unless ATP can be resynthesized, inorganic phosphate (Pi) will accumulate. Furthermore, since a proton is used in the creatine kinase reaction, intracellular pH will tend to increase with stimulation. This pH change will be counteracted by the formation of lactic acid through the glycolytic pathway.

Methods and Results

Healthy specimens of Narcine brasiliensis were obtained from Gulf Stream Specimen Company (Panacea, FL). They were acclimatized to a large seawater tank for a few days before use. The fish were transferred to a small bucket containing 0.1 g/liter 3-aminobenzoic acid ethyl ester (Sigma Chemical Co., St. Louis, MO) in seawater, which was used as an anesthetic agent. Type E2 subdermal electrodes (Grass Instrument Co., Quincy, MA) were implanted into the cranium over the electric cortex to induce discharge of the electric organ by stimulation through the nerve trunks controlling it. The fish was then placed in an NMR cradle (Johnson Foundation Biomedical Instrumentation Group, University of Pennsylvania, Philadelphia, PA) which allowed seawater to be pumped across its gills and to be collected beneath the fish. The cradle also held a two-turn surface coil used for NMR spectroscopy. The surface coil was placed over the electric organ. At the end of a session lasting several hours the fish were alive and could recover from the anesthesia.

Figure 1. Stimulation of the electric organ to discharge causes an immediate depletion of PCr and an increase of Pi through the coupled creatine kinase and [Na+,K+]-ATPase reactions.

The cradle and attachments were placed within the horizontal bore of a superconducting magnet operating at 2.4 T, associated with an Otsuka NMR spectrometer (Havertown, PA).

Application of a voltage pulse lasting a few milliseconds to the electrodes implanted adjacent to the electric cortex caused discharge of the electric organ. This could be monitored by pick-up electrodes in the periphery of the organ. A threshold voltage of ~ 50 V was needed to induce discharge. The pick-up voltage was ~ 15 V.

The 31P-NMR spectrum of the electric organ displayed PCr, Pi, ATP, and small amounts of phosphomonoester and phosphodiester. Intracellular pH was measured by the chemical shift (Jacobson and Cohen, 1982) between Pi and PCr. The discharge of the organ was accompanied by rapid depletion of the PCr and a concomitant increase of Pi. Fig. 2 shows the time course of a representative depletion curve. In this discharge, each pulse consisted of a train of three 3-ms pulses spaced 20 ms apart. The train was repeated every 20 s. This allowed rapid depletion of the PCr without causing any fatigue in the effectiveness of the discharging impulses. After termination of the stimulating pulses, the metabolites were stable over many minutes, with PCr and Pi neither increasing nor decreasing. pH was stable and did not decrease until depletion of PCr was essentially complete (data not shown).

Figure 2. Representative in vivo depletion of PCr and concomitant increase in Pi with stimulation of the electric cortex. The stimulation sequence is described in the text.

Figure 3. Typical in vivo time course of sodium (extracellular to intracellular ratio) and phosphorus (PCr/Pi) with stimulation. An intraarterial infusion of 120 mM Dy(TTHA) was started at time zero. Stimulation of the electric cortex was done between 111 and 225 min with a single pulse every 20 s. Each data point represents 75 free induction decays (FIDs) for 31P and 900 FIDs for 23Na over 6 min. Curves are drawn to help display the data and are not fitted, except that Na+ recovery after cessation of stimulation is least-squares fitted. The spectrometer interleaved between 31P and 23Na. The time resolution per point is 6 min. No change is observed in the Na NMR spectrum until PCr is virtually completely depleted. During recovery the PCr begins to recover only after the sodium transmembrane gradient has returned to normal.

A more extended time course of observation displayed in Fig. 3, shows that the ratio of PCr to Pi took several hours after stimulation had ceased before appreciable PCr resynthesis occurred. Full recovery of PCr after complete depletion took 24 h (data not shown).

Figure 4. In vivo recovery of the 23Na-NMR in Narcine after three rapid successive stimulations of the electric cortex. Each point is from a single FID acquired over 0.175 s. Data points are the result of a computer deconvolution of the incompletely resolved intra- and extracellular sodium signals.

More direct evidence of sodium pump activity after stimulated discharge was obtained from studies of the 23Na-NMR of the electric organ. Intracellular and extracellular sodium were distinguished (Springer, 1987) by intraarterial infusion of dysprosium triethylenetetraminehexaacetic acid (DyTTHA). This paramagnetic ion, which does not enter the intracellular volume, affects the chemical shift of the sodium in contact with it. Thus, intracellular sodium is unshifted and can be resolved from extracellular sodium. In Fig. 3, the ratio of extra- to intracellular sodium is shown. Stimulation forces this ratio down after depletion of cellular energy and consequent pump failure. After cessation of stimulation the sodium ratio recovers slowly toward its normal resting value. The rate of recovery is a measure of the maximum rate of synthesis of ATP in the electric organ. Fig. 4 shows this ratio obtained after a single stimulation sequence when the levels of PCr and ATP were adequate to allow full activity of the sodium pump. In this figure, recovery is virtually complete within a second.

Discussion

The in vivo data presented here show that the electric organ has little ability to synthesize ATP. PCr stores are therefore used as the immediate source of ATP, and the activity of the sodium pump can be monitored by its rate of depletion. Direct measurement of the sodium concentration within the electrocyte shows that the pump is extremely active immediately after a discharge but becomes inactive within seconds of cessation of stimulation and restoration of the normal transmembrane sodium gradient. The ratio of activities in the “on” and “off' states is greater than three orders of magnitude.

Additional studies have been done on dissected slices of the electric organ. These studies (Blum and Johnson, 1989; Blum et al., 1990) have demonstrated that activation of the sodium pump requires electrical or other depolarization of the electrocyte membrane. Increased intracellular sodium concentration is not, in itself, an adequate stimulus for pump activation.

These studies are consistent with the conclusion that the majority (> 95%) of the [Na+,K+]-ATPase molecules are in an inactive form in the resting electrocyte but rapidly activate during cholinergic stimulation of the cells via a mechanism independent of the sodium substrate concentration. The possible signal transduction mechanisms and Na+ pump regulation are under active investigation.

References

Blum, H., and R. G. Johnson, Jr. 1989. In vivo [Na+,K+]-ATPase activity in Narcine brasiliensis. Society for Neuroscience Abstracts. 15:1307.

Blum, H., S. Nioka, and R. G. Johnson, Jr. 1990. Activation of the Na+, K+-ATPase in Narcine brasiliensis. Proceedings of the National Academy of Sciences, USA. 87:1247–1251.

Jacobson, L., and J. S. Cohen. 1982. Intracellular pH measurements by NMR methods. In Noninvasive Probes of Tissue Metabolism. J. S. Cohen, editor. John Wiley & Sons, Inc., New York. 5–24.

Springer, C. S., Jr. 1987. Measurement of metal cation compartmentalization in tissue by high resolution metal cation nmr. Annual Review of Biophysics and Biophysical Chemistry. 16:375–399.

Defective Na Transport of Murine Ouabain-resistant Alpha-1 Na,K-ATPase

B. M. Anner, E. Imesch, and M. Moosmayer

Department of Pharmacology, University of Geneva Medical Center, CH-1211 Geneva 4, Switzerland

Introduction

The sensitivity of Na,K-ATPase to cardiac glycosides is species dependent (Detweiler, 1967). Table I shows some typical ouabain sensitivities of the animal kingdom. The toad Bufo marinus, for instance, is resistant to cardiac glycosides since it produces its own cardioactive compounds, the bufotoxins. Conversely, the reason for the murine resistance to cardiac glycosides is not known. The acquisition of resistance to cardiac glycosides is, in general, a mechanism for survival in a hostile environment (Reichstein et al., 1968). The question of whether other functional changes are associated with the ouabain resistance is not yet clear. No differences in the affinities for the enzyme substrates have been observed by comparing isolated renal rat and dog Na,K-ATPase (Periyasami et al., 1983). Conversely, transfection of ouabain-sensitive cells by the ouabain resistance (ouaR) gene appears to cause a Na transport defect since the Na/K coupling ratio decreases from 1.5 to 1.1 (Epstein and Lechene, 1988).

TABLE I
Species-dependent Variation of Cardiac Glycoside Sensitivity

Ouabain (s.c.)
LD_50/LD_50 cat*

Digoxin (i.v.)
LD_50

mg/kg

Cat

1

0.4

Rabbit

2

0.6

Frog

6

5.0

Toad

420

40.0

Rat

671

11.6

*Detweiler, 1967.

At least 99% of the renal Na,K-ATPase is composed of the alpha-1 Na,K-ATPase isoform (Lingrel et al., 1990). Comparison of the Na activation curves of a ouabain-sensitive (rabbit) and a ouabain-resistant (rat) alpha-1 Na,K-ATPase purified from renal outer medulla revealed a Na transport defect associated with ouabain resistance (Anner et al., 1989). The Na defect was found to be identical regardless of the experimental conditions used, such as measurement of the Na,K-ATPase activity of purified, unreconstituted enzyme by the linked-enzyme assay in maximal turnover conditions or of the ATP-activated 22Na uptake after reconstitution of the enzymes into liposomes (Anner et al., 1989).

In the present work we used another experimental assay, i.e., measurement of the Na,K-ATPase activity of enzymes under the conditions used for the active transport experiments in liposomes, i.e., at 25°C and in the presence of 50 mM KCl. In this condition we also observe the same Na defect as detected previously measuring either the unreconstituted Na,K-ATPase activity in maximal turnover conditions or the reconstituted active 22Na transport.

Materials and Methods

Na,K-ATPases purified from the outer medulla of rat, pig, or rabbit kidneys were then reconstituted into liposomes at equal protein/lipid ratios according to a standardized, calibrated, and highly reproducible cholate dialysis procedure (Anner and Moosmayer, 1981). The protein was determined by the Pierce BCA assay (Pierce Chemical Co., Rockford, IL). The ouabain sensitivity and the Na activation of the Na,K-ATPase activity were measured by the linked-enzyme assay as described (Anner et al., 1989). The ouabain sensitivity of the active 86Rb uptake was determined using a new test system developed in our laboratory: ATP-filled bifunctional Na,K-ATPase liposomes (Anner and Moosmayer, 1985; Rey et al., 1987; Anner et al., 1990).

Figure 1. Ouabain resistance of murine alpha-1 Na,K-ATPase. Rat (□), pig (), and rabbit () enzymes were incubated at increasing concentrations of ouabain either at 37°C in a solution for measuring the ATPase activity by the enzyme-linked assay at 100 mM NaCl and 10 mM KCl (), or after reconstitution into ATP and Na-filled liposomes () at 25°C in the presence of 10 µM external 86RbCl for measuring the active 86Rb accumulation by the techniques listed in Materials and Methods. Experimental data are averages of two to five determinations (SEM).

Results and Discussion

Fig. 1 shows the ouabain resistance of the purified murine alpha-1 Na,K-ATPase in comparison with enzymes purified from pig or rabbit outer medulla. The murine alpha-1 Na,K-ATPase is ~100 times less ouabain sensitive than the nonmurine enzymes, as revealed by an IC_50 of 100 µM vs. 1 µM. Importantly, the affinity of the rabbit enzyme for ouabain is not changed by reconstitution into liposomes. Similarly, the affinity for K ions (Rey et al., 1987) and Na ions (Anner et al., 1989) is not affected by reconstitution, indicating that the transport-active liposomes contain intact Na,K-ATPase molecules.

Table II shows the Na defect observed by measuring the Na,K-ATPase activity of rat and rabbit enzymes in transport conditions. Below 25 mM NaCl the enzyme activity of the ouabain-resistant Na,K-ATPase is 40–50% lower in comparison to the rabbit enzyme, in agreement with previous data obtained in different conditions and with other enzyme preparations (Anner et al., 1989). Thus, the high-affinity Na defect must be directly associated with the protein structure since it is found to be identical by three different experimental determinations: (a) the linked-enzyme assay before reconstitution at 37°C (10 mM KCl), (b) the linked-enzyme assay in transport conditions at 25°C (50 mM KCl), or (c) active 22Na accumulation at 25°C (50 mM RbCl). Further, the Na defect is not dependent on the specific Na,K-ATPase activities of the rat and rabbit enzymes.

TABLE II
Na Defect of Purified Ouabain-resistant Na,K-ATPase in Transport Conditions

Na,K-ATPases were purified from the outer medulla of rat (33 U/min) and rabbit (27 U/min) kidneys to equal purity as determined by laser densitometry after gel electrophoresis. The Na,K-ATPase activities were determined at increasing NaCl concentrations in active transport conditions (25°C, 50 mM KCl) by the linked-enzyme assay. The ATPase activities at 100 mM NaCl (100%) were 7.8 U/min (rat) and 5.5 U/min (rabbit). The values are averages of separate determinations. References for the techniques are indicated under Materials and Methods.

In conclusion, we have shown a Na defect of the murine alpha-1 Na,K-ATPase discovered by comparing the Na activation curves of the ouabain-sensitive and ouabain-resistant alpha-1 forms in the low Na region. Thus, two renal Na,K-ATPases, both of alpha-1 origin but with distinct ouabain sensitivity, transport Na ions differently at low NaCl concentrations. These results demonstrate a link between the cardiac glycoside sensitivity and the active Na transport of the Na,K-ATPase alpha-1 isoform.

Acknowledgments

This work was supported by grant 31–25666.88. from the Swiss National Science Foundation.

References

Anner, B. M., E. Imesch, and M. Moosmayer. 1989. Sodium transport defect of ouabain-resistant renal Na,K-ATPase. Biochemical and Biophysical Research Communications. 165:360–367.

Anner, B. M., and M. Moosmayer. 1981. Preparation of Na,K-ATPase-containing liposomes with predictable transport properties by a procedure relating the Na,K-transport capacity to the ATPase activity. Journal of Biochemical and Biophysical Methods. 5:299–306.

Anner, B. M., and M. Moosmayer. 1985. Right-side-out pumping Na,K-ATPase-liposomes: a new tool to study the enzyme's receptor function. Biochemical and Biophysical Research Communications. 129:102–108.

Anner, B. M., H. G. Rey, M. Moosmayer, I. Meszoely, and G. T. Haupert, Jr. 1990. Hypothalamic Na,K-ATPase inhibitor characterized in two-sided liposomes containing pure renal Na,K-ATPase. American Journal Physiology. 258:F144–F153.

Detweiler, D. K. 1967. Comparative pharmacology of cardiac glycosides. Federation Proceedings. 26:1119–1124.

Epstein, J. A., and C. Lechene. 1988. Ouabain-resistant, amiloride-sensitive Na,K-pumping activity and morphological changes are inducible. American Journal Physiology. 254:C847–C854.

Lingrel, J. B., J. Orlowski, M. M. Shull, and E. M. Price. 1990. Molecular genetics of Na,K-ATPase. Progress in Nucleic Acid Research and Molecular Biology. 38:37–83.

Periyasamy, S. M., W.-H. Huang, and A. Askari. 1983. Origins of different sensitivities of (Na + K)-dependent adenosinetriphosphatase preparations to ouabain. Comparative Biochemistry and Physiology. 76B:449–454.

Reichstein, T., J. von Euw, J. A. Parsons, and M. Rothschild. 1968. Heart poisons in the monarch butterfly. Science. 161:861–866.

Rey, H. G., M. Moosmayer, and B. M. Anner. 1987. Characterization of (Na + K)-ATPase-liposomes. III. Controlled activation and inhibition of symmetric pumps by timed asymmetric ATP, RbCl and cardiac glycoside addition. Biochimica et Biophysica Acta. 900:27–37.

Regulatory Role of ATP in the Na,K-ATPase Operation

A. A. Boldyrev, N. U. Fedosova, O. D. Lopina, and E. V. Pindel

Department of Biochemistry, Moscow State University, Moscow 119899, USSR

The nonhyperbolic dependence of the enzyme activity on the substrate concentration is a characteristic feature of Na,K-ATPase (Neufeld and Levy, 1969; Kanazawa et al., 1970; Boldyrev and Svinukhova, 1982). The widespread explanation of the enzyme function is the Post-Albers scheme (Albers, 1967; Post et al., 1969):

In agreement with this, the enzyme is able to exist in two main conformations differing in their affinity toward substrate and activating ions: E_1 is characterized by low affinity to K+, but high affinity to Na+ and ATP. The E_2 form has high affinity to K+, but low affinity to Na+ and ATP. E_1 and E_2 forms also differ in both fluorescence of probes covalently bound to the enzyme (Kapakos and Steinberg, 1986) and in intrinsic fluorescence (Karlish and Yates, 1978). By using this approach, Karlish and Yates were able to show that ATP may accelerate E_2 → E_1 transformation. According to these data, a modified scheme was proposed, which could explain nonhyperbolic kinetics of ATP hydrolysis by the enzyme (Smith et al., 1980):

The scheme suggested the dependence of enzymic activity on the substrate concentration to be described by the so-called “negative cooperativity curve,” being presented as a sum of two hyperbolas (in a double reciprocal plot it looks like two intersecting lines).

As a matter of fact, it is known from the literature that ATP binding to Na,K-ATPase in the presence of K+ is also described as a negative cooperative process (Ottolenghi and Jensen, 1983). Unfortunately, binding experiments were performed in the restricted range of ATP concentrations (0.1–10 μM), whereas enzymic activity was examined up to 1–2 mM ATP and under particular conditions (in binding experiments ATP hydrolysis was prevented by the Mg2+ and Na+ or K+ exclusion). Nevertheless, the negative cooperativity obtained for ATP binding with the E_2 form of Na,K-ATPase was explained in terms of a dimer consisting of two nonequivalently arranged protomers. Such a nonequivalence has to be induced by K+ ions, insofar as substitution of K+ by Na+ in binding experiments results in the detection of only one (high affinity) type of ATP binding site with Kd = 0.1–0.2 μM (Ottolenghi and Jensen, 1983).

In contrast to Ottolenghi and Jensen (1983), who used the enzyme from dog kidney, we used duck salt gland enzyme and compared both ATP binding and ATP hydrolysis by this enzyme over a broad range of substrate concentrations. The dependence of the hydrolytic activity of the enzyme on ATP concentration is described by a curve with an intermediary plateau (Fig. 1 a). In double reciprocal coordinates it is transformed into several intersecting lines. We have also investigated the interaction of Na,K-ATPase with ATP in the same concentration range using two independent methods: (a) 32ATP binding to the enzyme measured by the rate dialysis method (Colowick and Womack, 1969), and (b) measurement of ATP-induced E_2 → E_1 transition of the enzyme labeled with 5-iodacetamidofluorescein (IAF) (Lopina et al., 1990). The results of these experiments are presented in Fig. 1, b and c, respectively.

Figure 1. The dependences of some functional parameters of Na,K-ATPase on ATP concentration. (a) Na,K-ATPase activity, which was measured with the coupled enzymes method in the presence of piruvate kinase and lactate dehydrogenase. (b) 32ATP-binding measured by rate dialysis method. The data were obtained independently in three ranges of ATP concentrations: 0.2–10, 10–200, and 200–2,000 μM. Conditions: 100 μM KCl, 1 mM EDTA, 30 mM imidazole (pH 7.5 at 4°). 1.5 μCi of [32P]ATP was added to the protein (2 mg/ml) at the beginning of the experiment, and later different concentrations of cold ATP were added. (c) Fluorescence change of IAF-labeled Na,K-ATPase on ATP concentration. Conditions: 100 μM KCl, 1 mM EDTA, 50 mM imidazole (pH 7.5 at room temperature).

All of the three curves obtained are nearly identical and are characterized by the intermediary plateau at approximately the same ATP concentrations. As it is impossible to express these curves as a sum of two (or several) hyperbolas (and impossible to explain them by existence of independent ATP binding sites), we resolved an experimental curve on Fig. 1 a into hyperbolic and sigmoidal components and ascribed to them properties that reflect the interaction of ATP with protomeric and oligomeric forms of the enzyme, respectively (Boldyrev and Fedosova, 1991). When applied to the hydrolytic activity this curve corresponds to the elementary catalytic process and its activation by high ATP concentration.

Oligomeric complexes of Na,K-ATPase consist of at least two (αβ)-protomers; this has been demonstrated by a variety of different methods. However, comparison of molecular target size of Na,K-ATPase accomplishing the whole cycle with one for so-called “partial reactions” (Na-ATPase, Na-dependent phosphorylation of the enzyme, K-phosphatase) shows that the molecular size of the enzyme in the former case has to be 1.7–2-fold larger than that in the latter (Norby and Jensen, 1989; Boldyrev et al., 1990).

It is known that the E_2 form of the enzyme possesses a higher tendency to oligomerize than E_1. Thus we suggest that the overall cycle of ATP hydrolysis is performed by independent protomers, but the limiting step of the reaction (E_2 → E_1 transition) in the presence of high ATP concentrations could be accelerated due to the formation of a short-lived oligomer (Boldyrev and Fedosova, 1991).

With this in mind we propose the new scheme for Na,K-ATPase operation:

In connection with this scheme in the presence of K+ the enzyme has to consist of two different forms, E_2 (protomeric) and E_2n (oligomeric), in equilibrium with each other. They possess low and high affinity toward ATP, respectively. Computer simulation makes it possible to calculate the constants corresponding to this model: Ks for monomeric and oligomeric E_2 forms of the enzyme are equal to 21.2 and 0.18 mM, respectively; Km for the elementary catatylic cycle, performed by the protomeric form, is 0.07 mM.

It is nice to note that an independent approach gives strong support to our idea of a short-lived oligomer, regulated by ATP (Hayashi et al., 1990), taking part in the acceleration of the E_2 → E_1, transition. ATP acts at this step as a typical modifier: it regulates the process without being hydrolyzed itself (it is characteristic that ADP shifts the equilibrium between E_2 and E_1 forms in the same direction and to nearly the same extent as ATP).

The most important element in the modifying effect of the nucleotide is a N_1 atom in the purine base. From a number of nucleotides tested, only ATP and CTP are characterized by the complex dependence both for binding and for the hydrolytic reaction (for UTP and GTP these dependences are described by simple hyperbolas). We suggest that the nitrogen atom takes part in accepting a H+ from the active site of the enzyme (Boldyrev et al., 1984), which makes it possible to accelerate E_2 → E_1 transformation.

References

Albers, R. W. 1967. Biochemical aspects of active transport. Annual Review of Biochemistry. 36:727–756.

Boldyrev, A. A., and N. U. Fedosova. 1991. Comparison of the kinetic properties of membrane-bound and solubilized Na,K-ATPase. Biochemistry International. In press.

Boldyrev, A. A., O. D. Lopina, and N. U. Fedosova. 1990. Na,K-ATPase: radiation inactivation studies. Biochemistry International. 21:45–52.

Boldyrev, A. A., O. D. Lopina, N. N. Gulyaev, L. A. Baranova, and E. S. Severin. 1984. Na,K-dependent adenosine triphosphate phosphohydrolase: activation of the phosphatase reaction by ATP analogs. FEBS Letters. 175:303–306.

Boldyrev, A. A., and I. A. Svinukhova. 1982. Na+,K+-dependent adenosine triphosphatase: two types of kinetics. Biochimica et Biophysica Acta. 707:167–170.

Colowick, S. P., and F. C. Womack. 1969. Binding of diffusible micromolecules: rapid measurement by rate dialysis. Journal of Biological Chemistry. 244:774–777.

Hayashi, Y., K. Mimura, H. Matsui, and T. Takagi. 1990. Association and dissociation of solubilized Na,K-ATPase by phosphatidylserine and ATP. Journal of General Physiology. 96:27a–28a. (Abstr.)

Kanazawa, T., M. Saito, and Y. Tonomura. 1970. Formation and decomposition of a phosphorylated intermediate in the reaction of Na,K-dependent ATPase. Journal of Biochemistry. 67:693–711.

Kapakos, J. G., and M. Steinberg. 1986. Ligand binding to Na+,K+-ATPase labeled with 5-iodacetamidofluorescein. Journal of Biological Chemistry. 261:2084–2089.

Karlish, J. G., and D. W. Yates. 1978. Tryptophan fluorescence of (Na+,K+)-ATPase as a tool for study of the enzyme mechanism. Biochimica et Biophysica Acta. 527:115–130.

Lopina, O. D., E. V. Pindel, and A. A. Boldyrev. 1990. Na,K-ATPase labelled with 5-iodacetamidofluorescein: E_2-E_1 conformational transition induced by different nucleotides. FEBS Letters. 266:75–77.

Neufeld, A. H., and H. M. Levy. 1969. A second ouabain-sensitive sodium-dependent adenosine triphosphatase in brain microsomes. Journal of Biological Chemistry. 244:6493–6497.

Norby, J. G., and J. Jensen. 1989. A model for the stepwise radiation inactivation of the α_2-dimer of Na,K-ATPase. Journal of Biological Chemistry. 264:19548–19558.

Ottolenghi, P., and J. Jensen. 1983. The K+-induced apparent geterogeneity of high-affinity nucleotide-binding sites in (Na + K)-ATPase can only be due to the oligomeric structure of the enzyme. Biochimica et Biophysica Acta. 727:89–100.

Post, R. L., T. Kume, T. Tobin, B. Orcutt, and A. K. Sen. 1969. Flexibility of an active center in sodium-plus-potassium adenosine triphosphatase. Journal of General Physiology. 54:306s–326s.

Smith, R. L., K. Zinn, and L. C. Cantley. 1980. A study of the vanadate-trapped state of the (Na,K)-ATPase. Evidence against interacting nucleotide site models. Journal of Biological Chemistry. 255:9852–9859.

A Ouabain-sensitive Na,K-ATPase in the Sea Anemone Stoichactis helianthus: Preliminary Characterization

Roberto Lopez Rosado and Susan Corey

Institute of Neurobiology, University of Puerto Rico, San Juan, Puerto Rico 00901

Introduction

The Na,K-ATPase is a highly conserved integral membrane enzyme responsible for transmembrane transport of Na+ and K+. The enzyme has been cloned and sequenced from several diverse species: the brine shrimp Artemia salina (Baxter-Lowe et al., 1989), Drosophila, the chicken, Torpedo califomicus, and several mammalian species (reviewed in Sweadner, 1989). The sequences exhibit a high degree of identity and homology around functional sites, including the phosphorylation site and the putative ouabain binding site (Price and Lingrel, 1988).

To obtain information about the primitive origins of the Na,K-ATPase, we have examined several tropical marine coelenterates for evidence of a (Na,K)-stimulated, ouabain-inhibited Na,K-ATPase. Since the Na,K-ATPase is present in high concentration in the nervous system of higher animals, we examined the tentacles, which are known to possess an innervation that is important in the feeding response (Van Marle, 1977).

After examining several coelenterate species, we have evidence that at least one possesses an active ouabain-inhibited Na,K-ATPase. The enzyme has relatively low affinity for ouabain, although Na+ and K+ affinities appear to be as in higher animals.

Methods

The experiments were performed using a microsomal preparation of tentacles cut from the sea anemone Stoichactis helianthus. The anemone was collected from shallow water off the east coast of Puerto Rico and maintained at ambient temperature with flowing sea water. To obtain tissue for analysis, an anemone was removed to a glass dish with a small amount of sea water. The water was changed to remove discharged cnidocysts. A portion of the tentacles was trimmed off with a scissors and the animal was returned to the tank.

Tentacles were homogenized in 0.32 M sucrose with 1 mM EDTA-Tris (pH 7.4). A low speed precipitate containing contractile material was discarded. A microsomal pellet was prepared by centrifuging the supernatant at 13,000 rpm for 1 h in a JA-20 rotor (Beckman Instruments, Inc., Palo Alto, CA). The pellets were resuspended at a concentration of 6–8 mg/ml and maintained in ice, since freezing resulted in a large loss of enzyme activity. Resuspension in 20% glycerol provided only minimal protection against denaturation.

Na,K-ATPase activity was measured at 37°C. The liberation of inorganic phosphate was determined by the method of Brotherus et al. (1981). An incubation volume of 25 or 50 μl was used. Na+ and K+ concentrations were varied as indicated in the text. The enzyme was preincubated for 10 min with 3 mM Na_2ATP, 3 mM MgCl_2, and 50 mM histidine-HCl (pH 7.4) with or without 1 mM ouabain. Na+ and K+ or water were then added and the reaction was stopped after an additional 20 min by the addition of stop solution (Brotherus et al., 1981). When strophanthidin was used, it was dissolved in dimethylformamide (DMF); the final concentration of DMF in the reaction mixture (250 μl) was 2%. In some cases, enzyme activity was measured by the K-stimulated hydrolysis of p-nitrophenyl phosphate (pNPP) (Robinson, 1969) using the same protocol.

Results

The Na,K-dependent ATPase activity was superimposed on a large Mg-dependent activity and represented only 40–45% of total ATPase activity in the best preparations. Na+ affinity was determined in the presence of 20 mM KCl. When considered only in terms of stimulation over the basal Mg-ATPase activity, half-maximal activation was achieved at ~ 65 mM NaCl.

Figure 1. Na+ activation of Na,K-ATPase from tentacles of Stoichactis helianthus. Values are means of three to five determinations performed in duplicate.

The ouabain-inhibited activity had a higher affinity for Na+, K0.5 ~ 5 mM (Fig. 1). Since this activity represented only a small proportion of total ATPase activity, the K0.5 determination was not precise.

The K+ affinity of the ouabain-sensitive enzyme activity was determined in the presence of 130 mM Na+, a concentration that may have been suboptimal. The K0.5 was ~ 5 mM.

The Na,K-stimulated activity was inhibited to 70% of control activity by 0.1 mM ouabain. The Ki for ouabain was estimated to be 10–3 M (Fig. 2). Strophanthidin was used in initial studies of Na,K-ATPase activity, since it inhibited to 41 ± 9% of control (mean ± SE, n = 8) at 10–4 M. Nonetheless, at lower concentrations strophanthidin frequently had a stimulatory effect, whereas it inhibited (Na + K)-stimulated activity to less than the basal Mg-dependent level at higher concentrations. Due to this aberrant behavior, we were unable to determine a Ki for strophanthidin.

The K-stimulated, ouabain-inhibited p NPPase activity was determined routinely when screening activity of new preparations. Ouabain completely inhibited K-stimulated activity at 0.1 mM. The Ki was ~ 10–6 M.

Conclusions

The sea anemone Stoichactis helianthus was found to possess a ouabain-sensitive Na,K-ATPase. Its affinity for ouabain and strophanthidin was very reduced compared with the enzyme from higher animals (e.g., Artemia salina [Cortas et al., 1989]) and a variety of mammals (Sweadner, 1989). Ouabain had very minimal effect on Na,K-ATPase activity, but inhibited the K-p NPPase activity more strongly.

It was not possible to obtain reasonable results with the highly lipid-soluble cardiac glycoside strophanthidin dissolved in DMF, although it has been successfully used with mammalian enzymes (Sweadner, 1979). Very potent inhibition was found with high concentrations and marked stimulation at low concentrations. The excessive inhibition at high concentrations was probably a nonspecific effect of strophanthidin in the lipid. The stimulatory effect of low concentrations was reminiscent of that occasionally observed with Na,K-ATPase isolated from mammalian brain, but more powerful. Although it may also be a nonspecific effect, it may represent a partial agonism that is made more obvious due to the low affinity of the enzyme for cardiac glycosides at the inhibitory site.

Figure 2. Ouabain inhibition of Na,K-ATPase activity (open circles) and K-p NPPase activity (filled circle) from tentacles of the sea anemone Stoichactis helianthus. Values are means of three to five determinations performed in duplicate.

In the sea anemone, the ouabain affinity of the K-p NPPase activity was 1,000 times greater than that of the Na,K-ATPase. This differs from Na,K-ATPase isolated from rat kidney, which is 10 times more sensitive to ouabain in the presence of ATP than with p NPP (Soto, S., and S. Corey, unpublished observations).

The data clearly demonstrate that the affinity of the sea anemone Na,K-ATPase for cardiac glycosides is much lower than that seen in higher phyla (e.g., both vertebrates and arthropods). On the other hand, Na+ affinity seems to be very similar to that reported for higher animals (e.g., Sweadner, 1985). The K+ affinity is somewhat lower than mammalian values, but within the range reported for Artemia salina (Cortas et al., 1989).

Canfield et al. (1991) have reported the primary sequence of a fresh-water coelenterate Hydra attenuata. The putative H1–H2 segment, currently under investigation as a ouabain interaction site, is very different from previously reported sequences, compatible with our measurements of low ouabain affinity. These data suggest that the emergence of ouabain sensitivity may be a relatively recent development for the Na,K-ATPase.

Work is in progress to purify the enzyme in order to obtain more precise determinations of ligand affinities.

Acknowledgments

We are indebted to Dr. P. A. G. Anderson (Whitney Laboratories, University of Florida, St. Augustine, FL) for the gift of Cyanea capillata tissues used in initial studies.

This work was supported in part by NIH grants NS-07464 and RR-08224.

Note added in proof: The name Stoichactis helianthus has recently been changed to Stichodactyla helianthus.

References

Baxter-Lowe, L. A., J. Z. Guo, E. E. Bergstrom, and L. E. Hokin. 1989. Molecular cloning of the Na,K-ATPase α-subunit in developing brine shrimp and sequence comparison with higher organisms. FEBS Letters. 257:181–187.

Brotherus, J. R., J. V. Moller, and P. L. Jorgensen. 1981. Soluble and active renal Na,K-ATPase with maximum protein molecular mass 170,000 +/– 9,000 daltons: formation of larger units by secondary aggregation. Biochemical and Biophysical Research Communications. 100:146–154.

Canfield, V. A., K.-Y. Xu, T. D'Aquila, A. W. Shyjan, and R. Levenson. 1991. Molecular cloning of Na,K-ATPase α-subunit cDNA from Hydra vulgaris. In The Sodium Pump: Recent Developments. J. H. Kaplan and P. De Weer, editors. The Rockefeller University Press, New York. 31–36.

Cortas, N., M. Arnaout, J. Salon, and I. S. Edelman. 1989. Isoforms of Na,K-ATPase in Artemia salina. II. Tissue distribution and kinetic characterization. Journal of Membrane Biology. 108:187–195.

Price, E. M., and J. B. Lingrel. 1988. Structure-function relationships in the Na,K-ATPase α subunit: site-directed metagenesis of glutamine-111 to arginine and asparagine-122 to aspartic acid generates a ouabain-resistant enzyme. Biochemistry. 27:8400–8408.

Robinson, J. D. 1969. Kinetic studies on a brain microsomal adenosine triphosphatase. II. Potassium-dependent phosphatase activity. Biochemistry. 8:3348–3355.

Sweadner, K. J. 1979. Two molecular forms of (Na+ + K+)-stimulated ATPase in brain. Separation, and difference in affinity for strophanthidin. Journal of Biological Chemistry. 254:6060–6067.

Sweadner, K. J. 1985. Enzymatic properties of separated isozymes of the Na,K-ATPase: substrate affinities, kinetic cooperativity, and ion transport stoichiometry. Journal of Biological Chemistry. 260:11508–11513.

Sweadner, K. J. 1989. Isozymes of the Na+/K+-ATPase. Biochimica et Biophysica Acta. 988:185–220.

Van Marie, J. 1977. Contribution to the knowledge of the nervous system in the tentacles of some coelenterates (Anemonia sulcata, Metridium senile, Cerianthus membraneceus, Tealis felina, and Hydra vulgaris). Bijdragen tot de Dierkunde. 46:221–260.

Spectral Tagging of Reaction Substates in a Sodium Pump Analogue: The Plasma Membrane H+ -ATPase of Neurospora

Georg Nagel, Esther Bashi, and Clifford L. Slayman

Department of Cellular and Molecular Physiology, Yale School of Medicine, New Haven, Connecticut 06510

Introduction

In 1988, Klodos and Forbush discovered that certain fluorescent styryl dyes (so-called RH-dyes; Grinvald et al., 1982), when bound to Na+,K+-ATPase-rich membrane fragments from dog kidney outer medulla, undergo a fluorescence shift during the E_1—E_2 transition of the ATPase. Apart from noting the potential importance of such findings for conformational studies on P-type ATPases, the authors raised the question of whether the RH-dyes, which had been designed and tested primarily as trans-membrane voltage indicators, might in fact be indicating local changes of electric field, or charge displacements. This idea of an “electrochromic” mode of action elicited considerable supporting evidence from Läuger and his collaborators (Bühler et al., 1991; Stürmer et al., 1991), who systematically explored interactions of ATPase-related fluorescence changes and lipid-soluble ion-related fluorescence changes in the dye. That work, carried out mostly with RH-421, showed an ATP-dependent red shift of ~ 10 nm in the excitation spectrum, enhancement by lipid-soluble anions (e.g., tetraphenyl-boron), and suppression by lipid-soluble cations (e.g., tetraphenylphosphonium).

To test the RH-dyes for range of usefulness as conformational indicators, and to explore their mechanism of action, we undertook experiments in collaboration with Dr. Irena Klodos on the fungal plasma membrane H+ -ATPase/proton pump (Nagel et al., 1989). That enzyme closely resembles the Na+,K+ -ATPase in physical properties, in secondary and tertiary structure (Slayman et al., 1990), and regionally in sequence homology (Hager et al., 1986), but differs in a variety of functional properties, including ion specificity, stoichiometry, and practical electrogenicity (Slayman and Zuckier, 1989). Initial experiments with the dye RH-160 and the plasma membrane ATPase from Neurospora crassa confirmed the basic observations of Klodos and Forbush (1988), but with quantitative differences: specifically, the fungal enzyme shows a very small fluorescence change upon activation with ATP, but a very large change upon addition of the E_2-trapping reagent orthovanadate (Slayman et al., 1989). The finding is consistent with both biochemical and kinetic data on the Neurospora proton pump (Slayman and Sanders, 1985), which have shown low steady-state levels of phosphate-associated intermediates.

The experiments described below were carried out to examine key spectral characteristics of interactions between RH-160 and the Neurospora H+ -ATPase, and have led to two main conclusions: (a) that the dye changes are more complicated than expected for a simple conformational indicator; and (b) that the largest fluorescence shift is probably not electrochromic in nature.

Methods

Experiments were carried out with detergent-washed plasma membrane fragments of Neurospora, prepared by the method of Bowman et al. (1981). Such preparations are about fivefold enriched in the H+-ATPase, and consist morphologically of a mixture of flat sheets and broken vesicles, with a small admixture of protein and carbohydrate particles. Experiments were conducted in a standard buffer containing 100 mM MES-Tris at pH 6.7, and 1 μM RH-160. Measurements were made on an SLM DW-2000 spectrophotometer fitted with a differential fluorescence adaptor, and emitted fluorescence was recorded above 640 nm. Kinetic experiments were arranged to report the emission difference for excitation at 550 nm minus that at 440 nm on a single sample cuvette; spectral experiments were arranged to report the emission difference, over a spectral range, between the sample cuvette and a flat fluorophor. Spectra were analyzed by means of a graphics program written by Dr. Bliss Forbush III.

Results

Fluorescence curves for a typical experiment are shown in Fig. 1. The excitation maximum for the dye control occurred at 505 nm (Fig. 1 A), and the main effect of adding Mg2+ and ATP appeared to be a small reduction of excitation efficiency. Addition of the E_2-trapping agent orthovanadate produced a conspicuous increase of excitation efficiency, and a conspicuous red shift in the spectrum. Scale expansion showed the first vanadate curve and the dye-control curve to cross at ~ 470 nm (Fig. 1 A, inset). The spectral events are revealed more clearly after subtracting the dye–control curve from the three test curves, and plotting on a fivefold expanded scale (Fig. 1 B). Addition of Mg2+ and ATP can thus be seen to have produced an asymmetric deficit of excitation (bottom curve of Fig. 1 B), slightly larger in the blue than in the red, but with little or no shift of the peak wavelength. The initial excitation increase upon addition of vanadate, however, peaked 40 nm to the red, at 545 nm (middle curve of Fig. 1 B), with very strong asymmetry. Prolonged vanadate elicited a progressive blue shift and further increase in the excitation efficiency (upper curve of Fig. 1 B).

A more precise view of these spectral changes emerges from sequential difference calculations. Fig. 2 represents the same experiment as in Fig. 1, but shows the calculated sequential difference curves, including two previously omitted curves: 15 min vanadate, and Mg2+ alone (added to the enzyme/dye mixture). The dot plots are simple scaled versions of the dye-control curve in Fig. 1 A, for direct comparison with each of the sequential difference curves. The salient features of these curves are as follows. (a) Addition of vanadate to the steady-state energized enzyme produced a prompt increase in excitation efficiency at 540 nm (Fig. 2 C), with no “compensating” decrement at shorter wavelengths. (b) Prolonged vanadate yielded increased excitation at the characteristic peak (505 nm) for the dye-control curve (Fig. 2, A and B, upper plots). (c) Mg2+ resulted in asymmetrically decreased excitation near the dye-control peak, and further addition of ATP slightly enhanced the Mg2+ effect. Comparison with Fig. 1 B shows the exaggerated red shift (at 545 nm) to be the sum of (a) and (c), while the progressive changes after vanadate addition are the sum of (a) and (b).

Figure 1. Spectral changes in the styryl dye RH-160 associated with the plasma membrane H+ -ATPase of Neurospora. (A) Primary excitation spectra read after successive addition of 1 μM dye, 1 mM Mg2+ (not shown), 5 mM Mg-ATP, and 100 μM orthovanadate, to a suspension of washed membrane fragments (12.5 μg protein and 2 ml 100 mM MES-Tris buffer, pH 6.7). (Inset) Expanded scale to show crossing of first vanadate curve (VO_4) and dye–control. (B) Difference spectra, with dye–control subtracted. Total emission > 640 nm sampled at scan intervals of 0.25 nm = 125 ms.

Separate experiments have yielded the following additional information: (d) the Mg2+ effect largely persists after heat denaturation of the ATPase, but (e) the ATP effect and both vanadate effects are abolished. (f) In the absence of ATP, both vanadate effects occur at the slow rate (as for b), so the emergent peak lies at 520–525 nm. (g) No circumstances elicit the kind of symmetric difference spectrum that would be expected for a linear electrochromic response (i.e., similar in shape to the first derivative of the excitation spectrum, reflecting a parallel shift but little alteration in shape or amplitude of the excitation spectrum [Waggoner and Grinvald, 1977]).

Figure 2. Sequential difference curves, individually compared with the scaled dye-control curve (dot plots). Data from the full experiment that is partially shown in Fig. 1. Data filtered to 40 points/curve.

Interpretations

The simplest view of this ensemble of results is that the Mg2+ effect reports nonspecific interaction, either with protein or phospholipid; that ATP yields a dye–enzyme complex with slightly diminished excitation efficiency in the blue; and that vanadate yields two dye–enzyme complexes, both with enhanced excitation efficiency: a rapid complex peaking within 1 min at 540 nm, and a slow complex peaking after 15–20 min at 505 nm (near the dye–control maximum). If the rapid vanadate response is assumed to represent full trapping of the ATPase in E_2-V, and the ATP response is assumed to represent the steady-state level of phosphoenzyme forms, then the ratio of the two peak amplitudes roughly estimates the steady-state phosphoenzyme level: ~ 15%, in agreement with the previous calculation of Slayman and Sanders (1985).

Thus, it may be possible in principle to distinguish three different conformations of the H+-ATPase by spectral interactions with RH-160. The small size of the ATP effect, and the slowness of the second vanadate effect, however, clearly indicate that only the first vanadate effect is likely to be useful for dynamic conformational analysis. Unfortunately, none of the results suggest a simple indicator mechanism for the dye.

Acknowledgments

The authors are indebted to Dr. Irena Klodos for helping to initiate these experiments, and to Dr. Bliss Forbush III for many helpful discussions and for use of computer programs.

This work was supported by research grant GM-15858 from the National Institute of General Medical Sciences, and by a postdoctoral fellowship (to G. Nagel) from the James Hudson Brown–Alexander B. Coxe Fund (Yale University School of Medicine).

References

Bowman, B. J., F. Blasco, and C. W. Slayman. 1981. Purification and characterization of the plasma membrane ATPase of Neurospora crassa. Journal of Biological Chemistry. 256:12343–12349.

Bühler, R., W. Stürmer, H.-J. Apell, and P. Läuger. 1991. Charge translocation by the Na,K-pump. I. Kinetics of local field changes studied by time-resolved fluorescence measurements. Journal of Membrane Biology. 121:141–161.

Grinvald, A., R. Hildesheim, I. C. Farber, and L. Anglister. 1982. Improved fluorescent probes for the measurement of rapid changes in membrane potential. Biophysical Journal. 39:301–308.

Hager, K. M., S. M. Mandala, J. W. Davenport, D. W. Speicher, E. J. Benz, Jr., and C. W. Slayman. 1986. Amino acid sequence of the plasma membrane ATPase of Neurospora crassa: deduction from genomic and cDNA sequences. Proceedings of the National Academy of Sciences, USA. 83:7693–7697.

