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PUMPS CHANNELS AND TRANSPORTERS

CHEMICAL ANALYSISA SERIES OF MONOGRAPHS ON ANALYTICAL CHEMISTRY

AND ITS APPLICATIONS

EditorMARK F VITHA

Editorial BoardSTEPHEN C JACOBSON

STEPHEN G WEBER

VOLUME 183

A complete list of the titles in this series appears at the end of this volume

PUMPS CHANNELS AND TRANSPORTERSMethods of Functional Analysis

Edited By

RONALD J CLARkESchool of ChemistryUniversity of SydneySydney Australia

MOHAMMED A A kHALiDDepartment of ChemistryUniversity of TaifTurabah Saudi Arabia

Copyright copy 2015 by John Wiley amp Sons Inc All rights reserved

Published by John Wiley amp Sons Inc Hoboken New JerseyPublished simultaneously in Canada

No part of this publication may be reproduced stored in a retrieval system or transmitted in any form or by any means electronic mechanical photocopying recording scanning or otherwise except as permitted under Section 107 or 108 of the 1976 United States Copyright Act without either the prior written permission of the Publisher or authorization through payment of the appropriate per‐copy fee to the Copyright Clearance Center Inc 222 Rosewood Drive Danvers MA 01923 (978) 750‐8400 fax (978) 750‐4470 or on the web at wwwcopyrightcom Requests to the Publisher for permission should be addressed to the Permissions Department John Wiley amp Sons Inc 111 River Street Hoboken NJ 07030 (201) 748‐6011 fax (201) 748‐6008 or online at httpwwwwileycomgopermissions

Limit of LiabilityDisclaimer of Warranty While the publisher and author have used their best efforts in preparing this book they make no representations or warranties with respect to the accuracy or completeness of the contents of this book and specifically disclaim any implied warranties of merchantability or fitness for a particular purpose No warranty may be created or extended by sales representatives or written sales materials The advice and strategies contained herein may not be suitable for your situation You should consult with a professional where appropriate Neither the publisher nor author shall be liable for any loss of profit or any other commercial damages including but not limited to special incidental consequential or other damages

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Library of Congress Cataloging‐in‐Publication Data

Pumps channels and transporters methods of functional analysis edited by Ronald J Clarke Mohammed A A Khalid pages cm ldquoIon-transporting membrane proteins permanently embedded in the membranes of cells or cell organelles can be grouped into three broad categories channels pumps and transportersrdquondashChapter 1 introduction Includes index ISBN 978-1-118-85880-6 (cloth)1 Carrier proteins 2 Ion pumps 3 Ion channels 4 Membrane proteins 5 Biological transport I Clarke Ronald J (Ronald James) editor II Khalid Mohammed A A editor QP552C34P86 2015 572prime696ndashdc23

2015019124

Set in 1012pt Times by SPi Global Pondicherry India

Printed in the United States of America

10 9 8 7 6 5 4 3 2 1

1 2015

Contents

Preface xv

List of Contributors xix

1 Introduction 1Mohammed A A Khalid and Ronald J Clarke11 History 112 Energetics of Transport 613 Mechanistic Considerations 714 Ion Channels 8

141 Voltage‐Gated 8142 Ligand‐Gated 9143 Mechanosensitive 9144 Light‐Gated 9

15 Ion Pumps 10151 ATP‐Activated 10152 Light‐Activated 11153 Redox‐Linked 12

16 Transporters 13161 Symporters and Antiporters 13162 Na+‐Linked and H+‐Linked 14

17 Diseases of Ion Channels Pumps and Transporters 15171 Channelopathies 15172 Pump Dysfunction 17173 Transporter Dysfunction 18

18 Conclusion 18References 19

vi CoNTENTS

2 study of Ion Pump Activity Using Black Lipid Membranes 23Hans‐Juumlrgen Apell and Valerij S Sokolov21 Introduction 2322 Formation of Black Lipid Membranes 2423 Reconstitution in Black Lipid Membranes 25

231 Reconstitution of Na+K+‐ATPase in Black Lipid Membranes 25232 Recording Transient Currents with Membrane

Fragments Adsorbed to a Black Lipid Membrane 2624 The Principles of Capacitive Coupling 28

241 Dielectric Coefficients 2925 The Gated‐Channel Concept 3126 Relaxation Techniques 34

261 Concentration‐Jump Methods 34262 Charge‐Pulse Method 39

27 Admittance Measurements 3928 The Investigation of Cytoplasmic and Extracellular

Ion Access Channels in the Na+K+‐ATPase 4229 Conclusions 43References 45

3 Analyzing Ion Permeation in Channels and Pumps Using Patch‐Clamp Recording 51Andrew J Moorhouse Trevor M Lewis and Peter H Barry31 Introduction 5132 Description of the Patch‐Clamp Technique 52

321 Development of Whole‐Cell Dialysis with Voltage‐Clamp 5233 Patch‐Clamp Measurement and Analysis of Single

Channel Conductance 54331 Conductance and ohmrsquos Law 54332 Conductance of Channels versus Pumps 56333 Fluctuation Analysis 57334 Single Channel Recordings 61

34 Determining Ion Selectivity and Relative Permeation in Whole‐Cell Recordings 67341 Dilution Potential Measurements 67342 Bi‐Ionic Potential Measurements 69343 Voltage and Solution Control in Whole‐Cell

Patch‐Clamp Recordings 70344 Ion Shift Effects During Whole‐Cell Patch‐Clamp

Experiments 71345 Liquid Junction Potential Corrections 72

35 Influence of Voltage Corrections in Quantifying Ion Selectivity in Channels 74351 Analysis of Counterion Permeation in Glycine

Receptor Channels 74

CoNTENTS vii

352 Analysis of AnionndashCation Permeability in Cation‐Selective Mutant Glycine Receptor Channels 75

36 Ion Permeation Pathways through Channels and Pumps 76361 The Ion Permeation Pathway in Pentameric

Ligand‐Gated Ion Channels 763611 Extracellular and Intracellular Components of

the Permeation Pathway 783612 The TM2 Pore is the Primary Ion Selectivity Filter 79

362 Ion Permeation Pathways in Pumps Identified Using Patch‐Clamp 803621 Palytoxin Uncouples the occluded Gates of

the Na+K+‐ATPase 8137 Conclusions 82References 83

4 Probing Conformational transitions of Membrane Proteins with Voltage Clamp Fluorometry (VCF) 89Thomas Friedrich41 Introduction 8942 Description of The VCF Technique 90

421 Generation of Single‐Cysteine Reporter Constructs Expression in Xenopus laevis oocytes Site‐Directed Fluorescence Labeling 90

422 VCF Instrumentation 91423 Technical Precautions and Controls 93

43 Perspectives from Early Measurements on Voltage‐Gated K+ Channels 95431 Early Results obtained with VCF on Voltage‐Gated

K+ Channels 95432 Probing the Environmental Changes Fluorescence

Spectra Anisotropy and the Effects of Quenchers 9844 VCF Applied to P‐Type ATPases 100

441 Structural and Functional Aspects of Na+ K+‐ and H+K+‐ATPase 100

442 The N790C Sensor Construct of Sheep Na+K+‐ATPase α1‐Subunit 1024421 Probing Voltage‐Dependent Conformational

Changes of Na+K+‐ATPase 1034422 The Influence of Intracellular Na+ Concentrations 107

443 The Rat Gastric H+K+‐ATPase S806C Sensor Construct 1084431 Voltage‐Dependent Conformational Shifts of

the H+K+‐ATPase Sensor Construct S806C During the H+ Transport Branch 109

4432 An Intra‐ or Extracellular Access Channel of the Proton Pump 110

viii CoNTENTS

4433 Effects of Extracellular Ligands K+ and Na+ 111444 Probing Intramolecular Distances by Double Labeling

and FRET 11345 Conclusions and Perspectives 116References 117

5 Patch Clamp Analysis of transporters via Pre‐steady‐state Kinetic Methods 121Christof Grewer51 Introduction 12152 Patch Clamp Analysis of Secondary‐Active Transporter

Function 122521 Patch Clamp Methods 122522 Whole‐Cell Recording 124523 Recording from Excised Patches 124

53 Perturbation Methods 125531 Concentration Jumps 126532 Voltage Jumps 129

54 Evaluation and Interpretation of Pre‐Steady‐State Kinetic Data 130541 Integrating Rate Equations that Describe Mechanistic

Transport Models 131542 Assigning Kinetic Components to Elementary processes

in the Transport Cycle 13155 Mechanistic Insight into Transporter Function 133

551 Sequential Binding Mechanism 133552 Electrostatics 134553 StructurendashFunction Analysis 134

56 Case Studies 136561 Glutamate Transporter Mechanism 136562 Electrogenic Charge Movements Associated with the

Electroneutral Amino Acid Exchanger ASCT2 13757 Conclusions 139References 139

6 Recording of Pump and transporter Activity Using solid‐supported Membranes (ssM‐Based electrophysiology) 147Francesco Tadini‐Buoninsegni and Klaus Fendler61 Introduction 14762 The Instrument 148

621 Rapid Solution Exchange Cuvette 149622 Setup and Flow Protocols 150623 Protein Preparations 151624 Commercial Instruments 152

63 Measurement Procedures Data Analysis and Interpretation 152631 Current Measurement Signal Analysis and

Reconstruction of Pump Currents 152

CoNTENTS ix

632 Voltage Measurement 156633 Solution Exchange Artifacts 157

64 P‐Type ATPases Investigated by SSM‐Based Electrophysiology 159641 Sarcoplasmic Reticulum Ca2+‐ATPase 159642 Human Cu+‐ATPases ATP7A and ATP7B 163

65 Secondary Active Transporters 165651 Antiport Assessing the Forward and Reverse Modes

of the NhaA Na+H+ Exchanger of E coli 166652 Cotransport A Sugar‐Induced Electrogenic Partial

Reaction in the Lactose Permease LacY of E coli 168653 The Glutamate Transporter EAAC1 A Robust

Electrophysiological Assay with High Information Content 17066 Conclusions 172References 173

7 stopped‐Flow Fluorimetry Using Voltage‐sensitive Fluorescent Membrane Probes 179Ronald J Clarke and Mohammed A A Khalid71 Introduction 17972 Basics of the Stopped‐Flow Technique 181

721 Flow Cell Design 181722 Rapid Data Acquisition 181723 Dead Time 183

73 Covalent Versus Noncovalent Fluorescence Labeling 184731 Intrinsic Fluorescence 185732 Covalently Bound Extrinsic Fluorescent Probes 186733 Noncovalently Bound Extrinsic Fluorescent Probes 187

74 Classes of Voltage‐Sensitive Dyes 188741 Slow Dyes 188742 Fast Dyes 190

75 Measurement of the Kinetics of the Na+K+‐ATPase 193751 Dye Concentration 194752 Excitation Wavelength and Light Source 197753 Monochromators and Filters 198754 Photomultiplier and Voltage Supply 199755 Reactions Detected by RH421 200756 origin of the RH421 Response 202