Klodos, I., and B. Forbush III. 1988. Rapid conformational changes of the Na/K pump revealed by a fluorescent dye, RH-160. Journal of General Physiology. 92:46a. (Abstr.)

Nagel, G., C. Slayman, and I. Klodos. 1989. Fluorescence probing of a major conformation change in the plasma membrane H+ -ATPase of Neurospora. Biophysical Journal. 55:338a. (Abstr.)

Slayman, C. L., P. Kaminski, and D. Stetson. 1990. Structure and function of fungal plasma-membrane ATPases. In Biochemistry of Cell Walls and Membranes in Fungi. P. J. Kuhn, A. P. J. Trinci, M. J. Jung, M. W. Goosey, and L. G. Copping, editors. Springer-Verlag, Berlin. 299–316.

Slayman, C., I. Klodos, and G. Nagel. 1989. Probing for the molecular mechanism of active transport. In Plant Membrane Transport: The Current Position. J. Dainty, M. I. de Michelis, E. Marré, and F. Rasi-Caldogno, editors. Elsevier, Amsterdam. 467–472.

Slayman, C. L., and D. Sanders. 1985. Steady-state kinetic analysis of an electroenzyme. Biochemical Society Symposia. 50:11–29.

Slayman, C. L., and G. N. Zuckier. 1989. Differential functional properties of a P-type ATPase/proton pump. Annals of the New York Academy of Sciences. 574:233–245.

Stürmer, W., R. Bühler, H.-J. Apell, and P. Läuger. 1991. Charge translocation by the Na,K-pump. II. Ion-binding and release at the extracellular face. Journal of Membrane Biology. 121:163–176.

Waggoner, A. S., and A. Grinvald. 1977. Mechanisms of rapid optical changes of potential sensitive dyes. Annals of the New York Academy of Sciences. 303:217–241.

Interaction of NaK-ATPase Proteoliposomes with Palytoxin at Picomolar Concentrations

Atsunobu Yoda, Marsha Rich Rosner, Peter Morrison, and Shizuko Yoda

Department of Pharmacology, University of Wisconsin Medical School, Madison, Wisconsin, 53706; and Ben May Institute, University of Chicago, Chicago, Illinois 60637

Introduction

Palytoxin (PTX), from a coelenterate of the genus palythoa, is one of the most potent marine toxins known (LD_50 = 0.15 μg/kg i.v. in mice). This large hydrophilic polyalcohol (C_129H_223N_3O_54; 2,680 mol wt) is a powerful vasoconstrictor and tumor promoter (Uemura and Hirata, 1989). PTX's high potency and wide variety of effects are believed to be caused by an increase in alkali ion permeability through the cell membrane, but the cellular receptor for PTX has not yet been clearly identified.

Several researchers have assumed, based on the effects of PTX on ATP-hydrolysis and the interaction of ouabain with NaK-ATPase, that NaK-ATPase is the receptor for PTX (Ishida et al., 1983; Böttinger et al., 1986; Grell et al., 1988). However, the concentration of PTX required for inhibition of fragmental (nonvesicular) NaK-ATPase is 10,000 times greater than that required to produce biological effects (Böttinger et al., 1986; Muramatsu et al., 1988; Wattenberg et al., 1989).

Experimental Procedures

We measured the Na+- or Rb+- influx into the proteoliposomes (PLs) in the rightsideout form of the NaK-ATPase PLs to mimic PTX action in vivo with an in vitro system. Electric eel NaK-ATPase (specific activity 20 ± 3 μmol ADP/min per mg protein at 37°C) was reconstituted into the phospholipid–cholesterol liposomes as reported previously[1] (Yoda et al., 1984; Yoda and Yoda, 1990). The inside (cytoplasmic) medium contained 20 mM NaCl, 80 mM KCl, and 10 mM ATP with buffer A (5 mM CDTA and 25 mM histidine–arginine buffer, pH 7.35). The PLs were suspended (30–40 mg phospholipid/ml) in a solution containing 100 mM choline-HCl in buffer A.

Unless otherwise indicated, the reaction mixture consisted of a 1/5 volume of the PL suspension, 40 mM NaCl, 60 mM choline-HCl, 10 mM MgCl_2, 0.5 mM boric acid, 0.1% BSA, 1 μM CCCP, 20 μM A23l87, and buffer A. The reaction was initiated by adding Mg2+ and A_23187 (a Mg2+, Ca2+ ionophore), which induced Mg2+ penetration into the PLs. Boric acid and BSA were used as the activator (Böttinger et al., 1986; Grell et al., 1988) and stabilizer for PTX, respectively. CCCP (a proton ionophore) was used to clamp the membrane potential difference across the lipid bilayer (Yoda and Yoda, 1990). This voltage clamp enabled us to measure the ion flux through a channel.

After incubating for the indicated period at 30°C, the reaction was quenched with a 0.2 M sucrose–3 mM ouabain mixture. The reaction mixture was then promptly passed through a Dowex 50 (Tris-form, 200 mesh) column (1 ml column volume), and the radioactivity of the eluent was counted.

Results and Discussion

As shown in Fig. 1 and Table I, addition of 20 pM or 40 pM PTX enhanced the overall influx of Na+ by 30%.[2] The enhancement of Na+-influx by PTX was not observed in the absence of ATP, A_23187, or NaK-ATPase itself. These results indicate that PTX can induce Na+-influx into PLs containing the NaK-pump in an ATP-dependent, Na+-Na+ exchange mode. Pretreatment of the PLs with ouabain completely inhibited the PTX induction of the Na+ influx. The Na+ influx (It) into the PLs results from ouabain-sensitive transport via the NaK-pump (Ip) as well as ouabain-insensitive diffusion through the membrane (Id). Thus, the following equation completely described our system:

Figure 1. Palytoxin enhanced Na+-influx into PLs. The values obtained from two independent sets of experiments were normalized using the results of a 6-min incubation without PTX as control values. The control values of the Na+-influx for the two experiments were 1.70 and 1.55 nmol/min per 0.1 ml PLs, respectively. Each point represents the mean ± standard deviation from triplicate assays.

Under the present experimental conditions, the diffusion is negligible at the beginning of the PTX reaction and in the presence of PTX. The rate of diffusion into the PLs (ΔId/Δt) then increased as the PTX reaction progressed. As shown in Fig. 2, the rate of Na+ -diffusion was enhanced by 20 pM PTX and reached a maximum after 5 min at 30°C, although it remained near zero for more than 20 min in the absence of PTX, as described above. This ouabain-insensitive Na+-influx is presumably due to the formation of a new sodium channel by PTX as observed in vivo (Muramatsu et al., 1988; Wattenberg et al., 1989). The ouabain-sensitive pump activity decreased gradually to zero in 15 min in the presence of 20 pM PTX; this inactivation was not observed in the absence of PTX, however (see Fig. 1). If PTX converts the NaK-pump into a Na+ channel, the channel formation should coincide with pump inactivation. However, the PTX inhibition of pump activity was slower than the new channel formation (Fig. 2). A similar time discrepancy was also observed when 5 pM (instead of 20 pM) PTX was used. These kinetic differences for the Na+-influx and pump inactivation suggest that the inactivation of the active transport and creation of the new Na+ channel occur via different mechanisms.

TABLE I
Na+ Influx into Proteoliposomes Required an Activated NaK-ATPase

The experiments were performed as described in the text. The reaction time of experiments 1 and 6 was 6 min; for the other experiments it was 30 min.
*The relative Na+ -influx values were calculated against the control (the same as experiment 1, no PTX). The control values were between 1.05 and 1.77 nmol Na+/0.1 ml PLs per min. Each point represents the mean ± standard deviation from triplicate assays.
‡The medium was the same as the reaction medium cited in text.
§Experiments 3 and 4: The PLs lacking ATP or ATPase were prepared with the same procedures without ATP or NaK-ATPase. The assay of the Na+ -influx was started by adding PLs into the medium, and compared with the value for the PLs prepared with ATP or enzyme.
║Experiment 5: The ouabain treatment was performed in the standard medium containing ouabain and unlabeled NaCl instead of [22Na]NaCl for 3 min at 30°C. Then 1/10 volume of the standard medium containing [22Na]NaCl was added to initiate the assay.

Figure 2. PTX changed the rates of sodium diffusion and pump activity in the PLs. These time courses illustrate an increase in the rate of sodium diffusion and a decrease in the rate of active sodium transport by the NaK-pump after treatment with 20 pM PTX. The closed symbols indicate average pump activity (ouabain-sensitive Na+ -influx for 3 min from the indicated time), while the open symbols indicate the average diffusion rate (ouabain-insensitive Na+ -influx for 3 min from the indicated time). These points were derived from the results obtained in three independent sets of experiments. Within each experiment, values were normalized to the amount of total sodium influx between 0 and 9 min. Each point represents the mean ± standard deviation from three or six assays.

Figure 3. PTX inhibition of Rb+ uptake. Media in both sides of the PLs were the same as described in the text, except for the use of RbCl instead of KCl and the addition of 1 mM RbCl in the extracellular medium. Each point represents the mean ± standard deviation from triplicate assays. The specific activity of the [86Rb]RbCl was 150 cpm/nmol. The extracellular medium contained 1 mM [86Rb]RbCl with (Δ) or without (O) 20 pM PTX. 0.3 mM ouabain was added at the point marked by the arrow and the Rb+ uptake was measured after 3 min of continuing the incubation (closed symbols). Other experimental conditions were the same as described in Fig. 1.

To obtain a more accurate dose response for the PTX inhibition of pump activity, we monitored the uptake of Rb+ under conditions where it was transported by the Na+-K+ exchange mode of the pump. Although the high K+ concentration in the outside medium interfered with the PTX effect in vivo (Böttinger et al., 1986), the PTX-dependent enhancement of Na+ -influx in the PLs was still observed in the presence of 1 mM K+ (Table I). At this low K+ concentration we could assay the Na+-K+ exchange due to the NaK-pump. In this system, no diffusion of Rb+ from the outside medium was observed in the presence of PTX because the Rb+ concentration on the inside was much higher than that outside. PTX gradually inhibited the Na+-K+(Rb+) exchange, as shown in Fig. 3. When the Na+-K+ exchange was measured after 10 min in the presence of PTX, the ID_50 of PTX was ~ 30 pM (Fig. 4). This value is ~ 10,000 times lower than the reported ID_50 for inhibition of the fragmental NaK-ATPase (Ishida et al., 1983) and is within the physiological range of PTX action.

Figure 4. Dose response for inhibition of Rb+ uptake by PTX. The PLs were incubated with the indicated PTX concentration for 10 min at 30°C in the same extracellular medium as in Fig. 3, except that 1 mM unlabeled RbCl was used instead of [86Rb]RbCl. At Vio volume of the extracellular medium containing [86Rb]RbCl was then added to start the Rb+ uptake assay. After a 5-min incubation the reaction was quenched with 0.2 M sucrose-3 mM ouabain solution and the [86Rb]Rb+ uptake was measured.

The results obtained strongly suggest that the NaK-pump is the receptor for PTX in vivo. The experiments using PLs demonstrate that physiological (picomolar) concentrations of PTX are sufficient for the creation of a new sodium channel and the inhibition of ion transport in both the Na+-Na+ and Na+-K+ exchange mode of the NaK-pump. The time courses of both reactions suggest that PTX may induce two independent reactions: channel formation and inhibition of ion transport. Since channel formation occurs more rapidly than the inhibition of the NaK-pump, PTX may at first be anchored on the specific site located on the extracellular side of the NaK-ATPase molecule. It then could form a channel either nearby or on the interface between NaK-ATPase and the lipids. The inhibition of the NaK-pump may be caused by an independent and subsequent interaction with a second site.

Although PTX clearly inhibits ion transport by the NaK-pump in picomolar concentrations, more than 10 nM was necessary to inhibit ATP-hydrolysis in the fragmental NaK-ATPase. This discrepancy between the PLs and the fragmental enzyme could be due to uncoupling between ion transport and ATP-hydrolysis. Another possibility is that the membrane potential plays an important role in the PTX-NaK-pump interaction, as observed previously (Yoda and Yoda, 1990). This question remains unsolved at the moment.

Acknowledgments

This work was supported by NIH grants HL-16549 (to A. Yoda) and CA-315541 (to M. R. Rosner).

References

Böttinger, H., L. Beress, and E. Harbermann. 1986. Involvement of (Na+-K+)-ATPase in binding and actions of palytoxin on human erythrocytes. Biochimica et Biophysica Acta. 861:165–176.

Grell, E., E. Lewitzki, and D. Uemura. 1988. Interaction between palytoxin and purified NaK-ATPase. In The Na+, K+-pump. Part B: Cellular Aspects. J. C. Skou, J. G. Nørby, A. B. Maunsbach, and H. Esmann, editors. Alan R. Liss, Inc., New York. 393–400.

Ishida, Y., K. Takagi, M. Takahashi, N. Satake, and S. Sibata. 1983. Palytoxin isolated from marine coelenterates. Journal of Biological Chemistry. 258:7900–7902.

Muramatsu, I., M. Nishio, S. Kigoshi, and D. Uemura. 1988. Single ionic channel induced by palytoxin in guinea-pig ventricular myocytes. British Journal of Pharmacology. 93:811–816.

Uemura, D., and Y. Hirata. 1989. Antitumor polyethers from marine animals. In Studies in Natural Products Chemistry. Atta-ur-Rahman, editor. Elsevier Science Publishers Ltd., Essex, UK. 377–401.

Wattenberg, E. V., P. L. Neil, H. Fujiki, and M. R. Rosner. 1989. Palytoxin down-modulates the epidermal growth factor receptor through a sodium-dependent pathway. Journal of Biological Chemistry. 264:213–219.

Yoda, A., A. W. Clark, and S. Yoda. 1984. Reconstitution of (Na+-K+)-ATPase proteoliposomes having the same turnover rate as the membranous enzyme. Biochimica et Biophysica Acta. 778:332–340.

Yoda, A., and S. Yoda. 1990. CCCP activation of the reconstituted NaK-pump. Journal of Membrane Biology. 117:153–161.

Palytoxin Forms Ion Channels through Na,K-ATPase

Soung Y. Kim, Chau H. Wu, and Lászlo Béress

Department of Pharmacology, Northwestern University Medical School, Chicago, Illinois 60611; and Abteilung Toxikologie, Christian-Albrechts University, D-2300 Kiel, Germany

Introduction

Palytoxin is a potent marine toxin isolated from coelenterates of the genus Palythoa. It has a wide variety of actions on the mammalian system, including K+ efflux and hemolysis of red blood cells, contraction of cardiac, skeletal, and smooth muscles, inositol phosphate production, norepinephrine release, and non-TPA-type tumor promotion (for review, see Habermann, 1989). Ouabain and other cardiac glycosides are known to antagonize palytoxin action. Böttinger et al. (1986) have found that ouabain and radiolabeled palytoxin compete for a specific binding site in human erythrocytes. They have also found that palytoxin binding is inhibited by high K+ and promoted by ATP, though Mg2+ was not required. These observations have led to the hypothesis that the Na,K-ATPase is the site of palytoxin action.

Palytoxin has also been found to depolarize all cells investigated. Patch clamp studies on cardiac cells have shown that playtoxin forms a new type of channel which is selective for Na+ and other small monovalent cations (Ikeda et al., 1988; Muramatsu et al., 1988). We have found palytoxin-induced single channels in frog red blood cells, and furthermore, ouabain inhibits the formation of these channels.

Methods

Palytoxin from Palythoa caribaeorum was dissolved in distilled water and kept as stock solution at –60°C. Final dilutions with the desired solutions were made immediately before use.

Fresh red blood cells from frog (Rana pipiens) were collected in frog Ringer solution composed of (in mM): 100 NaCl, 5.4 KCl, 1.8 CaCl_2, and 3 HEPES, pH 7.3 and 300 mosM. The erythrocytes were continuously perfused with the solution during patch clamping. In some experiments, NaCl in the patch pipette was replaced with choline chloride or NH_4Cl. Microelectrodes of 10 MΩ were used to form cell-attached patches according to the method of Hamill et al. (1981). Patch pipettes contained palytoxin in frog Ringer solution with or without ouabain. Currents were filtered at 0.2 kHz (4302; Ithaco Inc., Ithaca, NY) and recorded with a patch clamp amplifier (EPC5; List-Electronic, Darmstadt, Germany) on a digital oscilloscope (4094; Nicolet Instrument Corp., Madison, WI). Experiments were conducted at room temperature (20–23°C).

Figure 1. Palytoxin-induced single channel currents from a cell-attached patch of frog red blood cell. Current traces are records at increasing time from the appearance of the first current step. The patch was held at –50 mV from the resting potential (r .p. = –10 mV to –20 mV). Dashed lines represent the holding current (closed channel) level. Downward deflection indicates inward current through an open channel.

Results and Discussion

We examined palytoxin action on frog red blood cells at the single channel level. Fig. 1 shows single channel currents induced by palytoxin in a cell-attached patch held at –50 mV from the resting potential. Several minutes after contact of the patch pipette with the membrane, a current step was observed. Additional current steps of the same amplitude appeared over time. The rate at which they appeared seemed to depend on the concentration of palytoxin. At 10–10 M palytoxin the first current step appeared 0–5 min after contact of the patch pipette with the membrane; at 10–14 M the channel current appeared at 5–10 min; and at 10–16 M it appeared at 20–30 min. When NaCl in the patch pipette was replaced with choline chloride, no current steps were observed. Substitutions with NH_4Cl, however, produced single channel currents of about the same amplitude.

Figure 2. Current-voltage relation of palytoxin-induced single channels. Data points were obtained from a cell-attached patch with 10–14 M palytoxin and Na+ as the major cation in the patch pipette. They were fit by a straight line to give a conductance of 9.6 pS and a reversal potential at –10 mV for the induced channels.

The amplitude of the current steps changed linearly with voltage in the range of –80 to –10 mV from the resting potential (Fig. 2). The single channel conductance calculated from the slope of the current-voltage relation was 9.6 pS. The reversal potential, estimated by extrapolation, was at –10 mV. These channel characteristics were similar to those of palytoxin-induced single channels in cardiac cells found by Ikeda et al. (1988) and Muramatsu et al. (1988).

Ouabain at concentrations equal to that of palytoxin did not affect the palytoxin-induced channel currents. At higher concentrations, however, it effectively inhibited channel formation. With 10–5 M ouabain and 10–14 M palytoxin, current steps were never observed over the duration of the experiment, which was on average 30 min (Fig. 3). These results agree with the binding data that ouabain and palytoxin compete for the same binding sites. Böttinger et al. (1986) have found that in human erythrocytes the Kd for ouabain is 2.6 × 10–9 M and that for palytoxin is 2 × 10–11 M. The 100-fold lower affinity of ouabain can account for the greater ouabain concentration required for inhibition of channel formation when applied simultaneously with palytoxin.

Figure 3. Ouabain inhibition of palytoxin-induced single channel currents. The cell-attached patch was held at –50 mV from the resting potential. Channel currents were never observed with 10–5 M ouabain and 10 –14 M palytoxin in the patch pipette.

Ouabain antagonism of palytoxin action suggests that the Na,K-ATPase is the site of palytoxin action. The present results suggest that the interaction between Na,K-ATPase and palytoxin creates ion channels in cell membranes. Palytoxin may be converting the Na,K-ATPase from a pump into an open channel, or alternatively, the channel may be formed adjacent to the Na,K-ATPase molecule.

Acknowledgments

This work was supported by a Predoctoral Fellowship (F31 MH10004) from NIMH to S. Y. Kim.

References

Böttinger, H., L. Béress, and E. Habermann. 1986. Involvement of Na+,K+ -ATPase in binding and actions of palytoxin on human erythrocytes. Biochimica et Biophysica Acta. 861:165–176.

Habermann, E. 1989. Palytoxin acts through Na,K-ATPase. Toxicon. 27:1171–1187.

Hamill, O. P., A. Marty, E. Neher, B. Sakmann, and F. J. Sigworth. 1981. Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflügers Archiv. 391:85–100.

Ikeda, M., K. Mitani, and K. Ito. 1988. Palytoxin induces a nonselective cation channel in single ventricular cells of rat. Naunyn-Schmiedeberg's Archives of Pharmacology. 337:591–593.

Muramatsu, I., M. Nishio, S. Kigoshi, and D. Uemura. 1988. Single-ion channels induced by palytoxin in guinea-pig ventricular myocytes. British Journal of Pharmacology. 93:811–816.

Properties of the Ion Channel Formed in Bilayer Lipid Membranes by α-Subunits of (Na+ + K+)-ATPase

Galina D. Mironova and Nina I. Zolotarjova

Institute of Biological Physics, Academy of Sciences of the USSR, Pushchino, Moscow Region, USSR

Introduction

Despite the importance of understanding sodium pump regulation, little is known about its regulation at the molecular level. Currently, much evidence supports the view that all the ion-transporting systems are channels. Using a new patch-clamp technique this can be shown directly (Conti et al., 1975; Geletyuk and Kazachenko, 1989). However, this technique allows detection of only passive channels, when the rate of transport is ~ 10^8 ions/s. In the case of active transport (e.g., the sodium pump), the rate of ion transport is five orders of magnitude less, and the methods available do not allow us to detect the ion transport through this channel.

A number of authors have suggested that the sodium pump contains three components: a large, nonselective channel, an ion-selective gate, and an energy transducer (Shamoo and Goldstein, 1977). In this paper we present data on the characteristics of an isolated ion channel from (Na+ + K+)-ATPase. We have developed a technique that makes it possible to isolate the native cation-transporting protein from microsomal membranes (Mironova and Mirsalikhova, 1981). The advantage of this method is that the isolation procedure does not require detergents, which not only disturb the native protein but also complicate the assay with the bilayer lipid membrane (BLM). The reconstitution of isolated protein in a lipid bilayer showed that it forms the monovalent cation-selective channels that are inhibited by the ouabain. The protein incorporated in BLM also possesses ouabain-inhibited ATPase activity (Mironova et al., 1986).

Results

This work, using immunoblotting analysis with monospecific polyclonal and monoclonal antibodies, identified the ion-transporting protein isolated from microsomes by water–alcohol extraction as the α-subunit of (Na+ + K+)-ATPase (Fig. 1). Polyclonal antibodies were raised in rabbits against α-subunit, which was isolated using standard procedures from purified (Na+ + K+)-ATPase. The isolated protein is homogeneous according to SDS-PAGE, and there are no other protein bands on the gel. The molecular mass of this protein is 96 kD and it reacts with α-subunit-specific antisera. Since this band and the entire gel do not stain with Schiff reagent, we conclude that there is no β-subunit of the (Na+ + K+)-ATPase in the isolated protein preparation. Purified (Na+ + K+)-ATPase α-subunit obtained by water–alcohol extraction formed conductance channels in BLM (Fig. 2). The channels were selective for monovalent cations and sensitive to ouabain, ATP, and p-nitrophenyl phosphate (Mironova and Mirsalikhova, 1981; Mironov et al., 1984). The main state conductance of this channel was 40 pS in 0.1 M NaCl (Fig. 2), and the subconductance state was 8–12 pS (Mironova et al., 1986). This latter value is close to the minimal conductance step of 4–8 pS (in 0.1 M KCl) obtained for a wide variety of ion channels by the patch-clamp method (Geletyuk and Kazachenko, 1989). As shown by Reinhardt et al. (1984) and Kumazawa et al. (1986), sodium pump consisting of both α- and β-subunits formed ouabain-sensitive ion channels in BLM with a minimum conductance of 40 pS in 0.1 M NaCl. Since a similarity exists between channels formed by α-subunit only and channels formed by the whole enzyme, and because these channels possess the same properties as the whole enzyme, we further conclude that in the cell membrane the α-subunit also forms the channel for Na+ and K+ ion transport.

Figure 1. Identification of the α-subunit of (Na+ + K+)-ATPase. (A) 7.5% SDS-PAGE. (B) Western blot analysis with monospecific polyclonal antibodies.

The conductance of the channels formed by α-subunits of the (Na+ + K+)-ATPase in BLM was characterized by a pronounced voltage dependence. The current–voltage characteristics of the modified membrane are normally asymmetric when protein is added to one side of the membrane (Fig. 3). The conductance properties of these rectifying cation channels show the following: (a) that the channel formed by the α-subunit has an ion-selective gate, and (b) that vectorial protein incorporation has been achieved. This vectorial (Na+ + K+)-ATPase incorporation in BLM has been shown previously (Last et al., 1983).

Figure 2. Conductivity channels of BLM modified with 1 μg/ml α-subunit of (Na+ + K+)-ATPase.

The dependence between the conductance of the channel and the concentration of the added protein is sigmoid (data not shown). This type of conductance dependence on protein concentration points out that more than one molecule of the α-subunit forms the channel. The analysis of current–voltage characteristics of these conductance channels showed that when the potential is > 100 mV, the channels are mostly closed (Fig. 3). As we can see from Fig. 2, a single channel at a potential of ~ 50 mV is in an open state. When the membrane potential is increased (at 75 mV) the current going through the single channel is increased but the channel tends to close, and at higher potentials the channels spend more time in the closed state than in the open state. When we added a larger amount of protein we observed that the number of channels and total conductance of the modified membrane were increased with increasing potential from 0 to 50 mV. However, beyond 50 mV the number of conductance channels is decreased, and at ~ 75–100 mV most of the channels are closed (Fig. 4). The conductance of the single channel, formed in BLM by α-subunits of (Na+ + K+)-ATPase was not always characterized by a pronounced voltage dependence (Fig. 5). Thus, the present data indicated that the incorporated α-subunits of (Na+ + K+)-ATPase formed cation-selective, ouabain-sensitive, voltage-dependent, ion-gated channels in BLM.

Figure 3. Current–voltage characteristics of the BLM conductance with the incorporated (Na+ + K+)-ATPase α-subunit. The aqueous medium contained 30 mM imidazole-HCl buffer (pH 7.0), 10 mM KCl, and 100 mM NaCl. Protein was added to the cis side (A) and the trans side of the membrane (B) to a final concentration of 5 μg/ml. The sign of potential is denoted for the cis compartment. The rate of voltage change was 20 mV/min.

Figure 4. Effect of potential on the conductance of BLM modified with (Na+ + K+)-ATPase α-subunit (3 μg/ ml). The conditions are the same as in Fig. 2. A and B are different membranes.

Discussion

Earlier Anner (1985) assumed that the three functional components of the (Na+ + K+)-ATPase molecule (i.e., the channel, gating unit, and catalytic part) can be uncoupled and recoupled under appropriate experimental conditions. Isolation of the α-subunits probably led to uncoupling the channel from the energy transducer. This allowed us to study the properties of the passive channel. Kim et al. (1990), using patch-clamp techniques on frog red blood cells, showed that palytoxin may convert the (Na+ + K+)-ATPase into an ouabain-sensitive ion channel with a conductance of 10 pS. The palytoxin treatment might also lead to uncoupling of the channel from the energy transducer.

Study of the passive channel properties shows that the membrane potential influences the function of BLM-reconstituted α-subunit of (Na+ + K+)-ATPase. The oligochannels are voltage dependent and at potentials > 100 mV they are mostly closed. Such behavior of ion channels might play an essential role in preventing membrane hyperpolarization.

As can be seen from Fig. 5, the conductance of the single channel was not always characterized by a pronounced voltage dependence. Currently, using the patch-clamp method, much evidence supports the view that oligochannels have a greater degree of potential dependence than single channels, and that oligochannels correspond to the physiologically observed channels (Fox, 1987; Hymel et al., 1988). The data presented here are in agreement with a hypothesis that the oligomerization of channel subunits has an important role in the acquisition of their voltage sensitivity (Hymel et al., 1988). We suggest that clustering and disaggregation of sodium pump channels or molecules to protochannels has an important role in the regulation of this enzyme.

Figure 5. Current–voltage relationships of a single channel. The conditions are the same as in Fig. 3. Protein was added to both sides of the membrane. Concentration, 1 μg/ml.

Voltage dependence of the channel might be due to specific properties of the structure of the protein and the character of its interaction with lipids. It was shown (Lakos et al., 1990) that changes of transmembrane potential are accompanied by local alterations in membrane lipid dynamics and structure and can lead to conformational changes of various membrane proteins. Recently it was also shown that electrical stimulation increases the (Na+ + K+)-ATPase activity 2,000-fold (Blum et al., 1990). This burst of activity cannot be explained by changes in internal Na+ concentration alone acting on membrane pump, but also depends, as Blum et al. (1990) suggested, on recruitment of additional pumps. We propose that the opening of the channels we have described may be essential for the activation of (Na+ + K+)-ATPase after electrical stimulation, when the membrane potential becomes low.

References

Anner, B. M. 1985. Interaction of (Na+ + K+)-ATPase with artificial membranes. II. Expression of partial transport reactions. Biochimica et Biophysica Acta. 832:335–353.

Blum, H., S. Nioka, and R. G. Johnson. 1990. Activation of the (Na+ + K+)-ATPase in Narcine brasiliensis. Proceedings of the National Academy of Sciences, USA. 87:1247–1251.

Conti, F., L. F. De Felice, and E. Wanke. 1975. Potassium and sodium ion current noise in the membrane of the squid giant axon. The Journal of Physiology. 248:45–82.

Fox, J. A. 1987. Ion channel subconductance states. The Journal of Membrane Biology. 97:1–8.

Geletyuk, V. I., and V. N. Kazachenko. 1989. Single potential-dependent K+ channels and their oligomers in molluscan glial cells. Biochimica et Biophysica Acta. 981:343–350.

Hymel, L., J. Striessnig, H. Glossman, and H. Shindler. 1988. Purified skeletal muscle 1,4-dihydropyridine receptor forms phosphorylation-dependent oligomeric calcium channels in planar bilayers. Proceedings of the National Academy of Sciences, USA. 85:4290–4294.

Kim, S. Y., C. H. Wu, T. Narahashi, and L. Berress. 1990. Modification of Na+,K+ -ATPase by palytoxin. Journal of General Physiology. 96:74a-75a. (Abstr.)

Kumazawa, N., T. Tsujomoto, and Y. Fukushima. 1986. Influence of voltage and ATP on ion channel of (Na+ + K+)-ATPase incorporated into solvent-free phospholipid planar bilayers. Biochemical and Biophysical Research Communications. 136:767–772.

Lakos, Z., B. Somosyi, M. Balars, J. Matko, and S. Damjanovich. 1990. The effect of transmembrane potential on the dynamic behavior of cell membranes. Biochimica et Biophysica Acta. 1023:41–16.

Last, T., M. L. Gantzer, and C. D. Tyler. 1983. Ion-gated channel induced in planar bilayers by incorporation of (Na+ + K+)-ATPase. The Journal of Biological Chemistry. 258:2399–2404.

Mironov, G. P., T. A. Mirzabekov, N. I. Bocharnikova, N. M. Mirsalikhova, and G. D. Mironova. 1984. Ionic channels formed in bilayer lipid membranes by large subunits of Na+,K+ -ATPase and activated by ATP and p-nitrophenyl phosphate. Biofizika. 29:688–690.

Mironova, G. D., and N. M. Mirsalikhova. 1981. Channel-forming protein of Na+,K+ -ATPase preparation of the medullary layer of pig kidney. Biofizika. 26:731–734.

Mironova, G. D., N. I. Bocharnikova, N. M. Mirsalikhova, and G. P. Mironov. 1986. Ion-transporting properties and ATPase activity of (Na+ + K+)-ATPase large subunit incorporated into bilayer lipid membranes. Biochimica et Biophysica Acta. 861:224–236.

Reinhardt, R., B. Lindemann, and B. M. Anner. 1984. Leakage-channel conductance of single (Na+ + K+)-ATPase molecules incorporated into planar bilayers by fusion of liposomes. Biochimica et Biophysica Acta. 774:147–150.

Shamoo, A. E., and D. A. Goldstein. 1977. Isolation of ionophores from ion transport systems and their role in energy transduction. Biochimica et Biophysica Acta. 472:13–53.

Lack of K+ Stimulation of the Na-ATPase Activity of Red Cell Ghosts from the LK Sheep Ovis aries

Fulgencio Proverbio, José A. Duque, and Reinaldo Marín

Centro de Biofísica y Bioquímica, Instituto Venezolano de Investigaciones Científicas, Caracas, Venezuela

Introduction

The Na,K-ATPase is an enzyme present in the plasma membrane of practically every animal cell. However, its activity can be very low or even absent in the membranes of red blood cells of several mammals such as sheep (LK), cows (LK), cats, dogs, and ferrets (Chan et al., 1964; Gupta et al., 1974). Consequently, these cells have Na+ and K+ concentrations very similar to those of the blood plasma (Bernstein, 1954; Tosteson and Hoffman, 1960). The LK mammal red cells possess the ability to volume regulate, which has been associated with a cellular mechanism of Na+ efflux (Parker et al., 1975). The LK sheep Ovies aries, like other LK mammals, maintains a very low gradient of Na+ and K+ across its red blood cell membranes. In fact, it has 147 ± 8 mM Na+ and 4.8 ± 0.3 mM K+ in its blood plasma, and 110 ± 7 mM Na+ and 13 ± 1 mM K+ in its red cells.

The ci/co ratio for Cl– in these red cells is 0.72. Considering that they are highly permeable to Cl–, a transmembrane potential of 8.8 mV (negative inside) is expected. This potential, nearly identical to the potassium equilibrium potential, enhances the inward driving force on Na+. These circumstances generate a net passive influx of Na+ which must be actively extruded to maintain steady state. Under these physicochemical conditions, the cells need ~ 400 cal to extrude 1 mol of Na+.

Consequently, and considering that a K+ -independent, active Na+ efflux has been measured in LK mammal red cells (Parker et al., 1975), we examined the possibility of the existence of a K+ -independent, Na+ -ATPase activity in these cells, similar to the one described for other mammalian tissues such as kidney and small intestine (Proverbio and del Castillo, 1981; del Castillo et al., 1982; Luppa and Müller, 1982; del Castillo and Robinson, 1985; Marín et al., 1985a, b; Proverbio, 1985; Orsenigo et al., 1988; Tosco et al., 1988).

Results and Conclusions

Table I shows the ATPase activity of the red cell ghosts measured in the presence of Mg2+ + ouabain, Mg2+ + Na+ + ouabain, Mg2+ + Na+ + K+, and Mg2+ + Na+ + K++ouabain. It is evident that in addition to a basal Mg2+ -dependent ATPase activity, there is a Na+ -stimulated activity that is ouabain-insensitive and K+ -independent. On the other hand, it may also be seen that K+ does not exert any further stimulation of the ATPase activity when added together with Na+. This may be taken as an indication that these membranes do not possess Na,K-ATPase protomers.

TABLE I
ATPase Activity of LK Sheep Red Cell Ghosts Measured in the Presence of Mg2+ +Ouabain, Mg2+ + Na+ +Ouabain, Mg2+ + Na+ + K+, and Mg2+ + Na+ + K+ +Ouabain

Final concentrations (in mM): 0.2 Mg2+, 0.4 ATP, 100 Na+, 0.24 EGTA. Ouabain, when present, 0.1 mM. The values are expressed as the mean ± SE (n = 6).

Besides its ouabain and K+ refractoriness, the Na-ATPase activity described for mammalian tissues is inhibited by diuretics such as ethacrynic acid, furosemide, and bumetanide. Table II shows the effect of these drugs on the Na-ATPase activity of the LK sheep red cell ghosts. All of them, at the indicated concentrations, inhibited nearly 100% the activity of the Na-ATPase.

Whether this Na-ATPase is an isoform of the Na,K-ATPase or a different ATPase has yet to be determined. However, it is ouabain-insensitive, does not require K+ to work, and is inhibited by furosemide, ethacrynic acid, and bumetanide, poor inhibitors of the Na,K-ATPase under our assay conditions.

These characteristics have been taken by several authors to differentiate between the Na,K-ATPase and a Na-ATPase, or in some cases a monovalent cation-stimulated ATPase, which has already been described in many organisms and tissues. Furthermore, a ouabain-insensitive, Na-stimulated ATPase activity has been described for membranes from mammals, birds, iguanas, fish, toads, squid, shrimp, and crabs. It has also been shown in plasma membranes from kidney cortex, small intestine, liver, brain cortex, and gills (see review by Proverbio et al., 1990). It is interesting that the Na-ATPase is inhibited by such agents as furosemide, ethacrynic acid, and bumetanide, known inhibitors of the Na+,K+,2Cl– cotransport system. If there is any relationship between these two systems, it has yet to be determined.

TABLE II
Effect of Ethacrynic Acid, Furosemide, and Bumetanide on the Na-ATPase Activity of LK Sheep Red Cell Ghosts

Incubation conditions

ATPase activity

nmol Pi/mg protein per min

Control

0.43 ± 0.01

+0.5 mM ethacrynic acid

0.04 ± 0.02

+0.5 mM furosemide

0.06 ± 0.01

+50 μM bumetanide

0.07 ± 0.02

Final concentrations (in mM): 0.2 Mg2+, 0.4 ATP, 100 Na+, 0.24 EGTA. All the experiments were carried out in the presence of 0.1 mM ouabain. The values are expressed as the means ± SE (n = 6).

Acknowledgments

We would like to thank Mrs. Teresa Proverbio for technical assistance.

References

Bernstein, R. E. 1954. Potassium and sodium balance in mammalian red cell. Science. 120:459–460.

Chan, P. C., V. Calabrese, and L. S. Theil. 1964. Species differences in the effect of sodium and potassium ions on the ATPase of erythrocyte membranes. Biochimica et Biophysica Acta. 69:424–426.

del Castillo, J. R., and J. W. L. Robinson. 1985. Na+ -stimulated ATPase activities in basolateral plasma membranes from guinea-pig small intestinal epithelial cells. Biochimica et Biophysica Acta. 812:413–422.

del Castillo, J. R., R. Marín, T. Proverbio, and F. Proverbio. 1982. Partial characterization of the ouabain-insensitive, Na+ -stimulated ATPase activity of kidney basal lateral plasma membranes. Biochimica et Biophysica Acta. 692:61–68.

Gupta, J. D., V. J. Peterson, and J. D. Harley. 1974. Erythrocytic ouabain-sensitive and ouabain-insensitive adenosine triphosphatase in various mammalian species. Comparative Biochemistry and Physiology. 47A:1123–1126.

Luppa, D., and F. Müller. 1982. Nachweis einer Na+ -aktivierbaren, durch Furosemid hemmbaren ATPase-Aktivität in der Dünndarmmukosa der Ratte. Acta Biologica et Medica Germanica. 41:1037–1043.

Marín, R., T. Proverbio, and F. Proverbio. 1985a. Characterization of the Na+ + K+ -ATPase activity of basolateral plasma membranes of kidney proximal tubular cells from young and old rats. Biochemical Pharmacology. 34:4197–4201.

Marín, R., T. Proverbio, and F. Proverbio. 1985b. Active sodium transport in basolateral plasma membrane vesicles from rat kidney proximal tubular cells. Biochimica et Biophysica Acta. 814:363–373.

Orsenigo, M. N., M. Tosco, G. Esposito, and A. Faelli. 1988. Ouabain-insensitive Na-ATPase activity in the basolateral membrane from rat jejunum. International Journal of Biochemistry. 20:1411–1415.

Parker, J. C., P. S. G. Gitelman, and D. L. Leonard. 1975. Role of calcium in volume regulation by dog red blood cells. Journal of General Physiology. 65:84–96.

Proverbio, F. 1985. Expulsión de Na+ en células renales de mamífero. Archivos Venezolanos de Farmacología y Terapéutica. 4:361–370.

Proverbio, F., and J. del Castillo. 1981. Na+ -stimulated ATPase activities in kidney basal-lateral plasma membranes. Biochimica et Biophysica Acta. 646:99–109.

Proverbio, F., R. Marín, and T. Proverbio. 1991. The ouabain-insensitive sodium pump. Comparative Biochemistry and Physiology. In press.

Tosco, M., M. N. Orsenigo, G. Esposito, and A. Faelli. 1988. Ouabain-insensitive active sodium transport in rat jejunum: evidence from ATPase activities, Na uptake by basolateral membrane vesicles and “in vitro” transintestinal transport. Cell Biochemistry and Function. 6:155–164.

Tosteson, D. C., and J. F. Hoffman. 1960. Regulation of cell volume by active cation transport in high and low potassium sheep cells. Journal of General Physiology. 44:169–194.

Ouabain, Amiloride, and Gossypol Inhibit the Channels Formed by α-Subunit of Na+,K+ -ATPase in Bilayer Lipid Membranes

Nina I. Zolotarjova, Larisa V. Ivkova, Galina D. Mironova, Nailya M. Mirsalikhova, and Svetlana F. Sokolova

Institute of Biological Physics, Academy of Sciences of the USSR, and Institute of Physiology, Academy of Sciences of the Uzbek SSR, USSR

Introduction

We have previously reported that the α-subunit of Na+,K+ -ATPase forms conductance channels in bilayer lipid membranes (BLM), the channels selective for monovalent cations (Mironova et al., 1986).

We now present data on the influence of a number of inhibitors of Na+,K+ -ATPase (ouabain, amiloride, gossypol) on the ion-transporting properties of α-subunit incorporated into BLM. The effects of gossypol on the conductance of the channels and ATPase activity of Na+,K+ -ATPase have been compared. Such inhibitors are effective tools for studying the structure-function relations of the Na+,K+ -ATPase. Moreover, some of these inhibitors have significant pharmacological applications.

Figure 1. Channel conductance of BLM modified with α-subunit of Na+,K+ -ATPase (1 μg/ ml) aqueous phase contained l0mM Tris-HCl (pH 7.5), 100 mM NaCl, and 10 mM KCl. Membrane potential, 15 mV.

Methods

The α-subunit of Na+,K+ -ATPase was isolated from microsomes by water–alcohol extraction and then purified using Sephadex column chromatography and Polyacrylamide gel electrophoresis (Mironova et al., 1986).

The ion-transporting activity of the protein was measured using the BLM technique (Mueller et al., 1963). Current and potential were measured with a Y-5–9 electrometric amplifier. The output current of the amplifier was registered with an Endim-b.21.02 recorder.

Figure 2. Ouabain inhibition of conductance channels. Aqueous phase contained 20 mM Tris-HCl (pH 7.5), 80 mM NaCl, and 16 mM KCl. Protein concentration, 5 μg/ml.

Results and Discussion

α-Subunit of Na+,K+ -ATPase formed conductance channels in the bilayer lipid membrane (Fig. 1). Ouabain, a specific inhibitor of the Na+,K+-ATPase, increased the conductance of the modified BLM at a concentration of 1 μM (data not shown). Increasing of ouabain concentration to 0.1 mM caused inhibition of conductance channels (Fig. 2).

Addition of K+ (80–100 mM) to the medium prevented the inhibitory effect of ouabain (Fig. 3). Presumably potassium reduces the ouabain binding to Na+,K+ -ATPase.

Amiloride is a specific blocking agent of epithelial sodium channels and is used clinically as a potassium-sparing diuretic. At high concentrations (1–2 mM) amiloride inhibits Na+,K+ -ATPase of the intact proximal tubule (Soltoff and Mandel, 1983). Investigation of amiloride effects on the conductance of the channels formed by α-subunit showed that amiloride inhibited this conductance (Fig. 4).

Gossypol is a natural polyphenol extracted from cotton plants. It has a broad spectrum of action on various enzymes and cell functions and produces antifertile (Qian and Wang, 1984), immunosuppressive, and antiviral effects (Aripov et al., 1982). Presently more than 100 derivatives have been synthesized from gossypol. Among them is batriden, which, like gossypol, possesses immunosuppressive activity. Another new immunosuppressive preparation is megosin (Sahibov et al., 1986). Though gossypol and its derivatives are used in medicine, the mechanism of their action still remains unclear. We observed the inhibitory effect of gossypol and its derivatives on the ATPase activity of Na+,K+ -ATPase (Table I). The influence of gossypol on the conductance of the channels, formed by α-subunit of Na+,K+ -ATPase in BLM was studied. Gossypol at the concentration of 1–3 μM decreased the conductance of these channels (Table II). As shown in Fig. 5, gossypol effects on ATPase activity of the Na+,K+ -ATPase and ion-conducting activity of incorporated α-subunit are similar with respect to gossypol concentration.

The data presented show that the ion-conducting activity of α-subunit incorporated into BLM is inhibited by the same inhibitors that affect the whole enzyme. Ouabain, amiloride, and gossypol probably bind to the α-subunit of Na+,K+ -ATPase. Data obtained on amiloride inhibition of the incorporated channels support the supposition of the similarity between Na+ sites of Na+ channels and those of Na+,K+ -ATPase (Reznik and Myazina, 1985).

Figure 3. Elimination of the ouabain effect by potassium ions. The conditions are the same as in Fig. 2.

Figure 4. Inhibition of the conductance of the channels by amiloride. Conditions are the same as in Fig. 1.

Our results may be important for understanding the molecular mechanisms of the biological action of gossypol. We suggested that the antifertility activity of gossypol is associated with its direct effect on Na+,K+ -ATPase. The changes in intracellular Na+ and K+ concentrations in the presence of gossypol may immobilize sex cells and be one of the reasons for the antifertile activity.

Thus, incorporation of the α-subunit of Na+,K+ -ATPase into BLM provides a convenient model for the investigation and screening of drugs that affect the Na+,K+ -ATPase.

TABLE I
The Inhibitory Effect of Gossypol and Its Derivatives on the ATPase Activity of Na+,K+ -ATPase

Na+,K+-ATPase was isolated from pig kidney medulla by the modified version of Jorgensen's method (Chetverin et al., 1979). Na+,K+-ATPase activity was determined in medium containing 30 mM Tris-HCl (pH 7.5), 130 mM NaCl, 5 mM KCl, 4 mM MgCl_2, and 2 mM ATP by the method of Panusz et al. (1970).

TABLE II
Gossypol Inhibition of Conductance of BLM Modifled by α-subunit of Na+,K+ -ATPase

Aqueous phase bathing of BLM contained 10 mM Tris-HCl (pH 7.5), 5 mM ATP, 5 mM MgCl_2,100 mM NaCl, and 10 mM KCl.

Figure 5. Comparison of gossypol influence on ATPase activity of Na+,K+ -ATPase (1) and ion-transporting activity of α-subunit of Na+,K+ -ATPase reconstituted in BLM (2).

References

Aripov, U. A., K. G. Urazmetov, and P. I. Ismailov. 1982. Actual questions of transplantology and immunosuppressive therapy. Tashkent. FAN. 28–37.

Chetverin, A. B., E. V. Brazhnikov, and Yu. N. Chirgadze. 1979. Isolation of membrane-bound Na+,K+-adenosine triphosphatase from pig kidneys and study of its structural properties. Biokhimiya. 44:741–747.

Mironova, G. D., N. I. Bocharnikova, N. M. Mirsalikhova, and G. P. Mironov. 1986. Ion-transporting properties and ATPase activity of (Na+ + K+)-ATPase large subunit incorporated into bilayer lipid membranes. Biochimica et Biophysica Acta. 861:224–236.

Mueller, P., D. D. Rudin, H. T. Tien, and W. C. Wescott. 1963. Methods for the formation of single bimolecular lipid membranes in aqueous solution. The Journal of Physical Chemistry. 67:534–535.

Panusz, H. T., G. Graczyk, D. Wilmanska, and J. Skarzynski. 1970. Analysis of orthophosphate-pyrophosphate mixtures resulting from weak pyrophosphatase activities. Analytical Biochemistry. 35:494–504.

Qian, S.-Z., and Z.-G. Wang. 1984. Gossypol: a potential antifertility agent for males. Annual Review of Pharmacology and Toxicology. 24:329–360.

Reznik, G. V., and E. M. Myazina. 1985. Inhibition of Na,K-ATPase activity by sodium channel blocking agents. Voprosy Meditsinskoi Khimii. 31:122–124.

Sahibov, A. D., S. F. Sokolova, and N. M. Mirsalikhova. 1986. Investigation of mechanism of Na+,K+-ATPase inhibition by gossypol and megosin. Voprosy Meditsinskoi Khimii. 3:111–114.

Soltoff, S. P., and G. J. Mandel. 1983. Amiloride directly inhibits the Na,K-ATPase activity of rabbit kidney proximal tubules. Science. 220:957–959.

[1] The PL used in this study contained one molecule of NaK-ATPase per vesicle and had a diameter of - 120 nm; a single molecule contained in the inside medium of a PL of that size corresponds to a concentration of 1.5 ILM (Yoda and Yoda, 1990). The PLs made with the above method were a mixture of the rightside-out form, the inside-out form, and liposomes lacking NaK-ATPase molecules. Only the rightside-out form of these PLs can be used for studying a ligand like PTX, which binds on the extracellular side at such low picomolar concentrations. Since ATP was limited to the inside medium, only the rightside-out form functioned as a NaK-pump under our experimental conditions.

[2] The Na+-influx is composed of ouabain-sensitive, active Na+-transport and the ouabain-insensitive Na+-diffusion. Since PTX inhibits the active Na+-transport, as shown in Fig. 2, the increase in the Na+-influx through the PTX-induced channel is significantly higher than 30%.

Electrogenic Aspects

Correlation of Charge Translocation with the Reaction Cycle of the NaK-ATPase

K. Fendler, J. Froehlich, S. Jaruschewski, A. Hobbs, W. Albers, E. Bamberg, and E. Grell

Max-Planck-Institut für Biophysik, D-6000 Frankfurt 70, Germany; National Institute on Aging, National Institutes of Health, Baltimore, Maryland 21224; National Institute of Neurological and Communicative Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892

Introduction

Correlation of the electrical and chemical events during the reaction cycle of an ion translocating ATPase represents an important step toward an understanding of its transport mechanism. A considerable body of information about the chemical partial reactions during the cycle of NaK-ATPase from eel electric organ has been accumulated by rapid mixing quenched-flow techniques. For a comparison of the chemical and electrical events we therefore investigated the pre–steady state charge translocation of the NaK-ATPase from the electric organ of Electrophorus electricus.

Results and Discussion

Current Measurements and Analysis

The NaK-ATPase-containing membrane fragments were adsorbed to a planar lipid bilayer forming a compound membrane as described elsewhere (Fendler et al., 1985). A rapid ATP concentration jump was generated by photolysis of an inactive photolabile derivative of ATP (caged ATP) with a UV laser flash. The released ATP activates the NaK-ATPase contained in the adsorbed membrane fragments, and a transient current can be measured. The electrolyte contained 130 mM NaCl, 3 mM MgCl_2, 1 mM DTT, 25 mM imidazole, pH 6.2, and different amounts of caged ATP. If not otherwise indicated the experiments were performed at 24°C.

Good quality fits to the recorded signals could be obtained with the function:

using an automatic fit program (SPLMOD; Provencher and Vogel, 1983).

As reported previously, the largest observed time constant τ_3 has to be assigned to the characteristic time constant of the compound membrane (Fendler et al., 1985, 1987; Borlinghaus et al., 1987). The remaining time constants, τ_1 and τ_2, describe the electric activity of the enzyme and will be discussed below.

ATP and Caged ATP Binding

Caged ATP has been shown to be a competitive inhibitor of the NaK-ATPase (Forbush, 1984; Nagel et al., 1987). To investigate the effect of caged ATP on the enzyme, signals were recorded at different ATP concentrations and different ATP/caged ATP ratios. The ATP/caged ATP ratio was varied by applying different UV-laser energies to the membrane. This results in a variable fraction η of caged ATP being transformed to ATP.

In Fig. 1 the effect of caged ATP on the inverse of the time constants τ_1–1and τ_2–1 is shown. Only τ_2–1 is strongly affected by the variation of ATP and caged ATP. The dependency of τ_2–1 on the concentration of caged ATP after the flash follows a hyperbolic saturation curve. A fit with a function of this type is included in the figure. A similar observation was reported for the NaK-ATPase from pig kidney (Fendler et al., 1987). These findings led to the conclusion that τ_2–1 corresponds to ATP released after a UV-flash binding to the enzyme and exchanging for the caged ATP already bound. In the following a model is discussed that explains the hyperbolic ATP dependence in the presence of caged ATP.

Figure 1. Inverse time constants τ–1 of the electric signal at different concentrations of caged ATP. Conditions: η = 0.2, T = 24°C.

ATP and caged ATP binding to the enzyme is described by the model:

The dependence of the eigenvalue describing ATP and caged ATP binding can be approximated by:

ka+ is the rate constant of ATP binding, whereas kc+ and kc– are the rate constants of caged ATP binding and dissociation, respectively. η is the fraction of ATP released after the UV light flash and cc^0 is the concentration of caged ATP present before illumination.

From Eq. 3 the ATP and caged ATP binding rate constants can be expressed as functions of the parameters (τ–1)max and K0.5:

The dissociation of caged ATP from the NaK-ATPase was measured with the rapid mixing technique. After incubation with caged ATP the enzyme was diluted 20-fold and mixed with 100 μM [γ^32P]ATP. Under these conditions the formation of the phosphoenzyme is limited by the dissociation of caged ATP. From these measurements (data not shown) a dissociation rate constant kc– of at least 1,000 s–1 can be estimated.

Figure 2. Effect of different fractions of released ATP on the time constants. Conditions: [caged ATP] = 300 μM, T = 24°C.

Using Eq. 4 together with the experimentally determined values K0.5 = 38 μM, (τ–1_2)max = 54 s–1, and η = 0.2 yields the ATP binding rate constant ka+ = 7.1∙10^6 M–1s–1 and the binding constant for caged ATP Kc0.5 = 30 μM. Similar values for these constants were reported in the literature: k+a = 0.8 – 1∙10^7 M–1s–1 (Froehlich et al., 1976), Kc0.5 = 43 μM (Forbush, 1984), and Kc0.5 = 30 μM (Nagel et al., 1987).

At high caged ATP concentrations c^0c Eq. 3 reduces to:

Eq. 5 describes the dependence of the time constant of ATP and caged ATP binding and exchange on the fraction of released ATP at high caged ATP concentrations. Inserting the values determined above in Eq. 5 gives a straight line with a slope of 216 s–1. For comparison with the experimental data this line is plotted together with the experimental results in Fig. 2. It is clear that the slope of the theoretical curve agrees well with the experimental data. The shift of the data points to larger values of τ–1 by ~ 9 s–1 is due to the fact that the ATPase reaction cycle is closed, a fact that is not taken into account by the model.

The analysis given above shows that at different ATP and caged ATP concentrations τ_2 behaves as predicted by the simple model of Eq. 2. In addition, reasonable values for ka+ and Kc0.5 are obtained. This gives confidence that the assignment of τ_2 to caged ATP and ATP binding and exchange is correct.

Phosphorylation in the Presence of Caged ATP

To test the competitive influence of caged ATP, rapid mixing quenched flow experiments with [γ^32P]ATP as a substrate were performed in the absence and presence of caged ATP. For comparison, electrical experiments were made at the same enzyme preparation.

In the electrical measurements a fraction of released ATP of η = 30% was determined. The inverse time constants obtained by the fitting procedure described above were τ_1–1 198 s–1 and τ_2–1 = 88 s-1.

The phosphorylation measurements were designed to match the conditions of the electrical measurements. In particular, the concentrations of ATP and caged ATP before and after starting the reaction were the same in both types of experiments. In Fig. 3 the time course of phosphoenzyme formation with and without caged ATP is shown.

Figure 3. Formation of the phosphoenzyme with and without caged ATP. Conditions: [ATP] = 90 μM, if required, [caged ATP] = 210 μM, T = 24°C.

A fit with a monoexponentially rising function EP(t) = A(1 – e–t/τ) yields a value of τ–1 = 91 s–1 for phosphorylation in the presence of caged ATP. This agrees well with the inverse time constant τ_2–1 = 88 s–1 obtained with the simultaneous electrical measurement and lends further support to the assignment of τ_2 to ATP and caged ATP binding and exchange.

The Electrogenic Step

As shown in the previous section, the decay of the electrical signal (τ_2) is assigned to ATP and caged ATP binding and exchange. As in the case of the NaK-ATPase from pig kidney (Fendler et al., 1987) the rise of the signal (τ_1–1 = 265 s–1) is not limited by the release of ATP from the caged precursor (τ–1 ≈ 1,400 s–1; McCray et al., 1980) or filtering time constants of the amplifier (τ_1–1 ≈ 3,000 s–1). It must therefore also be due to the pumping activity of the enzyme.

In studies with NaK-ATPase from rabbit kidney (Apell et al., 1987; Borlinghaus et al., 1987) and pig kidney (Fendler et al., 1987) the electrogenic step was assigned to the conformational transition E_1P E_2P. The same is probably true for NaK-ATPase in eel electric organ and τ_1 corresponds to the E_1P E_2P transition. It is interesting to compare the rate constant of the E_1P E_2P transition τ_1–1 = 265 s–1 to data obtained by other techniques. From rapid mixing experiments this rate constant was estimated to be 350 s–1 – 1,000 s–1 at 21°C (Hobbs et al., 1985; Froehlich et al., 1983). This is somewhat larger than τ–1_1. However, the fitting procedure used here tends to underestimate τ–1_1. Analysis with a complete kinetic model can give values for τ–1_1 as high as 400 s–1 (see Froehlich and Fendler, 1991).

Conclusions

The issue of the present study was to compare electric events during the reaction cycle of NaK-ATPase from eel electric organ with partial reactions of the Albers-Post scheme. Therefore a rigorous assignment of the different phases of the observed current is necessary.

Comparison of the time constants with a simple kinetic model that accounts for the interaction of the ATPase with the nucleotides shows that the decaying phase of the signal has to be assigned to ATP binding and exchange with caged ATP. The rise of the signal corresponds to the electrogenic step. The data presented here are consistent with a simple Albers-Post scheme where the electrogenic step is the E_1P E_2P transition.

References

Apell, H.-J., R. Borlinghaus, and P. Läuger. 1987. Fast charge translocations associated with partial reactions of the Na,K-Pump. II. Microscopic analysis of transient currents. Journal of Membrane Biology. 97:179–191.

Borlinghaus, R., H.-J. Apell, and P. Läuger. 1987. Fast charge translocations associated with partial reactions of the Na,K-Pump. I. Current and voltage transients after photochemical release of ATP. Journal of Membrane Biology. 97:161–178.

Fendler, K., E. Grell, and E. Bamberg. 1987. Kinetics of pump currents generated by the Na+,K+-ATPase. FEBS Letters. 224:83–88.

Fendler, K., E. Grell, M. Haubs, and E. Bamberg. 1985. Pump currents generated by the purified Na+K+-ATPase from kidney on black lipid membranes. EMBO Journal. 4:3079–3085.

Forbush, B., III. 1984. Na+ movement in a single turnover of the Na pump. Proceedings of the National Academy of Sciences, USA. 81:5310–5314.

Froehlich, J. P., and K. Fendler. 1991. The partial reactions of the Na+- and Na+ + K+-activated adenosine triphosphatases. In The Sodium Pump: Structure, Mechanism, and Regulation. J. H. Kaplan and P. De Weer, editors. The Rockefeller University Press, New York. 227–247.

Froehlich, J. P., A. S. Hobbs, and R. W. Albers. 1983. Evidence for parallel pathways of phosphoenzyme formation in the mechanism of ATP hydrolysis by Electrophorus Na,KATPase. Current Topics in Membranes and Transport. 19:513–535.

Hobbs, A. S., R. W. Albers, and J. P. Froehlich. 1985. Quenched-flow determination of the E_1P to E_2P transition rate constant in electric organ Na,K-ATPase. In The Sodium Pump. I. Glynn and C. Ellory, editors. The Company of Biologists Ltd., Cambridge, UK. 355–361.

McCray, J. A., L. Herbette, T. Kihara, and D. R. Trentham. 1980. A new approach to time-resolved studies of ATP-requiring biological systems: laserflash photolysis of caged ATP. Proceedings of the National Academy of Sciences, USA. 77:7237–7241.

Nagel, G., K. Fendler, E. Grell, and E. Bamberg. 1987. Na+ currents generated by the purified (Na+ + K+)-ATPase on planar lipid membranes. Biochimica et Biophysica Acta. 901:239–249.

Provencher, S. W., and R. H. Vogel. 1983. Regularisation techniques for inverse problems in molecular biology. In Progress in Scientific Computing. P. Deuflhard and E. Hairer, editors. Birkhäuser, Boston. 304–319.

Charge Translocation by the Sodium Pump: Ion Binding and Release Studied by Time-resolved Fluorescence Measurements

W. Stürmer, R. Bühler, H.-J. Apell, and P. Läuger[†]

Department of Biology, University of Konstanz, D-7750 Konstanz, Germany

Introduction

Operation of the Na,K-pump involves a sequence of conformational transitions and ion-binding and -release reactions. These may be described by a modified Post-Albers cycle (Fig. 1). An important question in the understanding of the pumping mechanism is: in which step(s) of the cycle is charge actually moved? Recently, Klodos and Forbush (1988) reported that a fluorescent styryl dye, RH 160, incorporates into open Na,K-ATPase membrane fragments and monitors transitions between states of the pumping cycle. Styryl dyes (Fig. 2) have been introduced by Loew et al. (1982) and Grinvald et al. (1982) for the optical recording of membrane potentials. Fluorescent signals from styryl dyes bound to the membrane fragments, which are observed upon activation of the pump, may have at least two different origins: (a) the dye may bind directly to the protein and in this way become sensitive to conformational changes; (b) dye molecules incorporated into lipid bilayer regions of the fragments may sense local field changes resulting from charge movements in the protein.

Figure 1. Modified Post-Albers cycle.

Experimental Methods

Na,K-ATPase was prepared from the outer medulla of rabbit kidneys using procedure C of Jørgensen (1974). This method yields membrane fragments containing several thousand ATPase molecules per square micrometer and ~ 1 mg lipid per mg protein. Using this preparation, some styryl dyes (RH 237, RH 160, RH 421) have been characterized with respect to their sensitivity and mechanism. In particular, we used these dyes to investigate charge translocation associated with binding and release of K+ and Na+ by the pump. For detailed description see Bühler et al. (1991) and Stürmer et al. (1991).

Figure 2. Structure of styryl dyes, according to Grinvald et al. (1982).

Results

Phosphorylation of the Na,K-ATPase by ATP in the presence of Na+ and in the absence of K+ leads to a change of the fluorescence of all dyes we tested. In Fig. 3 the time-resolved fluorescence change is shown for dye RH 421.

Mechanism of the Dye Response

The notion that the styryl dyes respond mainly to changes in the electric field strength in the membrane is based on the following observations: (a) The spectral characteristic of the dye response essentially agrees with the predictions for an electrochromic shift of the absorption spectrum. (b) Adsorption of lipophilic anions (TPB–) to membrane fragments leads to an increase, adsorption of lipophilic cations (TPP+) to a decrease, of dye fluorescence. This is in agreement with the fact that these ions bind to the hydrophobic interior of the membrane and change the electric field strength in a narrow zone close to the interface. (c) The fluorescence change that is normally observed upon phosphorylation of the enzyme is abolished at high concentrations of lipophilic ions. These ions are thought to redistribute between the adsorption layer and water upon the change of the electrical field and to neutralize in this way the change of field strength caused by ion translocation. (d) Changes of the fluorescence of RH 421 correlate with known electrogenic transitions in the pumping cycle (Na+ translocation), but are absent during electrically silent reactions (E_2(K_2) → E_1 conformational change).

Figure 3. Relative fluorescence change ∆F/F_0 ≡ (F – F_0)/F_0 in the presence of RH 421, as a function of time t after photochemical ATP release at t = 0. The solution contained 0.7 μM RH 421, 20 μM “caged” ATP, MgCl_2 as indicated, 1 mM EDTA, 30 mM imidazole chloride, pH 7.2, and 150 mM NaCl.

Figure 4. Fluorescence changes of RH 421 bound to Na,K-ATPase membranes. Open membrane fragments (9 μg protein/ml) were equilibrated with 0.23 μM RH 421 in the presence of 5 mM MgSO_4, 1 mM EDTA, and 30 mM imidazole sulfate, pH 7.2. The fluorescence was excited at 580 nm and recorded at 660 nm. 20 mM NaCl, 10 μM ATP, and 20 mM KCl were added successively to the medium. ∆F/F_0 is the relative fluorescence change referred to the fluorescence intensity F_0 before the addition of sodium.

Release of Sodium Ions

Previous experiments have shown that phosphorylation of the enzyme with ATP and occlusion of Na+ are electroneutral steps (Borlinghaus et al., 1987). The release of Na+, however, is associated with translocation of charge. The transition from state Na_3∙E_1 to state P-E_2 leads to a large increase of dye fluorescence (Fig. 4). The sign of the fluorescence change corresponds to the creation of a negative electrostatic potential inside the membrane dielectric. At high Na+ concentrations (> 100 mM) the ATP-induced fluorescence change is diminished. This is consistent with the assumption that the equilibrium (Na_3)E_1-P ↔ P-E_2 + 3Na+ is shifted to the left at high sodium concentrations (Fig. 5). This effect of Na+ is reduced in the presence of lipophilic cations (TPP+) and enhanced in the presence of lipophilic anions (TPB–). A likely interpretation of the effects of TPP+/TPB– is that the change of electrical potential in the membrane affects the electrostatic energy of Na+ at its binding sites (Läuger, 1991).

Figure 5. Fluorescence change of RH 421 upon ATP-induced phosphorylation as a function of Na+ concentration cN. A suspension of membrane fragments (30 μg protein/ml) was added to a solution of 0.7 μM RH 421, 20 μM “caged” ATP, 10 mM MgCl_2, 1 M (NaCl + choline chloride), 1 mM EDTA, 30 mM imidazole chloride, pH 7.2, and 0 mM (TPP+/TPB–), 300 μM TPP+, or 3.3 μM TPB–.

Binding of Potassium Ions

The transition from P-E_2 to E_2(K_2), which is induced by addition of K+ (Rb+, Cs+) to the phosphorylated enzyme is associated with a decrease of fluorescence (Fig. 4), corresponding to the creation of a positive potential inside the membrane. The half-saturating concentration of K+ in this experiment (c1/2 = 0.25 mM) is shifted toward larger values in the presence of TPP+ (Fig. 6), thus paralleling the decrease of Na+ affinity observed in the presence of TPP+.

Figure 6. Fluorescence change of RH 421 upon addition of K+ to the phosphoenzyme as a function of K+ concentration ck. ∆F was evaluated as the difference of the stationary signal amplitudes with and without addition of K+. A suspension of membrane fragments (30 μg protein/ml) was added to a solution of 0.7 μM RH 421, 20 μM “caged” ATP, 10 mM MgCl_2, 1 M choline chloride, 1 mM EDTA, 30 mM imidazole chloride, pH 7.2, and various concentrations of KCl and TPP+. In the absence of choline chloride the results were virtually identical.

Binding of Sodium Ions

The fluorescence decrease upon the initial addition of Na+ (Fig. 4), which can be observed under conditions where the enzyme is predominantly in state E_1, indicates that the binding of the third (?) Na+ at the cytoplasmic side is another electrogenic step.

Location of the Binding Sites

A likely explanation of these experimental findings is that the dye responds mainly to variations of electric field strength resulting from changes of the charge state of the protein (–3e_0 for Na+ release, +2e_0 for K+ binding). It is important for the understanding of the mechanism of ion binding and release to know where in the protein these changes of electric charge occur. The ion-binding sites may be located at the surface of the protein, or they may be buried in its interior. These possibilities can be distinguished by studying the effects of ionic strength on the dye response since, at high ionic strength, surface charges should be shielded by counterions. The fluorescence signals associated with release of Na+ or binding of K+ remain virtually unchanged upon addition of 1 M choline chloride to the medium. This leads to the conclusion that the ion-binding sites are located inside the protein. This agrees with the notion that the binding sites are connected to the extracellular medium by a narrow access channel (ion well).

Summary

Our findings may be described by the following assumptions, as visualized in Fig. 7: (a) There are two negatively charged sites and one neutral binding site for Na+. (b) In the E_1 conformation, the charged binding sites are located at the surface of the protein, whereas the neutral side is buried inside the protein. (c) In the E_2 conformation, at least the negative binding sites are located in the protein interior. At this binding sites (2?) Na+ ions are bound with low, 2 K+ ions with high affinity. (d) An access channel (ion-well) exists at the extracellular side, which traverses part of the membrane dielectric and connects the binding sites with the aqueous medium. (e) The release of Na+ ions from their binding sites is a major rate-limiting step during sodium translocation. (f) The transition P-E_2(K_2) → E_1 is electrically silent.

Figure 7. Simplified electrostatic model for charge translocation by the Na,K pump. See text for explanation.

Acknowledgments

This work has been supported by the Deutsche Forschungsgemeinschaft (Sonderforschungsbereich 156).

References

Borlinghaus, R., H.-J. Apell, and P. Läuger. 1987. Fast charge-translocation associated with partial reactions of the Na,K-pump. I. Current and voltage transients after photochemical release of ATP. Journal of Membrane Biology. 97:161–178.

Bühler, R., W. Stürmer, H.-J. Apell, and P. Läuger. 1990. Charge translocation by the Na,K-pump. I. Kinetics of local field changes studied by time-resolved flurescence measurements. Journal of Membrane Biology. 121:141–161.

Jørgensen, P. L. 1974. Isolation of the (Na+ + K+)-ATPase. Methods in Enzymology. 32:277–290.

Klodos, I., and B. Forbush III. 1988. Rapid conformational changes of the Na/K pump revealed by fluorescent dye, RH 160. Journal of General Physiology. 92:46a. (Abstr.)

Läuger, P. 1991. Kinetic basis of voltage dependence of the Na,K-pump. In The Sodium Pump: Structure, Mechanism, and Regulation. J. H. Kaplan and P. De Weer, editors. The Rockefeller University Press, New York. 303–315.

Loew, L. M. 1982. Design and characterization of electrochromic membrane probes. Journal of Biochemical and Biophysical Methods. 6:243–260.

Grinvald, A., R. Hildesheim, I. C. Farber, and L. Anglister. 1982. Improved fluorescent probes for the measurement of rapid changes in membrane potential. Biophysical Journal. 39:301–308.

Stürmer, W., R. Bühler, H.-J. Apell, and P. Läuger. 1991. Charge translocation by the Na.K-pump. II. Ion binding and release at the extracellular face. Journal of Membrane Biology. 121:163–176.

Comparison of the Na/K and the Na-only Mode of the Sodium Pump

Hans-Jürgen Apell and Milena Roudna

Department of Biology, University of Konstanz, D-7750 Konstanz, Germany

Introduction

In the absence of K+ ions the Na,K-ATPase performs Na-only pumping modes in addition to its physiological mode. In the literature electroneutral Na-Na exchange, electrogenic Na-Na exchange, and uncoupled Na pumping are described (for review see Glynn, 1985). Under the condition of high Na on both sides of the membrane an electrogenic Na-Na exchange of the Na,K-ATPase has been reported (Forgac and Chin, 1982; Cornelius and Skou, 1985; Cornelius, 1989; Apell et al., 1990). The aim of this investigation has been to identify common steps in the Post-Albers pumping cycle and to define a comprehensive scheme for both pumping modes.

Experimental Methods

The electrogenic activity of the Na,K-ATPase in vesicular systems can be investigated by the potential-sensitive dye oxonol VI, which detects the charging of the membrane capacitance by increasing the fluorescence intensity with inside positive potentials. Na,K-ATPase was isolated from rabbit kidney and reconstituted in unilamellar lipid vesicles by a detergent dialysis method. Experimental methods are described in detail by Apell and Bersch (1987). Vesicles were prepared in buffer containing 75 mM Na_2SO_4 and no K+ ions. Thereafter, part of the preparation was dialyzed in buffer with different concentrations of K+ to compare the two pumping modes from the same batch of vesicles. Since vesicles have a very limited internal volume of ~5∙10–16 ml in our preparation, the intravesicular potassium concentration changes rapidly upon pumping. Starting with 100 mM K+ and maintaining the average number of five active pumps at a turnover rate of 30 s–1 would nominally deplete the vesicles completely within 200 s. This led to the conclusion that information about the Na,K mode can be obtained only during the time when the internal potassium concentration is not significantly reduced. For investigating the electrogenic action of the pump by monitoring the fluorescence of oxonol VI, the initial slope of the fluroescence signal after addition of ATP, dF/dt, turned out to be an easily accessible and reliable parameter (Fig. 1). Using an appropriate calibration curve (Apell and Bersch, 1987), the fluorescence can be transformed into a voltage signal, dUAV/dt ≡ , representing the mean voltage change averaged over all vesicles, since they are heterogeneous in size and number of active pumps. Since the vesicle membrane can be represented in electrical terms as a capacitor, can be correlated with the pumping activity by = np∙vp∙zp ∙e_0/Cm, where np is the number of active pumps, vp the pumping rate, zp the number of net charges transported per cycle, e_0 the elemetary charge, and Cm the capacitance of the vesicle.

Results and Discussion

Both Pumping Modes Are Electrogenic

Upon addition of ATP to a vesicle suspension in Na buffer, an inside positive potential is generated (Fig. 1). When the intravesicular (extracellular) K+ concentration is > 5 mM, the initial slope, which is proportional to the pumping rate, is almost saturated. This is in good agreement with an equilibrium dissociation constant for K+ of ~ 0.1 mM. At a (cytoplasmic) Na concentration of 20 mM the ratio of the initial slopes for both pumping modes has been determined. This number is also the ratio of the products of turnover rate and number of net charges transported:

Figure 1. Membrane potential generated by the Na,K-pump in the presence and absence of intravesicular (extracellular) potassium. Vesicles contain 150 mM Na+ or 15 mM K+ + 135 mM Na+. The anion is sulfate. The cuvette contains buffer with 10 mM Na_2SO_4 + 140 mM Tris-H_2SO_4, pH 7.5. 100 nM oxonol, vesicles (18 μg/ml lipid), and 2.5 mM ATP were added successively. From the initial rise of fluorescence intensity, ∆F/(F_0∙∆t), the initial slope of membrane potential, dU/dt ≡ , can be obtained by the appropriate calibration curve according to Apell and Bersch (1987). (Inset) Initial slope of the membrane potential as a function of the intravesicular K+ concentration. The apparent equilibrium dissociation constant was found to be in the range of 0.2 mM.

Ratio of ATP Hydrolysis

When K+-containing and K+-free vesicles under otherwise identical conditions are tested for ATP hydrolysis, the amount of liberated inorganic phosphate under steady-state conditions represents a measure of the phosphorylation and dephosphorylation rate. The comparison of both pumping modes results in a ratio of (Apell et al., 1990):

Since one ATP is split per each turnover, Rhydrolysis can also be interpreted as the ratio of the turnover rates. Comparing Relectrogenic and Rhydrolysis shows that the ratio of the number of net charges is one. For the Na,K mode it is well known that the stoichiometry is 3 Na to 2 K. Since the first half of the pumping cycle should be identical for both pumping modes (3 Na+ out of the cell), the second half can be interpreted as the transport of 2 K+ or 2 Na+, respectively, into the cell. That would mean that Na+ can replace K+ in the pumping cycle with a sixfold smaller efficiency.

Ratio of 22Na Tracer Flux

In tracer flux experiments (Apell et al., 1990) 22Na was used to measure the amount of Na+ that is transferred from the cytoplasmic to the extracellular side. Under otherwise identical conditions the tracer flux could be determined for both transport modes:

Rtracer can be assumed to be equal to Rhydrolysis. This shows that in both transport modes the amount of Na+ pumped out of the cell is the same. Therefore it can be claimed that the reaction steps E_1ATP + 3 Na+cyt Na_3E_1ATP (Na_3)E_1−P P−E_2 + 3 Na+ext are used by both transport modes and that all 3 Na+cyt are released on the extracellular side. These findings strongly support a 3 Na/2 Na stoichiometry for the Na-Na exchange under high Na conditions. In addition, these results show that all 3 Na+ are released into the extracellular medium. If one or two of the Na+ ions would stay bound, Rtracer would be significantly greater than Rhydrolysis.

Figure 2. Extended Post-Albers scheme for the pumping cycle of the Na,K-ATPase, which combines the Na,K and the Na-only modes. E_1 and E_2 are conformations of the enzyme with ion-binding sites exposed to the cytoplasmic and extracellular membrane interface, respectively. In the “occluded” states the exchange of ions with the aqueous phases is assumed to be disabled [(Na_3)E_1–P] or at least slowed (P–E_2(Kn), P–E_2(Nan); n = 1, 2). Dashes indicate covalent bonds and dots indicate noncovalent bonds. The double lines show the cycle under physiological conditions.

Extended Post-Albers Scheme

Putting these findings together, an extended Post-Albers scheme that includes both transport modes can be formulated (Fig. 2). The physiological pumping cycle is indicated by double lines in Fig. 2. At low concentrations of ATP transitions E_2(K_2) → K_2E_1 would be preferred. In the absence of K+, but with Na+ on both sides of the membrane, the Na transport would be described by a cycle through the two upper limbs of the scheme, at which the states NaxE_1 or NaxE_1ATP are in a fast equilibrium and the transition P–E_2(Na_2) → E_2(Na_2) is slow compared with the ion dissociation and binding steps at the extracellular sites to explain the tracer experiments. The states E_2(Na_2) and ATP∙E_2(Na_2) are putative and are drawn in analogy to the K+ limb.

Figure 3. Initial increase of membrane potential dUAV/dt as a function of ATP concentration. This parameter is proportional to the turnover rate. Virtually no ADP is present. The filled circles show the concentration dependence of the pump working in the Na-only mode, and the open circles represent the pump working in the Na,K mode (normalized to the maximum value of the Na-only mode). Vesicles were suspended in buffer containing 150 mM Na+, pH 7.5. The lines are calculated according to a Michaelis-Menten kinetics for one binding site (Km = 54 nM) in the Na-only mode and two binding sites (Km,1, = 60 nM, Km,2 = 13 μM) in the Na,K mode.

ATP Concentration Dependence

In Fig. 3 the ATP concentration dependence of the initial turnover rate is shown for both pumping modes. In the Na,K mode the well-known two binding sites with Km values of ~60 nM and 13 μM were observed. These findings are in agreement with previously published data with the same vesicle system (Apell et al., 1987). In the Na-only mode only one Km value can be observed at Km = 54 nM, which corresponds to the high affinity binding E_1 + ATP → E_1ATP. This implies that the E_2 → E_1 transition is not rate limiting under any ATP concentration in the Na-only mode. Possible explanations are that (a) the transition P–E_2(Na_2) → E_2(Na_2) is slower than any reaction from E_2(Na)2 to E_1, or (b) the analogy of the low affinity binding site for ATP in the K+ limb is not true in the Na-only mode.

Figure 4. Initial increase of membrane potential dUAV/dt as a function of ADP concentration. The ATP concentration was 250 μM. The filled circles show the concentration dependence of the pump in the Na-only mode. The line follows a kinetic behavior according to two inhibiting sites (KI,1 = 2.2 μM and KI,2 = 4 mM). The dashed line shows the concentration dependence of the pump working in the Na,K mode (normalized to the maximum value of the Na-only mode) and can be described by a single inhibition constant KI = 140 mM. Data points representing the Na,K mode are removed for the sake of clarity.

ADP Concentration Dependence

The pumping activity is inhibited by ADP in both transport modes. In Fig. 4 the initial turnover rate is plotted as a function of the ADP concentration. In both cases a noncompetitive inhibition is observed, but the half-inhibition concentrations are different by a factor of 20. Since the inhibition is noncompetitive with ATP binding, the interaction of ADP is restricted on the reaction Na_3E_1ATP (Na_3)E_1–P + ADP. The difference in ADP sensitivity can be explained easily by the assumption that the state (Na_3)E_1–P is populated 20 times higher under pumping conditions in the Na-only mode compared with the Na,K mode.

Conclusions

The presented findings can be explained by the modified Post-Albers scheme presented in Fig. 2:

The Na+ transfer from cytoplasm to the extracellular compartment is the same pathway in both transport modes. The rate constants can be assumed to be the same.

The cycle is branched at the state PE_2, dependent on the presence or absence of K+:

In the presence of K+, these ions are bound to the extracellular binding moieties with a higher affinity than Na+. When 2 K+ are bound, a spontaneous transition to the dephosphorylated state (E_2)K_2 occurs as the first step in moving the enzyme through the “K+-limb” back to the initial state of pumping, E_1 ± ATP.

In the absence of external K+ the enzyme is slowly dephosphorylated from state P–E_2(Na_2) to E_2(Na_2), which is similar to state E_2(K_2). This step has to be rate limiting. An acceleration of the E_2 → E_1 transition by low affinity ATP binding cannot be detected.

Acknowledgments

The authors want to thank Dr. Peter Läuger for interesting discussion.

This work was supported by the Deutsche Forschungsgemeinschaft (Sonderforschungsbereich 156).

References

Apell, H.-J. and B. Bersch. 1987. Oxonol VI as an optical indicator for membrane potentials in lipid vesicles. Biochimica et Biophysica Acta. 903:480–494.

Apell, H.-J., V. Häring, and M. Roudna. 1990. Na,K-ATPase in artificial lipid vesicles. Comparison of Na,K and Na-only pumping mode. Biochimica et Biophysica Acta. 1023:81–90.

Apell, H.-J., M. M. Marcus, M. T. Nelson, B. M. Anner, H. Oetliker, and P. Läuger. 1987. Optical measurements of ATP-driven cation fluxes in artificial lipid vesicles. Topics in Molecular Pharmacology. 4:53–79.

Cornelius, F. 1989. Uncoupled Na+-efflux on reconstituted shark Na,K-AtPase is electrogenic. Biochemical and Biophysical Research Communications. 160:801–807.

Cornelius, F., and J. C. Skou. 1985. Na+-Na+ exchange mediated by (Na+ + K+)-ATPase reconstituted into liposomes: evaluation of pump stoichiometry and response to ATP and ADP. Biochimica et Biophysica Acta. 818:211–221.

Forgac, M., and G. Chin. 1982. Na+ transport by the (Na+)-stimulated adenosine triphosphatase. Journal of Biological Chemistry. 257:5652–5655.

Glynn, I. M. 1985. The Na+,K+-transporting adenosine triphosphatase. In The Enzymes of Biological Membranes. Vol. 3: Membrane Transport. A. N. Martonosi, editor. Plenum Publishing Corp., New York. 35–114.

Na/K Pump Current in Giant Membrane Patches Excised from Ventricular Myocytes

Donald W. Hilgemann, Georg A. Nagel, and David C. Gadsby

Department of Physiology, University of Texas Southwestern Medical Center, Dallas, Texas 75235; and Laboratory of Cardiac Physiology, The Rockefeller University, New York 10021

Introduction

Stationary Na/K pump current provides a convenient on-line monitor of pump turnover rate and has previously been measured in several different preparations, each with its particular advantages and disadvantages. For example, Na/K pump currents have been measured from membrane fragments adhering to lipid bilayers (Fendler et al., 1985; Borlinghaus et al., 1987; Nagel et al., 1987), where ion and nucleotide concentrations at the cytoplasmic face can be accurately controlled, but where it is not possible to alter the membrane voltage, or to have different solutions bathing the cytoplasmic and extracellular surfaces of the membrane fragments. Those difficulties can be largely overcome in voltage clamped, intact cardiac myocytes, in which the compositions of intra- and extracellular solutions can be varied independently, but in which control of the concentrations of the pumped ions at intra- and extracellular membrane surfaces is limited (Bahinski and Gadsby, 1988; Nakao and Gadsby, 1989; Mathias et al., 1990) and access of large molecules that might modulate the pump (e.g., kinases) is restricted. In principle, pump current measurements using excised membrane patches (Hamill et al., 1981) could overcome all of these difficulties. However, because the turnover rate of the Na/K pump is several orders of magnitude smaller than that of ion channels, a much larger patch area is required to obtain measurable (several picoamperes) Na/K pump currents than is needed for investigations of channel currents. For a pump turnover rate of 55 s–1 (Gadsby and Nakao, 1989), a stationary current of 1 pA would be generated by 114,000 pump molecules which, for a pump site density of 1,200 μm–2 (Nakao and Gadsby, 1986), would require a patch of membrane with an area of 95 μm^2. An excised patch of that size dictates that the pipette tip diameter be 11 μm if the patch is a flat disc, or ~8 μm if the patch has a (more realistic) hemispherical shape. Recently it has become possible to measure currents from such “giant” excised patches (Hilgemann, 1989, 1990).

Materials and Methods

Inside-out patches of sarcolemmal membrane were excised from the large blebs that consistently form on guinea pig ventricular myocytes either during 8 h of incubation in Ca-free, isotonic KCl Tyrode's solution, or within minutes of exposure to that solution made hypotonic (60–80%).

Pipettes with tip diameters of 9–20 μm were pulled in two stages (Hamill et al., 1981) using a Narishige PP83 puller and fire polished, and their tips were then made hydrophobic by coating them with a mixture of mineral oil and Parafilm, as previously described (Hilgemann, 1989, 1990).

Pipette solutions (extracellular membrane surface) were Ca-free and included 10 mM K to activate the Na/K pump, and 2 mM Ba, 20 mM TEA, 20 mM Cs, and 0.5 mM Cd to diminish ion channel and Na/Ca exchange currents. After formation of a seal of resistance > 1 G, the patch was excised and the pipette tip was immersed in a miniature chamber in which rapid exchanges (mixing time ~ 0.3 s) of bathing solution could be achieved.

Bath solutions (cytoplasmic surface) were prewarmed to 35°C and included 20 mM TEA, 20 mM Cs, 10 mM EGTA, 1 mM MgCl_2, up to 2 mM MgATP, and additional CsCl and/or NaCl (sum = 90 mM). Internal and external pH were usually kept at 7.4 using 10 mM HEPES. The patch current was recorded with an AXOCLAMP 1C amplifier and displayed on a chart recorder.

Figure 1. Inhibition by Na ortho-vanadate identifies the Na- and MgATP-dependent current as Na/K pump current. Activation of pump current by switching from Na-free solution to either 90 mM Na or 1 mM Na and subsequent reduction of pump currents by exposure to 20 μM vanadate in the presence of 90 mM Na. All solutions contained 1 mM MgATP.

Results and Discussion

Forward (outward) Na/K pump current was monitored at 0 mV, and was activated by switching either from Na-free to Na-containing bath (cytoplasmic) solution in the presence of 1 mM MgATP, or from ATP-free to ATP-containing solution in the presence of Na. Substituting Na for Cs activated the outward current only in the presence of MgATP and, without cytoplasmic Na, even 1 mM MgATP did not alter patch current.

The Na- and MgATP-dependent outward current is identified as Na/K pump current because it could be virtually abolished either by adding 20 μM vanadate to the bath solution (e.g., Fig. 1) or, alternatively, by perfusing the pipette with 0.5 mM strophanthidin or K-free (and Cs-free) solution (not illustrated).

Examination of the dependence of the pump current on MgATP concentration yielded a K0.5 of ~ 80 μM MgATP. This is comparable to values for the low-affinity Km for ATP (e.g., Robinson, 1983), as might be expected for measurements of stationary pump current under optimal conditions, with no ADP or inorganic phosphate present.

The K0.5 for activation of the pump current by cytoplasmic Na in the absence of internal K was found to be ~ 5 mM, and was larger in the presence of cytoplasmic K. The dependence on cytoplasmic [Na] is thus comparable to that previously observed in whole cell experiments (e.g., Glitsch et al., 1989; Nakao and Gadsby, 1989).

Figure 2. Observed pump current amplitude as a function of pipette tip diameter. The large variation of pump current size for a given pipette tip opening could result from variable patch geometry inside the tip.

The amplitude of maximally activated outward pump current (up to 36 pA in the largest patch) at 0 mV was similar in Na-containing or Na-free (N-methyl-D-glucamine) pipette solutions and, from the observed geometry of the patches, corresponding pump current densities were estimated to range from 1 to 3 μA∙cm–2. Fig. 2 shows pump current amplitude plotted against the pipette tip diameter. The observed variation of pump current for the same tip diameter is possibly attributable to differences in patch geometry, and hence in patch area, resulting from small changes in the sealing process. Surprisingly, the pump current density was somewhat higher on average than that estimated in internally dialyzed cardiac myocytes (Gadsby and Nakao, 1989). Moreover, in contrast to those measurements in myocytes, virtually no rundown of pump activity with time was observed in these experiments with giant excised patches. This suggests that the slow loss of ~ 50% of the initial pump current observed in those whole cell experiments might not reflect the washout of some essential diffusible factor, but rather the activation of some regulatory process(es) in the dialyzed cell. In this regard, it is intriguing that dopamine has been found (Bertorello et al., 1990) to diminish Na/K pump rate by 50% in striatal neurons, via synergistic intracellular pathways.

Preliminary experiments indicate that, under certain conditions, cytoplasmic Ca ions can depress Na/K pump current. Because large Na/Ca exchange currents can be activated in these patches (Hilgemann, 1990) in the presence of extracellular Na and cytoplasmic Ca, it was essential to fully inhibit Na/Ca exchange current in these experiments. For this reason, 5 mM NiCl_2 was added (Kimura et al., 1987) to the Ca-free pipette solution, on top of the 0.5 mM CdCl_2 usually present. Fig. 3 shows that a change from nominally 0 to 3 μM [Ca2+] did not affect patch current in the absence of cytoplasmic Na, when there was no pump current, but did diminish the outward pump current activated by 4.5 mM cytoplasmic Na. This inhibitory effect of Ca was seen only in the presence of Na at the extracellular membrane surface (pipette). Further experiments will be needed to clarify whether this is an effect on the K0.5 for activation by Na, or whether the pump is inhibited by Ca2+ also at saturating cytoplasmic [Na+].