76 Conclusions 204References 204

8 nuclear Magnetic Resonance spectroscopy 211Philip W Kuchel81 Introduction 211

811 Definition of NMR 212812 Why So Useful 212813 Magnetic Polarization 212

x CoNTENTS

814 Larmor Equation 213815 Chemical Shift 213816 Free Induction Decay 214817 Pulse Excitation 215818 Relaxation Times 217819 Splitting of Resonance Lines 2178110 Measuring Membrane Transport 217

82 Covalently‐Induced Chemical Shift Differences 218821 Arginine Transport 218822 other Examples 220

83 Shift‐Reagent‐Induced Chemical Shift Differences 220831 DyPPP 220832 TmDTPA and TmDoTP 220833 Fast Cation Exchange 220

84 pH‐Induced Chemical Shift Differences 223841 orthophosphate 223842 Methylphosphonate 224843 Triethylphosphate 31P Shift Reference 224

85 Hydrogen‐Bond‐Induced Chemical Shift Differences 225851 Phosphonates DMMP 225852 HPA 225853 Fluorides 227

86 Ionic‐Environment‐Induced Chemical Shift Differences 229861 Cs+ Transport 229

87 Relaxation Time Differences 229871 Mn2+ Doping 229

88 Diffusion Coefficient Differences 231881 StejskalndashTanner Plot 231882 Andraskorsquos Method 231

89 Some Subtle Spectral Effects 233891 Scalar (J) Coupling Differences 233892 Endogenous Magnetic Field Gradients 233

8921 Magnetic Induction and Magnetic Field Strength 2348922 Magnetic Field Gradients Across Cell Membranes

and Co Treatment of RBCs 2348923 Exploiting Magnetic Field Gradients in Membrane

Transport Studies 235893 Residual Quadrupolar (ν

Q) Coupling 235

810 A Case Study The Stoichiometric Relationship Between the Number of Na+ Ions Transported per Molecule of Glucose Consumed in Human RBCs 236


CoNTENTS xi

9 time‐Resolved and surface‐enhanced Infrared spectroscopy 245Joachim Heberle91 Introduction 24592 Basics of IR Spectroscopy 246

921 Vibrational Spectroscopy 246922 FTIR Spectroscopy 247923 IR Spectra of Biological Compounds 248924 Difference Spectroscopy 250

93 Reflection Techniques 250931 Attenuated Total Reflection 250932 Surface‐Enhanced IR Absorption 251

94 Application to Electron‐Transferring Proteins 252941 Cytochrome c 252942 Cytochrome c oxidase 253

95 Time‐Resolved IR Spectroscopy 254951 The Rapid‐Scan Technique 254952 The Step‐Scan Technique 255953 Tunable QCLs 255

96 Applications to Retinal Proteins 256961 Bacteriorhodopsin 256962 Channelrhodopsin 260


10 Analysis of Membrane-Protein Complexes by single‐Molecule Methods 269Katia Cosentino Stephanie Bleicken and Ana J Garciacutea‐Saacuteez101 Introduction 269102 Fluorophores for Single Particle Labeling 270103 Principles of Fluorescence Correlation Spectroscopy 271

1031 Analysis of Molecular Complexes by Two‐Color FCS 2751032 FCS Variants to Study Lipid Membranes 2751033 FCS Applications to Membranes 278

104 Principle and Analysis of Single‐Molecule Imaging 2791041 TIRF Microscopy 2801042 Single‐Molecule Detection 2821043 Single Particle Tracking and Trajectory Analysis 284

105 Complex Dynamics and Stoichiometry by Single‐Molecule Microscopy 2851051 Application to Single‐Molecule Stoichiometry Analysis 2851052 Application to Kinetics Processes in Cell Membranes 290

106 FCS Versus SPT 291References 291

xii CoNTENTS

11 Probing Channel Pump and transporter Function Using single‐Molecule Fluorescence 299Eve E Weatherill John S H Danial and Mark I Wallace111 Introduction 299

1111 Basic Principles 300112 Practical Considerations 300

1121 observables 3011122 Apparatus 3011123 Labels 3021124 Bilayers 303

113 SMF Imaging 3031131 Fluorescence Colocalization 3041132 Conformational Changes 3061133 Superresolution Microscopy 307

114 Single Molecule Foumlrster Resonance Energy Transfer 3081141 InteractionsStoichiometry 3081142 Conformational Changes 309

115 Single‐Molecule Counting by Photobleaching 312116 optical Channel Recording 314117 Simultaneous Techniques 315118 Summary 318References 318

12 electron Paramagnetic Resonance site‐Directed spin Labeling 327Louise J Brown and Joanna E Hare121 Introduction 327

1211 Development of EPR as a Tool for Structural Biology 3291212 SDSLndashEPR A Complementary Approach

to Determine StructurendashFunction Relationships 330122 Basics of the EPR Method 331

1221 Physical Basis of the EPR Signal 3311222 Spin Labeling 3331223 EPR Instrumentation 336

123 Structural and Dynamic Information from SDSLndashEPR 3361231 Mobility Measurements 3361232 Solvent Accessibility 341

124 Distance Measurements 3451241 Interspin Distance Measurements 3451242 Continuous Wave 3471243 Pulsed Methods DEER 349

125 Challenges 3531251 New Labels 3531252 Spin‐Label Flexibility 3551253 Production and Reconstitution Challenges Nanodiscs 355


CoNTENTS xiii

13 Radioactivity‐Based Analysis of Ion transport 367Ingolf Bernhardt and J Clive Ellory131 Introduction 367132 Membrane Permeability for Electroneutral Substances

and Ions 368133 Kinetic Considerations 370134 Techniques for Ion Flux Measurements 371

1341 Conventional Methods 3711342 Alternative Method 373

135 Kinetic Analysis of Ion Transporter Properties 375136 Selected Cation Transporter Studies on Red Blood Cells 376

1361 K+Clminus Cotransport (KCC) 3781362 Residual Transport 378

137 Combination of Radioactive Isotope Studies with Methods using Fluorescent Dyes 379


14 Cation Uptake studies with Atomic Absorption spectrophotometry (AAs) 387Thomas Friedrich141 Introduction 387142 overview of the Technique of AAS 389

1421 Historical Account of AAS with Flame Atomization 3901422 Element‐Specific Radiation Sources 3911423 Electrothermal Atomization in Heated Graphite Tubes 3921424 Correction for Background Absorption 394

143 The Expression System of Xenopus laevis oocytes for Cation Flux Studies Practical Considerations 395

144 Experimental outline of the AAS Flux Quantification Technique 395

145 Representative Results obtained with the AAS Flux Quantification Technique 3971451 Reaction Cycle of P‐Type ATPases 3981452 Rb+ Uptake Kinetics Inhibitor Sensitivity 3981453 Dependence of Rb+ Transport of Gastric H+K+‐ATPase

on Extra‐ and Intracellular pH 4001454 Determination of Na+K+‐ATPase Transport

Stoichiometry and Voltage Dependence of H+K+‐ATPase Rb+ Transport 403

1455 Effects of C‐Terminal Deletions of the H+K+‐ATPase α‐Subunit 404

1456 Li+ and Cs+ Uptake Studies 405146 Concluding Remarks 407References 408

xiv CoNTENTS

15 Long timescale Molecular simulations for Understanding Ion Channel Function 411Ben Corry151 Introduction 411152 Fundamentals of MD Simulation 412

1521 The Main Idea 4121522 Force Fields 4141523 other Simulation Considerations 4161524 Why Do MD Simulations Take So Much

Computational Power 41615241 Force Calculations 41715242 Time Step 417

153 Simulation Duration and Simulation Size 418154 Historical Development of Long MD Simulations 421155 Limitations and Challenges Facing MD Simulations 423

1551 Force Field and Algorithm Accuracy 4231552 Sampling Problems 424

156 Example Simulations of Ion Channels 4251561 Simulations of Ion Permeation 4251562 Simulations of Ion Selectivity 4281563 Simulations of Channel Gating 432


Index 443

Chemical Analysis A series of Monographs on Analytical Chemistry and its Applications 461

Ion-transporting membrane proteins which include ion pumps channels and trans-porters play crucial roles in all cellular life forms Not only do they provide path-ways linking the extracellular medium with the cytoplasm and the cytoplasm with the contents of intracellular organelles they are also intimately involved in energy transduction in all cells Furthermore in organisms with higher levels of cellular differentiation they provide sophisticated mechanisms for signal transduction for example in both electrical and chemical forms of nerve impulse transmission and in muscle contraction and relaxation

Although probably not the most objective criterion for judging the importance of pumps channels and transporters it is interesting and enlightening to consider the number of Nobel Prizes awarded for research directly related to membrane proteins The first Nobel Prize ever given for research on a specific membrane protein was awarded to Johann Deisenhofer Robert Huber and Hartmut Michel in 1988 for the determination of the first crystal structure of an integral membrane protein the pho-tosynthetic reaction center of the bacterium Rhodopseudomonas viridis Although the structure determination of membrane proteins is still not routine in comparison to water-soluble proteins this was a major breakthrough which paved the way for the crystallization of many other membrane proteins

A further significant contribution to the field was the development by Erwin Neher and Bert Sakmann of the patch-clamp technique which enables the activity of single ion channels to be detected For this important technical advance and its appli-cation (described in Chapter 3) together they were awarded the 1991 Nobel Prize in Physiology or Medicine A few years later in 1997 Paul Boyer John Walker and Jens Christian Skou were awarded the Nobel Prize in Chemistry Jens Christian Skou won the prize for his discovery of the Na+K+-ATPase Paul Boyer and John Walker

PREFACE

Mohammed A A Khalid1 and Ronald J Clarke2

1 Department of Chemistry University of Taif Turabah Saudi Arabia2 School of Chemistry University of Sydney Sydney NSW Australia

xvi PREFACE

were chosen for their research into the enzymatic mechanism of ATP biosynthesis The critical contribution of John Walker was his crystallization and structural deter-mination of the cytoplasmic domain of the ATP synthase Then in 2003 Peter Agre and Roderick MacKinnon were also awarded the Nobel Prize in Chemistry In the case of Peter Agre this was for the discovery of aquaporins Roderick MacKinnon on the other hand won the prize for his work on ion channels in particular the deter-mination of the structure of the K+ channel of Streptomyces lividans using X-ray crystallography

In this brief overview of Nobel Prizes in the membrane protein field it is notable that several Nobel Prizes have been awarded for structural determinations via X-ray crystallography This is certainly an important step toward the complete under-standing of any membrane protein However crystal structures alone do not immedi-ately explain how a protein functions After the discovery of the helical structure of DNA and its base pairing by Crick Watson Franklin and Wilkins the mechanism of information transfer via the genetic code was relatively soon unraveled and the sig-nificance of the structure is now obvious However in the case of proteins often after the publication of a crystal structure one is still none the wiser regarding how they function To understand how a protein functions a static picture is insufficient Time resolution is essential One needs to observe a protein in action