Figure 3. Influence of Ca2+ on the Na/K pump current. Switching between 0 Ca (10 mM EGTA, no added Ca) and 3 μM Ca (10 mM EGTA, 8.5 mM Ca, pH 7) solutions did not affect the patch current in the absence of cytoplasmic Na, but decreased outward current in the presence of 4.5 mM cytoplasmic Na. This apparent reduction of Na/K pump current was observed only in the presence of extracellular Na (125 mM Na in the pipette in this instance).

Because of the improved accessibility of the cytoplasmic membrane surface, while retaining both control of membrane voltage and sidedness, the “giant patch” method holds great promise for investigations of possible regulation of the Na/K pump by protein kinases, or by other final effectors of second messenger systems. The technique also has the potential to tackle more biophysical, mechanistic problems, such as the still unresolved question of precisely how charge is translocated across the membrane, especially if very fast (< 2 ms) changes of substrate concentration can be achieved.

Acknowledgments

We gratefully acknowledge the excellent technical assistance of David Cash.

The work was supported by the American Heart Association (D. W. Hilgemann) and NIH grant HL-36783 (to D. C. Gadsby). D. C. Gadsby is a Career Scientist of the Irma T. Hirschl Trust.

References

Bahinski, A., and D. C. Gadsby. 1988. [K]0-dependent outward current contaminates strophanthidin-sensitive current in cardiac myocytes dialyzed with K-containing fluid, but not with Cs-containing fluid. Journal of General Physiology. 92:46a. (Abstr.)

Bertorello, A. M., J. F. Hopfield, A. Aperia, and P. Greengard. 1990. Inhibition by dopamine of (Na+ + K+)ATPase activity in neostriatal neurons through D_1 and D_2 dopamine receptor synergism. Nature. 347:386–388.

Borlinghaus, R., H.-J. Apell, and P. Läuger. 1987. Fast charge-translocations associated with partial reactions of the Na/K-pump. I. Current and voltage transients after photochemical release of ATP. Journal of Membrane Biology. 97:161–178.

Fendler, K., E. Grell, M. Haubs, and E. Bamberg. 1985. Pump currents generated by the purified Na+,K+-ATPase from kidney on black lipid membranes. EMBO Journal. 4:3079–3085.

Gadsby, D. C., and M. Nakao. 1989. Steady-state current-voltage relationship of the Na/K pump in guinea pig ventricular myocytes. Journal of General Physiology. 94:511–537.

Glitsch, H. G., T. Krahn, and H. Pusch. 1989. The dependence of sodium pump current on internal Na concentration and membrane potential in cardioballs from sheep Purkinje fibres. Pflügers Archiv. 414:52–58.

Hamill, O. P., A. Marty, E. Neher, B. Sakmann, and F. J. Sigworth. 1981. Improved patch-clamp techniques for high resolution current recordings from cells and cell-free membrane patches. Pflügers Archiv. 391:85–100.

Hilgemann, D. W. 1989. Giant excised cardiac sarcolemmal membrane patches: sodium and sodium-calcium exchange currents. Pflügers Archiv. 415:247–249.

Hilgemann, D. W. 1990. Regulation and deregulation of cardiac Na+-Ca2+ exchange in giant excised sarcolemmal membrane patches. Nature. 344:242–245.

Kimura, J., S. Miyamae, and A. Noma. 1987. Identification of sodium-calcium exchange current in single ventricular cells of guinea-pig. Journal of Physiology. 384:199–222.

Mathias, R. T., I. S. Cohen, and C. Oliva. 1990. Limitations of the whole cell patch clamp technique in the control of intracellular concentrations. Biophysical Journal. 58:759–770.

Nagel, G. A., K. Fendler, E. Grell, and E. Bamberg. 1987. Na+ currents generated by the purified (Na+ + K+)-ATPase on planar lipid membranes. Biochimica et Biophysica Acta. 901:239–249.

Nakao, M., and D. C. Gadsby. 1986. Voltage dependence of Na translocation by the Na/K pump. Nature. 323:628–630.

Nakao, M., and D. C. Gadsby. 1989. [Na] and [K] dependence of the Na/K pump current-voltage relationship in guinea pig ventricular myocytes. Journal of General Physiology. 94:539–565.

Robinson, J. D. 1983. Kinetic analyses and the reaction mechanism of the Na,K-ATPase. Current Topics in Membranes and Transport. 19:485–512.

Sodium Pump Current in Cardiac Purkinje Cells Dialyzed with ATP-free Solutions

Helfried G. Glitsch and André Tappe

Department of Cell Physiology, Ruhr University, Bochum, Germany

Introduction

Little is known about the dependence of the current generated by the active Na+/K+ exchange (Na+ pump current, Ip) on the intracellular ATP supply and about the importance of oxidative phosphorylation and glycolysis for this supply in cardiac Purkinje cells. The aim of this work is to demonstrate the dependence of Ip on the ATP concentration of the solution within the patch pipette and to evaluate the effect of some metabolic inhibitors on Ip during whole-cell recording from sheep Purkinje cardioballs.

Methods

The preparation of the cardioballs and the technique of whole-cell recording (Hamill et al., 1981) used have been described elsewhere in detail (Glitsch et al., 1989). The measurements were performed at a holding potential of –20 mV in order to suppress the Na+ and Ca2+ conductances of the sarcolemma. The bathing solution contained (in mM): 100 Na+, 3 Mg2+, 2 Ba2+, 0.9 Ca2+, 60 OH–, 72 Cl–, 120 HEPES, and 20 TEA+ (pH 7.4). Ip was identified as current activated by external Cs+. For this purpose a bathing medium containing 20 mM Cs+ instead of 20 mM TEA+ was applied every 2 min for ~ 20 s to the cell under study via a nearby glass pipette. The Cs+-activated current was abolished by 10–4 M dihydroouabain, a specific blocker of the Na+/K+ pump (Fig. 2 B). The standard patch pipette solution had the following composition (in mM): 30 Na+, 85 Cs+, 11 Mg2+, 6 EGTA, 86 HEPES, 72 Cl–, 55 OH– with or without 5 ATP, and 15 glucose (pH 7.4). To avoid changes in osmolarity and Cl– concentration, the concentrations of HEPES and Mg2+ were adjusted correspondingly. If required, metabolic inhibitors were added to the pipette medium. The initial resistance of the pipettes filled with the solution ranged from 1 to 4 M. The experiments were carried out at 30–33°C. The surface areas of the cells were estimated from microscopic measurements (cf. Glitsch et al., 1989). Whenever possible, results are presented as means ± SD. Differences between means were checked for significance by Student's t test. They were deemed significant if P was ≤0.05.

Results

Time Course of Ip

Fig. 1 displays the time course of Ip in cardioballs internally perfused by the standard pipette solution (open circles). Ip first increases to a maximum (1.2 ± 0.4 μA∙cm–2) within 8.0 ± 4.4 min (n = 6), probably because the intracellular Na+ concentration rises to an unphysiologically high level. Afterward Ip declines to 50% of its maximum value 27 ± 8.5 min after gaining access to the cell interior. The decline of Ip is significantly enhanced when cardioballs are dialyzed with an ATP- and glucose-free patch pipette solution (diamonds). The maximum pump current (1.32 ± 0.46 μA∙cm–2) is obtained 5.0 ± 3.5 min after establishing the whole-cell recording configuration. Ip declines to half of its maximum value within 21.5 ± 4.7 min (n = 6). Addition of 2 or 20 μM carbonylcyanide m-chlorophenylhydrazone (CCCP), an uncoupler of oxidative phosphorylation (Heytler and Prichard, 1962), to the ATP-and glucose-free pipette solution marginally enhanced the Ip decline (not illustrated). If, in order to further inhibit glycolysis, 2 mM iodoacetic acid was added to an ATP- and glucose-free pipette solution containing 2 μM CCCP, maximum Ip (1.42 ± 0.59 μA∙cm–2) was obtained within 5.3 ± 1.1 min (n = 6). The current decreased significantly faster to 50% of its maximum within 16.2 ± 7.4 min (data not shown). An even more rapid decline of Ip was observed in cardioballs perfused with an ATP- and glucose-free pipette solution containing 5 mM 2-deoxyglucose (2-DG), an inhibitor of glycolysis and an “ATP-sink” (Higgins et al., 1981). Further addition of 2 μM CCCP to the pipette medium causes only a small additional effect and diminishes Ip to 50% of its maximum (0.65 ± 0.39 μA∙cm–2 after 2 min) within 6.1 ± 0.6 min (n = 8; squares). The more rapid decline of Ip is prevented if the glucose-free pipette solution containing CCCP and 2-DG is supplemented with 15 mM ATP (triangles).

Figure 1. Time course of Ip during whole-cell recording from cardioballs under control conditions (O; pipette solution containing 5 mM ATP and 15 mM glucose), during internal perfusion with an ATP- and glucose-free medium , and during internal perfusion with an ATP- and glucose-free solution containing 2 μM CCCP and 5 mM 2-DG . Addition of 15 mM ATP to the latter medium prevents the fast Ip decline (∆). Ip normalized to its maximum. For clarity only the standard deviations of three means are shown. Holding potential, –20 mV.

Figure 2. (A) Ip-V curves under control conditions (●) and after 5–10 min of internal perfusion with an ATP- and glucose-free solution containing 2 μM CCCP and 5 mM 2-DG (O, different cells). Voltage steps of 800 ms duration performed from the holding potential of –20 mV to the potentials indicated. Ip values normalized to their values at +20 mV. (B) I-V relationships of a cardioball in dihydroouabain (10–4 M)-containing bathing solution with (O) or without (●) 20 mM CsCl. Standard pipette solution containing 5 mM ATP and 15 mM glucose. Lines in A and B are drawn by eye to connect the data points.

Ip-V Relationship

The mean Ip-V curve of five cardioballs internally perfused with the ATP- and glucose-containing pipette solution is shown in Fig. 2 A (solid circles). Surprisingly, little voltage dependence of Ip is observed. The Ip density amounted to 1.13 ± 0.55 μA∙cm–2. The figure also displays the mean Ip-V relationship of four different cells dialyzed with ATP- and glucose-free medium containing 5 mM 2-DG and 2 μM CCCP (open circles). The Ip density decreased to 0.41 ± 0.29 μA∙cm–2. However, the shape of the Ip-V curve remains essentially unchanged. Fig. 2 B demonstrates that under our experimental conditions the I-V relationship of a cardioball is little altered by 20 mM Cs+ after blockade of the Na+/K+ pump by 10–4 M dihydroouabain.

Discussion

The experiments described above reveal a decline of the Ip amplitude under a variety of experimental conditions. The cause of the decline in cells dialyzed with ATP- and glucose-containing solution (control condition) is not yet understood. Enhanced endocytosis of pump molecules or the wash-out by internal perfusion of a large molecule essential for Na+/K+ pumping might be involved (Gadsby and Nakao, 1989). On the one hand, the blockade of oxidative phosphorylation by CCCP does not markedly enhance the Ip decline in cells perfused with an ATP- and glucose-free pipette solution, but further addition of iodoacetic acid does. On the other hand, 2-DG alone abolishes Ip and this effect is only slightly (but significantly) accelerated by adding CCCP to the ATP- and glucose-free pipette medium containing 2-DG. These results are in line with observations of various authors emphasizing the important role of glycolysis in fueling the Na+/K+ pump (Dydynska and Harris, 1966; Paul et al., 1979). This might be due to the existence of cell membrane–bound, glycolytic enzymes which produce ATP in a pool preferentially used by the Na+/K+ -ATPase (Proverbio and Hoffman, 1977; Mercer and Dunham, 1981). The shape of the Ip-V relationship in cardic Purkinje cells remained unaffected even if the Ip density dropped to ~35% of that under control conditions after metabolic inhibition. Obviously, a diminished intracellular ATP concentration per se is of minor importance for the voltage dependence of Ip.

Acknowledgments

This work was supported by DFG, FG “Konzell”.

References

Dydynska, M., and E. J. Harris. 1966. Consumption of high-energy phosphates during active sodium and potassium interchange in frog muscle. Journal of Physiology. 182:92–109.

Gadsby, D. C., and M. Nakao. 1989. Steady-state current-voltage relationship of the Na/K pump in guinea pig ventricular myocytes. Journal of General Physiology. 94:511–537.

Glitsch, H. G., T. Krahn, and H. Pusch. 1989. The dependence of sodium pump current on internal Na concentration and membrane potential in cardioballs from sheep Purkinje fibres. Pflügers Archiv. 414:52–58.

Hamill, O. P., A. Marty, E. Neher, B. Sakmann, and F. J. Sigworth. 1981. Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflügers Archiv. 391:85–100.

Heytler, P. G., and W. W. Prichard. 1962. A new class of uncoupling agents—carbonyl cyanide phenylhydrazones. Biochemical and Biophysical Research Communications. 7:272–275.

Higgins, T. J. C., P. J. Bailey, and D. Allsopp. 1981. The influence of ATP depletion on the action of phospholipase C on cardiac myocyte membrane phospholipids. Journal of Molecular and Cellular Cardiology. 13:1027–1030.

Mercer, R. W., and P. B. Dunham. 1981. Membrane-bound ATP fuels the Na/K pump. Studies on membrane-bound glycolytic enzymes on inside-out vesicles from human red cell membranes. Journal of General Physiology. 78:547–568.

Paul, R. J., M. Bauer, and W. Pease. 1979. Vascular smooth muscle: aerobic glycolysis linked to sodium and potassium transport processes. Science. 206:1414–1416.

Proverbio, F., and J. F. Hoffman. 1977. Membrane compartmentalized ATP and its preferential use by the Na,K-ATPase of human red cell ghosts. Journal of General Physiology. 69:605–632.

Study of the Voltage Dependence of the Na,K Pump in Cardiac Cells Using Simulation

D. Renald Lemieux and J. Mailen Kootsey

Institut de Génie Biomédical, Université de Montréal/Ecole Polytechnique, Montréal, Québec, Canada; and National Biomedical Simulation Resource, Department of Cell Biology, Duke University Medical Center, Durham, North Carolina

Introduction

Published experimental studies have demonstrated the voltage-dependence of the Na,K pump under steady-state and transient conditions (Bahinski et al., 1988; Gadsby and Nakao, 1989; Gadsby et al., 1989). It was suggested that voltage dependence was limited to the Na+translocational step and described by an asymmetric energy barrier with ~ 10% of the applied voltage affecting the forward rate of reaction and the remainder affecting the reverse rate. Using simulation, we addressed two questions: (a) can the voltage-dependent steps be identified from measurements of the voltage dependence of the net rate, and (b) can the specific influence of voltage on the forward and reverse fluxes in those steps be determined

Methods

We used two consecutive mechanisms for the Na,K pump of stoichiometry of 3Na+:2K+:1ATP, GV3 and MGV3, based on the reduced Post-Albers scheme used by Chapman and co-workers (1983). GV3 has six steps, including a single voltagedependent step (step 3) which combines Na+translocation and Na+release into the extracellular medium (see Fig. 1 of Chapman et al., 1983). In MGV3, these two processes are allocated to two separate consecutive steps, but only Na+translocation (again step 3) is voltage dependent. The charge transfer across the membrane is expressed by the Butler-Volmer electrodic equation with a symmetry factor (β) varying between 0.5 and 1 to force the rate constant of decay (1/τ) of the transient current to decrease with depolarization as reported by Nakao and Gadsby (1986). We used step 3 to calculate the net flux (i.e., the forward flux) affected by e(1–β)FV/RT, minus the reverse flux, affected by e–βFV/RT.

With the optimization software SCoPfit on a SUN-4/280 mini-computer, we numerically solved the set of six (for GV3) or seven (for MGV3) differential equations to find values for rate coefficients, β, and pump site density so that the models could reproduce the steady-state I-V relationships for both forward- and backward-running of the pump, and [Na+]in and [K+]out activation curves (Nakao and Gadsby, 1986, 1989; Bahinski et al., 1988; Gadsby and Nakao, 1989). Initial parameter values and standard free energy of ATP splitting (–31.9 kJ/mol) were taken from Chapman et al. (1983). We used these optimized parameter values and simulated the voltage-clamp experiment of Nakao and Gadsby (1986) with both models running in their Na-Na exchange mode. Voltage jumps from –40 mV in both hyperpolarizing and depolarizing directions during 100 ms, from –120 mV to +80 mV by steps of 20 mV. We computed transient currents and curve-fit them with an exponential relation to find the time constant of decay (τ), which was inverted to obtain the rate constant of decay, which was then plotted against voltage. All simulations were done under conditions duplicating those used experimentally.

Results and Discussion

Behavior of the Models under Steady-State Conditions

Fig. 1, A-C, shows the published data points and the computed steady-state I-V relationships when β = 0.884 for GV3 (solid curves) and 0.932 for MGV3 (broken curves). Computed Na+and K+activation curves are hyperbolic with a constant of half-activation for external K+(KK0.5) of 1.69 mM for GV3 and 1.0 mM for MGV3 (published value is 1.5 mM), and for internal Na+, KNA0.5 = 10.3 mM for GV3 and 9.5 mM for MGV3 (published value is 11 mM).

Behavior of the Models under Transient Conditions

Fig. 1D shows the 1/τ - V curves computed by models GV3 (solid curve) and MGV3 (broken curve), along with the results published by Nakao and Gadsby (1986) (solid circles). The general feature of the computed 1/τ – V curve is a concave upward shape with a minimum varying in location along the voltage axis, according to the value of β (β ≈ 0.9 brings the minimum to +35 mV). The fit to published data was reasonable at potentials positive to −20 mV with both models. The discrepancy between published data and the curve computed by GV3 at potentials negative to −20 mV was reduced by using model MGV3, but it persisted at potentials negative to −60 mV.

Values of the Rate Coefficients

Values of the rate coefficients found by SCoPfit are reasonable for both models. fi and bi are, respectively, the forward and backward rate coefficients of the ith step. For GV3, f_2 = 138.66 s–1 is similar to those reported (180 s–1 at 21°C in Mårdh and Zetterqvist, 1974; 270 s–1 in Läuger and Apell, 1986; 200 s–1 in Reynolds et al., 1985). b_2 = 9 × 10^3 M–1 ∙ s–1 was imposed by the shape and amplitude of the backward-running Na,K pump I-V curve measured by Bahinski et al., 1988. b_2 × [ADP] = 45 s–1 at 36°C corresponds to the value of 20–30 s–1 reported by Karlish and co-workers (1978) at 20–25°C and a saturating [ADP] of ~5 mM.

For MGV3, the value of f_3 = 120.61 s–1 is comparable to 100 s–1 reported by Fendler et al. (1988), and lies within the values 104 and 490 s–1 reported by Gadsby and Nakao (1989). b_3 = 65.63 s–1 is somewhat higher than the values reported by Gadsby and Nakao (1989) (6 and 24 s–1), but it was imposed by the steady-state I-V curve reported by Bahinski et al., 1988.

Transient Currents

The discrepancy between published experimental and computed values of 1/τ at potentials negative to −60 mV (Fig. 1 D) may be eliminated by considering the following hypotheses, given by order of kinetic complexity: (a) A more detailed mechanism with a single voltage-dependent step containing separate steps to describe ion binding/unbinding, and ion occlusion/deocclusion. (b) A reaction cycle having more than one voltage-dependent step to reduce the increase with hyperpolarization of the net rate of increase of the intermediates involved in the voltage-dependent steps. (c) A reaction cycle having a single voltage-dependent step with a voltage-dependent symmetry factor, varying from < 0.5 at negative potentials to 0.5 at 0 mV and to > 0.5 at positive potentials. (d) A reaction cycle having more than one voltage-dependent step, each with a voltage-dependent symmetry factor.

Figure 1. The theoretical curves generated by both models are displayed as solid curves (GV3) and broken curves (MGV3). (A) Na,K pump current versus membrane potential. Experimental data points are from Gadsby et al. (1989; Fig. 1 A). Two values of [Na+]out are used: 1.5 mM (filled circles) and 150 mM (open circles). (B) Backward-running Na,K pump current versus membrane potential. Data points (filled circles) are from Bahinski et al. (1988; Fig. 2 D). The nominally zero [K+]out and [Na+]in were assumed to be 100 μM. (C) Na,K pump current versus membrane potential. Experimental data points are from Bahinski et al. (1988; Fig. 1 B). Three values of [K+]out are used: 5.4 mM (open circles), 1 mM (filled circles), and nominally zero (100 μM; solid triangles). The nominally zero [K+]in was assumed to be 100 μM. (D) 1/τ – V curve during Na-Na exchange. Simulation of the voltage-clamp experiment of Nakao and Gadsby (1986) with both models running in the Na-Na exchange mode. The nominally zero [K+]in and [K+]out were assumed to be 100 μM.

Given the limited information available on the voltage dependence of the Na,K pump, the fourth alternative is too complex to be considered at this time. The third alternative is simpler mathematically, but rather complex kinetically. It might indicate that membrane potential would change the conformation of the enzyme, which would displace the energy barrier inside the membrane. The second alternative is simpler and needs to be considered following Läuger and Apell (1988). Because model MGV3 considerably improved the fit of the 1/τ − V curve computed by GV3, we may conclude that we need to start the study using the first alternative, considering a very complex distribution of the increased population of intermediates. When model MGV3 was reduced to a pseudo-two-state mechanism, the published 1/τ − V curve was well fitted when b_3 ≈ 11.5 s–1 while imposing f_3= 120.61 s–1. The use of these values introduces more important problems: (a) it reduces the amplitude of the backward-running Na,K pump current, and (b) it results in a loss of the reduction of voltage dependence of the Na,K pump current with reduction of [Na+]out (the two curves of Fig. 1 A become parallel). This suggests that the starting point should be the second alternative. Consequently, the answer to the basic questions posed in the introduction is no, and the dilemma persists. To clarify it, we would need measurements of unidirectional efflux and influx, using labeled Na+ ions, when the Na,K-pump is working in its Na-Na exchange mode.

References

Bahinski, A., M. Nakao, and D. C. Gadsby. 1988. Potassium translocation by the Na+/K+ pump is voltage insensitive. Proceedings of the National Academy of Sciences, USA.85:3412–3416.

Chapman, J. B., E. A. Johnson, and J. M. Kootsey. 1983. Electrical and biochemical properties of an enzyme model of the sodium pump. Journal of Membrane Biology. 74:139–153.

Fendler, K., G. Nagel, and E. Bamberg. 1988. Pump currents generated by renal Na+K+-ATPase on black lipid membranes. Progress in Clinical and Biological Research. 273:225–232.

Gadsby, D. C., and M. Nakao. 1989. Steady-state current-voltage relationship of the Na/K pump in guinea-pig ventricular myocytes. Journal of General Physiology. 94:511–537.

Gadsby, D. C., M. Nakao, and A. Bahinski. 1989. Voltage dependence of transient and steady-state Na/K pump currents in myocytes. Molecular and Cellular Biochemistry. 89:141–146.

Karlish, S. J. D., D. W. Yates, and I. M. Glynn. 1978. Elementary steps of the (Na++ K+)-ATPase mechanism, studied with formycin nucleotides. Biochimica et Biophysica Acta. 525:230–251.

Läuger, P., and H.-J. Apell. 1986. A microscopic model for the current-voltage behavior of the Na,K-pump. European Biophysics Journal. 13:309–321.

Läuger, P., and H.-J. Apell. 1988. Transient behavior of the Na+/K+-pump: microscopic analysis of nonstationary ion-translocation. Biochimica et Biophysica Acta. 944:451–464.

Mårdh, S., and O. Zetterqvist. 1974. Phosphorylation and dephosphorylation reactions of bovine brain (Na+-K+)-stimulated ATP phosphohydrolase studied by a rapid-mixing technique. Biochimica et Biophysica Acta. 350:473–483.

Nakao, M., and D. C. Gadsby. 1986. Voltage dependence of Na translocation by the Na/K pump. Nature. 323:628–630.

Nakao, M., and D. C. Gadsby. 1989. [Na] and [K] dependence of the Na/K pump currentvoltage relationship in guinea-pig ventricular myocytes. Journal of General Physiology. 94:539–565.

Reynolds, J. A., E. A. Johnson, and C. Tanford. 1985. Incorporation of membrane potential into theoretical analysis of electrogenic ion pumps. Proceedings of the National Academy of Sciences, USA. 82:6869–6873.

Effects of Intra- and Extracellular pH on Na+/K+ Pump Current in Single Rabbit Cardiac Purkinje Cells

Frieda V. Bielen and Fons Verdonck

Interdisciplinary Research Centre, Catholic University of Leuven, Campus Kortrijk, 8500 Kortrijk, Belgium

Introduction

The effect of pH on the Na+/K+pump has been extensively studied in various tissues, but not the heart (Breitwieser et al., 1987). During hypoxia and reperfusion of the heart, pH changes do occur and can have important electrical and mechanical consequences. Intracellular acidification stimulates the Na+/K+ pump in rabbit cardiac Purkinje fibers (Bielen et al., 1990). The activation is caused by the increase in intracellular Na+(Na+i) due to Na+/H+exchange activation. It may mask an eventual direct inhibitory effect of low intracellular pH on the Na+/K+pump. To study the direct effect of pH on the Na+/K+pump, the intracellular [Na+] has to be controlled. Single cells, internally perfused via wide-tipped patch pipettes, are thought to fulfill this requirement. The experiments described below were designed to analyze the direct, independent effects of intracellular pH (pHi) and extracellular pH (pHo) on the Na+/K+pump current in single cardiac rabbit Purkinje cells.

Materials and Methods

Cell Isolation

The procedure used to isolate the Purkinje cells is modified from the method described by Scamps and Carmeliet (1989). After Langendorff perfusion for 10 min with a Ca2+-free Krebs Ringer solution, perfusion was continued for 12 min with the same solution containing 0.4 mg/ml collagenase and 0.15 mg/ml hyaluronidase. For the final 3 min, protease (0.1 mg/ml) was added to the latter solution. Purkinje fibers were dissected from both ventricles and stirred in the last enzymatic solution for 30 min to 1 h to dissociate the cells from the fibers.

Solutions and Drugs

The standard superfusion solution (31–34° C) contained (in mM): 144 NaCl, 20 CsCl, 0.5 MgCl_2, 1.8 CaCl_2, 10 glucose, and 10 HEPES, adjusted to pH 7.4 by NaOH. Whole-cell voltage clamp was performed with wide-tipped, fire-polished pipettes (resistance 2–4 MΩ) filled with (in mM): 80 Csaspartate, 5 MgCl_2, 5 MgATP, 5 Na_2 creatine phosphate, 20 EGTA, 5 HEPES, and 4.7 glucose, adjusted to pH 7.2 with 20 NaOH and CsOH. The holding potential was −20 mV throughout the experiments in order to inactivate Na+and Ca2+currents. To suppress K+currents, K+was replaced by Cs+, and Ba2+(1 mM) was added to all solutions. The superfusion could be changed at the cell within 250 ms via two multibarreled pipettes.

Results and Discussion

The Na+/K+pump current (Ip) was measured as the current activated by extracellular Cs+. Changing the superfusion solution from 0 to 20 mM CsCl induced an outward current (Fig. 1 A) which could be reversibly blocked by dihydro-ouabain (DHO, 10–4M). DHO is a well-known inhibitor of the Na+/K+pump. The pipette solution contained 30 mM Na+, which nearly maximally activates the Na+/K+pump (Glitsch et al., 1989; Nakao and Gadsby, 1989). To analyze the effects of pHo on the Na+/K+pump, the outward current induced by 20 mM Cs+was measured at pHo 6.0, 6.5, 7.4, and 8.2. The average pump currents relative to the pump current at pHo 7.4 are plotted as a function of pHo in Fig. 1 B. The pump current is not significantly affected by changes in pHo in the range 6.0–8.2. The pump current amounted to 113 ± 14 pA at pHo 7.4 (mean ± SEM, n = 12).

Figure1. Effect of pHo on Na+/K+pump current. (A) Chart recording of whole-cell current during addition and withdrawal of Cs+at pHo 7.4 and 8.2. (B) Na+/K+pump current (Ip) measured at pHo 6.0, 6.5, 7.4, and 8.2 relative to Ip at pHo 7.4 (means ± SE).

To study the effect of pHi on the Na+/K+pump, pHi was changed by addition and withdrawal of 20 mM NH_4Cl (Boron and De Weer, 1976). Addition of NH_4Cl, after a period in K+- and Cs+-free solution, generated an outward current (Fig. 2). This current could be blocked by DHO. Upon removal of NH_4Cl, which is assumed to acidify the cell, the holding current shifted inward. The 20-mM Cs+ pulses induced an outward current which increased with time to reach a plateau value after ~ 2.5 min. This suggests an inhibition of the pump by a NH_4Cl prepulse. However, a simple, straightforward conclusion cannot be drawn from these experiments. Using short activator pulses to measure Ip, a similar, although less pronounced, pattern of increasing pump current could be seen when the NH_4Cl prepulse was replaced by prolonged pump activation by other activator ions, e.g., K+ or Cs+. The current rapidly declined when activated for a longer period with maximal activator concentrations. These results suggest that the concentration of activator ions rises during pump inhibition and declines during pump activation. Because the latter effect became more prominent by increasing external K+ or Cs+, it was probably not due to external activator ion depletion but to a decrease in subsarcolemmal [Na+], The outward shift of the zero pump current trace (Fig. 2) supports the idea of an ineffectively controlled [Na+i]. It could be blocked by Ni2+ (5 mM), a blocker of Na+/Ca2+ exchange. This is also in accordance with an increase in Na+i. Changes in Na+i will interfere with direct effects of pHi on the Na+/K+ pump current. Therefore, the test procedure was changed. The pump was continuously activated with 20 mM Cs+. Pump amplitude was measured by applying short 0 mM Cs+pulses. In control conditions, a stable pump current could be measured. After NH_4Cl wash-out, there was a transient increase in DHO-sensitive outward current (Fig. 3). It reached a maximum after ~ 2 min. If at the end of the NH_4Cl prepulse pHo was lowered to 6.5,Ip was smaller than the steady-state Ip and the amplitude of the transient was markedly lower (not shown). Na+/H+ exchange is depressed at low pHo, slowing the restoration of pHi and decreasing the gain in Na+i(Bielen et al., 1990). The smaller initial Ip, measured during the first tens of seconds after NH_4Cl wash-out, points to an inhibition of the Na+/K+ pump by intracellular acidification. The time course of the increase in Ip, with a maximum after ~ 2 min, does not mirror the time course of pHi restoration (Boron and De Weer, 1976; Bountra et al., 1990), but does reflect the time course of the gain in Na+i during recovery from intracellular acidification in multicellular preparations (Bielen et al., 1990). The effect of low pHo and the time course of the Ip changes suggest a gain in Na+i due to Na+/H+ exchange activation, causing a secondary stimulation of the Na+/K+ pump.

Figure 2. Effect of NH_4Cl pulse on whole-cell current and on Na+/K+ pump current after NH_4Cl wash-out. Ip is measured as the Cs+-activated current.

In conclusion, the use of single cells did not allow us to accurately measure the direct effect of pHi on the Na+/K+ pump current. Direct effects of pHi may be masked by Na+i -related changes in pump current. These variations in pump current amplitude indicate that during measurement of Ip in our conditions, subsarcolemmal [Na+] deviates substantially from a concentration that saturates the Na+/K+ pump. The lack of an effect of external pH and the inhibitory effect of intracellular acidification on the Na+/K+ pump agree with results found previously in squid axon (Breitwieser et al., 1987).

Figure 3. Effect of NH_4Cl prepulse on whole-cell current. Downward deflections of the current were evoked by short 0 Cs+ pulses and represent the pump current amplitude.

References

Bielen, F. V., S. Bosteels, and F. Verdonck. 1990. Consequences of CO_2 acidosis for transmembrane Na+ transport and membrane current in rabbit cardiac Purkinje fibers. Journal of Physiology. 427:325–345.

Boron, W. F., and P. De Weer. 1976. Intracellular pH transients in squid giant axons caused by CO_2, NH_3, and metabolic inhibitors. Journal of General Physiology. 67:91–112.

Bountra, C., T. Powell, and R. D. Vaughan-Jones. 1990. Comparison of intracellular pH transients in single ventricular myocytes and isolated ventricular muscle of guinea-pig. Journal of Physiology. 424:343–365.

Breitwieser, G. E., A. A. Altamirano, and J. M. Russell. 1987. Effects of pH changes on sodium pump fluxes in squid giant axon. American Journal of Physiology. 253:C547–C554.

Glitsch, H. G., T. Krahn, and H. Pusch. 1989. The dependence of sodium pump current on internal Na concentration and membrane potential in cardioballs from sheep Purkinje fibers. Pflügers Archiv. 414:52–58.

Nakao, M., and D. C. Gadsby. 1989. [Na] and [K] dependence of the Na+/K+ pump current–voltage relationship in guinea pig ventricular myocytes. Journal of General Physiology. 94:539–565.

Scamps, F., and E. Carmeliet. 1989. Delayed K+ current and external K+ in single cardiac Purkinje cells. American Journal of Physiology. 257:C1086–C1092.

Current–Voltage Relation of the Na-K Pump–generated Current in A6 Cells

Jean-Daniel Horisberger and Marie-Christine Broillet

Institut de Pharmacologie, Université de Lausanne, Lausanne, Switzerland

Introduction

The Na-K pump transports an unequal number of Na+ and K+ ions and thus it must move a net number of charges in the electrical field existing across the membrane. The charge translocating step(s) of the pump cycle must have a rate that is dependent on the membrane potential. However, the effect of membrane potential on the overall pump activity will depend on how rate limiting these voltage-dependent steps are. Na-K pump current–voltage (I-V) curves have been established in several cell types and although voltage dependence has generally been observed, the shape of the I-V curve was different in excitable cells (Gadsby and Nakao, 1989; Rakowski et al., 1989), oocyte (Schweigert et al., 1988), and epithelial cells (Horisberger and Giebisch, 1989). We have studied the voltage dependence of the Na-K pump current in cultured A6 cells using the technique of apical membrane permeabilization with an ionophore antibiotic in order to voltage clamp the basolateral membrane (Lewis et al., 1977).

Methods

A6 cells were cultured on permeable support (Transwell®, 4.7 cm^2; Costar Corp., Cambridge, MA) in a standard amphibian culture medium supplemented with 5% FCS and 100 nM dexamethasone. Aldosterone (300 mM) was added 20–48 h before measurements to increase Na-K pump activity (Verrey et al., 1987). The epithelia were placed in a modified Ussing chamber allowing continuous perfusion of both sides of the epithelium. The apical membrane was permeabilized by exposure to 10 μM amphotericin B in a low Cl–/high K+ solution, while the transepithelial potential (Vt) was continuously clamped at –60 mV. We have shown that this maneuver fully permeabilizes the apical membrane, while the permselective properties of the basolateral membrane or of the paracellular “shunt” pathway are preserved (Broillet and Horisberger, 1990). The dominant basolateral membrane K+ conductance was reduced by addition of 2 mM barium (to block inward K+ currents) and 100 μM quinidine (to block outward K+ currents) to the basolateral solution. I-V curves were established by recording the transepithelial current (It) when Vt was stepped from the baseline –60 mV to 0, –20, −40, −80, −100, and −120 mV for 0.4-s periods, with return to −60 mV for at least 4 s between each step.

Results

The activity of the Na-K pump is dependent on the concentration of basolateral K+ ([K+]bl) and intracellular Na+ ([Na+]i). Under our experimental conditions [Na+]i was assumed to equilibrate with the apical Na+ concentration ([Na+]ap) across the permeabilized apical membrane. Increasing [Na+]ap in the presence of 12 mM [K+]bl induced an outward It of 3.8 ± 0.8μA·cm–2, while the same maneuver performed with a K-free basolateral solution resulted in a 1.5 ± 0.5 μA·cm–2 current. Similarly, adding 12 mM K+ to a previously K+ -free basolateral solution in the presence 20 mM [Na+]ap induced a 2.1 ± 0.6 μA-cm–2 outward It (example in Fig. 1), while the same maneuver in the presence of the 1 mM Na+ apical solution had no significant effect (−0.2 ± 0.2 μA-cm–2). Thus, the [Na+]i-dependent K+-activated current and the [K+]o-dependent Na+-activated current were 2.2 and 2.3 μA-cm–2, respectively, two similar estimates of the Na-K pump current.

Strophanthidin (20 μM), added to the basolateral side (in the presence of 20 mM [Na+]ap and 12 mM [K+]bl), reduced It (at −60 mV) by a similar amount, 2.4 ± 0.8 μA·cm–2 (n = 8). The effects of strophanthidin were completely reversible. I-V curves of the strophanthidin-sensitive current were computed from the It values before and after addition of 20 μM strophanthidin. The resulting I-V curve (Fig. 1) showed a distinct voltage dependence between −60 and −120 mV (equivalent to a “pump” conductance of 18 μS·cm^2), but the strophanthidin-sensitive current saturated at smaller potentials: between −40 and 0 mV there was practically no voltage dependence. Increasing [K+]bl from 0 to 12 mM in the presence of 20 μM strophanthidin produced only a small outward current (Fig. 1).

Figure 1. Changes in the transepithelial current (∆It) induced by (a) adding 12 mM K+ to the K+-free basolateral solution , (b) adding 20 μM strophanthidin to a 12 mM K+ basolateral solution (●), and (c) adding 12 mK+ in the presence of strophanthidin (∆) as a function of the membrane potential.

Discussion

These data demonstrate the presence of an electrogenic Na-K pump in the basolateral membrane of A6 cells. The small effect of a large change (0–12 mM) in basolateral K+ concentration in the presence of strophanthidin indicates that the strophanthidin-sensitive current is for the most part a Na-K pump–generated current and not a K+ current flowing through a conductive pathway due to changes of K+ concentration in the unstirred layers along the basolateral membrane.

In this preparation of A6 cells, equivalent to a physiologically isolated basolateral membrane, the I-V curve of the Na-K pump current is very similar to that established by a different technique in the isolated perfused collecting tubule of Amphiuma (Horisberger and Giebisch, 1989). There are, however, some differences between these pump I-V curves obtained in renal epithelial tissues and those observed in excitable cells (Gadsby and Nakao, 1989; Rakowski et al., 1989) or oocyte (Schweigert et al., 1988) where the voltage dependence was marked in the −60−0 mV membrane potential range.

These differences could be due to a number of causes: for instance, rate-limiting access of substrates (Na+,K+,ATP) to the pump could cause an apparent saturation at high levels of activity; the results reported for squid axon (Rakowski et al., 1989) and rat heart cells (Gadsby and Nakao, 1989) were obtained with internally dialyzed cells, allowing a more precise control of intracellular composition. Differences in experimental conditions (for instance, intracellular and extracellular K+ and Na+ concentrations) could also explain part of the difference. However, as different populations of Na-K pump isoforms are found in excitable tissues and in epithelia (Sweadner, 1989), it is tempting to speculate that some kinetic characteristics of the Na-K pump are isoform specific.

Summary

We have measured the intrinsic electrical activity of the Na-K pump present in the basolateral membrane of A6 cells. The I-V curve of the pump generated current showed a distinct voltage-dependence in the high negative potential range.

Acknowledgments

This work was supported by grant 31-27798.89 from the Swiss “Fonds National de la Recherche Scientifique.”

References

Broillet, M.-C., and J.-D. Horisberger. 1990. Blockers of basolateral K+ channels in A6 cells. Experientia. 46:A53. (Abstr.)

Gadsby, D. C., and M. Nakao. 1989. Steady-state current-voltage relationship of the Na/K pump in guinea pig ventricular myocytes. Journal of General Physiology. 94:511–537.

Horisberger, J.-D., and G. Giebisch. 1989. Na-K-pump current in the Amphiuma collecting tubule. Journal of General Physiology. 94:493–510.

Lewis, S. A., D. C. Eaton, C. Clausen, and J. M. Diamond. 1977. Nystatin as a probe for investigating the electrical properties of a tight epithelium. Journal of General Physiology. 70:427–440.

Rakowski, R. F., D. C. Gadsby, and P. De Weer. 1989. Stoichiometry and voltage dependence of the sodium pump in voltage-clamped, internally dialyzed squid giant axon. Journal of General Physiology. 93:903–941.

Schweigert, B., A. V. Lafaire, and W. Schwarz. 1988. Voltage dependence of the Na-K ATPase: measurements of ouabain-dependent membrane current and ouabain binding in oocytes of Xenopus laevis. Pflügers Archiv. 412:579–588.

Sweadner, K. J. 1989. Isozymes of the Na+/ K+-ATPase. Biochimica et Biophysica Acta. 988:185–220.

Verrey, F., E. Schaerer, P. Zoerkler, M.P. Paccolat, K. Geering, J.P. Kraehenbuhl, and B. C. Rossier. 1987. Regulation by aldosterone of Na+,K+ -ATPase mRNAs, protein synthesis, and sodium transport in cultured kidney cells. Journal of Cell Biology. 104:1231–1237.

Transport Properties of an Electrogenic Proton Pump from Enterococcus hirae

H.-J. Apell, C. Hölscher, and M. Solioz

Department of Biology, University of Konstanz, Konstanz, Germany; and Department of Clinical Pharmacology, University of Berne, Berne, Switzerland

Introduction

We recently described the purification of a novel ATPase from the gram-positive bacteria Enterococcus hirae, formerly called Streptococcus faecalis or faecium (Hugentobler et al., 1983). The enzyme has the following properties: (a) it consists of a single polypeptide component of Mr 78,000, (b) it is inhibited by micromolar concentrations of vanadate, and (c) it forms an aspartyl phosphate intermediate as part of the reaction cycle. Based on these properties, this bacterial ATPase appears to be a P-type ionmotive ATPase, chiefly represented by the eukaryotic Na,K-ATPase, Ca-ATPases, and the fungal and plant plasma membrane H-ATPase (Serrano, 1988).

Due to the relative ease of purification and its stability, the E. hirae ATPase appears to be a suitable model system for studying fundamental aspects of ATP-driven ion translocation. We have previously reported the reconstitution of the ATPase into proteoliposomes, their functional analysis (Fürst and Solioz, 1986), and a fluorescence method for investigating the electrogenic properties of this enzyme (Apell and Solioz, 1990).

Methods

The protein was isolated from E. hirae (ATCC 9790) as described previously (Solioz and Fürst, 1988). 50–125 μg protein solubilized with decyl maltoside and 2.5 mg asolectin or bacterial lipid extract solubilized in an octyl glucoside–containing buffer were mixed on ice and transferred to dialysis tubing. Dialysis was performed at 4°C against two changes of 1,000 vol of 50 mM Na_2SO_4, 10 mM Na-HEPES, pH 7.5, or the buffer specified in the experiment, respectively, for 6–8 and 50 h. For detailed description see Apell and Solioz (1990).

Fluorescence measurements were carried out on a 650–40 fluorescence spectrophotometer (Perkin-Elmer Corp., Norwalk, CT), using a thermostatically controlled cuvette holder equipped with a magnetic stirrer. The excitation wavelength was set to 580 nm (slit width 20 nm) and the emission wavelength to 660 nm (slit width 20 nm). Experiments were performed at a final concentration of 100 nM oxonol. Temperature and pH were controlled before and after the experiments.

Figure 1. Transient membrane potential generated by the ATPase. Vesicles were reconstituted in buffer containing 50 mM Na_2SO_4 and 10 mM HEPES, pH 7.5, and measured in buffer containing 50 mM Na_2SO_4, 5 mM MgSO_4, 10 mM HEPES, pH 7.5, and 100 nM oxonol VI. Vesicles and 1.25 mM ATP were successively added. F is the fluorescence intensity. The maximum rise in fluorescence intensity after addition of ATP corresponds to a membrane potential UAV 70 mV.

Results and Discussion

Electrogenicity of the Pump

Fig. 1 shows a typical experiment. Upon ATP addition the inside-out oriented pumps start to work and create an inside positive potential. The fluorescence reaches a maximum within ~ 100 s and then declines to the initial value. The maximum value corresponds to voltages between 30 and 150 mV depending on the activity of the preparation. The transient nature of the pumping activity was examined. It was caused by successively switching off pumps, and was not influenced by the membrane potential and the ionic composition of the buffer on either face of the membrane.

The pH dependence is presented in Fig. 2. In this plot the fluorescence signal is transformed into membrane potential, UAV, applying the method of Apell and Bersch (1987). The potential has to be interpreted as average potential since the vesicle preparation is known to be heterogeneous in size and number of active pumping molecules. The data of Fig. 2 show that with decreasing extravesicular (cytoplasmic) pH the transient is prolonged and at pH < 5.6 the pump works continuously. Above pH 6.2 the transient is so short that the high possible potential is no longer obtained, although the initial pumping rate is still maximal.

Figure 2. Influence of the pH on the membrane potential generated by the ATPase. With the exception of pH, the conditions of the experiment were as in Fig. 1. The pH was adjusted to the indicated value with H_2SO_4 or NaOH and confirmed to be stable during the time of the experiment. (A) Time course of the average membrane potential, UAV, at different pH values. (B) Initial slope of the potential signal dUo/dt, as a function of the pH. This parameter is proportional to np·vp·zp: the product of the number of active pumps, np, the turnover rate, vp, and the pump stoichiometry factor, zp. npcan be assumed to be constant in a vesicle preparation.