Without the crystal structure of a protein or one closely related to it certainly no meaningful theoretical simulations of the proteinrsquos activity via molecular dynamics (MD) methods are possible Although the timescales of MD simulations are gradu-ally increasing as described in Chapter 15 many protein conformational changes important for the functions of pumps channels and transporters occur over milli-seconds or seconds beyond the reliability of any current MD methods Therefore it is clear that experimental methods for the direct detection of the activity of ion-transporting membrane proteins are essential and will likely remain so for many years to come

The emphasis of this book is therefore on experimental methods for resolving the kinetics and dynamics of pumps channels and transporters Structural methods such as X-ray crystallography or electronmicroscopy although clearly important for a complete understanding of membrane protein function down to the atomic level are specifically excluded The experimental methods treated in the book are divided into three main groups electrical (Chapters 2ndash6) spectroscopic (Chapters 7ndash12) and radioactivity-based and atomic absorption-based flux assays (Chapters 13 and 14) Finally the book concludes with a chapter on computational tech-niques (Chapter 15)

Many pumps channels and transporters are electrogenic that is their activity involves a net transport of charge across the membrane Hence it is logical that a variety of techniques have been developed to detect the currents and membrane voltage changes that these proteins produce For this reason after an introduction overviewing pumps channels and transporters their energetics and mechanisms the following five chapters of the book are devoted to electrical methods

However not all ion-transporting membrane proteins are electrogenic There are many which function in an electroneutral fashion This is the case when the current

PREFACE xvii

produced by the transport of one ion across the membrane is compensated by the transport of another ion with the same charge in the opposite direction Therefore electrical techniques alone are insufficient to quantify the activity of all pumps chan-nels and transporters Complementary techniques are necessary Chapters 7ndash14 are therefore devoted to either spectroscopic or radioactivity-based methods Apart from allowing the detection of protein activity independent of the proteinrsquos electrogenicity some spectroscopic techniques in particular IR spectroscopy (Chapter 9) and EPR spectroscopy (Chapter 11) have the added advantage of providing time-resolved structural information not accessible by any electrical method

We thank all chapter authors for their contributions to the book the series editor Mark Vitha for his valuable comments and suggestions and Michael Leventhal of Wiley for his assistance in the preparation of the book for publication We hope that readers with an interest in dynamic aspects of membrane protein function find the book interesting and of value for their own research

RONALD J CLARKEBerlin Germany

February 2015

MOHAMMED A A KHALIDTurabah Saudi Arabia

February 2015

Hans-Juumlrgen Apell Department of Biology University of Constance Constance Germany

Peter H Barry Department of Physiology School of Medical Sciences UNSW Australia (The University of New South Wales) Sydney New South Wales Australia

Ingolf Bernhardt Laboratory of Biophysics Saarland University Saarbruumlcken Germany

Stephanie Bleicken Max Planck Institute for Intelligent Systems Stuttgart Germany Interfaculty Institute of Biochemistry University of Tuumlbingen Tuumlbingen Germany

Louise J Brown Department of Chemistry and Biomolecular Sciences Macquarie University Sydney New South Wales Australia

Ronald J Clarke School of Chemistry University of Sydney Sydney New South Wales Australia

Ben Corry Research School of Biology Australian National University Canberra Australian Capital Territory Australia

Katia Cosentino Max Planck Institute for Intelligent Systems Stuttgart Germany Interfaculty Institute of Biochemistry University of Tuumlbingen Tuumlbingen Germany

John S H Danial Department of Chemistry Chemical Research Laboratory University of Oxford Oxford UK

J Clive Ellory Department of Physiology Anatomy and Genetics University of Oxford Oxford UK

LIST OF CONTRIBUTORS

xx LIST OF CONTRIBUTORS

Klaus Fendler Department of Biophysical Chemistry Max Planck Institute of Biophysics FrankfurtMain Germany

Thomas Friedrich Institute of Chemistry Technical University of Berlin Berlin Germany

Ana J Garciacutea-Saacuteez Max Planck Institute for Intelligent Systems Stuttgart Germany Interfaculty Institute of Biochemistry University of Tuumlbingen Tuumlbingen Germany

Christof Grewer Department of Chemistry Binghamton University Binghamton NY USA

Joanna E Hare Department of Chemistry and Biomolecular Sciences Macquarie University Sydney New South Wales Australia

Joachim Heberle Institute of Experimental Physics Department of Physics Free University of Berlin Berlin Germany

Mohammed A A Khalid Department of Chemistry Faculty of Applied Medical Sciences University of Taif Turabah Saudi Arabia

Philip W Kuchel School of Molecular Bioscience University of Sydney Sydney New South Wales Australia

Trevor M Lewis Department of Physiology School of Medical Sciences UNSW Australia (The University of New South Wales) Sydney New South Wales Australia

Andrew J Moorhouse Department of Physiology School of Medical Sciences UNSW Australia (The University of New South Wales) Sydney New South Wales Australia

Valerij S Sokolov A N Frumkin Institute of Physical Chemistry and Electrocemistry Russian Academy of Sciences Moscow Russia

Francesco Tadini-Buoninsegni Department of Chemistry ldquoUgo Schiffrdquo University of Florence Sesto Fiorentino Italy

Mark I Wallace Department of Chemistry Chemical Research Laboratory University of Oxford Oxford UK

Eve E Weatherill Department of Chemistry Chemical Research Laboratory University of Oxford Oxford UK

Pumps Channels and Transporters Methods of Functional Analysis First Edition Edited by Ronald J Clarke and Mohammed A A Khalid copy 2015 John Wiley amp Sons Inc Published 2015 by John Wiley amp Sons Inc