Figure 3. Schematic reaction cycle for the Enterococcus hirae ATPase. E_1 and E_2 are conformations of the enzyme with the proton binding site(s) exposed to the cytoplasm and the extracellular medium, respectively. In the “occluded” states (H)E_1 ~ P and ()E_1 ~ P the proton cannot directly exchange between binding site and aqueous phase.

Model of the Pumping Cycle

Fig. 3 outlines a minimal model for the reaction cycle of the E. hirae ATPase: the high affinity form of the enzyme, E_1, binds a cytoplasmic proton, H+cyt, to form HE_1; ATP is then used to generate the high energy intermediate (H)E_1~ P, where the parentheses indicate that the proton may be “occluded” (i.e., not readily exchangeable with bulk protons). After transition to P-E_2H with relaxation of the high energy intermediate ~ P to –P and conversion of the enzyme to the low affinity form E_2, the proton can be expelled to the exterior. Finally, inorganic phosphate, Pi, is released and E_2 returns to the initial form, E_1. A simple mechanism for the downregulation of the ATPase at high cytoplasmic pH would be deprotonation of a functional group. Since at pH 7.5 ~ 20 turnovers can take place before the activity is inhibited, this deprotonation would have to occur either in a short-lived intermediate or from a slowly exchanging site such as the “occluded” protein-binding site in (H)E ~ P. This would result in the resting state ()E_1 ~ P, which could be reactivated by acidic pH. Prolonged storage of the ATPase in the absence of ATP (as during reconstitution) could, even at high pH, conceivably lead to a slow depopulation of the state ()E_1~ P by direct pH-independent conversion of inactive ()E_1~ P to the active E_1,-form (Fig. 3, dashed arrow). Accumulated E_1 would then be responsible for the observed transient activity at neutral pH values. Since the resting form of the enzyme, ()E_1 ~ P, is not an obligatory intermediate of the reaction cycle, an active enzyme molecule could go through several reaction cycles until it is bypassed into the ()E_1~ P-trap.

This model can be tested by a pH jump experiment as shown in Fig. 4. When most of the pumps had reached the inactivated state ()E_1~ P after ~ 10 min, the activity could be regained by lowering the pH to 5.8 as suggested by the model in Fig. 3 and by numerical simulations of the cyle (not shown).

Figure 4. Influence of the pH on the electrogenic pump activity of the ATPase. An experiment performed in buffer of pH 5.85 (upper dashed line) is compared with an experiment at pH 7.5 (solid line). When the pumping transient had almost ended after ~10 min, H_2SO_4was added to lower the external pH to 5.8. The lower dashed line indicates the proton diffusion potential in the absence of ATP, generated by proton leakage due to the pH gradient.

Figure 5. Initial slope of voltage rise, dUo/dt as a function of ATP concentration. Data fitted with a Michaelis-Menten type kinetics of first order. The Km values and the maximum slope (under saturating ATP concentrations) are pH dependent.

Interactions with ATP, ADP, and Pi

To investigate the influence of the ATP concentration on the pumping activity, the initial slope of the fluorescence was measured and transformed into the initial slope of membrane potential, dUo/dt. At all pH values measured between 5.8 and 8.4 the concentration dependence could be fitted by a first order Michaelis-Menten type kinetics with Km values between 9 (pH 5.8) and 40 (pH 8), as indicated in Fig. 5. Unlike the Na,K-ATPase, there seems to be only one ATP-binding step in the cycle. The enzyme becomes more senstivie to ATP at low pH values. This observation can be interpreted as a preference of the ATP-binding site for the protonated form of ATP.

Under the condition of saturating ATP concentrations the initial slope still exhibits a strong pH dependence. This fact is a strong indication that the ratelimiting step of the pump cycle is dependent on binding/dissociation of a proton on the cytoplasmic side. The deprotonated form is the more active form. ADP inhibits the pump as presented in Fig. 6. Analysis of the kinetic behavior leads to the conclusion that ATP and ADP compete for the same binding site. At low pH the enzyme is more sensitive to ADP. Since the pH dependences for ADP and ATP binding are different (shifted by > 1 pH unit to high pH values for ADP), it is most likely that the whole process is controlled by protons in a very complex way.

Figure 6. Initial slope of membrane potential, dUo/dt as a function of ADP concentration at pH 5.8 and 7.9 ATP concentration was in all experiments 300 μM. Half-inhibition concentrations of ADP are 160 μM. (pH 5.8) and 650 μM. (pH 7.9). Lines were drawn according to MichaelisMenten-type kinetics for competitive substrate binding.

Inorganic phosphate in the cytoplasmic buffer could also influence the pumping activity by “low energy” phosphorylation of the enzyme in the assumed E_2→ P-E_2 transition (Fig. 3). In the pH range between 5.8 and 8 and phosphate concentrations up to 40 mM no significant effect could be detected. This result can be interpreted such that the transition P-E_2 E_2 is not limiting.

Conclusion

All experiments performed so far can be explained in terms of the scheme of Fig. 3 and support it as an acceptable description of the pumping mechanism.

Acknowledgments

We gratefully acknowledge the stimulating discussions with Peter Läuger.

This work was supported by the Deutsche Forschungsgemeinschaft (Sonderforschungs-bereich 156) and grant 3.600.087 from the Swiss National Foundation.

References

Apell, H.-J. and B. Bersch. 1987. Oxonol VI as an optical indicator for membrane potentials in lipid vesicles. Biochimica et Biophysica Acta. 903:480–494.

Apell, H.-J., and M. Solioz. 1990. Electrogenic transport by the Enterococcus hirae ATPase. Biochimica et Biophysica Acta. 1017:221–228.

Fürst, P., and M. Solioz. 1986. The vanadate-sensitive ATPase of Streptococcus faecalis pumps potassium in a reconstituted system. Journal of Biological Chemistry. 261:4301–4308.

Hugentobler, G., I. Heid, and M. Solioz. 1983. Purification of a putative K+ -ATPase from Streptococcus faecalis. Journal of Biological Chemistry. 258:7611–7617.

Serrano, R. 1988. Structure and function of proton translocating ATPase in plasma membranes of plants and fungi. Biochimica et Biophysica Acta. 947:1–28.

Solioz, M., and P. Fürst. 1988. Purification of the ATPase of Streptococcus faecalis. Methods in Enzymology. 157:680–689.

[†] Deceased 13 September 1990.

Regulation and Adaptation

Regulation of Expression and Polar Distribution of Na,K-ATPase in Renal Epithelium during Recovery from Ischemic Injury

M. Kashgarian, S. K. Van Why, F. Hildebrand, A. S. Mann, K. M. Gaudio, and N. J. Siegel

Departments of Pathology and Pediatrics, Yale University School of Medicine, New Haven, Connecticut 06510

Introduction

Renal ischemia results in a decrease in renal tubule transport of sodium and marked structural alterations of the renal tubular epithelial cells, which are associated with a clinical syndrome of acute renal failure. Previous work from our laboratory has shown a decrease in the oxygen consumption rate of renal proximal tubules in suspension after in vivo ischemic injury (Gandio et al., 1989). The decline in respiration is related predominantly to a decrease in functional Na/K-ATPase activity. However, when Na/K-ATPase activity was measured enzymatically under Vmax conditions, no significant difference was found when compared with non-ischemic controls. Recovery is associated with a return to a normal metabolic profile, and that process is accelerated by treatment with thyroxin or MgCl_2-ATP (Sutter et al., 1988). Since the discrepancy between functional and enzymatic Na/K-ATPase activity might be related to a maldistribution of the enzyme (Molitoris et al., 1989), and since the cortical cytoskeleton may play an important role in the maintenance of cell polarity (Morrow et al., 1989; Nelson and Hammerton, 1989), the cellular distribution of Na/K-ATPase subunits and cortical cytoskeletal elements was determined immunocytochemically after ischemia and during recovery. To determine the relative contribution of the synthesis of new pumps versus recycling of abnormally distributed pumps, mRNA abundance for Na/K-ATPase subunits are determined.

Methods

Renal ischemia was induced in Sprague-Dawley rats with a vascular clamp placed on the aorta proximal to both renal arteries for 45 min. After the ischemic interval, the kidneys were allowed to reperfuse for either 15 min, 2 h, 6 h, 12 h, or 24 h before study. In two separate groups of rats, after 45 min of ischemia rats were infused with 25 μmol of MgCl_2-ATP or thyroxin during the first 15 min of reperfusion after the ischemic period. Kidneys were harvested at each time period and a portion was fixed in periodate lysine paraformaldehyde, and Na/K-ATPase subunits, fodrin, and ankyrin were localized using methods previously described (Morrow et al., 1989). The remaining portion of the kidney was decapsulated, and snap-frozen in liquid nitrogen, and pulverized. RNA was isolated by standard methods (Chomczynski and Sacchi, 1987) and subjected to formaldehyde gel electrophoresis and RNA was transferred by capillary transfer onto Hybond. Detection of the target mRNA was accomplished using full-length cDNA probes labeled by [32P]ATP. Differences in loading were assessed by ethidium bromide staining and examination under ultraviolet lights. Control for loading was further assessed using a cDNA probe for glyceradehyde phosphate dehydrogenase.

Results

Immunocytochemical Studies

After ischemia and 15 min of recovery there were marked alterations of the proximal tubular epithelial cells. There was loss of the brush border with internalization of the apical membranes and the formation of subapical membrane vesicle profiles. Immunocytochemical localization of Na/K-ATPase α- and β-subunits showed a loss of the polar distribution of the enzyme. Na/K-ATPase was no longer confined to the lateral and basal infoldings of the plasma membrane. The subunits were also present on the apical membrane, apical membrane blebs, and subapical membrane vesicle profiles (Fig. 1). Since previous work from our laboratory and others (Morrow et al., 1989; Nelson and Hammerton, 1989) has demonstrated that the polar distribution of Na/K-ATPase is due to an interaction of the α-subunit with ankyrin and the cortical cytoskeletal protein fodrin, the distribution of these two proteins was also examined. There was a similar loss of the polar distribution of ankyrin, with ankyrin present in the apical membranes and in the subapical vacuoles. The distribution of fodrin was also markedly altered in that it was no longer closely associated with Na/K-ATPase and ankyrin along the lateral and basal infoldings of the plasma membrane, but was now found diffused throughout the cytosol. After 24 h of recovery, Na/K-ATPase distribution was once again associated with the basal and lateral membranes (Fig. 2). Ankyrin codistributed with Na/K-ATPase. Fodrin, however, was incompletely restored to a normal distribution. It associated with the plasma membrane and codistributed with Na/K-ATPase and ankyrin along the basal and lateral membranes, but continued to be present in a diffuse cytosolic pattern in the region of the terminal web of the apical membrane.

Figure 1. Electron micrographs of renal proximal tubular cells after 45 min of ischemia and 15 min of reflow. Immunocytochemical localization of the α-subunit Na/K-ATPase (A) shows a loss of the normal polar basolateral distribution. There is dense reaction product associated with the apical membrane subapical vesicles and apical blebs. The loss of polarity of the enzyme is accompanied by disruption of the cortical cytoskeleton (B) with fodrin no longer associated with the plasma membrane and is present diffusely in the cytosol.

Figure 2. Electron micrograph of renal proximal tubule cells after 24 h of reflow. The distribution of the α-subunit of Na/K-ATPase has returned to its normal polar basolateral distribution (A). Fodrin (Sutter et al., 1988) codistributes with the α-subunit in the basolateral membranes but has not been completely restored to the preischemic state. There is persistent diffuse staining in the subapical region.

mRNA Analysis

RNA was extracted from the renal cortex of animals at 15 min, 6 h, 12 h, and 24 h. After RNA extraction, Northern analysis was performed using cDNA probes for the α-1 and β-1 subunits of Na/K-ATPase and compared with control nonischemic animals. mRNA expression for both subunits was parallel (Fig. 3). After ischemia and 2 h of reflow, mRNA abundance for α- and β-subunits fell (at a time when functional pump activity is reduced, but Na/K-ATPase activity at Vmax is unchanged). In contrast, mRNA for actin was increased. The severity of the decrease of mRNA abundance for Na/K-ATPase subunits was modulated by treatment with MgCl_2-ATP and thyroxin. By 24 h of reflow, untreated animals had decreased message compared with both nonischemic controls and MgCl_2-ATP- or thyroxin-treated animals.

Discussion

These results demonstrate that the decrease in ouabain-inhibitable oxygen consumption seen after ischemic injury is associated with a loss of the polar distribution of Na/K-ATPase. The loss of polarity appeared to be the result of disruption of the α-subunit-ankyrin-fodrin interaction, since the polar distribution of these proteins was also lost. The restoration of polarity paralleled the gradual return of functional Na/K-ATPase activity toward preischemic levels. Since our hypothesis suggests that ankyrin complexed to mature Na/K-ATPase oligomers associates with laterally organized fodrin due to the high affinity ankyrin-fodrin interaction, the ability of fodrin to assemble and bind actin is critical. The assembly of fodrin and its interaction with Na/K-ATPase may involve changes in intercellular calcium and/or the expenditure of metabolic energy which is reduced during the ischemic period.

Figure 3. mRNA expression for α- and β-subunits of Na/K-ATPase. There is a coordinate decrease in mRNA for both α- and β-subunits after ischemic injury. The decrease is blunted by treatment with MgCl_2-ATP and thyroxin. Solid bars, control; stippled bars, ATP treated; hatched bars, thyroxine treated.

The fall in mRNA abundance for the subunits of Na/K-ATPase could be attributed either to a decrease in transcription or, more likely, to a change in mRNA stability, in particular, an increase in mRNA degradation. Since the treatment with the ameliorative agents MgCl_2-ATP and thyroxin attenuated this decrease, and since these agents have been demonstrated to accelerate ATP recovery after ischemia (Sutter et al., 1988), it is likely that preservation of normal transcription and prevention of mRNA degradation may be the mechanism involved. Overall, the studies demonstrate that the renal tubule conserves its metabolic effort by reducing transport activity. This is accomplished by a reduction of functional Na/K-ATPase activity while total enzymatic Na/K-ATPase is unchanged. The discrepancy between ouabain-inhibitable oxygen consumption and enzymatic activity determined in membranes at Vmax conditions appears to be associated with the loss of the polar distribution of Na/K-ATPase. The loss of polarity is due to a disruption of the cortical cytoskeleton and the normal interaction of the α-subunit of Na/K-ATPase with ankyrin and fodrin. Recovery of transport activity after reperfusion is accelerated by the treatment with MgCl_2-ATP or thyroxin and the polar distribution of Na/K-ATPase is restored. The mRNA abundance for Na/K-ATPase subunits is reduced after ischemia and the reduction is blunted by these ameliorative agents. These results suggest that the improvement in functional pump activity in the first 24 h of recovery from ischemia may be related to recovery of cellular integrity and may involve the recycling of maldistributed pump units.

References

Chomczynski, P., and N. Sacchi. 1987. Single step method for RNA isolation by acid quanidinium thiocyanate-phenol-chloroform extraction. Analytical Biochemistry. 162:156–159.

Gaudio, K. M., G. Thulin, T. Ardito, M. Kashgarian, and N. J. Siegal. 1989. Metabolic alterations in proximal tubule suspensions obtained from ischemic kidneys. American Journal of Physiology. 256:F383–F390.

Molitoris, B. A., L. K. Chan, J. J. Shapiro, J. D. Conger, and S. A. Falk. 1989. Loss of epithelial polarity: a novel hypothesis for reduced proximal tubule Na+ transport following ischemic injury. Journal of Membrane Biology. 107:119–127.

Morrow, J. S., C. D. Cianci, T. Ardito, A. S. Mann, and M. Kashgarian. 1989. Ankyrin links fodrin to the α-subunit of Na/K-ATPase in Madin-Darby canine kidney cells and in intact renal tubule cells. Journal of Cell Biology. 108:455–465.

Nelson, J. J., and R. W. Hammerton. 1989. A membrane cytoskeletal complex containing Na/K-ATPase ankyrin and fodrin in Madin-Darby canine kidney cells. Journal of Cell Biology. 108:893–902.

Sutter, P. M., G. Thulin, M. Stromski, T. Ardito, K. M. Gaudio, M. Kashgarian, and N. J. Siegal. 1988. Beneficial effect of thyroxin in the treatment of ischemic acute renal failure. Pediatric Nephrology. 2:1–7.

Regulation of the Myocardial Na+,K+-ATPase and Ca2+ during Ischemia and Reperfusion

Tai Akera and Takashi Koyama

Department of Pediatric Pharmacology, National Children's Hospital Medical Research Center, Setagaya-ku, Tokyo 154, Japan; and Department of Medicine, Keio University School of Medicine, Shinanomachi, Tokyo 160, Japan

Introduction

Ischemia and subsequent reperfusion of the heart damages the sarcolemmal sodium pump, increases intracellular Ca2+ concentrations, and disrupts the sarcolemma. Because contracture develops after the start of reperfusion of the ischemic heart, but not during the ischemia, an increase in intracellular Na+ during ischemia and the exchange of Na+ with Ca2+ after reperfusion have been proposed to be the mechanisms of increase in intracellular Ca2+ concentrations, which in turn causes contracture and dysfunction of the cardiac muscle characterized as “stunned myocardium” (Shen and Jennings, 1972; Bourdillon and Poole-Wilson, 1981; Braunwald and Kloner, 1982). Thus, the role of changes in sodium pump activity in this sequence of events was examined.

Ischemia and Reperfusion

In electrically paced Langendorff preparations obtained from guinea pig heart, complete global ischemia causes a gradual increase in resting tension and a rapid loss of twitch tension. However, the increase in resting tension (contracture) is slow and the magnitude of the increase is relatively minor during the initial 20–30-min period of complete global ischemia. In these preparations, reperfusion causes a marked increase in resting tension. Similar results were observed when Langendorff preparations were perfused with “ischemic solution” (Fig. 1). The “ischemic solution” has a composition that mimics the interstitial fluid of the ischemic heart (Table I).

Ferrier et al. (1985) have demonstrated that perfusion of the isolated heart with ischemic solution produces essentially the same results as complete ischemia because the individual myocyte is influenced by the interstitial fluid surrounding the cell, instead of only the event that occurred above the inlet cannula. Therefore, when the cells are exposed to a solution that has a composition equivalent to the interstitial fluid of the ischemic heart, the biochemistry and physiology of the myocyte undergo the same changes as the true ischemia. The only difference is that metabolites, which accumulate in the ischemic heart, were removed when Langendorff preparations or myocytes were perfused with the ischemic solution.

Upon reperfusion, the resting tension increases rapidly (Fig. 1). Electrically evoked twitches, which are relatively large during the initial “hypercontraction period,” decrease as the resting tension increases. The muscle may recover after a few hours if the ischemic period resulting from either a complete ischemia or the perfusion of an ischemic solution is less than 30 min. When the ischemic period is longer than 1 h, the resting tension increases markedly and the subsequent reperfusion usually fails to restore twitches responding to electrical stimulation. Reperfusion after 2–5 h of ischemia causes no twitch tension development, and markedly increases resting tension, which is already increased during ischemia.

Figure 1. Resting and developed tension of Langendorff preparations obtained from guinea pig heart. The preparation was perfused at 36°C. During a 20-min period indicated as ischemic condition, the preparation was perfused with the ischemic solution.

Na+,K+-ATPase

Global ischemia of Langendorff preparations up to 2 h at 36°C fails to cause reduction of Na+,K+-ATPase activity estimated with cardiac muscle homogenates (Kim and Akera, 1983). 1-h reperfusion of Langendorff preparations after 2-h ischemia, however, caused a marked decrease in Na+,K+-ATPase activity. This was caused by a decrease in the number of functioning Na+,K+-ATPase units. Their affinity for the dihydrodigoxin was unchanged, indicating that properties of the remaining enzyme units are probably unchanged. Global ischemia for 6 h at 36°C caused a significant reduction in Na+,K+-ATPase activity estimated with cardiac muscle homogenates. Again, the reduction in Na+,K+-ATPase activity results from a decrease in the number of functioning Na+,K+-ATPase units, indicating that either a long ischemia or a reperfusion following ischemia damages a fraction of Na+,K+-ATPase units in the heart muscle.

Reduction in Na+,K+-ATPase activity observed after 1-h reperfusion following the 2 h of global ischemia was partially protected by either 100 U/ml superoxide dismutase, 150 U/ml catalase, 50 mM dimethylsulfoxide, 10 mM histidine, or 1 mM allopurinol, and completely abolished by 50 μg/ml α-tocopherol, when these agents were added to the perfusing solution 5 min before the start of ischemia (Kim and Akera, 1987). All of these agents reduced oxygen free radicals or prevented their generation as estimated from a reduction in tissue malon dialdehyde concentrations in the same Langendorff preparations. The degree of protection of the Na+,K+-ATPase paralleled the degree of reduction in malon dialdehyde production (Kim and Akera, 1987).

TABLE I
Composition of the Simulated Extracellular Fluid of Ischemic Heart

NaCl

38.5

KCl

10.0

NaH_2 PO_4

0.9

NaHCO_3

6.0

CaCl_2

1.2

MgSO_4

1.2

Sodium lactate

100.0

The solution is saturated with a 95% N_2–5% CO_2 gas mixture, and the pH was adjusted to 6.5 using HCl.

The decrease in sodium pump activity, caused by ischemia/reperfusion and estimated from ouabain-sensitive 86Rb+ uptake by ventricular muscle slices, was greater than the decrease in Na+,K+-ATPase activity observed with ventricular muscle homogenates that were obtained from the same Langendorff preparations (Kim and Akera, 1987). Moreover, α-tocopherol protected sodium pump activity only partially even though it completely reversed reperfusion-induced reduction in Na+,K+-ATPase activity.

These results indicate that a prolonged ischemia or ischemia and reperfusion of isolated heart damages sarcolemmal Na+,K+-ATPase resulting from the generation of oxygen free radicals. Compared with Na+,K+-ATPase inhibition, the degree of sodium pump inhibition is greater. The finding that α-tocopherol completely reverses reperfusion-induced inhibition of Na+,K+-ATPase but only partially reverses sodium pump inhibition indicates that other factors, such as the depletion of tissue ATP, may be responsible for the dysfunction of the sodium pump in addition to oxygen radical–induced injury to the Na+,K+-ATPase enzyme. These changes in Na+,K+-ATPase, however, occur with cardiac muscle that has lost its ability to contract, and probably represent a partial phenomenon of general cell death.

Intracellular Ca2+ Concentrations

Intracellular Ca2+ concentrations, estimated from the fluorescence ratio observed with fura 2–loaded myocytes, increased gradually in a resting myocyte during exposure to ischemic solution (Table I). Under the ischemic conditions, the magnitude of the Ca2+ transient was markedly depressed and the time-to-peak Ca2+ concentration was delayed. The decrease in Ca2+ concentration during the relaxing phase is markedly suppressed in myocytes under the ischemic conditions. During the period of hypercontractions (Fig. 1), diastolic Ca2+ concentration decreases. The magnitude of the Ca2+ transient is slightly greater than that during the ischemic period; however, the magnitude was less than that observed in control preparations. At this time, the time-to-peak Ca2+ concentrations and the rate of recovery of the Ca2+ transient returned toward the control values.

It should be noted that Ca2+ transients are attenuated but not abolished in myocytes exposed to ischemic conditions at the time when visual examination with a microscope indicates that the myocyte does not respond to electrical stimulation. Apparently the Ca2+ transient was uncoupled from muscle contraction in ischemic heart.

Diastolic intracellular Ca2+ concentration increased gradually during the 20-min period when myocytes were exposed to a solution mimicking the interstitial fluid of ischemic heart. Subsequent reperfusion with an oxygenated Tyrode solution caused an immediate reduction in diastolic intracellular Ca2+ concentration toward the control value. Despite the decrease in intracellular Ca2+ concentration, contracture developed in myocytes during the reperfusion.

These results indicate that the increase in resting tension observed during reperfusion of ischemic myocytes is not associated with an increase in intracellular Ca2+ concentration. Even though diastolic Ca2+ concentration was higher than normal values, development of contracture does not parallel the changes in Ca2+ concentration. Thus, the development of contracture is not caused directly by an increase in intracellular Ca2+ concentration. In view of the fact that the Ca2+ transients fail to cause twitch contractions during ischemia, the coupling mechanism that links an increase in intracellular Ca2+ concentration to a contraction is apparently impaired in the ischemic heart. This also appears to be the reason why contracture does not develop in ischemic cardiac muscle despite the fact that intracellular Ca2+ is elevated. Upon reperfusion of ischemic heart muscle, sensitivity of the contractile mechanism to Ca2+ should be restored, resulting in the development of contracture responding to elevated intracellular Ca2+. Twitch contractions responding to Ca2+ transients are also restored.

Summary

The prolonged ischemia of cardiac muscle damages sarcolemmal Na+,K+-ATPase. The injury to the Na+,K+-ATPase is markedly enhanced by reperfusion through mechanisms involving oxygen free radical generation. This injury to the Na+,K+-ATPase is a partial phenomenon of the extensive cell damage, and therefore a reduction in Na+,K+-ATPase activity or specific [3H]ouabain binding can be used as an estimate of the extent of general cell injury. These events, however, are not the cause of stunned myocardium observed during reperfusion of the heart after a relatively short ischemia. During short ischemia and subsequent reperfusion, changes in diastolic intracellular Ca2+ concentration parallels neither Na+,K+-ATPase activity observed with muscle homogenates nor the resting tension observed with electrically stimulated muscle preparations. Functional changes in the sodium pump activity, caused by the depletion of ATP, changes in pH or other factors, changes in other cell membrane functions such as the sarcolemmal Ca2+ pump, and changes in extra- and intracellular pH would appear to be more direct causes of dysfunctions of the stunned myocardium.

Acknowledgments

This work was supported by a Grant-in-Aid for Cancer Research (1-23) from the Ministry of Health and Welfare, and a research grant (63624515) from the Ministry of Education, Science and Culture, Japan.

References

Bourdillon, P. D. V., and P. A. Poole-Wilson. 1981. Effects of ischaemia and reperfusion on calcium exchange and mechanical function in isolated rabbit myocardium. Cardiovascular Research. 15:121–130.

Braunwald, E., and R. A. Kloner. 1982. The stunned myocardium: prolonged, postischemic ventricular dysfunction. Circulation. 66:1146–1149.

Ferrier, G. R., M. P. Moffat, and A. Lukas. 1985. Possible mechanisms of ventricular arrhythmias elicited by ischemia followed by reperfusion. Circulation Research. 56:184–194.

Kim, K. H., and T. Akera. 1983. Ischemia-induced enhancement of digitalis sensitivity in isolated guinea-pig heart. Journal of Pharmacology and Experimental Therapeutics. 226:335–342.

Kim, M. S., and T. Akera. 1987. O_2 free radicals: cause of ischemia-reperfusion injury to cardiac Na+,K+-ATPase. American Journal of Physiology. 252:H252–H257.

Shen, A. C., and R. B. Jennings. 1972. Myocardial calcium and magnesium in acute ischemic injury. American Journal of Pathology. 67:417–440.

Quantification of Rat Cerebral Cortex Na+,K+-ATPase with High Recovery: Evaluation of Age and K+-Depletion

Jim S. Larsen, Thomas A. Schmidt, and Keld Kjeldsen

Department of Medicine B 2142, Division of Cardiology, Rigshospitalet, University of Copenhagen School of Medicine, DK-2100 Copenhagen, Denmark

Introduction

Conventional studies of Na+,K+-ATPase are usually based on membrane fractions isolated by differential centrifugation. In skeletal muscle (Kjeldsen, 1986; Hansen and Clausen, 1987) and myocardium (Jones and Besh, 1984; Schmidt et al., 1990) such fractions contain typically less than a few percent of the total Na+,K+-ATPase content in the original material. From a quantitative point of view such membrane preparations are inappropriate, because variations in recovery of the Na+,K+-ATPase may give rise to misleading interpretations. However, methods based on [3H]ouabain binding to intact tissue samples and K+-dependent 3-O-methylfluorescein phosphatase (3-O-MFPase) activity of crude tissue homogenates have been developed and have proven adequate for quantification of Na+,K+-ATPase with complete recovery in skeletal muscle (Nørgaard et al., 1983; Kjeldsen, 1986), heart ventricular muscle (Nørgaard et al., 1985), and peripheral nerve (Kjeldsen and Nørgaard, 1987). Also in brain tissue loss of membrane material during purification (Klodos et al., 1975) may be a problem in studies estimating quantitative changes of the enzyme. Thus, we have evaluated the potentiality of using [3H]ouabain binding to intact samples and K+-dependent 3-O-MFPase activity of crude homogenates in studies on quantitative aspects of cerebral cortex Na+,K+-ATPase.

Methods

The assays were only slightly modified from those described for muscle tissue. In short, for standard [3H]ouabain binding measurements cerebral cortex samples in the range 2–8 mg wet wt were incubated twice for 3 h in a vanadate buffer containing 1 × 10−6 M [3H]ouabain and ouabain at 37°C, followed by a washout in unlabeled buffer four times for 30 min at 0°C. The methodological errors of the method were evaluated and where possible corrections were made. For K+-dependent 3-O-MFPase measurements cerebral cortex tissue was homogenized and exposed to 0.08% sodium-deoxycholate (DOC). The 3-O-MFPase assay contained 19.5 μM 3-O-MPF and DOC-activated homogenate corresponding to 100 μg wet wt of cerebral cortex tissue. The K+-dependent activity was measured by the addition of 10 mM KCl. The K+-dependent 3-O-MFPase activity was completely ouabain sensitive, thus confirming association with the Na+,K+-activated ATPase.

Estimates of Cerebral Cortex Na+,K+-ATPase Concentration

The [3H]ouabain binding to intact cerebral cortex samples obtained from 12-wk-old rats gave a total concentration of 12,221 ± 190 pmol/g wet wt (n = 5; mean ± SEM). The K+-dependent 3-O-MFPase activity obtained from cerebral cortex of age-matched rats was 7.22 ± 0.14 μmol/min per g wet wt (n = 5). Based on a hydrolytic activity of 593 cycles/min determined on purified ox brain (Nørgaard et al., 1985), the K+-dependent phosphatase activity could be converted into a concentration of Na+,K+-ATPase of 12,175 ± 236 pmol/g wet wt, thus demonstrating good accordance with [3H]ouabain binding measurements.

The concentration of cerebral cortex Na+,K+-ATPase obtained in this study is higher than is usually reported. In a recent study of the effect of hypertension, a Na+,K+-ATPase activity of 33.42 μmol/mg protein per h, a [3H]ouabain binding of 66.7 pmol/mg protein, and a protein yield of 13.42 mg/g wet wt of tissue were obtained in purified membrane fractions of cerebral cortex obtained from 14-wk-old rats (Chen and Lin-Shiau, 1989). Using a molecular activity of 9,300 cycles/min (Klodos et al., 1975) the Na+,K+-ATPase activity could be converted to a corresponding Na+,K+-ATPase concentration of 804 pmol/g wet wt. The [3H]ouabain binding site concentration corresponds to a Na+,K+-ATPase concentration of 895 pmol/g wet wt, showing a good agreement between the two different measurements. However, the values obtained may be insufficient to draw conclusions about quantitative regulatory changes of Na+,K+-ATPase. According to our measurements the values only represent ~7% of the total tissue Na+,K+-ATPase concentration and it cannot be ensured that this fraction is representative for the enzyme content of the original material. Variable recovery may give rise to misleading interpretations. Yet in most studies using membrane preparations in investigation of the regulatory aspects of cerebral cortex Na+,K+-ATPase, the protein yield of the membrane fractions is often not given (Matsuda and Iwata, 1987; Pylova et al., 1989). Hence, the recovery of Na+,K+-ATPase cannot be estimated and appraised. Klodos et al. (1975) have reported a method for preparation of Na+,K+-ATPase from ox cerebral cortex aimed at obtaining high recovery of enzyme. The protein and Na+,K+-ATPase contents were carefully evaluated through each step of the purification procedure. Using the molecular activity of 9,300 min−1 the measurements of Na+,K+-ATPase activity and protein content of the initial crude homogenate and the final membrane preparation could be converted to enzyme concentrations of 5,270 and 3,213 pmol/g wet wt, respectively, hence showing a Na+,K+-ATPase recovery in the final membrane preparation of 60%. Thus, even when going for high recovery, a problem in quantitative studies using Na+,K+-ATPase measurements on membrane fractions remains. Furthermore, measurements of Na+,K+-ATPase activity in crude homogenates may be imprecise due to large amounts of unspecific ATPases (Klodos et al., 1975). The lower Na+,K+-ATPase concentration obtained in the crude homogenate of ox cerebral cortex as compared with our measurements may be due to differences between species.

Na+,K+-ATPase Concentration of Cerebral Cortex Compared with Other Excitable Tissues

The table compares the Na+,K+-ATPase content in cerebral cortex with heart and other excitable tissues obtained from adult rats and guinea pigs as estimated from [3H]ouabain binding to intact samples and measurement of K+-dependent 3-O-MFPase activities in crude homogenates. To aid comparison, all values have been converted to Na+,K+-ATPase activities using molecular activities of 593 and 9,300 min−1 for the K+-dependent 3-O-MFPase and Na+,K+-ATPase, respectively (Nørgaard et al., 1985; Klodos et al., 1975). The total Na+,K+-ATPase activity in rodent heart ventricle is in the range 10–20 μmol/min per g wet wt (Nørgaard et al., 1985; Kjeldsen et al., 1985), and in skeletal muscle (Clausen et al., 1982; Kjeldsen et al., 1984, 1985), resistance vessels (Aalkjaer et al., 1985), and uterine muscle (Everts et al., 1990) in the range 2–4 μmol/min per g wet wt. Thus, compared with a total Na+,K+-ATPase activity of ~100 μmol/min per g wet wt in the cerebral cortex, it can be seen that the brain cortex contains the largest concentration of Na+,K+-ATPase of tissues evaluated (~33 and 7 times the concentration seen in skeletal and heart ventricular muscle). The low Na+,K+-ATPase concentration seen in sciatic nerve (Kjeldsen and Nørgaard, 1987) may be explained by its relative high amount of Na+,K+-ATPase-poor myelin. The difference in Na+,K+-ATPase content between the various tissues shown may be related to different demands for cation transport.

TABLE I
Comparison of Na+,K+-ATPase Activity in Rat and Guinea Pig Excitable Tissue

Total Na+,K+-ATPase activity per gram wet weight in adult rat and guinea pig excitable tissue as estimated from [3H]ouabain binding to intact samples and K+-dependent 3-O-MFPase activity in crude homogenates. EDL, extensor digitorum long.

Regulatory Aspects: Age and K+-Depletion

During maturation the concentration of rat cerebral cortex Na+,K+-ATPase increases steeply from ~1,000 pmol/g wet wt to reach a plateau after 5 wk of age of ~12,000 pmol/g wet wt. K+-depletion induced by keeping young rats on potassium-deficient fodder for 2 wk caused no significant reduction in K+-content (P > 0.5) or Na+,K+-ATPase concentration (P > 0.4) in the cerebral cortex, whereas the soleus muscle K+-content and Na+,K+-ATPase concentration decreased by 30% (P < 0.02) and 32% (P < 0.001), respectively. These observations may be of importance from a developmental point of view and valuable for understanding potassium homeostasis during potassium depletion.

Conclusion

The present methods using intact tissue and crude homogenates in determination of Na+,K+-ATPase concentration seem to be applicable to studies considering quantitative aspects of cerebral cortex Na+,K+-ATPase.

Acknowledgments

This study was in part supported by NOVO's Foundation, The Danish Heart Foundation, The Weimann Foundation, and the Foundation for the Advancement of Medical Science.

References

Aalkjaer, C., K. Kjeldsen, A. Nørgaard, T. Clausen, and M. J. Mulvaney. 1985. Ouabain binding and Na+,K+-content in resistance vessels and skeletal muscle of spontaneously hypertensive rats and K+-depleted rats. Hypertension. 7:277–286.

Chen, C. C., and S. Lin-Shiau. 1989. Decreased Na+,K+-ATPase activity and 3H-ouabain binding sites in various tissues of spontaneously hypertensive rats. European Journal of Pharamcology. 122:311–319.

Clausen, T., O. Hansen, K. Kjeldsen, and A. Nørgaard. 1982. Effect of age, potassium depletion and denervation on specific displaceable 3H-ouabain binding in rat skeletal muscle in vivo. Journal of Physiology. 333:367–381.

Everts, M. E., K. Skajaa, and K. Hansen. 1990. The concentration of ouabain binding sites in uterine muscle. Acta Physiologica Scandinavica. 139:503–510.

Hansen, O., and T. Clausen. 1987. Quantitative determination of Na+,K+-ATPase and other sarcolemmal components in muscle cells. American Journal of Physiology. 254:C1–C7.

Jones, L. R., and H. R. Besh, Jr. 1984. Isolation of canine cardiac sarcolemmal vesicles. Methods in Pharmacology. 5:1–12.

Kjeldsen, K. 1986. Complete quantification of the total concentration of rat skeletal-muscle Na+,K+-dependent ATPase by measurements of 3H-ouabain binding. Journal of Biochemistry. 240:725–730.

Kjeldsen, K., and A. Nørgaard. 1987. Quantification of rat sciatic nerve Na+,K+-ATPase by measurements of 3H-ouabain binding to intact nerve samples. Journal of Neurological Sciences. 79:205–219.

Kjeldsen, K., A. Nørgaard, and T. Clausen. 1984. The age-dependent changes in the number of 3H-ouabain binding sites in mammalian skeletal muscle. Pflügers Archiv. 402:100–108.

Kjeldsen, K., A. Nørgaard, O. Hansen, and T. Clausen. 1985. Significance of skeletal muscle digitalis receptors for 3H-ouabain distribution in guinea pig. Journal of Pharmacology and Experimental Therapeutics. 234:720–727.

Klodos, I., P. Ottolenghi, and A. Boldyrev. 1975. Large-scale preparation of Na+,K-ATPase from ox brain. Analytical Biochemistry. 67:397–403.

Matsuda, T. and H. Iwata. 1987. Decrease of high affinity ouabain binding in rat cerebellum and hypothalamus by thiamin deficiency. Brain Research. 437:375–378.

Nørgaard, A., K. Kjeldsen, and O. Hansen. 1985. K+-dependent 3-O-methylfluorescein phosphatase activity in crude homogenate of rodent heart ventricle: effect of K+-depletion and changes in thyroid status. European Journal of Pharmacology. 113:373–382.

Nørgaard, A., K. Kjeldsen, O. Hansen, and T. Clausen. 1983. A simple and rapid method for the determination of the number of 3H-ouabain binding sites in biopsies of skeletal muscle. Biochemical and Biophysical Research Communications. 111:319–325.

Pylova, S. I., J. Majkowska, W. Hilgier, A. Kapuscinski, and J. Albrecht. 1989. Rapid decrease of high affinity ouabain binding sites in hippocampal CA1 region following short-term global cerebral ischemia in rat. Brain Research. 490:170–173.

Schmidt, T. A., J. H. Svendsen, S. Haunsø, and K. Kjeldsen. 1990. Quantification of the total Na+,K+-ATPase concentration in atria and ventricles from mammalian species by measuring 3H-ouabain binding to intact samples. Stability to short term ischemia-reperfusion. Basic Research in Cardiology. 85:411–427.

Adaptation of Brain Na,K-ATPase to Changes in Neural Activity

Alan C. Swann

Department of Psychiatry, University of Texas Medical School, Houston, Texas 77225

Na,K-ATPase restores the alterations in cation gradients that occur during neural activity. Active transport of Na+ and K+ by Na,K-ATPase accounts for the increase in energy utilization that occurs with electrical activity in the brain (Yarowski et al., 1986). Yet there is little information about the manner in which Na,K-ATPase adapts to changes in neural activity in vivo.

A physiologically important aspect of Na,K-ATPase is its response to large changes in internal Na+. This can be ascertained by the degree of Na,K-ATPase stimulation by the Na+ ionophore monensin (Kennedy et al., 1986), which supports Na+ influx via electroneutral Na+-H+ exchange, thereby stimulating Na,K-ATPase activity. The ability of Na,K-ATPase to respond to Na+ influx is a major possible cellular adaptation to altered electrical activity.

In an initial investigation into the role of electrical activity in regulating brain Na,K-ATPase, we have examined effects of increased and decreased activity-mediated Na+ influx on the number of Na,K-ATPase sites and on the response of Na,K-ATPase to increased internal Na+. To reduce Na+ influx we infused tetrodotoxin (TTX), which blocks voltage-dependent Na+ channels. We selected this agent because it would block Na+ influx regardless of the neurotransmitter system involved. To increase Na+ influx, we infused the excitatory amino acid aspartate. We chose aspartate because it would stimulate all classes of excitatory amino acid receptors, but would be less potent than more toxic excitatory amino acid analogues. Agents were infused into the cerebral ventricles using osmotic minipumps. Intraventricular infusions were used to produce a general, widespread effect throughout the brain, minimizing local toxicity or regional variations.

Methods

Intracerebroventricular Infusions

Intracerebroventricular infusions were carried out as we have described (Swann and Steketee, 1989). Intraventricular (icv) cannulas were connected to osmotic minipumps (Alzet 2001; Alza Corp., Palo Alto, CA) containing TTX or aspartate dissolved in artificial CSF. The tubing contained a sufficient amount of vehicle to allow 24 h to elapse between surgery and the initial delivery of active drug. Doses were based on preliminary studies showing lack of behavioral toxicity (Swann, 1991).

Figure 1. Effect of TTX infusions on ouabain binding in cerebral cortex. The vertical axis shows ouabain binding in picomoles per milligram protein, ±SE. Difference from vehicle infusion by Dunnett's test: *P < 0.05; **P < 0.01.

Tissue Preparation

Synaptoneurosomes were prepared immediately from half of the cerebral cortex tissue for use in transport experiments (Swann and Steketee, 1989). This tissue preparation affords maximal preservation of functional intracellular relationships (receptor-coupled second messenger systems, etc.) (Hollingsworth et al., 1985). Membranes were prepared as previously described (Swann, 1984a).

Biochemical Procedures

Ouabain binding and Na,K-ATPase activity were determined as we have described (Swann, 1984a). Under these conditions, ouabain binding detects enzyme sites with high affinity for ouabain. To determine the relative proportion of enzyme with high and low affinity for ouabain, we carried out ouabain inhibition curves of Na,K-ATPase activity (Swann, 1984b). Active transport by Na,K-ATPase was assayed using ouabain-sensitive uptake of 86Rb+ into synaptoneurosomes (Swann, 1990). Transport was measured over a 2-min period after 60 min preincubation with or without 1 mM ouabain at a series of monensin concentrations.

Figure 2. Effect of TTX on ouabain-sensitive 86Rb+ influx. The vertical axis shows the rate of ouabain-sensitive transport in nanomoles per milligram protein per minute ± SE. Tissue was pooled from rats receiving the same treatment to give four independent experiments per dose of TTX. Difference from tissue from vehicle-treated rats by Dunnett's test: *P < 0.05; **P < 0.01.

Figure 3. Effect of aspartate infusions on ouabain binding in cerebral cortex, hippocampus, and corpus striatum. The vertical axis shows ouabain binding in picomoles per milligram protein, ±SE. Difference from vehicle infusion by Dunnett's test: *P < 0.05; **P < 0.01.

Results and Discussion

Effects of TTX

The data in Fig. 1 show that ouabain binding was decreased in cerebral cortex by 2-or 4-d infusions of TTX at 3 μg/h and by 4 d at 1 μg/h (F(2, 66) = 13.9, P < 0.0001) (Swann, 1991). The data in Fig. 2 show that TTX infusion at 4 d reduced both basal and monensin-stimulated 86Rb+ influx into synaptoneurosomes prepared from cerebral cortex. The reduction in monensin-stimulated transport was greater than that in ouabain binding or in basal transport, suggesting that the reserve capacity of Na,K-ATPase to respond to a sodium load was reduced by TTX infusions.

Figure 4. Effect of aspartate infusion on ouabain-sensitive 86Rb+ influx. The vertical axis shows the rate of ouabain-sensitive transport in nanomoles per milligrams protein per minute ± SE. Tissue was pooled from rats receiving the same treatment to give four independent experiments per dose of aspartate. Difference from tissue from vehicle-treated rats by Dunnett's test: *P < 0.05; **P < 0.01.

Effects of Aspartate

Infusion of aspartate increased the number of ouabain binding sites in hippocampus and cerebral cortex, as shown in Fig. 3 (F(2, 41) = 3.6, P < 0.05). Fig. 4 shows that aspartate infusion more than doubled the stimulation of ouabain-sensitive 86Rb+ uptake into cerebral cortex synaptoneurosomes. The increase in stimulation by monensin was much larger than the relatively modest effect of aspartate infusion on ouabain binding or basal transport rate. This is consistent with a marked increase in the reserve capacity of Na,K-ATPase to respond to a sodium load.