11 HISTORY

Modern membrane science can be traced back to 1748 to the work of the French priest and physicist Jean‐Antoine (Abbeacute) Nollet who in the course of an experiment in which he immersed a pigrsquos bladder containing alcohol in water accidentally discovered the phenomenon of osmosis [1] that is the movement of water across a semipermeable membrane The term osmosis was however first introduced [2] by another French scientist Henri Dutrochet in 1827 The movement of water across a membrane in osmosis is a passive diffusion process driven by the difference in chemical potential (or activity) of water on each side of the membrane The diffusion of water can be through the predominant matrix of which the membrane is composed that is lipid in the case of biological membranes or through proteins incorporated in the membrane for example aquaporins As the title of this book suggests here we limit ourselves to a discussion of the movement of ions and metabolites through membranes via proteins embedded in them rather than of transport through the lipid matrix of biological membranes

The fact that small ions in particular Na+ K+ and Clminus are not evenly distributed across the plasma membrane of cells (see Fig 11) was first recognized by the physiological chemist Carl Schmidt [3] in the early 1850s Schmidt was investigating the pathology of cholera which was widespread in his native Russia at the time and

INTRODUCTION


1Department of Chemistry Faculty of Applied Medical Sciences University of Taif Turabah Saudi Arabia2School of Chemistry University of Sydney Sydney New South Wales Australia

1

2 INTRODUCTION

discovered the differences in ion concentrations while comparing the blood from cholera victims and healthy individuals By the end of the nineteenth century it was clear that these differences in ionic distributions occurred not only in blood but existed across the plasma membrane of cells from all animal tissues However the origin of the concentration differences remained controversial for many years

In the 1890s at least two scientists Rudolf Heidenhain [5] (University of Breslau) and Ernest Overton [6] (then at the University of Zuumlrich) both reached the conclusion that the Na+ concentration gradient across the membrane was produced by a pump situated in the cell membrane which derived its energy from metabolism Although we know now that this conclusion is entirely correct it was apparently too far ahead of its time In 1902 Overton even correctly proposed [7] that an exchange of Na+ and K+ ions across the cell membrane of musclemdashnow known to arise from the opening and closing of voltage‐sensitive Na+ and K+ channelsmdashwas the origin of the change in electrical voltage leading to muscle contraction This proposal too was not widely accepted at the time or even totally ignored It took another 50 years before Overtonrsquos hypothesis was rediscovered and finally verified by the work of Hodgkin and Huxley [8] for which they both received the Nobel Prize in Physiology or Medicine in 1963 According to Kleinzeller [9] Andrew Huxley once said that ldquoIf people had listened to what Overton had to say about excitability the work of Alan [Hodgkin] and myself would have been obsoleterdquo

Unfortunately for Heidenhain and Overton their work did not conform with the Zeitgeist of the early twentieth century At the time much fundamental work on the theory of diffusion was being carried out by high‐profile physicists and physical chemists among them vanrsquot Hoff Einstein Planck and Nernst Of particular releshyvance for the distribution of ions across the cell membranes was the work of the Irish physical chemist Frederick Donnan [10] on the effect of nondialyzable salts Therefore it was natural that physiologists of the period would try to explain membrane transport in terms of passive diffusion alone rather than adopt Overtonrsquos and Heidenhainrsquos controversial hypothesis of ion pumping or active transport

Mndash

K+ K+

Clndash Clndash

Na+

Na+

FIgURe 11 Ionic distributions across animal cell membranes Mminus represents impermeant anions for example negatively charged proteins Typical intracellular (int) and extracellular (ext) concentrations of the small inorganic ions are [K+]

int = 140ndash155 mM [K+]

ext = 4ndash5 mM

[Clminus]int

= 4 mM [Clminus]ext

= 120 mM [Na+]int

= 12 mM [Na+]ext

= 145ndash150 mM (Note In the speshycial case of red blood cells [Clminus]

ext is lower (98ndash109 mM) due to exchange with HCO

3minus across

the plasma membrane which is important for CO2 excretion and the maintenance of blood

pH This exchange is known as the ldquochloride shiftrdquo) Adapted from Ref 4 with permission from Wiley

HISTORY 3

Donnan [10] suggested that if the cytoplasm of cells contained electrolytically dissociated nondialyzable salts (eg protein anions) which it does small permeable ions would distribute themselves across the membrane so as to maintain electroneushytrality in both the cytoplasm and the extracellular medium Thus the cytoplasm would naturally tend to attract small cations whereas the extracellular medium would accumulate anions Referring back to Figure 11 one can see that this idea could explain the distribution of K+ and Clminus ions across the cell membrane However the problem is that the so‐called Donnan equilibrium doesnrsquot explain the distribution of Na+ ions Based on Donnanrsquos theory any permeable ion of the same charge should adopt the same distribution across the membrane but the distributions of Na+ and K+ are in fact the opposite of one another To find a rational explanation for this inconshysistency many physiologists concluded that whereas cell membranes were permeshyable to K+ and Clminus ions they must be completely impermeable to Na+ ions The logical consequence of this was that the Na+ concentration gradient should have origshyinated at the first stages of the cell division and persisted throughout each animalrsquos entire life This view was an accepted doctrine for the next 30 years following the publication of Donnanrsquos theory [11]

In the late 1930s and early 1940s however evidence was mounting that the idea of an impermeant Na+ ion was untenable In this period radioisotopes started to become available for research which greatly increased the accuracy of ion transport measurements A further stimulus at the time was the development in the United States of blood banks and techniques for blood transfusion during which researchers were again investigating the distribution of ions across the red blood cell membranes and the effects of cold storage Researchers in the United States in particular at the University of Rochester Yale University and the State University of Iowa were now the major players in the field among them Fenn Heppel Steinbach Peters Danowski Harris and Dean Details of the experimental evidence that led to the universal discarding of the notion of an impermeant Na+ ion and the reemergence of the hypothesis of an active Na+ pump located in the cell membrane of both excitable and nonexcitable cells are described elsewhere [4 12 13] Here it suffices to say that by the middle of the twentieth century the active transport of Na+ had become an established fact