General Discussion

These data suggest that, over a period of several days, the number of brain Na,K-ATPase sites adapts to changes in sodium influx associated with neural activity. The effect on the ability to respond to a sodium load appeared greater than the effects on the number of enzyme sites or on basal transport. This could reflect the fact that, under resting conditions, the internal sodium sites of Na,K-ATPase are not saturated. A relatively small increase in the number of sites over those needed for basal sodium transport requirements would therefore produce a disproportionately large effect on the ability to respond to higher sodium concentrations. Alternatively, changes in Na+ influx over time could produce a shift in isoforms toward enzyme with lower affinity for Na+, which would also produce a disproportionately large increase in transport at high cell Na+ (Brodsky and Guidotti, 1990). Results of ouabain inhibition curves suggest roughly equivalent changes in enzyme with high and low affinity for ouabain, but direct measurements of functional isoforms of Na,K-ATPase and of internal sodium affinity will be required to establish which of these possibilities is the case.

Acknowledgments

I thank Ms. Debora Astorga and Ms. Uzoma Williams for technical assistance.

References

Brodsky, J. L., and G. Guidotti. 1970. Sodium affinity of brain Na+,K+-ATPase is dependent on isozyme and environment of the pump. American Journal of Physiology. 258:C803–C811.

Hollingsworth, E. B., E. T. McNeal, J. L. Burton, R. J. Williams, and J. W. Daly. 1985. Biochemical characterization of a filtered synaptoneurosomal preparation from guinea pig cerebral cortex: cyclic 3':5'-monophosphate-generating systems, receptors, and enzymes. Journal of Neuroscience. 5:2240–2253.

Kennedy, R. H., E. Seifen, and R. Kafiluddi. 1986. Can maximal ouabain-sensitive 86Rb+ uptake rate be obtained by increasing Na+ influx? European Journal of Pharmacology. 129:77–85.

Swann, A. C. 1984a. Brain (Na+,K+)-ATPase and noradrenergic function: recovery of enzyme activity after norepinephrine depletion. Brain Research. 321:323–327.

Swann, A. C. 1984b). (Na+,K+)-ATPase regulation by the sympathetic nervous system: effects of noradrenergic stimulation and depletion in vivo. Journal of Pharmacology and Experimental Therapeutics. 228:304–311.

Swann, A. C. 1990. Ethanol-inhibition of active 86Rb+ transport: evidence for enhancement by sodium or calcium influx. Journal of Pharmacology and Experimental Therapeutics. 254:864–871.

Swann, A. C. 1991. Brain Na,K-ATPase regulation in vivo: reduction in activity and response to sodium by intracerebroventricular tetrodotoxin. Brain Research. 543:251–255.

Swann, A. C., and J. D. Steketee. 1989. Subacute noradrenergic agonist infusions in vivo increase Na+,K+-ATPase and ouabain binding in rat cerebral cortex. Journal of Neurochemistry. 52:1598–1604.

Yarowski, P., A. F. Boyne, R. Wierwille, and N. Bookes. 1986. Effect of monensin on deoxyglucose uptake in cultured astrocytes: energy metabolism is coupled to sodium entry. Journal of Neuroscience. 6:859–866.

Changes in Responses of Na Pump Isoforms to Glutamate Stimulation of Cerebral Neurons during Maturation in Culture

Nobuo Inoue and Hideo Matsui

Department of Biochemistry, Kyorin University School of Medicine, Mitaka, Tokyo 181, Japan

Introduction

The Na pump (Na,K-ATPase) is essential for neuronal functions because it generates and maintains concentration gradients of Na+ and K+ across plasma membranes. Three different isoforms (αl, α2, and α3) of the Na pump are known in the brain (Shull et al., 1986; Hara et al., 1987; Sweadner, 1989). We have reported that the Na pump isoforms are present in cultured rat cerebral neurons and that a “brain type” (α2 and α3) isoform appears and increases during the maturation of the cultured neurons in addition to a pre-existing “common type” (αl) isoform (Inoue et al., 1988c, d). The physiological significance of the isoforms in neurons is as yet unknown. However, we have suggested a hypothesis that the brain type isoform plays an important role in restoring concentration gradients of Na+ and K+ after neuronal excitation, because the K+ uptake activities of the Na pump isoforms are differently regulated and the activity of the brain type isoform is selectively activated in response to the stimulation of the mature neurons with the excitatory neurotransmitter, glutamate (Inoue and Matsui, 1990). Here we report that the responses of the Na pump isoforms to the glutamate stimulation of the neurons changed during the maturation of neurons in culture.

Methods

Cerebral neurons generated from 17-d-old rat embryos were cultured on 24-well plates for 2–11 d, and K+ uptake by the Na pump was assayed essentially as described previously (Inoue et al., 1988b, c; Inoue and Matsui, 1990). After washing the neurons three times with a buffer solution containing 130 mM NaCl, 2 mM KCl, 0.8 mM MgCl_2, 1.8 mM CaCl_2, 1.8 mM CaCl_2, 5 mM glucose, and 5 mM HEPES-imidazole, pH 7.3, the cells were preincubated and incubated in an assay solution, the buffer solution containing various concentrations of ouabain (0–1 mM), throughout the assay. The cells were preincubated with 450 μl/well of the assay solution for 20 min and incubated in the solution with or without 100 μM glutamate for 12 min at 37°C. The K+ uptake assay was carried out for 12 min at 37°C in the assay solution (475 μl) containing 42K+ (2.7–6.2 kBq/well), which we obtained from a 42Ar-42K generator (185 kBq). The cells were washed twice with the ice-cold buffer solution after incubation and the 42K radioactivity and amount of total cellular protein were measured. The Na pump activities were the differences between the K+ influxes with the various concentrations of ouabain and with 1 mM ouabain. The activities were expressed on the basis of total cellular protein.

Results and Discussion

Activation of the Brain Type Na Pump and Inhibition of the Common Type Na Pump after Glutamate Excitation of Mature Cerebral Neurons

The Na pump isoforms in rat could be distinguished by the different affinities for ouabain; that is, the brain type isoform is highly sensitive and the common type only weakly sensitive (Sweadner, 1979, 1989; Inoue et al., 1988b, c). Therefore, dose-response studies for inhibition of the Na pump activities by ouabain were performed in the presence and absence of glutamate (Fig. 1). Corresponding to the presence of the Na pump isoforms in the mature neurons (Inoue et al., 1988a, c), the inhibition curves by ouabain of the K+ uptake into the mature neurons cultured for 11 d were biphasic (Fig. 1 C). After the stimulation of the matured neurons with glutamate, the K+ uptake activities of the Na pump isoforms were differently regulated (Fig. 1 C). The glutamate stimulation increased the K+ uptake activity by the total Na pump about twofold via a remarkable increase in the activity of the brain type isoform and a slight decrease in that of the common type isoform. The differential regulation of the Na pump isoforms shown here has been reported previously in the mature neurons cultured for more than 10 d (Inoue and Matsui, 1990). We have also reported that the effects of glutamate were produced in response to an enhancement of [Na]i in the neurons, which resulted from passive Na+ influx through glutamate receptor-mediated cation channels (Inoue and Matsui, 1990).

Figure 1. Inhibition of K+ uptake by the Na pump into primary cultured cerebral neurons by ouabain. After the cerebral neurons were incubated in the assay solutions in the absence () or presence () of 100 μM glutamate for 12 min at 37°C, K+ uptake was measured over the next 12 min of incubation. K+ uptake into the cells was inhibited by addition of ouabain at the concentrations (molar) indicated on the abscissa. (A) Immature neurons cultured for 2 d. (B) Neurons cultured for 6 d. (C) Mature neurons cultured for 11 d.

Different Responses of Na Pump Isoforms to Glutamate Stimulation of Cerebral Neurons during Maturation in Culture

The total Na pump activity and the proportion of the brain type isoform activity to the total Na pump activity of the neurons without the glutamate stimulation increased during maturation of the neurons in culture, as reported previously in the assay of Na,K-ATPase activity (Inoue et al., 1988c). The responses of the Na pump isoforms to the glutamate stimulation of the neurons changed during the maturation of the neurons (Fig. 1), although the total Na pump activity increased about twofold after the stimulation throughout maturation. In the immature neurons cultured for 2 d shown in Fig. 1 A, glutamate stimulation induced increases in the activities of both the brain- and common type isoforms. In the neurons cultured for 6 d (Fig. 1 B), glutamate increased the brain type isoform but had little effect on the common type isoform. Along with the maturation of the neurons, it was shown that the increase in the brain type isoform activity induced by the glutamate stimulation became slightly larger; in contrast, the increase in the common type isoform activity became smaller and vanished. These results support our hypothesis that the Na pump isoforms differ in their physiological significance in the mature neurons, and the brain type isoform plays an important role in restoring concentration gradients of cations after neuronal excitation during neuronal maturation.

Acknowledgments

We thank Ms. Yoshiko Ishihara and Ms. Hiromi Takai for technical assistance, and Dr. Haruhiko Morinaga, München University, and The Japan Radioisotope Association for allowing us to make use of the 42Ar-42K generator.

References

Hara, Y., O. Urayama, K. Kawakami, H. Nojima, H. Nagamune, T. Kojima, T. Ohta, K. Nagano, and M. Nakao. 1987. Primary structure of two types of alpha-subunit of rat brain Na+,K+-ATPase deduced from cDNA sequences. Journal of Biochemistry. 102:43–58.

Inoue, N., Y. Hara, M. Nakao, H. Tsukui, H. Hatanaka, and H. Matsui. 1988a. Three types of mRNAs encoding α-subunit isoforms of Na,K-ATPase are present in primary cultured rat cerebral neurons. Neurochemical Research. 13:1081.

Inoue, N., and H. Matsui. 1990. Activation of a brain type Na pump after glutamate excitation of cerebral neurons. Brain Research. 534:309–312.

Inoue, N., H. Matsui, and H. Hatanaka. 1988b. Nerve growth factor induces Na+,K+-ATPase in a nerve cell line. Journal of Neurochemistry. 50:230–236.

Inoue, N., H. Matsui, H. Tsukui, and H. Hatanaka. 1988c. The appearance of a highly digitalis-sensitive isoform of Na+,K+-ATPase during maturation in vitro of primary cultured rat cerebral neurons. Journal of Biochemistry. 104:349–354.

Inoue, N., H. Matsui, H. Tsukui, and H. Hatanaka. 1988d. The appearance of Na+,K+-ATPase isozymes in rat neuronal cell cultures. Progress in Clinical and Biological Research. 268B:91–96.

Shull, G. E., J. Greeb, and J. B. Lingrel. 1986. Molecular cloning of three distinct forms of the Na+,K+-ATPase α-subunit from rat brain. Biochemistry. 25:8125–8132.

Sweadner, K. J. 1979. Two molecular forms of (Na+ + K+)-stimulated ATPase in brain. Separation, and difference in affinity for strophanthidin. Journal of Biological Chemistry. 254:6060–6067.

Sweadner, K. J. 1989. Isozymes of the Na+/K+-ATPase. Biochimica et Biophysica Acta. 988:185–220.

Induction of α2 by Growth-promoting Concentrations of Insulin in 3T3-L1 Preadipocytes

James J. Russo, K. J. Sweadner, and I. S. Edelman

Department of Biochemistry & Molecular Biophysics, College of Physicians & Surgeons of Columbia University, New York 10032; and Department of Cellular & Molecular Physiology, Massachusetts General Hospital, Neurosurgical Research/Harvard Medical School, Boston, Massachusetts 02114

Introduction

The 3T3-L1 cell line has the ability to spontaneously differentiate from a fibroblast to an adipocyte phenotype in culture (Green and Kehinde, 1976). The conversion can be accelerated by dexamethasone and the phosphodiesterase inhibitor isobutylmethyl-xanthine (IBMX) (Rosen et al., 1979; Reed and Lane, 1980). If 1.6 μM insulin is also provided, there is a proliferation of preadipocytes, along with a substantially increased conversion frequency (Reed and Lane, 1980; Zezulak and Green, 1986; Smith et al., 1988). This concentration of insulin binds to the IGF-I receptors as well as to insulin receptors (Froesch and Zapf, 1985). Thus the hyperplasia observed in the 3T3-L1 cells is probably a growth factor–related response. We examined the difference in the Na pump repertoire in preadipocytes maintained as fibroblasts or converted to adipocytes in the presence or absence of insulin. Cells were grown to confluence and 2 d later exposed to medium containing (a) no additions, (b) 1.6 μM insulin, (c) 0.25 μM dexamethasone + 0.5 mM IBMX, or (d) dexamethasone + IBMX + insulin. The proportions of adipocytes in the four populations were 5, 20, 50, and 95%, respectively.

Results and Discussion

In previous studies (Russo et al., 1990) we demonstrated that mature adipocytes (condition c) were substantially different from mature fibroblasts (condition a) with regard to Na,K-ATPase activity and its isoform composition. For instance, differentiated adipocytes had a 45% lower enzyme specific activity than fibroblasts; in addition, there was a switch from only αl polypeptide and mRNA in fibroblasts to both α1 and α2 in adipocytes, and β1 mRNA was detected only in the fibroblast state. In agreement with the appearance of the α2 polypeptide, a significant percentage of a form of the enzyme with a higher affinity for ouabain was evident.

As shown in Table I, the changes in isoform profile seen with differentiation were mimicked by growing fibroblasts to maturity in the presence of proliferation-inducing concentrations of insulin. In the latter case, αl protein and mRNA declined slightly though not significantly, α2 mRNA and protein were induced nearly to the extent they were in adipocytes, and the β mRNA was substantially reduced. These changes were not quantitatively attributable to the additional number of adipocytes in the insulin-treated fibroblast population.

Whereas conversion to adipocytes in the earlier study (Russo et al., 1990) led to decreased Na,K-ATPase specific activity and 86Rb uptake, in the present study the Na,K-ATPase specific activity was identical in fibroblasts grown with or without insulin, or for that matter in adipocytes converted in the presence or absence of the hormone. There are as yet no reports of induction of α2 by growth factors such as IGF-I. Other investigators report increased enzyme activity and α1- and β1-subunit mRNA abundances with serum (possibly related to its growth factor complement [Bhutada et al., 1990]), though addition of serum did not stimulate Na,K-ATPase activity in a liver cell line in our hands (see Cayanis et al., 1991). Brief exposure to insulin has been reported to stimulate Na,K-ATPase activity by several different mechanisms (reviewed by Gick et al., 1988, and Clausen and Everts, 1989) in several cells, including 3T3-L1 cells (Resh, 1982). Interestingly, the work of Lytton (1985) and Lytton et al. (1985) demonstrated that the Na affinity of the α2 isoform in adipocytes was increased by short-term exposure to physiological concentrations of insulin. Further work will be necessary to reconcile these disparate observations with each other and with the work presented here.

TABLE I
Comparison of Fibroblasts and Adipocytes

There are several possible interpretations of the changes induced by high concentrations of insulin in the 3T3-L1 cells. One is that the changes occurring are an early differentiation event: that preadipocytes during a growth phase preceding frank differentiation begin to express some proteins necessary for subsequent phenotypic alterations. A second triggering event downstream of the initial stimulus might permit assembly of α2 into a functional enzyme unit. If the α2-containing molecule has lower endogenous activity than that containing α1, the decreased enzyme activity seen after complete conversion may ensue. If the β1 protein declines as does its mRNA, one might envision it playing a rate-limiting role in such a process (Geering et al., 1989). Additionally, a previously undescribed isoform of the β-sub-unit could be present in the 3T3-L1 cells at one or more stages. Alternatively, the changes accompanying treatment of fibroblasts with growth-promoting concentrations of insulin and those accompanying differentiation may be entirely unrelated. It will be important to examine the isoform content and enzyme activity at various times after initiation of the differentiation program with and without insulin, as well as to attempt to dissect the insulin component of the response from that portion associated with other factors.

References

Bhutada, A., C. Perez, D. Y. Chon, and F. Ismail-Beigi. 1990. Induction of Na+-K+-ATPase and its subunit mRNAs by serum in a rat liver cell line. American Journal of Physiology. 258 (Cell Physiology): C1044–C1050.

Cayanis, E., J. J. Russo, Y.-S. Wu, and I. S. Edelman. 1991. Serum-independent induction of Na,K-ATPase and c-fos by low K+ in a rat liver cell line. In The Sodium Pump: Recent Developments. J. H. Kaplan and P. De Weer, editors. The Rockefeller University Press, New York. 79–81.

Clausen, T., and M. A. Everts. 1989. Regulation of the Na,K-pump in skeletal muscle. Kidney International. 35:1–13.

Froesch, E., and J. Zapf. 1985. Insulin-like growth factors and insulin: comparative aspects. Diabetologia. 28:485–493.

Geering, K., I. Theulaz, F. Verrey, M. T. Hauptle, and B. C. Rossier. 1989. A role for the beta-subunit in the expression of functional Na+-K+-ATPase in Xenopus oocytes. American Journal of Physiology. 257 (Cell Physiology). C851–C858.

Gick, G. G., F. Ismail-Beigi, and I. S. Edelman. 1988. Overview: hormonal regulation of Na,K-ATPase. In The Na+,K+-Pump. Part B: Cellular Aspects. Alan R. Liss, Inc., New York. 277–295.

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Lytton, J. 1985. Insulin affects the sodium affinity of the rat adipocyte (Na+,K+)-ATPase. The Journal of Biological Chemistry. 260:10075–10080.

Lytton, J., J. C. Lin, and G. Guidotti. 1985. Identification of two molecular forms of (Na+,K+)-ATPase in rat adipocytes: relation to insulin stimulation of the enzyme. The Journal of Biological Chemistry. 260:1177–1184.

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Chronic Diabetes Increases Na,K-ATPase Activity, α1 Isoform Protein, and mRNA Abundance in Rat Small Intestinal Mucosa

Kassem Barada, Charles Okolo, Michael Field, and Nadim Cortas

Department of Medicine, Columbia University, New York 10032

Introduction

Na,K-ATPase has been implicated in the development of complications and adaptive changes in diabetes mellitus (Winegrad, 1986; Green et al., 1987). Changes in maximal Na,K-ATPase activity have been reported in the heart, retina, peripheral nerve, kidneys, and skeletal muscle in experimental diabetes (Brismar and Sima, 1981; Kjeldsen et al., 1987; Cohen and Klepser, 1988). The magnitude and direction of the changes depend on the duration of diabetes and the organ involved. Although the physiologic significance of these changes is not clear, they were sometimes reversed by sorbinil, an aldose reductase inhibitor (Yeh et al., 1987), myoinositol (Green and Lattimer, 1983), and insulin (Ku et al., 1986). Experimental diabetes results in several adaptive changes in the small intestine. Mucosal hypertrophy, increased sugar and amino acid absorption, and increased number of glucose carriers in brush border membranes have all been reported (Fedorak et al., 1987, 1989). We report here our findings on the effects of chronic streptozocin-induced diabetes on Na,K-ATPase activity, isoform expression, and abundance in the small intestinal mucosa of the rat.

Methods

300–500-g male Sprague-Dawley rats were made diabetic by streptozocin (75 mg/kg) administered intraperitoneally. Rats with a blood sugar > 300 mg/dl were considered diabetic. Age-matched controls were injected with vehicle. At 40–60 d of diabetes and immediately after sacrifice, small intestinal mucosa of diabetic and age-matched control rats were scraped separately into ice-cold 2 M NaCl, washed five times with 50 mM Tris and 1 mM EDTA, pH 7.2, homogenized, and fractionated to prepare microsomal membranes. Microsomal Na,K-ATPase assays were done in the presence of 1–4 mg SDS/10 mg protein or 1 mg sodium deoxycholate per mg protein. Assays for ouabain inhibition were done in the presence of varying ouabain concentrations and 1–2 mg SDS/10 mg protein to stabilize the enzyme. Partially purified enzymes were prepared according to the methods of Jorgensen (Jorgensen, 1974). To quantitate Na,K-ATPase abundance, equal amounts of partially purified enzyme protein were blotted on PVDF membranes, probed with monospecific anti-αl (McK1) and anti-α2 (McB2) antibodies (provided by K. Sweadner, Harvard Medical School, Boston, MA), and treated with radioiodinated strepavidin. Autoradiograms were then quantitated by densitometry. Northern blots were performed on total mucosal RNA prepared by the method of Chirguin et al. (1979). Equal amounts of mucosal RNA were blotted on nitrocellulose and then hybridized for 3–4 d with 32P-labeled cDNA α1,α2,α3, and β (provided by J. Lingrel, University of Cincinnati College of Medicine, Cincinnati, OH). Autoradiograms were then quantitated by densitometry. To study the effects of insulin treatment, isophane insulin suspension (NPH) was used and was administered subcutaneously to maintain a blood sugar < 130 mg/dl.

Results and Discussion

Na,K-ATPase activity in mucosal homogenate in the absence of detergent was 4.1 ± 1.0 and 10.1 ± 0.5 U for control and diabetic rats, respectively (P < 0.001; 1 U = 1 μmol Pi/mg protein per h). Na,K-ATPase activity in the microsomal membranes in the presence of detergents was 28.0 ± 5.6 and 61.4 ± 14.1 U for control and diabetic rats, respectively (P < 0.05). Results of experiments done on non–detergent-treated microsomes indicate a similar increase in Na,K-ATPase activity in diabetic rats (data not shown). There was no difference in Mg-ATPase activity between control and diabetic rats in either homogenate or microsomal membranes.

Experiments done on rats with acute diabetes (defined as diabetes of less than 2 wk duration) indicate that the change in Na,K-ATPase activity begins as early as 10 d after diabetes onset and reaches steady state in 15 d.

To determine if chronic diabetes results in the upregulation of one isoform or expression of a different isoform in the small intestinal mucosa, ouabain inhibition curves were constructed on microsomal membranes and mucosal homogenate from control and diabetic rats. A single component inhibition curve was obtained with an apparent Ki of 2.6 ± 1.4 × 10−4 and 2.0 ± 1.2 × 10−4 M for control and diabetic rats, respectively, in both homogenate (n = 3) and microsomal membranes (n = 4).

To investigate further the existence of Na,K-ATPase isoform(s) in the intestinal mucosa, Western blots of partially purified preparations with activities of 150 and 105 U for diabetic and control enzymes, respectively, were probed with anti-α1 (McKl) and α2 (McB2) antibodies. Only α1 was detected. Brain and kidney enzymes were used as controls. Quantitation of Na,K-ATPase in the above preparations was done by treating the immunoblots with radioiodinated strepavidin and then quantitating autoradiograms by densitometry. There was a 1.7-fold increase in Na,K-ATPase abundance in the diabetic preparation, which can account for the increased activity. Northern blots probed with 32P-labeled cDNA α1, α2, α3, and β revealed a 2.5-fold increase in mRNAαl (P < 0.03) and a 3.0-fold increase in mRNAβ in the mucosa of diabetic rats. Blots probed with cDNA α2 and α3 showed very faint bands. Because of the very high abundance of mRNAαl, this could reflect cross-hybridization. To determine if insulin treatment prevents the increase in Na,K-ATPase activity in the mucosa, 2–10 U of NPH insulin was administered subcutaneously daily to three rats immediately after diabetes onset and for 2–3 wk thereafter. Na,K-ATPase activity in mucosal homogenate was 6.6 ± 1.7 and 5.6 ± 1.5 U, while activity in non–detergent-treated microsomes was 22.8 ± 3.4 and 22.2 ± 4.4 U for diabetic and control rats, respectively (NS).

Our data indicate that the small intestinal mucosa of the rat has predominantly one isoform, namely αl. This is supported by the apparent Ki of 2–3 × 10−4 M for ouabain and by the absence of α2 on immunoblots. The existence of α3 cannot be excluded at this time. Chronic streptozocin-induced diabetes more than doubled Na,K-ATPase activity in the small intestinal mucosa. This seems to be caused by an increase in the number of pumps in the cell membrane. This may be achieved by (a) increased migration of already existing pumps from inside the cell to the membrane, (b) increased de novo synthesis of new pumps, or (c) decreased degradation of already existing pumps, or any combination thereof. The increased abundance of mRNAα1 suggests either increased transcription of the α1 gene or decreased degradation of the message in the diabetic mucosa.

The β-subunit plays a role in stabilizing the α-subunit and in the migration of the αβ dimers from the Golgi apparatus to the plasma membranes (Geering et al., 1989; Noguchi et al., 1990). Although we have not measured the abundance of the β-subunit protein in the mucosa, the increased abundance of mRNAβ may play a role in increasing the number of pumps in the cell membranes. Our data are in agreement with those of Luppa and Muller (1982), who showed an increase in Na,K-ATPase activity in rat small intestinal mucosal homogenate and basolateral membranes in alloxan- and streptozocin-induced diabetes.

Increased Na,K-ATPase activity in the small intestinal mucosa has also been described in hyperthyroid rats (Liberman et al., 1979), in rats fed taurocholate (Simon et al., 1988), and in fasting rats (Murray and Wild, 1980). The increases in Na,K-ATPase activity with T3 and taurocholate were associated with increases in pump abundance.

In conclusion, α1 is the predominant Na,K-ATPase isoform in rat small intestinal mucosa. In streptozocin-treated chronically diabetic rats the activity of this isoform, its abundance, and the abundance of its mRNA message are increased. These changes are prevented by insulin treatment.

Acknowledgments

This work was supported by NIH grant DK-39105-01. K. Barada is supported by the Juvenile Diabetes Foundation International.

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Cohen M. P., and H. Klepser. 1988. Glomerular Na,K-ATPase activity in acute and chronic diabetes and with aldose reductase inhibition. Diabetes. 37:558–562.

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Fedorak, R. N., M. D. Gershon, and M. Field. 1989. Induction of intestinal glucose carriers in streptozocin-treated chronically diabetic rats. Gastroenterology. 96:37–44.

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Green, D. A., and S. A. Lattimer. 1983. Impaired rat sciatic nerve sodium potassium adenosine triphosphatase in acute streptozocin diabetes and its correction by dietary myoinositol supplementation. Journal of Clinical Investigation. 72:1058–1063.

Green, D. A., S. A. Lattimer, and A. A. Sima. 1987. Sorbitol, phosphoinositides, and sodium-potassium-ATPase in the pathogenesis of diabetic complications. The New England Journal of Medicine. 316:599–623.

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Glucagon Changes the Kinetics of Ouabain Inhibition of Na,K-ATPase in Caco2 Cells: An Effect Reversed by Insulin

S. L. Lucak, R. W. Infantino, C. Okolo, M. Field, and N. Cortas

Department of Medicine, Columbia University, New York 10032

Introduction

Transepithelial glucose-evoked short circuit currents (Isc) in ileal loops from streptozocin-induced chronically diabetic rats (90 d), mounted in Ussing-like chambers, were more resistant to inhibition by ouabain than those in loops from age-matched controls. Glucagon, when added in vitro to nondiabetic rat ileum, mimicked this effect (Fedorak, R. N., N. Cortas, and M. Field, manuscript submitted for publication). In diabetic rats, plasma insulin concentration decreases and glucagon concentration increases (Unger, 1971). We report our findings on the effects of glucagon on the regulation of Na,K-ATPase in Caco2 cells in culture. This eliminates variables encountered in whole animal preparations. In this study we also characterized the changes in Na,K-ATPase, alkaline phosphatase, and Mg-ATPase in different stages of development. We found that glucagon induces a two-component ouabain inhibition curve of Na,K-ATPase as measured in cell homogenates by Na,K-ATPase assay and in intact cells by ouabain-sensitive 86Rb uptake. This effect was prevented by insulin. The magnitude of the response to glucagon was dependent on the stage of cell development.

Materials and Methods

Caco2 cells were derived from a well-differentiated human adenocarcinoma of the colon (Fogh et al., 1977). The monolayers were grown on collagen-free filters as modified from Hidalgo et al. (1989). The cells were exposed on the basolateral side for 24 h to 10−5 M glucagon (provided by Dr. Ron Chance of Eli Lilly, Indianapolis, IN), glucagon plus insulin (1.7 × 10−6 M), or diluent. Assays for ouabain inhibition curves in cell homogenates were performed in the presence of varying ouabain concentrations and sodium deoxycholate. The reaction mixture was supplemented with 0.1 mM PMSF, 10 μM pepstatin A, 10 μM iodoacetamide, 2 μM leupeptin, and 50 μg/ml antipain. Na,K-ATPase assays were performed by the method of Baginski et al. (1967) as modified by Cortas and Walser (1971). Ouabain inhibition curves of 86Rb uptake in intact Caco2 cells were performed as described by Pressley et al. (1986).

Results and Discussion

Na,K-ATPase, Mg-ATPase, and alkaline phosphatase activities were measured in homogenates of Caco2 cells at different stages of development (Fig. 1). Na,K-ATPase activity reached a steady state at 14 d, which was maintained until 21 d and then gradually declined.

The maximal activities of Na,K-ATPase (Vmax) in homogenates of Caco2 cells exposed to diluent (control), glucagon, glucagon plus insulin, and insulin alone were 8.5 ± 0.7, 8.5 ± 0.6, 8.2 ± 0.9, and 8.9 μmol Pi/h per mg protein, respectively. Cells treated with diluent alone (controls) showed a one-component ouabain inhibition curve with an apparent Ki of 4 × 10−7 M. Cells exposed to glucagon demonstrated a two-component ouabain inhibition curve with apparent Ki's of 4 × 10−9 and 4 × 10−7 M. The proportion of the glucagon-induced high affinity ouabain component increased with development from 3 ± 2% (NS) of total Na,K-ATPase in preconfluent cells to 31 ± 3% (P < 0.005) at 21–28 d after seeding (Table I). Insulin added along with glucagon prevented the development of the glucagon-induced high affinity ouabain component and showed a one-component ouabain inhibition curve with an apparent Ki of 4 × 10−7 M, similar to that in control.

Figure 1. Caco2 cells were grown as described in Materials and Methods and assayed for Na,K-ATPase (), Mg-ATPase (), and alkaline phosphatase (∆) activities. Na,K-ATPase activity starts increasing at confluence (7 d after seeding) and reaches a steady state from day 14 to day 21. In contrast, alkaline phosphatase and Mg-ATPase activities continuously increased at different rates with development.

To determine whether this effect of glucagon also occurs in a functional transport assay, 86Rb uptake into intact Caco2 cells (21 d in culture) was measured. Basal ouabain-sensitive 86Rb uptake was found to be 740 ± 40 nmol K+/h per mg protein in control (diluent-treated) cells, which was not different from uptake into glucagon- and glucagon plus insulin-treated cells. Ouabain inhibition curves of 86Rb uptake showed a single component with an apparent Ki of 7 × 10−7 M in control monolayers. Glucagon-treated cells showed a two-component ouabain inhibition curve with apparent Ki's of 1 × 10−9 and 7 × 10−7 M. The proportion of the glucagon-induced high affinity ouabain component comprised 49 ± 12% of total ouabain-sensitive 86Rb uptake. Insulin added along with glucagon prevented this effect. A one-component ouabain inhibition curve with an apparent Ki of 7 × 10−7 M was obtained with glucagon plus insulin.

The glucagon-induced high affinity ouabain component of Na,K-ATPase, demonstrated in both cell homogenates and intact monolayers, was abolished by insulin.

To determine whether the glucagon effect in Caco2 cells resulted from a change in relative Na,K-ATPase isoform abundance, Western blots of Caco2 cell homogenates and microsomal membranes were probed with monospecific antibodies for α1 (McKl) and α2 (McB2) provided by Dr. K. Sweadner (Harvard Medical School, Boston, MA). Only αl was seen. The presence of α3 was not excluded. Hara et al. (1990) found a glucagon-responsive element in the 5' flanking region of rat Na,K-ATPase α3 isoform.

Although we have not excluded the induction of a Na,K-ATPase α isoform by glucagon, factors other than isoforms have been implicated in causing changes in ouabain sensitivity (Geny et al., 1982). Cardiac hypertrophy in rats is associated with an increase in high affinity ouabain binding sites (Charlemagne et al., 1990); however, mRNAα3 was not detected and mRNAα2 was decreased. Furthermore, three β isoforms of Na,K-ATPase are known to exist (Shull et al., 1986; Martin-Vasallo et al., 1989; Jaunin et al., 1990) and may affect the affinity of Na,K-ATPase for ouabain and Na+.

TABLE I
Proportion of the High Affinity Ouabain Component of Na,K-ATPase with Development in Caco2 Cells

Ouabain inhibition curves were generated for Caco2 cell homogenates at different stages of development. Caco2 cells reach confluence at 7 d in culture. *P < 0.005.

Acknowledgments

This work was supported by NIH grant DK-39105-01.

References

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Clinical Aspects

Clinical Implications of Digitalis Glycoside Receptor Studies in Human Heart and Skeletal Muscle

Keld Kjeldsen, Thomas A. Schmidt, and Jim S. Larsen

Department of Medicine B 2142, Division of Cardiology, Rigshospitalet, University of Copenhagen School of Medicine, DK-2100 Copenhagen, Denmark

Introduction

Digitalis glycosides have been used in clinical practice for more than 200 years without much knowledge of their cellular and molecular function. Within the last decades it has, however, become generally accepted that their inotropic action is mediated by binding to and subsequent inhibition of a fraction of the Na,K-pumps in the heart (Smith, 1988). Furthermore, it has recently become possible to quantify the digitalis glycoside receptors (i.e., the Na,K-pumps) in specimens obtained shortly after death or in biopsies from human myocardial tissue (Nørgaard et al., 1986,1988) and skeletal muscles (Nørgaard et al., 1984, 1985) with high accuracy and precision using vanadate-facilitated (Hansen, 1979) [3H]ouabain binding intact samples (Hansen, 1984). The skeletal muscle studies are of interest since the skeletal muscle pool of Na,K-pumps is of major importance for extrarenal potassium homeostasis (Clausen and Everts, 1989; Nørgaard and Kjeldsen, 1991). Presently, clinical implications from studies of quantitation of human myocardial tissue and skeletal muscle Na,K-ATPase are beginning to emerge. It is particularly in relation to the therapeutic use of digitalis glycosides and aspects of extrarenal potassium homeostasis that such studies may have importance. Our main studies of human myocardial tissue and skeletal muscle Na,K-pump levels in relation to these aspects are shown in Table I.

Heart Disease

In studies on left ventricle endomyocardial biopsies taken during heart catheterization from patients with dilated cardiomyopathy or ischemic heart disease, a reduction in digitalis glycoside receptors of 41% (P < 0.001) or 34% (P < 0.025) was observed. Furthermore, a linear correlation between Na,K-pump concentration and angiographically determined left ventricle ejection fraction was obtained (r = 0.76, P < 0.001) (Nørgaard et al., 1988; Nørgaard and Kjeldsen, 1989).

Whereas digitalis glycosides may be beneficial in the early stages of dilated cardiomyopathy and ischemic heart disease, the further reduction in active digitalis glycoside receptors by digitalization may be less beneficial or even deleterious at later stages of these diseases where receptor concentration per se may be down regulated. In such situations digitalization may increase intracellular calcium concentration to levels causing cell deterioration. It may be that under these conditions digitalization at low serum levels could be the best choice, should this therapy be mandatory.

Age-dependent Changes

The digitalis glycoside receptor concentration was quantified at autopsy in left ventricle myocardial tissue obtained from 0–8-yr-old human subjects with no heart disease. Within the first 6 mo of life the Na,K-pump concentration showed a rapid decrease from ~ 1,200 to 600 pmol/g wet wt (Kjeldsen and Grøn, 1990). The large concentration of Na,K-pumps in the myocardium of small children may explain the clinical observation that such patients should be maintained at higher plasma levels of cardiac glycosides than adults. Furthermore, the rapid decrease in myocardial Na,K-ATPase concentration within the first 6 mo of life may in part explain why such patients can be difficult to digitalize.

TABLE I
Studies of Human Heart and Skeletal Muscle Na,K-ATPase Levels in Relation to Digitalization and Potassium Homeostasis

Long-Term Digitalis Therapy

Digitalis glycoside receptor concentration was determined in left ventricle myocardial samples obtained at autopsy from patients who had been on long-term digitalization and was compared with subjects not given this drug. This showed 39% lower (P < 0.001) digitalis glycoside binding in samples from digitalized as compared with undigitalized subjects. After removal of previously bound digoxin from the receptors (Schmidt and Kjeldsen, 1991), it was found that digitalization was associated with a 34% occupancy (P < 0.001) of digitalis receptors by digoxin. Furthermore, no upregulation of the digitalis glycoside receptor concentration due to long-term digitalization was found. On the contrary, a trend to a lower value in patients exposed to digoxin compared with the controls was observed (Schmidt et al., 1991).

This study for quantifies the first time at the receptor level the occupancy of myocardial Na,K-pumps with digoxin during therapeutic digitalization in human subjects. The study gave no evidence for development of tolerance to long-term digitalization in human subjects with heart disease. Thus, the regulation of the Na,K-ATPase concentration in the human failing heart seems to be different from that in cultured cells and peripheral blood cells, where an upregulation of receptor concentration in response to digoxin exposure has been reported.

Hyperthyroidism

In biopsies of human vastus lateralis muscle, an increase in Na,K-pump concentration of 68% was found with hyperthyroidism. The change was fully reversible with curative treatment of the disease (Kjeldsen et al., 1984). The increase in skeletal muscle Na,K-pump concentration with hyperthyroidism may in part explain the development of periodic thyreotoxic hypokalemic paralysis that can be seen in this disease. Plasma potassium has been found to decrease to 1.2 mmol/liter during such attacks without change in total body potassium content. The attacks can be prevented not only by potassium supplementation but also by beta-adrenoceptor antagonists blocking catecholamine-induced, Na,K-pump-mediated potassium uptake in skeletal muscle cells.

Potassium/Magnesium Depletion

In patients receiving long-term diuretic treatment, measurements of potassium and Na,K-pumps in biopsies of the vastus lateralis muscle showed reductions of 19% (P < 0.001) and 18% (P < 0.005), respectively, compared with healthy controls. Only 1 of these patients had reduced serum potassium concentration (Dørup et al., 1988). One of the observations of major importance in the study is that a major loss of muscle potassium and Na,K-pumps can occur without any major decrease in plasma potassium values. Thus, potassium depletion may be manifest without hypokalemia. Taken together with the fact that hypokalemia can occur without potassium depletion when potassium is translocated away from plasma into skeletal muscles, these observations may explain why there is still debate over whether there is a need for potassium supplementation in mild diuretic treatment. If studies to elucidate this problem are based on measurements of plasma potassium values, subjects may be erroneously allocated to the groups of potassium-depleted and control subjects. Hence, the results of such studies may be blurred. Our study also offers an explanation for the muscle fatigue often experienced in patients on diuretic therapy. Diminished muscle potassium concentration will reduce exercise-induced release of potassium and subsequently cause less vasodilation in skeletal muscles. Muscle function may also be reduced since protein synthesis may be reduced and protein degradation enhanced during potassium depletion. The study may also point to situations where mild hypokalemia or potassium depletion may change from being well compensated and well tolerated to being decompensated and perhaps dangerous. Thus, for example, a stress-induced catecholamine response may lower plasma potassium concentration to dangerous levels in such patients. In addition, acute normalization of a potassium deficit in hypokalemic patients may not entirely normalize the potassium homeostasis system. Several days may be required to normalize muscle Na,K-pump concentration. Thus, such treatment might actually reduce the capacity of the potassium homeostasis to handle a sudden potassium load.

Physical Conditioning

A major challenge to the potassium homeostasis system occurs during exercise (Kjeldsen, 1991). Potassium pours out of working muscles, causing increase in plasma potassium values (Kjeldsen et al., 1990b; Medbø and Sejersted, 1990). Training may reduce this exercise-induced rise in plasma potassium concentration significantly without change in muscle Na,K-pump concentration (Kjeldsen et al., 1990a, b). This may be due to training-induced increased catecholamine response to exercise and a concomitant stimulation of Na,K-pump-mediated potassium uptake in skeletal muscles. Moreover, it may be that major changes in physical conditioning may be associated with changes in human skeletal muscle Na,K-pump concentration. Thus, the Na,K-pump concentration in skeletal muscles has been found to be higher in trained compared with untrained subjects (Klitgaard and Clausen, 1989). Furthermore, ongoing studies seem to indicate that a major increase in capacity for performing muscle work can be associated with an increase in skeletal muscle Na,K-pump concentration and vice versa.

Conclusion

Human myocardial and skeletal muscle Na,K-pump concentration as well as plasma potassium values are dynamic factors undergoing modulation. A recognition of this and further studies of Na,K-pumps in these tissues as well as the dynamic aspects of plasma potassium may have increasing clinical importance.

Acknowledgments

This study was supported in part by NOVO's Foundation, The Danish Heart Foundation, and The Danish Sports Research Foundation.

References

Clausen, T., and M. E. Everts. 1989. Regulation of the Na,K-pump in skeletal muscle. Kidney International. 35:1–13.

Dørup, I., K. Skajaa, T. Clausen, and K. Kjeldsen. 1988. Reduced concentrations of potassium, magnesium and sodium-potassium pumps in human skeletal muscle during treatment with diuretics. British Medical Journal. 67:455–458.

Hansen, O. 1979. Facilitation of ouabain binding to (Na+,K+)-ATPase by vanadate at in vivo concentrations. Biochimica et Biophysica Acta. 568:265–269.

Hansen, O. 1984. Interaction of cardiac glycosides with (Na+,K+)-activated ATPase: a biochemical link to digitalis-induced inotropy. Pharmacological Reviews. 36:143–163.

Kjeldsen, K. 1991. Dysfunction of skeletal muscle Na,K-pumps may expose the heart to dangerous potassium concentrations during exercise. Canadian Journal of Sports Sciences. 16:33–39.

Kjeldsen, K., and P. Grøn. 1990. Age-dependent change in myocardial digitalis glycoside receptor (Na,K-pump) concentration in children. Journal of Cardiovascular Pharmacology. 26:185–190.

Kjeldsen, K., A. Nørgaard, C. O. Gøtzsche, A. Thomassen, and T. Clausen. 1984. The effect of thyroid status on the number of Na,K-pumps in human skeletal muscle. Lancet. ii:8–10.

Kjeldsen, K., A. Nørgaard, and C. Hau. 1990a. Human skeletal muscle Na,K-ATPase concentration quantified by 3H-ouabain binding to intact biopsies before and after moderate physical conditioning. International Journal of Sports Medicine. 11:304–307.

Kjeldsen, K., A. Nørgaard, and C. Hau. 1990b. Exercise-induced hyperkalemia in human subjects can be reduced by moderate training without change in skeletal muscle Na,K-ATPase concentration. European Journal of Clinical Investigation. 20:642–647.

Klitgaard, H., and T. Clausen. 1989. Increased total concentration of Na,K-pumps in vastus lateralis muscles of old trained human subjects. Journal of Applied Physiology. 67:2491–2494.

Medbø, J. I., and O. M. Sejersted. 1990. Plasma potassium changes with high intensity exercise. Journal of Physiology. 421:105–122.

Nørgaard, A., J. P. Bagger, P. Bjerregaard, U. Baandrup, K. Kjeldsen, and P. E. B. Thomsen. 1988. Regulation of left ventricular function and Na,K-pump concentration in suspected idiopathic dilated cardiomyopathy. American Journal of Cardiology. 61:1312–1315.

Nørgaard, A., and K. Kjeldsen. 1989. Human myocardial Na,K-pumps in relation to heart disease. Journal of Applied Cardiology. 4:239–245.

Nørgaard, A., and K. Kjeldsen. 1991. Hypokalemia and potassium depletion: interrelations and implications. A reevaluation based on skeletal muscle Na,K-pump studies. Clinical Science. In press.

Nørgaard, A., K. Kjeldsen, and T. Clausen. 1984. A method for the determination of the total number of 3H-ouabain binding sites in biopsies of human skeletal muscle. Scandinavian Journal of Clinical and Laboratory Investigation. 44:509–518.

Nørgaard, A., K. Kjeldsen, O. Hansen, T. Clausen, C. G. Larsen, and F. G. Larsen. 1986. Quantification of the 3H-ouabain binding site concentration in human myocardium: a post mortem study. Cardiovascular Research. 20:428–435.

Nørgaard, A., K. Kjeldsen, J. S. Larsen, C. G. Larsen, and F. G. Larsen. 1985. Estimation of stability of 3H-ouabain binding site concentration in rat and human skeletal muscle post mortem. Scandinavian Journal of Clinical and Laboratory Investigation. 45:139–144.

Schmidt, T. A., P. Holm-Nielsen, and K. Kjeldsen. 1991. No upregulation of digitalis glycoside receptor (Na,K-ATPase) concentration in human heart left ventricle samples obtained postmortem after long-term digitalization. Cardiovascular Research. In press.