The enzyme responsible for active Na+ transport the Na+K+‐ATPase which is powered by the energy released from ATP hydrolysis was isolated by Jens Christian Skou of the University of Aarhus Denmark in 1957 [14] This was the first ever ion‐transporting enzyme to be identified Almost 40 years later in 1997 when all possible doubt that Skoursquos Na+K+‐ATPase incorporated the complete active transport machinery for sodium and potassium ions and the broad significance of his discovery was clear he received the Nobel Prize in Chemistry

One reason why the concept of the active transport of Na+ took so long to be accepted was probably the perception that it represented a waste of a cellrsquos valuable energy resources However rather than think of the pumping of ions across a membrane as energy expenditure in fact it is more helpful and more accurate to describe it as an energy conversion process In the case of the Na+K+‐ATPase the energy released by ATP hydrolysis is stored as Na+ and K+

4 INTRODUCTION

electrochemical potential gradients across the membrane Therefore the energy can be released again whenever Na+ or K+ diffuse passively across the membrane The Na+ and K+ electrochemical potential gradients established by the Na+K+‐ATPase across the plasma membrane of all animal cells thus provide the driving force for diffusion of Na+ and K+ through all plasma membrane Na+‐ and K+‐selective ion channels which for example is the basis of the production of action potentials in nerve and muscle The Na+ electrochemical potential gradient created by the Na+K+‐ATPase also serves as a secondary source of energy to drive the active uptake or extrusion of other ions or metabolites across the plasma membrane by transporter membrane proteins For example the reabsorption of glucose into the bloodstream in the kidney is driven by the energy released by the simultaneous coupled passive flow of Na+ into the cytoplasm of the epithelial cells lining the kidney collecting tubules As these examples demonstrate the realization that the cell membrane is permeable to Na+ ions and requires a sodium pump to keep the ions out was pivotal for the understanding of membrane transshyport processes in general and the change in thinking that this realization genershyated no doubt contributed to the later discovery of many other membrane protein transport systems including channels and transporters

Now we will concentrate for the moment on channels alone When Hodgkin and Huxley proposed [8] consecutive changes in Na+ and K+ membrane permeability of nerve as the origin of the action potential in 1952 their hypothesis was based on the mathematical fitting of kinetic equations to their recorded data Impressive as their conclusions were their data still provided no clue as to the molecular origin of the changes in Na+ or K+ permeability A major step forward occurred in 1964 when Narahashi et al [15] discovered that tetrodotoxin (TTX) a paralytic poison found in some edible (with caution) puffer fish blocks the action potential in nerve axons by inhibiting the Na+ conductance but without any effect on the K+ conductance This clearly demonstrated that there must be separate pathways or channels for Na+ and K+ ions in the membrane Still the chemical nature of the channels was unclear but after this discovery TTX became an invaluable tool for the identification of the source of the Na+ conductance

The next major advance in the channel field occurred through the application of biochemical purification procedures The electric eel Electrophorus electricus is capable of producing voltages as high as 600 V along the whole animal As one might imagine its specialized electrical properties made it a prime source for the isolation of the molecules responsible for voltage changes across the cell membranes In 1978 Agnew et al [16] succeeded in extracting and purifying a 230 kDa protein that had a high affinity for TTX After it was shown in 1984 [17] that synthetic vesicles in which the purified protein had been reconstituted displayed Na+ currents that could be inhibited by TTX there was no longer any doubt that the Na+ channel had been isolated and that it was indeed a membrane protein More details on the history of ion channel research including more recent developments can be found in a fine review by Bezanilla [18]

Now finally in this brief historical overview we turn our attention to transporters Particularly in the intestine and in the kidney many metabolites including sugars

HISTORY 5

and amino acids need to be absorbed or reabsorbed respectively into the bloodstream In the early 1960s shortly after Skoursquos discovery of the Na+K+‐ATPase [14] Robert Crane first suggested [19 20] that the intestinal absorption of sugar was coupled to the influx of Na+ into the cell that is that the energy released by the passive diffusion of Na+ into the cell was utilized to absorb sugars His hypothesis was based in part on the fact that sugar absorption was already known to be dependent on the presence of Na+ in the medium Roughly 10 years later using isolated intestinal epithelial cells Kimmich [21] showed the sugar uptake system was located in the plasma membrane of the cells and not between the cells of an intact tissue or epithelium That the Na+glucose coupled transport system is in fact a membrane protein was shown in a similar way to that described above for the Na+ channel that is by isolashytion of the protein from tissue reconstitution in vesicles and the demonstration that the reconstituted system carried out Na+‐dependent active transport of glucose across the vesicle membrane [22] For these experiments kidney tissue was used because of the higher concentration of the protein that could be isolated in comparison to intestine

A useful question to ask here is why such coupled transport systems at least in animals all utilize the Na+ gradient across the membrane and not the K+ gradient The answer is quite simple The distribution of K+ ions across the plasma membrane is quite close to equilibrium that is the normal resting electrical potential across the membrane is quite close to what one would calculate theoretically based on the equilibrium theory of Nernst for electrical diffusion potentials In contrast the disshytribution of Na+ is far from equilibrium Therefore the passive diffusion of Na+ in through a transporter protein releases much more energy that can be used for metabshyolite uptake or extrusion than the passive diffusion of K+ out