Schmidt, T. A., and K. Kjeldsen. 1991. Enhanced clearance of specifically bound digoxin from human myocardial and skeletal muscle samples by specific digoxin antibody fragments: subsequent complete digitalis glycoside receptor (Na,K-ATPase) quantification. Journal of Cardiovascular Pharmacology. 17:670–677.

Smith, T. W. 1988. Digitalis: mechanisms of action and clinical use. New England Journal of Medicine. 318:358–365.

Preferential Occupancy of High-affinity Cardiac Na,K-ATPase Isoenzymes (α2 or α3) by LND-796 during Positive Inotropy in the Dog

Jean-Michel Maixent, Richard M. Kawamoto, James R. Skuster, Bruce M. Halpryn, Antoine Baggioni, and Simone Fenard

Laboratoire Nativelle, 92134 Issy Les Moulineaux Cedex, France; and Norwich Eaton Pharmaceuticals, Inc., Norwich, New York 13815

Introduction

Ventricular cardiomyocytes from various species (dog, ferret, and rat) contain several isoforms of Na,K-ATPase (Matsuda et al., 1984; Charlemagne et al., 1987; Maixent et al., 1987; Maixent and Lelièvre, 1987; Ng and Akera, 1987a, b). Digitalis interactions cause increases both in the force of contraction and in the severity of toxic effects by elevating intracellular concentrations of Na+ and Ca2+ (Schwartz et al., 1988). However, the toxic arrhythmogenic effects are seen only after an isoform with a low-affinity receptor has been occupied (Erdmann et al., 1980; Lelièvre et al., 1986; Maixent et al., 1987; Ng and Akera, 1987a). In contrast, the physiological role of an isoform with a high affinity for digitalis remains less clear. It has not yet been determined whether occupancy of this second type of isoform by low concentrations of digoxin or ouabain leads to positive inotropy.

To avoid kinetic problems caused by drug distribution and the slow rate of onset of action with such drugs, and to determine the extent to which the high-affinity isoform could be inhibited during inotropy, we measured in vivo blood levels of LND-796, a semisynthetic cardiac glycoside that has a rapid onset of activity. Circulating blood levels of LND-796 were determined at characteristic times of change in inotropy in the dog by an enzymatic assay that measures the inhibition of Na,K-ATPase activity (Maixent et al., 1987). The same assay has been used to correlate the blood levels of LND-796 with inhibition of specific isoforms in cardiac sarcolemma preparations.

Materials and Methods

Purified Na,K-ATPase from dog kidney (EC 3.6.1.3, grade IV) was purchased from Sigma Chemical Company (La Verpillière, France). LND-796 ([3b, 5b, 14b, 17b]-14-amino-3-[(6-deoxy-α-L-mannopyranosyl)oxy]androstane-17-carboxylic acid, methyl ester) was synthesized at Laboratoire Nativelle (Longjumeau, France).

Figure 1. Time course of changes in cardiac contractility (dP/dt; squares) and Na,K-ATPase inhibition (circles) after administration of 3.3 nmol/kg per min LND-796 in 7 of 11 anesthetized dogs. The index of contractility (positive dP/dt) at time 0 was 1,803 ± 394 mm Hg s−1. Data points represent mean ± SD. Values for Na,K-ATPase inhibition were determined with Na,K-ATPase from dog kidney and LND-796 that was extracted as indicated in the Methods. Statistically significantly different from the respective control samples from each dog at time 0 for the period of time between 15 and 60 min (P < 0.05).

Hemodynamic Studies

11 mongrel dogs (6 males, 5 females; 8–15 kg) were anesthetized with an intravenous 30-mg/kg bolus dose of sodium pentobarbital. The dogs were intubated with a cuffed endotracheal tube and artificially respirated with room air with a Bird Mark VII respirator. A pressure transducer-tipped catheter (Millar Instruments, Inc., Houston, TX) was placed in the left ventricle. The output was routed into a ES1000 differentiator (Gould Inc., Cleveland, OH) to determine positive dP/dt, an index of myocardial contractility. To ensure systemic mixing of drug before blood collection, LND-796 was administered through the saphenous vein at 3.3 nmol/kg per min for 20 min, and samples were withdrawn at various times from the right femoral artery. The samples were then centrifuged at room temperature for 10 min at 3,000 rpm.

LND-796 was extracted from plasma by a chromatographic method, lyophilized, and resuspended at appropriate concentrations. The chromatographic method used the Baker 10 SPE system and disposable columns prepacked with a selective reverse-phase octadecylsilane-bonded (C18) silica gel, which selectively retained steroid compounds. The purification factor of the extraction method ranged from 10- to 12-fold.

ATP Hydrolysis Studies

Na,K-ATPase activity (or ATP hydrolysis) was measured by continuously recording β-nicotinamide adenine dinucleotide, reduced form (NADH) oxidation with an ATP-regenerating assay medium previously described by Maixent et al. (1987). Duplicate aliquots (0.1 ml) of the concentrated extract from plasma that contained LND-796 were incubated with Na,K-ATPase from dog kidney in a spectrophotometric cuvette. The same procedure was used for four LND-796 concentrations (in triplicate) to establish a calibration curve that related drug concentration to Na,K-ATPase inhibition. IC_50 values for LND-796 were calculated from the abscissa intercept in a logit plot. Results are presented as mean ± SD for five preparations. ATPase activity insensitive to 30 μM of LND-796 or ouabain was <5% of the total Na,K-ATPase activity.

TABLE I
Computed Apparent Affinities and Percent Occupancy of Canine Isoforms by LND-796 In Vivo

Canine ventricular muscle sarcolemma (Maixent et al., 1987) was used to determine the effects of LND-796 on cardiac Na,K-ATPase. The enzymatic activity was determined with vesicles permeabilized with sodium dodecyl sulfate (0.2 mg/mg protein for 30 min at 20°C).

Figure 2. Positive correlation between inotropy in vivo and LND-796 concentrations in dogs. Circulating blood concentrations were measured in a total of 11 dogs (including the seven that were described in Fig. 1). Samples taken at times different than those shown in Fig. 1 were also included for the four additional dogs. Drug concentrations in the samples were measured as in Fig. 1. A relatively good relationship existed between contractile responses induced by LND-796 and plasma concentrations of LND-796 in dogs (r = 0.78, P < 0.01).

Results and Discussion

We used LND-796, a semisynthetic cardiac glycoside, to investigate the pharmacological significance of an isoform with a high-affinity receptor for digitalis drugs. This compound, which exhibits a rapid onset and reversal of inotropic activity, was chosen so that some of the problems such as slow tissue uptake and reactivity that have been associated with digoxin could be avoided.

Circulating blood levels of LND-796 were measured with a Na,K-ATPase enzymatic assay during the development and dissipation of positive inotropy in dogs. The onset and decline of positive inotropy after perfusion of 3.3 nmol/kg per min of LND-796 for 20 min in dogs paralleled that of Na,K-ATPase inhibition observed in vitro (Fig. 1). Concentrations of LND-796 in the blood (2–120 nM) correlated closely with positive changes in inotropy in vivo. The concentration–inhibition curves for LND-796 were constructed with values taken at equilibrium (less than 1 min). Only one inhibitory process caused by low-affinity binding was observed with the kidney enzyme source. The IC_50 value of LND-796 for Na,K-ATPase inhibition in this instance was 110 ± 10 nM, and the plasma concentrations of LND-796 ranged from 2 to 120 nM.

The sensitivity of Na,K-ATPase to LND-796 was tested in microsomes and sarcolemmal fractions isolated from canine left ventricular myocardium. The IC_50 values averaged 4 ± 0.6 nM for the high-affinity process and 600 ± 200 nM for the low-affinity one (Table I). The proportional contribution of the high-affinity process ranged from 50 to 70%, depending on the preparation. The LND-796 biphasic dose-response curve was not significantly different from those of digoxin and ouabain (P > 0.05).

The Na,K-ATPase isoenzymes (α1 in the kidney and α1 and α2 or α3 in the myocardium) used in our study have different affinities for digitalis drugs, and only the high-affinity myocardial Na,K-ATPase isoenzymes (α2 or α3) were expected to be occupied by LND-796. The lowest detectable concentration of LND-796 that corresponded to an inotropic effect (2 nM) represented an average of 33% occupancy of the high-affinity Na,K-ATPase isoenzymes isolated with in vitro measurements (Fig. 2, Table I).

The close correlation between the extent of Na,K-ATPase inhibition and increased inotropy supports the hypothesis that cardiac occupancy of high-affinity Na,K-ATPase isoenzymes is the primary target for LND-796 in vivo. Our results, combined with those of Rasmussen et al. (1990), also suggest that therapeutic blood levels of LND-796 can inhibit the Na,K pump in vivo.

Acknowledgments

The authors gratefully acknowledge Carrie Nicastro for editing this manuscript and Jocelyne Savornin for technical assistance.

References

Charlemagne, D., E. Mayoux, M. Poyard, P. Oliviero, and K. Geering. 1987. Identification of two isoforms of the catalytic subunit of Na,K-ATPase in myocytes from adult rat heart. Journal of Biological Chemistry. 262:8941–8943.

Erdmann, E., G. Philipp, and H. Scholz. 1980. Cardiac glycoside receptor, (Na+,K+)-ATPase activity and force of contraction in rat heart. Biochemical Pharmacology. 29:3217–3229.

Lelièvre, L. G., D. Charlemagne, C. Mouas, and B. Swynghedauw. 1986. Respective involvements of high and low affinity digitalis receptors in the inotropic response of isolated rat heart to ouabain. Biochemical Pharmacology. 35:3449–3455.

Maixent, J. M., D. Charlemagne, B. de la Chapelle, and L. G. Lelièvre. 1987. Two Na,K-ATPase isoenzymes in canine cardiac myocytes. Journal of Biological Chemistry. 262:6842–6848.

Maixent, J. M., and L. G. Lelièvre. 1987. Differential inactivation of inotropic and toxic digitalis receptors. Journal of Biological Chemistry. 262:12458–12462.

Matsuda, T., H. Iwata, and J. R. Cooper. 1984. Specific inactivation of alpha (+) molecular form of (Na+ + K+)-ATPase by pyrithiamin. Journal of Biological Chemistry. 259:3858–3863.

Ng, Y. C., and T. Akera. 1987a. Two classes of ouabain binding sites in ferret heart and two forms of Na+-K+-ATPase. American Journal of Physiology. 252:H1016–H1022.

Ng, Y. C., and T. Akera. 1987b. Relative abundance of two molecular forms of Na+,K+-ATPase in the ferret heart: development changes and associated alterations of digitalis sensitivity. Molecular Pharmacology. 32:201–205.

Rasmussen, H. H., G. T. Okita, R. S. Hartz, and R. E. Ten Eick. 1990. Inhibition of electrogenic Na+-pumping in isolated atrial tissue from patients treated with digoxin. The Journal of Pharmacology and Experimental Therapeutics. 252:60–64.

Schwartz, A., G. Grupp, E. Wallick, I. L. Grupp, and W. J. Ball, Jr. 1988. Role of the Na+K+ ATPase in the cardiotonic action of cardiac glycosides. In The Na+K+-Pump. Part B: Cellular Aspects. J. C. Skou, J. G. Nørby, A. B. Maunsbach, and M. Esmann, editors. Alan R. Liss, Inc., New York. 321–338.

An Outward K Current in Cat Ventricular Myocytes Shows Sensitivity to Dihydroouabain

Ruth L. Martin and Robert E. Ten Eick

Department of Pharmacology, Northwestern University Medical School, Chicago, Illinois 60611

Introduction

During studies on inward rectifying K+ current (IKI) in isolated cat ventricular myocytes, we found a decaying outward tail current that developed over a half-hour time period. Such a current (to our knowledge) has not been previously described. We refer to it as I?. I? can be elicited from a holding potential of –40 mV by conditioning the cell with a hyperpolarizing pulse (e.g., Vc = –140 mV) followed by test steps (Vt = –40 to +60 mV).

I? was investigated to determine if its electrophysiological and pharmacological characteristics were similar to those of any previously described current found in ventricular myocytes. Specifically, we sought to determine the following: (a) Is I? a K+ current? (b) Does it exhibit kinetic properties or a pharmacology similar to either Ito, IKI, or IK(ATP)? (c) Is I? development associated with Ca2+ overload? and (d) Does intra- or extracellular [Na+] influence I?? In the course of these studies we determined that I? resembles none of the presently known K+ currents and that it can be inhibited with low efficacy by the cardiac glycoside, dihydroouabain.

Methods

Membrane current (Im) was recorded using the whole-cell patch voltage clamp technique (Hamill et al., 1981) on single adult cat left ventricular myocytes isolated with a collagenase solution using a modification of the methods developed by Silver et al. (1983). The composition of external solution was (in mM): 30 or 140 NaCl, 5.4 KCl, 2.5 CaCl_2, 1.2 MgCl_2, 11.1 glucose, 5.5 HEPES, and 0 or 110 TMA-C1, pH 7.4. The pipette or internal solution had the following composition (in mM): 120 K glutamate, 10–30 NaCl, 0–6 MgCl_2,1 KH_2PO_4, 0–20 EGTA, 0–5 EDTA, 5 HEPES, 5 K_2ATP, and 0–20 TMA-C1, pH 7.2. Experiments were performed at 33 ± 1°C. In some experiments 0.5 mM Cd2+ and 10 μM TTX were added to the external solution to block Ca2+ and Na+ currents, respectively.

Results and Discussion

Fig. 1, A and B, shows the effect of time on the Im.A is a control current trace elicited 6.5 min after obtaining intracellular access. When the pulse protocol was repeated 100 min later (B), an outward transient current had developed. C shows the subtraction of current B – A, revealing I?, the current that developed with time after obtaining intracellular access. Peak and steady-state current–voltage (I-V) curves for I? are shown in D. Peak data are well fit by a straight line with a slope of 110 nS (correlation coefficient = 0.997). I? has ohmic voltage dependence and decays in ~ 200 ms, suggesting that it undergoes time-dependent inactivation. The I-V relationship in Fig. 1 D was determined in the presence of 0.5 mM Cd2+; therefore, the Cd2+-sensitive Ca2+ channels do not play a role in I?. In addition, altering external Ca2+ (0–2.5 mM) did not appreciably influence I?, suggesting that neither Ca2+ nor Ca2+-dependent processes are involved.

Figure 1. Effect of time on Im. See panel C inset for pulse protocol. At the arrows, time scale changes from 10 to 50 ms per division. (A) 6.5 min after obtaining intracellular access (control). (B) 106.5 min after obtaining intracellular access. (C) Subtraction current of trace A –trace B. (D) Peak () and steady state () (i.e., at 200 ms) current measured as illustrated in C. Vt = –40 to +60 mV.

We investigated whether I? was carried by K+ by examining if the reversal potential for I? was similar to EK, calculated from the transmembrane K+ gradient. Utilization of subtraction currents eliminated contributions from other K+ currents, which were present when intracellular access was obtained and were stable with time. Fig. 2 shows the I-V relationship of the I? peak tail currents from a cell with [K+]0 = 5.4 mM and [K+]i = 133 mM. The x intercept for a straight line fit to these data is –84 mV (correlation coefficient = 0.996). The calculated EK for this experiment is –83.5 mV, suggesting that I? is carried by K+. Using the protocol shown in Fig. 2, the x intercept was determined from the linear I-V relationships of the I? peak tail currents at [K+]0 = 2.4, 5.4, 10.4, and 25.4 mM. From these data, a slope of 58 mV/log [K+]0 was determined. The line calculated for K+ electrode behavior has a slope of 60 mV/log [K+]0. This suggests that I? is carried almost exclusively by K+.

The transient outward current (Ito) in heart is carried by K+. Since I? is a kinetically similar outward transient current, the pharmacological sensitivities of the two were compared. In cat, Ito was completely blocked by 0.5 mM 3,4-diaminopyridine, yet I? was virtually unaffected. On the other hand, I? was completely blocked by 0.2 mM Ba2+, while Ito was insensitive to Ba2+. Thus, I? does not appear to be the conventional Ito. Other evidence for this conclusion includes the observations that the initial presence or absence of Ito does not influence whether I? develops, and Ito remains unchanged with time whether or not cells develop I?.

A Ca2+-sensitive Ito also exists in ventricle (Hiraoka and Kawano, 1989). I? is probably not this current because I? developed in the presence of 20 mM EGTAi. This experimental condition presents the cell with pCa2+ (free) > 11.13 (calculated using the algorithm kindly provided by A. Fabiato [1988]), so the Ca2+-sensitive Ito is not expected to activate.

Figure 2. Dependence of I? on K+. Subtraction currents and pulse protocol are displayed in the insets. EK = –84 mV for this experiment.

Internal Mg2+ mediates the inwardly rectifying properties of IKI in guinea pig myocytes (Matsuda et al., 1987). When a cell is depolarized, Mg2+ blocks the IKI channel, allowing only a minimal number of ions to pass outward. At more hyperpolarized potentials, Mg2+ does not block the channel, allowing a maximal number of ions to pass through the membrane in the inward direction, thus causing inward rectification. If Mgi2+ were being chelated by EGTAi over time, one might expect the loss of inward rectification and display of ohmic conductance. This idea might explain I? development over time.

Fig. 3 demonstrates that varying [Mg2+]i had no effect on I? development. Fig. 3 A shows current traces elicited 40 min after obtaining intracellular access, with experimental conditions such that pMg2+ (free) was 3.00. This concentration of Mgi2+ would be enough to cause inward rectification of IKI in guinea pig. In Fig. 3 B, current traces elicited 30 min after obtaining intracellular access with experimental conditions such that pMg2+ (free) > 18.18 are displayed. I? developed even with more than 15 orders of magnitude difference in [Mg2+]i. Thus, I? does not appear to be induced by reduction of Mgi2+ due to gradual chelation by EGTA. This suggests that I? development is not due to conversion of IKI voltage dependence from inward rectifying to ohmic.

Hyperpolarization increases the fraction of IKI undergoing steady-state inactivation. If I? were flowing through the IKI channel, one would expect to see a decrease in current amplitude as inactivation increased at more negative Vc. However, I? amplitude did not decrease at Vc negative to –100 mV (Fig. 3). I? is probably not a time-modified IKI because I? completely inactivates within 200 ms in a voltage range in which IKI is an instantaneously activating, noninactivating outward current. Also, IKI varies little with time and is unaffected by the development of I?.

Because the effects of our experimental conditions over time on the kinetics of IK(ATP) are unknown, we examined the notion that I? might be a form of IK(ATP). An ATP-sensitive K+ current might develop over time if intracellular ATP became depleted or channel sensitivity to ATP changed. Glyburide, a sulphonylurea, is known to block IK(ATP), yet no glyburide-sensitive current was detected when 10 μM glyburide was added to the external solution. In addition, IK(ATP) demonstrates “run-down” over time in the whole-cell patch configuration (Belles et al., 1987), whereas I? develops over time after obtaining intracellular access. Thus, I? does not appear to be IK(ATP).

Figure 3. Dependence of I? on [Mg2+]i. I?'s shown here were elicited at Vt = –40 mV after a 200-ms conditioning pulse (Vc = –100 to –140 mV). Only 100 ms of the Vc is displayed. (A) pMg2+ (free) = 3.00. (B) pMg2+ (free) > 18.18.

Other K+ currents that have been described in heart and could possibly identify I? include IKP, described by Yue and Marban (1988), and IK. I? is probably neither IKP nor IK since both are noninactivating currents positive to EK, whereas I? completely inactivates within 200 ms in this voltage range.

The possibility that I? develops due to a Ca2+ overload induced by our experimental conditions over time was explored. Addition of dihydroouabain (DHO) should promote Ca2+ overload and enhance I?. Fig. 4 shows that 100 μM DHO decreased peak amplitude of I? by ~ 10% in this cell. In two other cells, 100 μM DHO decreased peak amplitude by ~ 5 and 15%. Thus, I? development does not appear to result from Ca2+ overload. Fig. 4 also illustrates a difference in the pharmacological sensitivities of I? and IKI. The current inset shows no change in IKI in the presence of 100 μM DHO, while I? peak amplitude is decreased. Two possibilities for the inhibitory action of DHO on I? come to mind. (a) DHO could decrease I? by directly blocking this novel channel, or (b) if the Na+ pump is involved in I? development, DHO could decrease the current indirectly by its well-known effect of inhibiting the Na+ pump.

If Na-dependent processes such as INa or Na+-Ca2+ exchange were involved, a change in I? might be seen with alteration of [Na+]. I? development was not affected by different [Na+]i (10–30 mM). Two levels of [Na+]0 used in the same cell, selected to obtain values of ENa of 0 and +40 mV, did not affect I? occurrence. Furthermore, I? voltage dependence was not influenced by [Na+]; peak amplitudes at Vt = –40 mV and Vt = +40 mV were not altered by the change in [Na+]0. A nearly fivefold difference in [Na+]0, which would be expected to affect the Na+-Ca2+ exchange process, does not alter I?. In addition, the absence of had no affect on I?. These data support the notion that Na+-Ca2+ exchange is not involved. Since [Na+]i and Na+-Ca2+ exchange are effected by Na+ pump inhibition, the results provide indirect evidence suggesting that the Na+ pump is not involved.

Figure 4. Sensitivity of I? to 100 μM DHO. , Control , in the presence of DHO; , washout of DHO. Current traces displayed in the inset were obtained at Vt = +40 mV.

Conclusions

An outward current (I?) with a linear I-V relationship develops with time after obtaining intracellular access in many but not all cat ventricular myocytes; 50% of cells develop I? within 100 min using physiological solutions. I? has the following characteristics: (a) appears to be carried exclusively by K+, (b) differs with Ito, IK(ATP), IKP, IK, and IKI in pharmacological sensitivity and/or kinetics, (c) is not dependent on [Mg2+]i, [Ca2+], or [Na+], and (d) is DHO sensitive.

I? does not appear to be the conventional Ito, Ca2+-sensitive Ito, IK(ATP), IKP, IK, or IKI. Thus I? appears to be a previously undescribed K+ current found in cat ventricular myocytes. We speculate that DHO may have other effects at high concentrations, in addition to its well-known effect of blocking the Na+ pump. Specifically, DHO may be able to block the ion channel responsible for I?.

Acknowledgments

This work was supported in part by Grants-In-Aid from the United States Public Health Service (HL-27026 and HL-38041).

References

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Na Pump in Vascular Smooth Muscle: Regulation of Cell Proliferation

J. C. Allen, C. L. Seidel, S. S. Navran, T. A. Pressley, and R. M. Medford

Department of Medicine, Baylor College of Medicine, Houston, Texas 77030; Department of Physiology, University of Texas Medical School at Houston, Houston, Texas 77030; and Division of Cardiology, Emory University, Atlanta, Georgia 30322

The density of Na pumps in vascular smooth muscle (VSM) sarcolemma is much lower than in other contractile tissues (Allen et al., 1989). However, this does not suggest that its importance is in any way diminished. Quite the contrary. It has become apparent that since the pump exists in such low abundance, the maximal pumping capacity of a VSM cell can easily be exceeded by circumstances that increase Na influx. In other tissues this capacity is rarely exceeded, and many studies have now addressed both short- and long-term regulatory processes (Rossier et al., 1987). This discussion addresses one of the manifestations of long-term upregulation of the low Na pump site abundance in VSM: proliferative control.

In most cell types studied, the addition of serum mitogens induces cell proliferation which may involve stimulation of Na-H exchange, resulting in cellular alkalinization with a concomitant small transient increase in intracellular Na. This increase is transient because the plasmalemmal Na pump is present in sufficient quantities to prevent a large intracellular accumulation of the ion. However, in VSM the events are slightly different as a result of the low Na pump site number (Allen et al., 1989). The addition of mitogens to canine VSM cells in primary culture induces an increase in intracellular Na via an amiloride-insensitive pathway (Seidel et al., 1989). The high Na in turn induces the increase of Na pump site content. The newly incorporated pump sites are then capable of diminishing the high intracellular Na concentration, thereby allowing proliferation to occur (Fig. 1). The discussion presented here is an attempt to begin to understand the control of Na pump site density in canine vascular smooth muscle cells in primary culture (i.e., long-term regulation) and the control the pump exerts on proliferation. A first step is an assessment of α- and β-subunit message content in primary cultures of VSM.

We have shown that canine saphenous vein and femoral artery contain disproportionate amounts of Na pump subunit messages (Allen et al., 1991). While the β-subunit message was readily demonstrable on Northern analysis, an α1-like mRNA could be shown only with the use of the polymerase chain reaction, using primers synthesized to sequences of bona fide rat αl cDNAs. Thus, in canine VSM there is a relatively large abundance of β-subunit message and a very low abundance of α-subunit message. The mechanism whereby these two messages produce the two subunits that are expressed as functional pump sites is the subject of much speculation. This message disparity does not appear to hold for rat aortic VSM (Herrera et al., 1988).

In most other tissues examined, although there seems to be a large variation in the α- and β-subunit message content, the major postulate is that the β-subunit acts as a membrane “acceptor” for the α-subunit, enhancing the appropriate conformational (functional) structure. Its abundance therefore may regulate the overall rate of assembly and maturation (Geering, 1990). However, in view of the relative overabundance of β-subunit mRNA observed in canine VSM, any proposed mechanism for control of pump site synthesis must address this quite different quantitative aspect.

The control of the upregulation of functional Na pump subunits has received much attention in recent years. While specific control mechanisms vary between different tissues, specific experimental protocols that can accomplish such upregulation seem to fall into several categories:

(a) Hormonal: thyroxine, aldosterone (Chaudhury et al., 1987; McDonough et al., 1988; Gick and Ismail-Beigi, 1990; Horowitz et al., 1990; Orlowski and Lingrel, 1990). This mechanism may involve direct activation of gene transcription, and may or may not require extracellular Na. Many laboratories have examined this issue in great detail. The reader is referred directly to these papers.

Figure 1. Proposed role of Na pump site control of vascular smooth muscle cell proliferation. See text for details.

(b) Na demand, no Na pump inhibition. Many compounds, such as nystatin, monensin, and veratridine increase Na influx without pump inhibition, but their action on pump site number is variable. Veratridine seems to activate gene transcription in chick myotubes (Taormino and Fambrough, 1990).

(c) Na demand, pump inhibition (Pressley, 1989; Rayson, 1989). In a number of tissues, increasing intracellular Na by pump inhibition (either low K or ouabain) induces pump site upregulation. It is not yet certain if low K+ or ouabain are in fact identical perturbations. Furthermore, in many tissues a simple increase of the Na+ load is not sufficient to induce upregulation of pump expression, since Na pump site number is sufficient to prevent Nai accumulation. Concomitant pump inhibition is required. What makes the study of the upregulation of Na pump site number in VSM different is that because of the low number of pump sites the induction of Na influx by mitogens occurs in a cell where Na pump capacity is already limited. It is interesting to speculate what similarities in control of pump site number may exist between veratridine-stimulated chick myotubes and VSM. In the former there clearly are sufficient sites to handle the increase in Na+, but the drug induces an upregulation of β-subunit mRNA before α-subunit mRNA (Taormino and Fambrough, 1990). It is postulated that overproduction of β-subunits may “drive” more efficient complex assembly.

An understanding of the relationship between the Na pump and vascular smooth muscle proliferation is of great functional significance, since in two very important disease states, atherosclerosis and hypertension, proliferation is presumed to be involved. Indeed, a number of hypotheses of the etiology of hypertension directly involve the pump (Bohr and Webb, 1988; Herrera and Ruiz-Opazo, 1990).

In hypertension it has been postulated that a natriuretic factor inhibits the VSM pump, inducing an increase in intracellular Na and, via Na-Ca exchange, a resulting increase in tension (Blaustein and Hamlyn, 1984). Since VSM has a low number of Na pump sites, such pump inhibition could lead to increased site synthesis. The subsequent decrease in Na would then trigger proliferation. If, however, the elevated Na did not induce site synthesis and subsequent proliferation, and thus remained elevated, then increased tension would be the result. Understanding the control of VSM proliferation is also key to understanding activating processes in atherosclerosis and the development of restenosis after coronary angioplasty.

In this short discussion we have not addressed at least two important issues which could also play a major role in the upregulation of Na pump sites: degradation rate (Pollack et al., 1981), and the presence of preformed sites (Barlet-Bas et al., 1990; Blot-Chabaud et al., 1990).

In summary, it is our hypothesis that in canine VSM there is an upregulation of Na pump sites in response to the mitogen-induced increase in intracellular Na. The addition of serum induces a large amiloride-insensitive increase in Na influx, which in turn activates a mechanism to increase functional pump site number. The high cytoplasmic Na in turn prevents proliferation from occurring. When the decreasing Na reaches a critical level, proliferation occurs. While at present we have not proposed a mechanism, we feel that the large mRNAβ to mRNAα ratio in some manner reflects this response. In no other tissue does pump synthesis appear to be so closely linked to cellular proliferation.

Because of the relatively low number of Na+ pump sites present in VSM, and because of the observed relationship between Na pump upregulation and proliferation, canine VSM may provide a unique model to study control of Na pump site synthesis.

References

Allen, J. C., R. M. Medford, X. Zhao, and T. A. Pressley. 1991. Disproportionate alpha and beta mRNA content in canine vascular smooth muscle. Circulation Research. In press.

Allen, J. C., S. S. Navran, C. L. Seidel, D. K. Dennison, J. M. Amann, and S. K. Jemelka. 1989. Intracellular Na+ regulation of Na+ pump sites in cultured vascular smooth muscle cells. The American Journal of Physiology. 256(Cell Physiology):C186–C792.

Barlet-Bas, C., C. Khadouri, S. Marsy, and A. Doucet. 1990. Enhanced intracellular Na concentration in kidney cells recruits a latent pool of Na-K-ATPase whose size is modulated by corticosteroids. The Journal of Biological Chemistry. 265:7799–7803.

Blaustein, M. P., and J. M. Hamlyn. 1984. Na transport inhibition, cell calcium, and hypertension: the natriuretic hormone/Na+-Ca2+ exchange/hypertension hypothesis. The American Journal of Medicine. 45–59.

Blot-Chabaud, M., F. Wanstok, J.-P. Bonvalet, and N. Farman. 1990. Cell Na-induced recruitment of Na+-K+-ATPase pumps in rabbit cortical collecting tubules is aldosterone-dependent. The Journal of Biological Chemistry. 265:11676-11681.

Bohr, D. F., and R. C. Webb. 1988. Vascular smooth muscle membrane in hypertension. Annual Review of Pharmacology and Toxicology. 28:389–409.

Chaudhury, S., F. Ismail-Beigi, G. G. Gick, R. Levenson, and I. S. Edelman. 1987. Effect of thyroid hormone on the abundance of Na,K-adenosin triphosphatase alpha-subunit messenger ribonucleic acid. Molecular Endocrinology. 1:83–89.

Geering, K. 1990. Subunit assembly and functional maturation of Na,K-ATPase. The Journal of Membrane Biology. 115:109–121.

Gick, G. G., and F. Ismail-Beigi. 1990. Thyroid hormone induction of Na+,K+-ATPase and its mRNAs in a rat liver cell line. The American Journal of Physiology. 258(Cell Physiology):C544–C551.

Herrera, V. L. M., A. V. Chobanian, and N. Ruiz-Opazo. 1988. Isoform-specific modulation of Na+,K+-ATPase alpha-subunit gene expression in hypertension. Science. 241:221–223.

Herrera, V. L. M., and N. Ruiz-Opazo. 1990. Alteration of alpha_1 Na+,K+-ATPase 86Rb influx by a single amino acid substitution. Science. 249:1023–1026.

Horowitz, B., C. B. Hensley, M. Quintero, K. K. Azuma, D. Putnam, and A. A. McDonough. 1990. Differential regulation of Na,K-ATPase alphal, alpha2, and beta subunit mRNA and protein levels by thyroid hormone. The Journal of Biological Chemistry. 265:14308–14314.

McDonough, A. A., T. A. Brown, B. Horowitz, R. Chiu, J. Schlotterbeck, J. Bowen, and C. A. Schmitt. 1988. Thyroid hormone coordinately regulates Na+-K+-ATPase alpha- and beta-subunit mRNA levels in kidney. The American Journal of Physiology. 254(Cell Physiology 23):C323–C329.

Orlowski, J., and J. B. Lingrel. 1990. Thyroid and glucocorticoid hormones regulate the expression of multiple Na,K-ATPase genes in cultured neonatal rat cardiac myocytes. The Journal of Biological Chemistry. 265:3462–3470.

Pollack, L. R., E. H. Tate, and J. S. Cook. 1981. Turnover and regulation of Na-K-ATPase in HeLa cells. American Journal of Physiology. 10:C173–C183.

Pressley, T. A. 1989. Ion concentration dependent regulation of Na,K-pump abundance. The Journal of Membrane Biology. 105:187–195.

Rayson, B. M. 1989. Rates of synthesis and degradation of Na+-K+-ATPase during chronic ouabain treatment. The American Journal of Physiology. 256(Cell Physiology 25):C75–C80.

Rossier, B. C., K. Geering, and J. P. Kraehenbuhl. 1987. Regulation of the Na pump: how and why? Trends in Biochemical Sciences. 12:483–487.

Seidel, C. L., J. C. Allen, C. L. Wallace, S. K. Jemelka, S. S. Navran, and K. K. Dennison. 1989. Relationship between cell Na, Na-pump number, myosin expression and proliferation in cultured canine vascular smooth muscle cells. In Molecular Biology of the Cardiovascular System. R. Roberts and J. Sambrook, editors. Alan R. Liss, Inc., New York. 269–276.

Taormino, J. P., and D. M. Fambrough. 1990. Pre-translational regulation of the (Na+ + K+)-ATPase in response to demand for ion transport in cultured chicken skeletal muscle. The Journal of Biological Chemistry. 265:4116–4123.

Isoforms of Na,K-ATPase in Myocardial Tissues of the Milan Hypertensive Rat

Christopher P. Cutler and Gordon Cramb

Department of Biology and Preclinical Medicine, University of St. Andrews, Fife, United Kingdom

Introduction

The involvement of Na,K-ATPase in the etiology of essential hypertension has been demonstrated in a limited number of cases (Diez et al., 1987). Changes in the regulation of Na,K-ATPase may be the cause or an effect of the hypertension. Alterations in Na,K-ATPase mRNA abundance in deoxycorticosterone (DOC)/high salt– or angiotensin II (A II)–treated animal models of hypertension have also been reported, where a 2–3-fold increase in the abundance of both αl and βl isoform mRNAs (in aorta and skeletal muscle tissues) and a 3–15-fold decrease in the abundance of α2 and α3 isoform mRNAs (in aorta and left ventricle) were found (Herrera et al., 1988). These results suggested that the increase in α1 and β1 isoforms was due to increased Na influx into the cells of the tissues concerned, whereas the decrease in the α2 and α3 isoforms was directly related to increases in vascular pressure.

In this study, measurements of Na,K-ATPase isoform mRNA abundance in the myocardial tissues of the Milan rat stain were used to reveal if these findings also apply more generally to other animal models of hypertension. The Milan hypertensive rat strain is a selectively inbred strain of Wistar rat that shows a mild variant of primary hypertension (Bianchi et al., 1973a, b). The genetic lesion associated with Milan hypertensive rats is manifested in an unknown renal malfunction, possibly associated with decreased GFR and/or enhanced proximal sodium absorption with an associated increase in blood volume (Genest et al., 1977). Glomerular filtration and blood volume are eventually returned to normal by the compensatory rise in blood pressure (Bianchi et al., 1979a, b). The systolic blood pressure of these hypertensive rats rises by 30–40% to 160–170 mmHg between 4 and 7 wk of life, although mean arterial blood pressure generally only increases by 20–25%. These rats also show a mild degree of left ventricular hypertrophy (~ 15–20%) and a reduced kidney size (10% smaller than normal).

Materials and Methods

Tissues were dissected from six 12–14-wk-old male hypertensive rats (MHS), and six age- and sex-matched normotensive controls (MNS) (Field Laboratories, University of Sheffield, Sheffield, UK). The mean kidney/body weight ratio for MHS was decreased by 28% compared with MNS (Table I).

Total RNA was extracted as outlined in Cutler et al. (1988) and blotted as described by Davis et al. (1986). Eight hypertensive and eight normotensive myocardial samples were blotted together with normotensive RNA samples from brain, kidney, and lung, as well as E. coli total RNA which served as a control. Four identical dot blots were hybridized with Na,K-ATPase isoform-specific DNA probes (αl, α2, α3, and β1 isoforms). The filters were hybridized as described by Anderson and Young (1985), except that hybridization was performed at 48°C. Northern blots, under identical conditions, resulted in mRNA bands of the correct size (Shull et al., 1986; Young et al., 1987), with no nonspecific background or cross-reactivity with other isoforms (data not shown). Blots were subsequently exposed to x-ray film and autoradiographs were quantified by a Shimadzu CS 9000 scanning densitometer (Shimadzu Corporation, Tokyo, Japan).

TABLE I
Total Body Weight and Wet Weight Body Weight Ratio (× 10 –3) of Various Tissues from Hypertensive and Normotensive Milan Rats

The figures represent ± SD (n = 6) except for normotensive myocardial tissues (n = 4) and kidney (n = 12). The statistical significance (produced using Student's t test, assuming normally distributed data) is indicated in parenthesis: ** and *** represent significance where P ≤ 0.01 and 0.001, respectively.

Results

In the ventricle samples, the amount of extractable total RNA was 25-28% lower in hypertensive than in normotensive rats (Fig. 1). In contrast, in atrial samples the amount of total RNA increased (but not significantly) in hypertensive rats.

The relative abundance of Na,K-ATPase isoform mRNAs in the myocardial tissues of normotensive and hypertensive rats was analyzed by Student's t test (Fig. 2). Assessing the relative isoform mRNA abundance per gram wet weight of tissue, all of the isoform mRNAs were decreased by between 1.5- and 3-fold in both hypertensive rat left and right ventricles compared with controls. In contrast, the abundance of the mRNA for the α1, α2 and β1 isoforms in the atria of hypertensive rats showed an increase of 1.5-2-fold over controls. The exception to this was the abundance of the α3 isoform mRNA, where levels in hypertensive atria showed a decrease of > 27%. These results are difficult to assess as an accurate measure of the mean mRNA expression per cell since no account of the extent of hypertrophy was made. Comparing the relative mRNA abundance per microgram of total RNA reduced the statistical significance of differences found between normotensive and hypertensive tissues, although most differences remained significant with the exception of the abundance of α1 mRNA in atria, α2 mRNA in ventricles, and α3 mRNA in right ventricle.

Figure 1. A chart showing the amount of total RNA extracted from normotensive and hypertensive rat tissues per gram wet weight of tissue. LV, left ventricle (n = 3); RV, right ventricle (n = 3); AT, atria (n = 2). The error bars represent standard deviations. The statistical significance of the data was assessed by Student's t test(**P ≤ 0.01) and the data were assumed to be normally distributed.

Discussion

Herrera et al. (1988) reported a two- to threefold increase in Na,K-ATPase α1 and β1 isoform mRNA abundance in the left ventricular myocardium of hypertensive rats, which was suggested to be a consequence of the increased sodium influx resulting directly from the treatments used (DOC and a high salt diet or A II administration). This is likely to result in an increased [Na]i, which would induce an upregulation of Na,K-ATPase mRNAs (Cramb et al., 1989). The same group also reported a 3-15-fold decrease in the abundance of the α2 and α3 isoform mRNAs in the left ventricle of hypertensive rats (Herrera et al., 1988); these decreases were similar although larger than those found in this study. The decrease in α2 and α3 mRNA abundance was suggested to be related to increases in vascular pressure in hypertensive rats as both aortic and ventricular tissue samples taken from hormone-treated but nonhypertensive rats (DOC treated, without a high salt diet, or subpressor doses of AII) did not show decreased expression of these isoform mRNAs. In the Milan hypertensive rat model, hypertension is thought to develop due to a decreased ultrafiltration in the kidney glomeruli and/or enhanced proximal tubule sodium absorption. After the onset of hypertension (~7 wk after birth), Na excretion, plasma volume, and plasma renin levels return to normal levels (Genest et al., 1977), suggesting that circulating mineralocorticoid concentrations, and consequently Na balance, also return to normal. As the increase in the α1 and β1 isoform mRNA abundance in the ventricles of the DOC-salt rat model was thought to be attributed to the action of DOC-salt treatment on [Na]i, the absence of this treatment from the Milan hypertensive model suggested that the α1 and β1 isoform mRNA abundances (just as with the α2 and α3 isoform mRNA abundances in the ventricles of the DOC-salt and All models) were then sensitive to changes in vascular pressure and were consequently decreased in hypertensive rats. This hypothesis, if true, would suggest that the decreases caused by the effect of vascular pressure on the α1 and β1 isoforms in the DOC-salt and A II models were masked by the increases in [Na]i. The magnitude of the decreases in isoform mRNAs (in the ventricles) in the Milan hypertensive model were less than those reported in the DOC-salt and A II models (α2 and α3 isoforms). The difference in magnitude may be related to the degree of hypertension. Milan hypertensive rats have systolic blood pressures in the range 160-170 mmHg, whereas the blood pressures of DOC-salt and A II hypertensive rats were reported to be within 168-220 mmHg (Herrera et al., 1988). The reduction in the abundance of the mRNA for the isoforms of Na,K-ATPase in hypertensive ventricular tissues may relate to a decrease in the number of sodium pump units and consequently Na,K-ATPase activity. This would effectively have the same result on ventricular cells as cardiac glycoside action, resulting in increased [Na]i and consequently [Ca]i, which increases contractility (Schwartz et al., 1988). The decreases in the abundance of Na,K-ATPase isoform mRNAs may therefore be part of the adaptive response of ventricular tissues to increased blood pressure in hypertensive rats. The reason for the increases in mRNA abundance of the α1, α2, and β1 isoforms in the atria of hypertensive rats is unknown.

Figure 2. The relative abundance of the mRNA for the α1 (a), α2 (b), α3 (c), and β1 (d) isoforms of Na,K-ATPase from the myocardium of normotensive and hypertensive rats. The abundances are expressed relatively per gram of tissue. Abbreviations are as in Fig. 1. The error bars are standard deviations of two dots of RNA from each of three left or right ventricles (i.e., n = 6) and two atrial (i.e., n = 4) samples. The column indicated with a dagger had no detectable hybridization signal. This column therefore indicates the maximum possible level of radioactivity that would have produced no signal under the film exposure conditions used. The asterisks indicate statistical significance of a Student's t test comparison between normotensive and hypertensive rat tissues: *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.

Acknowledgments

Na,K-ATPase probes were obtained from Dr. J. Lingrel, University of Cincinnati College of Medicine, and Dr. R. Levenson, Yale University School of Medicine.

This work was supported by BHF grants 87/46 and 89/56. C. P. Cutler is in receipt of an MRC studentship. We also gratefully acknowledge the receipt of a Wellcome Trust travel grant.

References

Anderson, M. L. M., and B. D. Young. 1985. Quantitative fiber hybridisation. In Nucleic Acid Hybridisation: A Practical Approach. B. D. Hames and S. J. Higgins, editors. IRL Press Ltd., Oxford, UK. 73–111.

Bianchi, G., P. G. Baer, U. Fox, L. Duzzi, A. M. Caravaygi, and J. Mohring. 1979a. Pathogenesis of hypertension in the Milan hypertensive strain of rats (MHS). Japan Heart Journal. 20(suppl 1):61.

Bianchi, G., D. Cusi, M. Gatti, G. P. Lui, P. Ferrari, C. Barlassina, G. B. Picotti, G. Bianchi, G. Colombo, D. Gori, O. Velis, and D. Mazzci. 1979b. A renal abnormality as a possible cause of “essential” hypertension Lancet, i: 173–177.

Bianchi, G., U. Fox, G. F. DiFrancesco, U. Bardi, and M. Radice. 1973a. Hypertensive role of the kidney in spontaneously hypertensive rats. Clinical Science and Molecular Medicine. 45(suppl 1):135.

Bianchi, G., U. Fox, and E. Imbasciati. 1973b. The development of a new strain of spontaneously hypertensive rats. Life Sciences. 14:339–347.

Cramb, G., C. P. Cutler, J. F. Lamb, T. McDevitt, P. H. Ogden, D. Owler, and C. Voy. 1989. The effects of monensin on the abundance of mRNA (α) and of sodium pumps in human cultured cells. Quarterly Journal of Experimental Physiology. 74:53–63.

Cutler, C. P., G. Cramb, and J. F. Lamb. 1988. Quantitative analysis of sodium pump-specific mRNA from human endothelial (HeLa) and canine kidney (MDCK) cell cultures. Progress in Clinical and Biological Research. 268B:59–64.

Davis, L. G., M. D. Dibner, and J. F. Battey. 1986. Basic Methods in Molecular Biology. Elsevier Science Publishing Co., Inc., New York. 147–149.

Diez, J., P. Hannaert, and R. P. Garay. 1987. Kinetic study of Na+-K+ pump in erythrocytes from essential hypertensive patients. American Journal of Physiology. 252:H1–H6.

Genest, J., E. Koiw, and O. Kuchel. 1977. Hypertension. McGraw-Hill Inc., New York. 522–526.

Herrera, V. L. M., A. V. Chobanian, and N. Ruiz-Opazo. 1988. Isoform-specific modulation of Na+,K+-ATPase α-subunit gene expression in hypertension. Science. 241:221–223.

Schwartz, A., G. Grupp, E. Wallick, I. L. Grupp, and W. J. Ball, Jr. 1988. Role of the Na+K+-ATPase in the cardiotonic action of cardiac glycosides. Progress in Clinical and Biological Research. 268B:321–328.

Schull, G. E., J. Greeb, and J. B. Lingrel. 1986. Molecular cloning of three distinct forms of the Na+,K+-ATPase α-subunit from rat brain. Biochemistry. 25:8125–8132.

Young, R. M., G. E. Shull, and J. B. Lingrel. 1987. Multiple mRNAs from rat kidney and brain encode a single Na+,K+-ATPase β-subunit protein. Journal of Biological Chemistry. 262:4905–4910.

Quantification of the Total Na,K-ATPase Concentration in Mammalian Heart: Regional Distribution and Regulatory Aspects

Thomas A. Schmidt, Jim S. Larsen, and Keld Kjeldsen

Department of Medicine B 2142, Division of Cardiology, Rigshospitalet, Copenhagen University School of Medicine, DK-2100 Copenhagen, Denmark

Introduction

Complete recovery is a prerequisite for evaluation of the quantitiative aspects of muscle Na,K-ATPase (Hansen and Clausen, 1988). Thus, we have measured vanadate-facilitated [3H]ouabain binding to intact tissue samples (Nørgaard et al., 1986) or K+-dependent 3-O-methylfluorescein phosphatase (3-O-MFPase) activities in crude homogenates (Nørgaard et al., 1985) to quantify the total Na,K-ATPase concentration in mammalian heart and to evaluate its regional distribution together with regulatory aspects of ischemia-reperfusion, acute hypertrophy, and ethanol exposure.

Quantification and Regional Distribution

Recently a relationship between Na,K-ATPase concentration in muscle tissue and the capacity for performing work has emerged (Clausen et al., 1981; Knochel et al., 1985; Kjeldsen et al., 1988). Thus, it was of interest to compare the Na,K-ATPase concentration in atria and ventricles since the higher pressure work performed by the left ventricle might be associated with higher Na,K-ATPase concentration. Experiments were carried out using vanadate-facilitated [3H]ouabain binding to intact right atrium (RA) and left ventricle (LV) samples obtained immediately after sacrifice from female Antonius pigs and male labrador retriever dogs (Schmidt et al., 1990). Evaluation of [3H]ouabain binding kinetics revealed no major difference between RA and LV. Binding equilibrium for the [3H]ouabain and ouabain concentrations used (1 × 10–8–1 × 10–3 mol/liter) was obtained after 2 h of incubation in RA and LV from porcine and canine heart. Unspecific uptake and retention of [3H]ouabain for porcine heart RA and LV and for canine heart RA and LV was 1.0–3.0% of total uptake and retention obtained in standard measurements, i.e., incubating 2 h in buffer containing 1 × 10–6 mol/liter [3H]ouabain and ouabain. The apparent dissociation constant (KD) was 1.4 × 1.0–8 and 1.9 × 10–8 mol/liter in porcine RA and LV and 2.6 × 10–8 and 6.1 × 10–8 mol/liter in canine RA and LV. Loss of specifically bound [3H]ouabain during the washout procedure at 0°C occurred with a half-life time (T½) of 17 and 29 h in RA and LV of porcine heart and 91 and 152 h in RA and LV of canine heart. Corrected for the errors of the method, total [3H]ouabain binding site concentrations are given in Table I. No significant variation in [3H]ouabain binding site concentration was seen through the left ventricle wall. This study confirms an earlier determination of [3H]ouabain binding site concentration of ~ 800 pmol/g wet wt in intact samples of porcine LV (Sejersted et al., 1985). This value was only corrected for unspecific uptake and retention and for incomplete saturation. If we apply only these two correction factors to our value it corresponds to 936 pmol/g wet wt. Hence, the two values for [3H]ouabain binding site concentration seem to be in good agreement. With respect to ouabain affinity, the present study gave no evidence of more than one population of [3H]ouabain binding sites. Thus, when evaluated in Scatchard-type plots the values for the saturation course of [3H]ouabain binding in both porcine and canine RA and LV showed a linear distribution. In the present study, mammalian heart Na,K-ATPase concentration was found to be about twofold higher in ventricles than in atria. The higher concentration in ventricles compared with atria may in association with difference in wall thickness be of importance for the higher pressure work performed by the ventricles.

Regulatory Aspects of Na,K-ATPase

Ischemia-Reperfusion

On the basis of the concept that ischemic myocytes may enter a reversible phase before irreversible injury occurs (Kloner and Braunwald, 1980; Harper and Lochner, 1989), we evaluated whether short-term myocardial ischemia followed by reperfusion has an influence on [3H]ouabain binding site concentration in the porcine heart (LV). Furthermore, this was of interest because earlier studies on membrane fractions have shown a reduced Na,K-ATPase activity after ischemia (Beller et al., 1976; Kim et al., 1983; Chemnitius et al., 1985; Kim and Akera, 1987). In our study the left anterior descending artery (LAD) was exposed and clamped for 20 min, inducing ischemia, and released for 60 min of reperfusion (Schmidt et al., 1990). Immediately after sacrifice, a myocardial sample from LV was taken from the center of the tissue supplied by LAD and another from outside this area. The ischemia-reperfusion did not alter the [3H]ouabain binding site concentration (P > 0.90) (Table I). This supports the opinion that myocytes exposed to ischemia-reperfusion may enter a reversible phase before irreversible injury occurs.

Acute Hypertrophy

The effect of isoproterenol-induced acute cardiac hypertrophy on Na,K-ATPase concentration was investigated to examine whether acute hypertrophy is associated with a change in Na,K-ATPase concentration. This was of interest because a downregulation of the Na,K-ATPase has been found in hypertrophic cardiomyopathy (Nørgaard et al., 1987). To induce acute cardiac hypertrophy, female Wistar rats received intraperitoneal injections of 0.2 mg isoproterenol three times daily for 1 wk. The LVs were taken for 3-O-MFPase activity measurements. Isoproterenol treatment of rats for 1 wk induced myocardial hypertrophy (30%, P < 0.01) without change in Na,K-pump concentration (P > 0.5). The values for K-activated 3-O-MFPase (means, n = 5) were ~0.87 μmol/g wet wt/min for both groups, corresponding to a Na,K-ATPase concentration of ~ 1,410 μmol/g wet wt (Table I). Thus, initially myocardial hypertrophy seems to be associated with a balanced synthesis of muscle mass and Na,K-ATPase.

Ethanol

The influence of ethanol administration on cardiac Na,K-ATPase concentration was evaluated. This was of interest because ethanol has been shown to induce an acute increase in Na-influx, which could be expected to cause Na overloading of the cells (Blachley et al., 1985; Blachley and Knochel, 1987). Thus, the intracellular Ca concentration might increase and cause dystrophic calcification. It was furthermore of interest to evaluate whether there were also changes in Na,K-ATPase concentration when estimated using methods with high recovery, because in membrane fractions an upregulation in response to ethanol has been reported (Ferguson et al., 1984; Brodie and Sampson, 1987). Initial experiments on the effect of ethanol on myocardial Na,K-ATPase concentration were performed using heart samples from female guinea pigs exposed to 4.7% ethanol for 2 wk followed by 10 and 20% ethanol each for a week in their drinking water. Atrium and ventricle samples were taken immediately after sacrifice for [3H]ouabain binding. This treatment induced a significant upregulation of the 3H-ouabain binding site concentration in atria as well as ventricles. Thus, it seems that ethanol ingestion can increase cardiac Na,K-ATPase concentration. This may compensate for the reported increase in Na influx induced by ethanol administration and thus perhaps for some time protect the heart from sodium overloading and concomitant dystrophic calcification.

TABLE I
Distribution and Regulatory Aspects of Porcine and Canine Heart Na,K-ATPase

Values are means ± SEM, n = 5. *Calculated from K-activated 3-O-MFPase measurements on the basis of molecular activity of the 3-O-MFPase of 620 cycles per mm.

Conclusion

Studies of the quantitative aspects of Na,K-ATPase, using the described methods, may be of importance for understanding pathophysiological aspects at cellular level of myocardial ischemia-reperfusion, myocardial hypertrophy, and alcoholic heart disease.

Acknowledgments

This study was supported in part by The Danish Heart Foundation and NOVO's Foundation.

References

Beller, G. A., J. Conroy, and T. W. Smith. 1976. Ischemia-induced alteration in myocardial (Na+–K+)-ATPase and cardiac glycoside binding. Journal of Clinical Investigation. 57:341–350.

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Dissociation Kinetics of [3H]Ouabain Na,K-ATPase Isoform Complexes

Alain De Pover and Gudrun Feige

Department of Cardiovascular Research, Ciba-Geigy Ltd., CH-4002 Basel, Switzerland

Introduction

The catalytic subunit (α) of Na,K-ATPase has a family of three isoform genes that are developmentally and hormonally regulated (Sweadner, 1989). In mammalian tissues, α1 is ubiquitous, α2 is expressed in cardiac and skeletal muscle and brain, and α3 is expressed in cardiac muscle and mostly in brain. Monoclonal antibodies, oligonucleotide probes, and [3H]ouabain binding have been used in these studies. A recurrent problem in the use of [3H]ouabain is its slow binding to Na,K-ATPase at low concentrations in very sensitive species. Furthermore, a clear separation between high and low affinity isoforms in equilibrium experiments is obtained only with some species, such as the rat, where the affinity ratio between low affinity α1 and high affinity α2 and α3 is > 100. At lower ratios, reliable determinations can be obtained in [3H]ouabain dissociation experiments. It is known that [3H]ouabain dissociation from Na,K-ATPase follows first-order kinetics and can be studied after stopping the binding reaction by dilution (Erdmann and Schoner, 1973). The wide disparity in ouabain sensitivities of the Na,K-ATPase among animal species has been related to differences in the dissociation rate of the ouabain receptor complex. Differences in affinities between isoforms could also be related to differences in dissociation rates. In this study we examined [3H]ouabain dissociation kinetics with Na,K-ATPase preparations from the kidneys (α1) and brains (α1, α2, and α3) of various mammalian species, and from rat heart ventricle (α2).

Methods

Na,K-ATPase Preparation and Assay

The enzyme was partially purified from NaI- and sodium desoxycholate-treated homogenates, and its activity was determined from released inorganic phosphate (Pi) as previously described (Feige et al., 1988). 0.5–3 μg of microsomal protein was incubated for 60 min at 37°C in 0.1 ml medium containing 5 mM ATP, 5 mM MgCl_2, 150 mM NaCl, 1 mM KCl (20 mM for maximal activity), 1 mM EGTA, and 10 mM maleic acid (pH = 7.4 adjusted with Tris). Na,K-ATPase activity was linear with time and protein, provided that ATP hydrolysis did not exceed 20%. The ATPase activities were in the range of 50–150 μmol Pi/mg protein per h for kidneys and brains, and 20–30 μmol Pi/mg protein per h for rat heart. Over 90% was ouabain sensitive.

Ouabain Binding Assay

[3H] Ouabain binding to Na,K-ATPase was assayed by a filtration technique as previously described (De Pover and Godfraind, 1979). 50–250 μg of microsomal protein was incubated at 37°C in 5 ml medium containing 3 mM MgCl_2, 3 mM phosphoric acid, 1 mM EGTA, 10 mM maleic acid (pH = 7.4 adjusted with Tris), and 10–6 M [3H]ouabain (3 Ci/mmol). For [3H]ouabain dissociation kinetics, 10–4 M ouabain was added after 10 min incubation, and 0.2-ml aliquots were rapidly filtered at various times on GF/F glassfiber filters (Whatman Inc., Clifton, NJ). Specific binding was 130–170 pmol/unit for kidneys and brains, and 10 pmol/unit for rat heart (1 enzyme unit hydrolyzes 1 μmol ATP/min).

Data Analysis

Dihydroouabain inhibition curves were fitted to the function:

where A is the inhibition for zero concentration, B the inhibition for infinite concentration, C the concentration for half-maximal inhibition (IC_50), X the variable concentration, and subscript n the number of independent sites (one or two). [3H]Ouabain dissociation kinetics were fitted to the function:

where A is the binding at infinite time, B the binding at time 0, D the first-order dissociation rate constant (k–1),X the variable time, and subscript n the number of independent sites (one or two). Nonlinear curve fitting was performed with a minicomputer VAX8350.

Results and Discussion

Fig. 1 shows the inhibition of kidney and brain Na,K-ATPase by dihydroouabain for various species. This glycoside, which is 10–100 times less potent than ouabain, was used in this experiment instead of ouabain because it quickly binds to Na,K-ATPase, allowing true equilibrium to be achieved at all concentrations. Fig. 2 shows dissociation kinetics of the [3H]ouabain receptor complex after isotopic dilution. In this experiment, > 90% of the specific sites, except for rat α1, had been saturated previously with a high [3H]ouabain concentration (10–6). Fig. 3 shows a comparison of dihydroouabain inhibition and [3H]ouabain dissociation kinetics between rat brain and rat heart. Adult rat heart ventricle was used here because it is known to contain 10% of ouabain high affinity α2 sites, with Kd of 2 × 10–7 M, and 90% of low affinity α_1 sites, with Kd of 2 × 10–5 M (Orlowski and Lingrel, 1988; Noel et al., 1990). [3H]Ouabain dissociation from rat α1 is extremely rapid and does not show up in Figs. 2 and 3.

Analysis of dihydroouabain inhibition and [3H]ouabain dissociation kinetics suggests three groups of species: (I) the very sensitive (dog, marmoset, pig, sheep); (II) the sensitive (guinea pig, rabbit); and (III) the resistant (rat). In kidney—whatever the species—the kinetics showed a single class of sites, most probably α1 as previously suggested (Sweadner, 1989). By contrast, in brain, inhibition kinetics showed one class of sites in group I and two classes in groups II and III, whereas [3H]ouabain dissociation kinetics showed two classes in groups I and II and three classes in group III. In group I, the rate constant k–1 of the slow phase in brain (0.007–0.013 min–1) was similar to the rate constant of kidney (0.005–0.006 min–1), suggesting that the high affinity site in brain could be α1. In group II, the rate constant k–1 of the kidney (0.5 min–1) was similar to that of the rapid phase and 40 times higher than that of the slow phase in brain, suggesting that the low affinity site in brain could be α1. In group III, two slow phases were observed. The comparison of rat brain with rat heart kinetics suggests that the more rapid phase in brain could be α2 and, by exclusion, the slowest phase α3. This conclusion is in agreement with Noel et al. (1990).

Figure 1. Na,K-ATPase inhibition by dihydroouabain. (Left) Kidney membranes: IC_50 = 10–7 M for marmoset , dog , sheep , and pig , 4 × 10–6 M for guinea pig and rabbit (A), and 5 × 10–4 M for rat . (Right) Brain membranes, same symbols as for kidney: IC_50 = 1.5 × 10–7 M for high affinity sites in all species, 5 × 10–6 M for low affinity sites (dotted lines) in guinea pig and rabbit, and 4 × 10–4 M in rat. For conditions see Methods.

Figure 2. [3H]Ouabain dissociation kinetics for kidney (left) and brain membranes (right). Rapid dissociation phases are indicated by dotted lines. For conditions see Methods. Same symbols as in Fig. 1.

Figure 3. Na,K-ATPase inhibition by dihydroouabain (left) and [3H]ouabain dissociation kinetics (right). Brain and heart membranes were from rat. For conditions see Methods.

References

De Pover, A., and T. Godfraind. 1979. Interaction of ouabain with Na,K-ATPase from human heart and from guinea-pig heart. Biochemical Pharmacology. 28:3051–3056.

Erdmann, E., and W. Schoner. 1973. Ouabain-receptor interactions in Na,K-ATPase preparations from different tissues and species: determination of kinetic constants and dissociation constants. Biochimica et Biophysica Acta. 307:386–398.

Feige, G., T. Leutert, and A. De Pover. 1988. Na,K-ATPase isozymes in rat tissues: differential sensitivities to sodium, vanadate and dihydroouabain. In The Na,K-Pump. Part B: Cellular Aspects. J. C. Skou, J. G. Nørby, A. B. Maunsbach, and S. Esmann, editors. Alan R. Liss, Inc., New York. 377–384.

Noel, F., M. Fagoo, and T. Godfraind. 1990. A comparison of the affinities of rat Na,K-ATPase isozymes for cardioactive steroids, role of lactone ring, sugar moiety and KCl concentration. Biochemical Pharmacology. 40:2611–2616.

Orlowski, J., and J. B. Lingrel. 1988. Tissue-specific and developmental regulation of rat Na,K-ATPase catalytic α isoform and β subunit mRNAs. Journal of Biological Chemistry. 263:10436–10442.

Sweadner, K. J. 1989. Isozymes of the Na,K-ATPase. Biochimica et Biophysica Acta. 988:185–220.

Ouabain-induced Changes in Intracellular Free Calcium Concentration Examined in Different Cell Types of Chick Heart Monolayer Cultures

Barbara Ahlemeyer, Horst Weintraut, Georg Seibold, and Wilhelm Schoner

Strahlenzentrum and Institut für Biochemie & Endokrinologie, Justus-Liebig-University, Giessen, Germany

Introduction

Cardiac glycosides produce positive inotropy of cardiac myocytes. This effect is the result of a sequence of events starting with the binding of the drug to its specific receptor, the Na,K-ATPase, which leads to the inhibition of the sodium pump. Consequently, intracellular sodium concentration increases, which is assumed to stimulate the Na-Ca exchanger (Blaustein and Nelson, 1982). This event results in an increase in the cytoplasmic calcium concentration, which is responsible for increased contractility. However, the relationship between sodium pump occupation by cardiac glycosides, inhibition of the enzyme, and positive inotropic action is still controversial. Recently, cells with high and low concentrations of intracellular calcium determined by fluorescent digital imaging microscopy have been found in cardiac muscle cells from rats (Wier et al., 1987) and guinea pigs (Williford et al., 1990). In addition, isoenzymes of the sodium pump have been described in heart muscle from dogs (Maixent et al., 1986) and ferrets (Ng and Akera, 1987). In chick heart, Sweadner and Farshi (1988) found only the α1 form of the Na,K-ATPase,whereas Lemas et al. (1991) found all three α forms of the enzyme. Controversial data have also been reported on the existence of high and low affinity ouabain binding sites in chick cardiac cells.

The aim of our work, using chick embryo heart cells in culture, was to learn (a) whether there are cells with high and low intracellular calcium concentration in embryonic chick heart, and (b) whether the intracellular calcium level responds to ouabain in all cells in a similar way or differently.

Materials and Methods

Preparation of the Cells

Monolayer cultures of chick embryo heart cells were prepared as described earlier by Renaud (1980). Heart tissue was disaggregated with trypsin (0.025% wt/vol at 37°C) for 10 min and mechanically minced through 20-μm-wide nylon meshes. The cells were seeded into glass petri dishes to a final density of 2 × 10^5 cells/dish. The semiconfluent cell monolayers were incubated at 37°C with 95% air and 5% CO_2. The culture medium was changed after 48 h. Experiments were carried out at the fourth day of cultivation.

Fura-2 Loading of the Cells

The cells were loaded with the Ca-sensitive indicator fura-2-AM (at a final concentration of 5 μM) by incubation for 30 min at 37°C in MEM (supplemented with 10% fetal calf serum), allowing the permeable acetoxymethylester form to enter the cells. The glass petri dish with the fura-2-loaded cells was washed three times with Hank's Balanced Salt Solution containing (in mM): 145 NaCl, 5.4 KCl, 1 MgCl_2,1.8 CaCl_2, 5 glucose, and 10 HEPES, adjusted to pH 7.40.

Fluorescence Microscopy

For fluorescence measurements, the glass petri dish was mounted in a temperature-controlled chamber of 37°C, which was placed on the stage of a scanning table of a modified Zeiss-IM 35 microscope. The light source was a 100-W HBO lamp (Osram, Berlin, Germany). The emitted light was focused through a lens and then divided into two excitation wavelengths. Excitation light was switched by sliding a computer-controlled filter chopper with narrow band pass filters centered at 334 and 366 nm. The filter chopper also functioned as a shutter: the cells were exposed to the excitation light for only 2 s to minimize photobleaching and phototoxicity. A 10% neutral filter was placed in front of the 366-nm channel to equalize the intensities of the two excitation wavelengths. The emitted fluorescence light of the fura-2 at 510 nm and the background fluorescence were focused through a UV-lens (Ultrafluar 32/0.4; Zeiss), a dichroic mirror FT460, and an LP480 to maximize light transmission. The fluorescent images were obtained with a C2400-08 SIT video camera (Hamamatsu, Japan) and digitized by a realtime frame grabber (LFS/AT Scan Vision, München, Germany) residing in a microcomputer (PC/AT, 16 MHz; Bargate, Taiwan) at eight bits per pixel. For the determination of the intracellular calcium concentration the mean pixel intensities of the ratioed images (334/366) were compared with those of the calibration solutions. Both were corrected for background fluorescence and noise by the following equation: [Ca2+]i = (Sf2/Sb2)kd (R – Rmin)/(Rmax – R) (Grynkiewicz et al., 1985)

Results

In all experiments with fura-2-loaded chick embryo heart cells we found cells with high and low [Ca2+]i Cells with low basal [Ca2+]i levels (mean value 60.23 ± 15.52 nM, n = 31) contributed to 30% of the cells. 70% of the cells had higher basal [Ca2+]i levels (mean value 132.36 ± 29.92 nM, n = 67).

When chick embryo heart cells were exposed to different concentrations of ouabain, both cell types responded with a rise in [Ca2+]i. The velocity of the increase in [Ca2+]i was slower when lower concentrations of ouabain were used and it was enhanced by using higher ouabain doses. The [Ca2+]i that was reached for each cell type under equilibrium conditions depended on the concentration of ouabain that was applied to the cell culture (Fig. 1). In both cell types, ouabain elevated the [Ca2+]i in a dose-dependent manner, but with different half-maximal saturation. Cells with higher [Ca2+]i showed a half-maximal effect at 5 × 10–8 M ouabain, and a second rise in [Ca2+]i was seen at ouabain concentrations >10–6 M. For heart cells with low basal [Ca2+]i levels no distinct half-maximal saturation could be evaluated. However, it is evident that the concentration of ouabain necessary to obtain a half-maximal effect is at least two orders of magnitude above that of the high [Ca2+]i cells.

Discussion

Cultures of embryonic chick heart cells contain cells with high and low [Ca2+]i as previously shown for heart cells of different species. Cells with high [Ca2+]i represent 70% of all cells, while those with low [Ca2+]i and lower affinity for ouabain represent only 30%. It is obvious that more detailed investigations are needed to clarify whether or not both cell types have different cellular functions. Moreover, the demonstration of the differences of the two cell types concerning the sensitivity to ouabain is an indication of different cell functions. Studies on binding characteristics of ouabain in chicken heart cells (Werdan et al., 1984) showed the existence of a single class of receptors with a binding constant of Kd = 1.5 × 10–7 M for muscle cells and kd = 1.9 × 10–7 M for nonmuscle cells. A single class of receptors was also found by Kim et al. (1984) with a kd of 4 × 10–7 M and Lobaugh and Lieberman (1987) with a kd of 6.6 × 10–6 M. These findings are inconsistent with our results of two types of cells with different affinities for ouabain. On the other hand, Kazazoglou et al. (1983) have described two classes of receptors, one with high affinity (kd = 4 × 10–8 M) and the other with low affinity for ouabain (kd = 4 × 10–6 M), but an effect was detectable only for the low affinity site. The obvious differences in results may be due to different experimental procedures and differences in cell culture conditions. An excessive increase in [Ca2+]i; has been seen at toxic concentrations of ouabain in both cell types. It is still unknown by which mechanism this excessive increase in [Ca2+]i takes place.

Figure 1. The effect of different ouabain concentrations on maximal [Ca2+]i elevation (under equilibrium conditions) in comparison to normal [Ca2+]i levels in heart cells with high basal [Ca2+]i levels (closed circles) and low basal [Ca2+]i levels (open circles). Mean values ± SD of 6–11 experiments are given.

Acknowledgments

This work was supported by SFB 249 Giessen Pharmakologie biologischer Makromoleküle and the Fonds der Chemischen Industrie Frankfurt/M.

References

Blaustein, M. P., and M. T. Nelson. 1982. Sodium-calcium exchange: its role in the regulation of cell calcium. In Membrane Transport of Calcium. E. Carafoli, editor. London. 217–236.

Grynkiewicz, G., M. Poenie, and R. Y. Tsien. 1985. A new generation of Ca2+–indicators with greatly improved fluorescence properties. Journal of Biological Chemistry. 260:3440–3450.

Kazazoglou, T., J. F. Renaud, B. Rossi, and M. Lazdunski. 1983. Two classes of ouabain receptors in chick ventricular cardiac cells and their relation to Na+,K+–ATPase-inhibition, intracellular Na+ accumulation, Ca2+ influx and cardiotonic effect. Journal of Biological Chemistry. 258:12163–12170.

Kim, D., W. H. Barry, and T. W. Smith. 1984. Kinetics of ouabain binding and changes in cellular sodium content, 42K-transport and contractile state during ouabain exposure in cultured heart chick cells. Journal of Pharmacology and Experimental Therapeutics. 231:326–333.

Lemas, V., J. Rome, J. Taormino, K. Takeyasu, and D. M. Fambrough. 1991. Analysis of isoform-specific regions within the α- and β-subunits of the avian Na+,K+–ATPase. In The Sodium Pump: Recent Developments. J. H. Kaplan and P. De Weer, editors. The Rockefeller University Press, New York. In press.

Lobaugh, L. A., and M. Lieberman. 1987. Na-K-pump site density and ouabain binding affinity in cultured chick heart cells. American Journal of Physiology. 253:C731–C743.

Maixent, J. M., D. Charlemagne, B. de la Chapelle, and L. G. Lelievre. 1986. Two Na K-ATPase isoenzymes in canine cardiac myocytes. Journal of Biological Chemistry. 262:6842–6848.

Ng, Y. C., and T. Akera. 1987. Two classes of ouabain binding sites in ferret heart and two forms of Na+,K+–ATPase. American Journal of Physiology. 252:H1016–H1022.

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Sweadner, K., and S. K. Farshi. 1988. Two forms of the Na,K-ATPase catalytic subunit detected by their immunological and electrophoretic differences: alpha and alpha+ in mammalian heart. In The Na-K-Pump. Part B: Cellular Aspects. J. C. Skou, G. Norby, A. B. Maunsbach, and M. Esmann, editors. New York. 149–156.

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Presence of an Inward Background Current Sensitive to Sodium in the Isolated Rabbit Sinoatrial Node Cells

N. Hagiwara, H. Irisawa, H. Kasanuki, and S. Hosoda

The Heart Institute of Japan, Tokyo Women's Medical College, Tokyo, 162 Japan

Deactivation of K+ conductance in the presence of an inward background current has been considered as one of the mechanisms of the pacemaker depolarization in the mammalian sinoatrial node cells (Brown, 1982; Irisawa, H., H. Brown, and W. Giles, manuscript submitted for publication). The inward background current has been assumed to exist in the simulated study as a necessary inward current for spontaneous rhythm (Yanagihara and Irisawa, 1980; Noble et al., 1989). The background inward current, however, has not been investigated experimentally.

Using the whole-cell patch clamp mode, we measured the time-independent inward background current sensitive to Na+ (Ib) in the pacemaker cell. Fig. 1 shows the current-voltage (I-V) relationship measured in 150 mM Tris/Na-free solution (A) and 150 mM Na solution (B). The slope resistance in 150 mM Na solution measured between –100 and –50 mV was 0.6 ± 0.3 GΩ (n = 18), whereas in Tris/Na-free solution it was 3.8 ± 0.5 GΩ (n = 18). The difference curve gives the Na+–sensitive current. Amplitude of Ib at –50 mV was –26.0 ± 8.9 pA (n = 64) and its current density was 0.77 ± 0.3 pA/pF.

As shown in Fig. 1 A, the Ib was approximately –30 pA at –50 mV and the conductance was 0.5 × 10–9 S. Assuming the surface area of the cell to be 2,000 μm^2, the current amplitude per 1 μm^2 would be 0.015 pA, which would be either too small to measure as a single-channel conductance, or the open probability of the channel would be too small to observe any channel event. In fact, we tried to record the single-channel events but failed to do so.

Fig. 1 B shows a similar experiment, except that the patch clamp mode is outside-out. The I-V curve in the isolated patch shows a similar pattern to that in Fig. 1 A, except that amplifier gain was 10 times larger. The amplitude of the Ib was 4.2 pA at –50 mV. Since the corresponding current in the whole-cell clamp mode was 30 pA, the membrane capacitance of the isolated membrane would be ~ 5 pF, suggesting that the patch must have a surface area larger than expected. The I-V curve in 150 mM Na intersected at 0 mV, whereas in 150 mM Tris/Na-free solution it intersected at a more negative potential. This finding also agrees well with those found previously in the whole-cell clamp mode. The slope resistance of the I-V current in Tris/Na-free solution in the whole-cell clamp mode was 3.8 ± 0.5 GΩ (n = 18) at –50 mV (Hagiwara, N., H. Irisawa, H. Kasanuki, and S. Hosoda, manuscript submitted for publication), whereas that in the outside-out patch was ~5 GΩ, indicating that the slope resistance in Tris is the resistance between the electrode and the extracellular space, which was apparently similar in spite of the large difference in the membrane surface. The inward current in Tris/Na-free solution was not changed significantly in the low temperature perfusate, whereas the Ib was reduced significantly at low temperature with a Q_10 of 3.2. The Ib is not sensitive to TTX, but it is significantly reduced in its amplitude under the influence of amiloride. Monovalent cations such as Li+, K+, Cs+, and Rb+ can be equally permeable via Ib, although the conductance is in the order of Rb+ > K+ > Cs+ > Na+ > Li+. Fig. 2 illustrates the effect of amiloride on the background inward current. In this example, instead of superfusing 150 mM Na+ solution, we superfused equimolar Cs+ solution.

Figure 1. (A) Panel 1, I-V relationship obtained by the ramp clamp method in the whole-cell clamp mode. (a) Control in Tris/Na-free solution, (b) in 150 mM Na+ solution. Panel 2, The difference curve between b and a, which indicates the inward background current sensitive to Na+ (Ib). The patch pipette contained 150 mM Cs+ aspartate and the external 150 mM Na+ solution contained 2 mM Ni2+, 2 mM Ba2+, 2 mM Cs+, and 10 μM ouabain. K+ and Ca2+ were not contained. Method of cell isolation and the electrical measurements were reported elsewhere (Denyer and Brown, 1990). Ramp clamp was made ±70 mV from the holding potential of –30 mV with a ramp duration of 300 ms. The falling phase of the current from +40 to –100 mV is illustrated in the figure. (B) Panel 1, Patch pipette contains 150 mM Cs+ aspartate solution. Outside-out preparation. Curve a illustrates that recorded in Tris/Na-free solution, whereas curve b illustrates that recorded in 150 mM Na+ solution. Panel 2 is the difference curve between b and a, indicating that it is Ib.

The conductance of the background inward current is much larger in Cs+ solution than in Na+ solution (Fig. 2). A significant reduction of the background inward current was also observed in Cs+ solution, suggesting that Cs+ and Na+ share a similar blocking site to amiloride in this background inward current. The amplitude of Ib is increased as the concentration of the extracellular Na+ increases. The presence of Ib in the S-A node cell indicates that the hypothesis of the contribution of Ib to the decay of IK appears to be one of the probable mechanisms of pacemaker potential. Due to the continuous current flow of Na+ through the Ib mechanism, the resting potential of the S-A node probably stays in a depolarization level, which contributes the major mechanism of the low resting potential and high pNa/pK ratio of the leading pacemaker cells of the S-A node region. In the atrial cells near the S-A node, the size of the cell was approximately three times larger than the S-A node cell, while the amplitude of the Ib in the atrial cell was not much different from that found in the S-A node cell, indicating that the current density of the Ib was approximately three times larger than in the atrial cell. In three different examples, we compared the data of the nodal cell with the ventricular cell, and found that the Ib in the ventricular cell was smaller than that of the nodal cell. Therefore, we concluded that the presence of Ib in the S-A node cell contributes to the high pNa/pK ratio of the nodal cell. Ib is voltage dependent; thus, when the membrane hyperpolarizes, the amplitude of Ib increases, and this tends to depolarize the membrane. When the membrane depolarizes, the amplitude of Ib will decrease; thus, the presence of Ib stabilizes the heart rate at a more or less constant rate.

Figure 2. Effect of amiloride on the background inward current sensitive to Na. In this example, 150 mM Cs+ was superfused in place of 150 mM Na+. Amiloride decreases the time- independent inward current caused by Cs+, thereby proving that the Ib is a nonselective cation current and amiloride is effective in blocking both Na+– and Cs+–sensitive inward currents.

The hyperpolarization-activated inward current (If) may also provide a similar role to Ib. In the present experiment, Ib was measured in the presence of 2 mM Cs+. The Ib could also be recorded under the condition of 150 mM Rb+ in the external solution and 150 mM Cs in the pipette solution. Therefore, it is unlikely that If is contributing the Ib in this observation. We directly compared the amplitude of Ib and If by using the ramp duration of 500 ms. Since the duration of the normal S-A node action potential was < 200 ms, our assessment of the relative amplitude of If must be an overestimate. Nevertheless, the amplitude of If at –70 mV was approximately one-third that of Ib. Thus, in the normal pacemaker potential, when the most negative potential lies more positive than –70 mV, the participation of If in the pacemaker potential is probably insignificant. This conclusion, however, does not exclude the physiological significance of If. The spontaneous heart rate is reduced by ~30% under the effect of Cs+ (Denyer and Brown, 1990), which suggests that a certain percentage of the normal low resting potential depends on activation of this current (Yanagihara and Irisawa, 1980). In the multicellular specimen, however, the spontaneous heart rate decreases less than 10% (Noma et al., 1983). It is still not certain whether the current If has a significant meaning on the cycle-to-cycle pacemaker potential. All these observations are consistent with the notion that If may play a role in maintaining the pacemaker activity by decreasing the resting potential of the nodal cell.

References

Brown, H. 1982. Electrophysiology of the sinoatrial node. Physiological Reviews. 62:505–530.

Denyer, J. C., and H. F. Brown. 1990. Pacemaking in rabbit isolated sino-atrial node cells during Cs+ block of the hyperpolarization-activated current if. Journal of Physiology. 429:401–409.

Noble, D., D. DiFrancesco, and J. C. Denyer. 1989. Ionic mechanisms in normal and abnormal cardiac pacemaker activity. In Neuronal and Cellular Oscillators. J. W. Jacklet, editor. Marcel Dekker, New York. 59–85.

Noma, A., M. Morad, and H. Irisawa. 1983. Does the “pacemaker current” generate the diastolic depolarization in the rabbit SA node cells? Pflügers Archiv. 397:190–194.

Yanagihara, K., and H. Irisawa. 1980. Inward current activated during hyperpolarization in the rabbit sinoatrial node cell. Pflügers Archiv. 385:11–19.

Author Index

Ahlemeyer, B., 653

Ahmed, K., 291, 297

Akera, T., 579

Akopyanz, N. S., 189

Albers, R. W., 137, 303

Albers, W., 525

Allen, J. C., 631

Anderson, W. R., 149

Anner, B. M., 479

Antolovic, R., 253, 405

Apell, H.-J., 531, 537, 567

Appelhans, H., 55

Araki, K., 221

Argüello, J. M., 199

Arystarkhova, E. A., 249

Azuma, K. K., 171, 177

Baggioni, A., 619

Balabanov, Yu. A., 189

Ball, W. J., Jr., 275

Bamberg, E., 525

Barada, K., 605

Barnstein, A. M., 11

Barrett, V., 447

Bashi, E., 493

Beaugé, L., 183, 389, 395

Becker, R. R., 297

Berberián, G., 389, 395

Béress, L., 505

Bessarab, D. A., 5

Bhutada, A., 75

Bielen, F. V., 559

Biemesderfer, D., 37

Blanco, G., 183

Bliss, D. P., Jr., 37

Blum, H., 475

Boldyrev, A. A., 483

Borm, G. F., 369

Boyd, K. L., 237

Broillet, M.-C., 563

Broude, N. E., 5, 189

Brüller, H.-J., 253

Bühler, R., 531

Burke, E., 165

Buxbaum, E., 405

Campbell, A. M., 107

Canessa, C., 19

Canfield, V. A., 31, 131

Caplan, M. J., 285

Cavieres, J. D., 401

Cayanis, E., 79

Champeil, P., 259

Clausen, T. N., 461

Coca-Prados, M., 157

Collins, J. H., 37

Corey, S., 489

Cornelius, F., 383

Cortas, N., 605, 609

Corthesy, I., 271

Covarrubias, Y. G.-M., 409

Cramb, G., 95, 635

Cutler, C. P., 95, 635

D'Aquila, T., 31

David, P., 427

De Pont, J. J. H. H. M., 369

De Pover, A., 647

De Tomaso, A. W., 69

De Weer, P., 409

Duque, J. A., 515

Eakle, K. A., 43, 125

Edelman, I. S., 79, 195, 601

Faller, L. D., 357

Fambrough, D. M., 11, 25, 49, 107, 111, 117, 265

Farley, R. A., 43, 61, 125, 357

Fedosova, N. U., 483

Feige, G., 647

Fenard, S., 619

Fendler, K., 525

Field, M., 605, 609

Filuk, P. E., 149

Fink, D. J., 143

Forbush, B., III, 37, 327

Froehlich, J., 525

From, A. H. L., 291, 297

Fukushima, Y., 423

Fullerton, D. S., 291, 297

Gadsby, D. C., 543

Gaeggeler, H. P., 19

Garrahan, P. J., 379

Gaudio, K. M., 573

Geering, K., 271, 465

Ghosh, S., 157

Glitsch, H. G., 549

Good, P., 271

Grell, E., 437, 441, 525

Grisham, C. M., 231

Grishin, A. V., 5

Haase, W., 465

Hagiwara, N., 657

Halpryn, B. M., 619

Hamer, E., 405

Hamrick, M., 11

Hansen, O., 461

Hatala, M. A., 195

Hayashi, Y., 281

Hebert, H., 243

Hensley, C. B., 171, 177

Hernando, N., 157

Hieber, V., 143

Hildebrand, F., 573

Hilgemann, D. W., 543

Hinz, H. R., 205

Hobbs, A., 525

Hoffman, J. F., 453

Hölscher, C., 567

Homareda, H., 217

Horisberger, J.-D., 563

Horowitz, B., 125, 165

Hosoda, S., 657

Huang, R. C. C., 49

Huston, E. E., 61

Imesch, E., 479

Infantino, R. W., 609

Inoue, N., 597

Irisawa, H., 657

Ismail-Beigi, F., 75, 195

Ito, E., 363, 413

Ito, O., 221

Ivkova, L. V., 519

Jaisser, F., 19

Jaruschewski, S., 525

Jaunin, P., 271

Johnson, R. G., Jr., 475

Kamo, Y., 413

Kaplan, J. H., 199, 309

Karlish, S. J. D., 427

Kasanuki, H., 657

Kashgarian, M., 573

Kavéus, U., 243

Kawakami, K., 1, 101, 217

Kawamoto, R. M., 619

Kawamura, M., 65, 225

Kessler, P. D., 107

Kim, K. S., 43, 125

Kim, S. Y., 505

Kirley, T. L., 205, 211

Kjeldsen, K., 585, 613, 641

Klodos, I., 321, 327, 333

Kone, B. C., 265

Kootsey, J. M., 553

Koyama, T., 579

Kraehenbuhl, J.-P., 19

Krämer, T., 253

Kröner, S., 55

Kudo, A., 363

Kuntzweiler, T. A., 231

Kuroki, J., 221

Kuwada, M., 221

Larsen, J. S., 585, 613, 641

Läuger, P., 531

le Maire, M., 259

Lehn, J. M., 437

Lemas, V., 117

Lemieux, D. R., 553

Lescale-Matys, L., 83

Levenson, R., 31, 125, 131

Lewitzki, E., 441

Link, W. T., 137, 303

Lopez Rosado, R., 489

Lopina, O. D., 483

Lucak, S. L., 609

Luckie, D. B., 237

Mädefessel, K., 89, 465

Mairbäurl, H., 453

Maixent, J.-M., 619

Maki, A. A., 149

Malyshev, I. V., 5

Mann, A. S., 573

Marín, R., 471, 515

Marjanovic, M., 457

Martin, R. L., 625

Martin-Vasallo, P., 157

Mata, M., 143

Matsui, H., 217, 281, 597

Maunsbach, A. B., 243

McDonough, A. A., 83, 171, 177

McGrail, K. M., 153

McParland, R. A., 297

Medford, R. M., 631

Mercer, R. W., 37, 69

Mimura, K., 281

Mironova, G. D., 509, 519

Mirsalikhova, N. M., 519

Mizushima, A., 11, 237

Modyanov, N. N., 5, 249

Moller, J. V., 259

Monastyrskaya, G. S., 5, 189

Moosmayer, M., 479

Morrison, P., 499

Mutoh, Y., 225

Nagamune, H., 423

Nagano, K., 1, 101, 217

Nagel, G., 493, 543

Nakamura, S., 363

Navran, S. S., 631

Noguchi, S., 65, 225

Nørby, J. G., 419

Okamoto, H., 101

Okolo, C., 605, 609

Okusa, M. D., 285

Omay, H., 55

Orlova, M. Y., 5

Osborn, C. P., 137

Pedemonte, C. H., 309

Peluffo, R. D., 379

Petrukhin, K. E., 5

Phillips, J. M., 153

Pindel, E. V., 483

Plesner, I. W., 339, 345, 351

Plesner, L., 321, 339

Post, R. L., 375

Potapenko, N. A., 249

Pratap, P. R., 433

Pressley, T. A., 631

Proverbio, F., 515

Proverbio, T., 471

Putnam, D. S., 177

Racine, L., 19

Rega, A. F., 379

Renaud, K. J., 25

Richter, U., 253

Robinson, J. D., 433

Rodríguez, A. J., 471

Rome, J., 117

Rosner, M. R., 499

Rossi, R. C., 419

Rossier, B. C., 19

Roudna, M., 537

Rowe, P. M., 137, 303

Ruf, H., 441

Russo, J. J., 79, 601

Sasaki, T., 413

Scheiner-Bobis, G., 43, 253, 357

Schmalzing, G., 55, 89, 465

Schmidt, T. A., 585, 613, 641

Schoner, W., 253, 405, 653

Schuurmans Stekhoven, F. M. A. H, 369

Schwarz, W., 55, 89

Seibold, G., 653

Seidel, C. L., 631

Serpersu, E. H., 405

Shao, Z., 237

Shinoguchi, E., 363, 413

Shyjan, A. W., 31, 131

Siegel, G. J., 143

Siegel, N. J., 573

Skou, J. Chr., 313, 317, 383

Skriver, E., 243

Skuster, J. R., 619

Slayman, C. L., 493

Sokolova, S. F., 519

Solioz, M., 567

Somlyo, A. P., 237

Stahl, W. L., 149

Steinberg, M. I., 433

Stürmer, W., 531

Sun, Y., 275

Suzuki, K., 375

Sverdlov, E. D., 5, 189

Sverdlov, V. E., 5

Swann, A. C., 591

Swarts, H. G. P., 369

Sweadner, K. J., 153, 601

Tachibana, S., 221

Takada, J., 423

Takagi, T., 281

Takeda, K., 65

Takeyasu, K., 11, 117, 237, 265

Tang, M.-J., 83, 171

Taniguchi, K., 363, 413

Taormino, J. P., 111, 117

Tappe, A., 549

Ten Eick, R. E., 625

Theeuwes, A. G. M., 369

Van Uem, T. J. F., 369

Van Why, S. K., 573

Vasilets, L. A., 89

Verdonck, F., 559

Vinogradova, N. G., 189

Vladimirova, N. M., 249

Volik, S. V., 5

Walton, T. J. H., 401

Wamberg, S., 461

Wang, G., 447

Wang, K., 61

Warmuth, R., 437, 441

Weintraut, H., 653

Willeke, M., 405

Willis, J. S., 457

Wu, C. H., 505

Wu, Y.-S., 79

Xu, K.-Y., 31

Yagawa, Y., 1, 101

Yano, M., 423

Ye-Hu, J., 447

Yingst, D. R., 447

Yoda, A., 499

Yoda, S., 499

Zdankiewicz, P., 69

Zolotarjova, N. I., 509, 519