At roughly the same time that Crane hypothesized the coupling of the energy stored in the Na+ gradient to glucose absorption the idea of energy storage in electrochemical potential gradients was also taken up by Peter Mitchell [23] when he proposed the chemiosmotic theory of oxidative and photosynthetic phosphorylation for which he received the 1978 Nobel Prize in Chemistry Central to Mitchellrsquos hypothesis was the existence of a membrane‐bound ATPase in mitochondria or chloroplasts that utilized the H+ gradient built up across their inner membranes for the conversion of ADP to ATP This enzyme now known as the ATP synthase or F

0F

1‐ATPase cannot strictly be classified as a transporter because the energy released

as H+ ions that flows through it across the membrane is not used for the transport of other ions or metabolites but rather it is converted into chemical energy in the form of ATP Closely related molecular machines are the bacterial flagellar motors which also use the energy of an H+ gradient but in this case the energy is released in mechanical form as flagellar rotation

Concluding this historical overview one can say that the existence of membrane‐embedded proteins that act as pumps channels and transporters and the means by which they gain their energy to carry out their transport processes were firmly established by the early 1980s Since that time further major advances have been made into the details of how they operate One significant advance was the development of patch‐clamp techniques by Neher and Sakmann [24] which enabled the opening and closing of single

6 INTRODUCTION

channels to be directly recorded and for which they received the Nobel Prize for Physiology or Medicine in 1991 Another major advance has been the resolution of the atomic structure of membrane proteins by X‐ray crystallography The first membrane protein to be crystallized and have its structure determined by X‐ray diffraction was that of a bacterial photosynthetic reaction center [25] for which Michel Deisenhofer and Huber received the Nobel Prize in Chemistry in 1988 After a slow start the structures of other membrane proteins at atomic resolution are now being determined at an increasingly rapid rate With structures becoming available this has allowed the application of molecular dynamics simulations and other theoretical techniques to obtain an improved chemical understanding of how pumps channels and transporters work Both patch‐clamp techniques and molecular simulations are topics of later chapters of this book

12 eNeRgeTICS OF TRANSPORT

How does one distinguish between pumps channels and transporters The decishysive criterion is whether or not energy is required for transport However to decide whether the transport of an ion requires energy or not it is not sufficient to consider its concentration c on each side of the membrane The electrical potential difference V

m across the membrane also contributes to the energetics of the process Therefore

one needs to define the electrochemical potential difference Δμ which for the transshyport of an ion into a cell is given by

RT

c

czFVln in

outm (11)

and for the transport of an ion out of a cell is given by

RT

c

czFVln out

inm (12)

In these equations R is the ideal gas constant T is the absolute temperature z is the valence of the ion (eg +1 for Na+ or +2 for Ca2+) F is Faradayrsquos constant and V

m

is the electrical potential difference across the membrane In both equations Vm is

defined as the potential inside the cell minus the potential outside the cell The movement of an ion across a membrane for which Δμ is calculated to be negative involves a loss of free energy If the movement is along an electrochemical potential gradient it is a spontaneous process and no energy is required If Δμ is calculated to be positive on the other hand then the movement of the ion requires energy the movement is against an electrochemical potential gradient the process is nonshyspontaneous and a source of energy would be required Of course if it is an uncharged metabolite that is moving across the membrane then the second term in Equations 11 and 12 disappears and it is only the direction of the chemical potenshytial gradient or the concentration gradient that determines whether or not the transshyport is spontaneous

MECHANISTIC CONSIDERATIONS 7

If no energy is required that is the transport occurs spontaneously along an electrochemical potential gradient (Δμ lt 0) then the transport is termed facilitated diffusion In this case the protein simply provides a pathway for the ions to move more easily through the membrane This is the situation that occurs with a channel If energy is required that is the transport occurs nonspontaneously up an electroshychemical potential gradient (Δμ gt 0) then the transport is termed active transport

There are a number of possible sources of energy in the case of active transport If the energy comes directly from light ATP or from the energy released in a redox reaction then this is termed primary active transport All pumps are primary active transporters If the energy is generated by the flow of an ion down an electrochemical potential gradient created by a pump then this is termed secondary active transport The Na+glucose cotransporter is an example of this In such a situation the transport of one species is down an electrochemical potential gradient (Δμ lt 0) and the transshyport of the other is up an electrochemical potential gradient (Δμ gt 0) When summed together as long as the overall Δμ is negative the transport proceeds For example in the case of the Na+glucose cotransporter as long as Δμ(Na+) + Δμ(glucose) lt 0 then both glucose and Na+ are taken up into the cytoplasm of the cell

13 MeCHANISTIC CONSIDeRATIONS

Apart from the difference in energetics there are also important mechanistic difshyferences between active transport and facilitated diffusion processes In active transport because the ions are transported against an electrochemical potential gradient the enzymersquos ion‐binding sites should not be open to both sides of the membrane simultaneously If this were to happen the efficiency of pumping would be drastically compromised The ions should first be bound from one side of the membrane become occluded within the protein via a conformational change and then be released to the other side of the membrane via a conformational change This is in contrast to the mechanism of ion channels which have their ion‐binding sites open to both sides of the membrane at once (see Fig 12) Because of these differences in mechanism the transport timescales of ion pumps and transporters are very different from those of ion channels

A channel that is open to both sides of the membrane at once allows a rapid flux of ions across the membrane For example the flux through open Na+ channels of the nerve membrane is approximately 107 ionss corresponding to an average time for the transport of a single ion of 01 micros In contrast ion pumps and transporters function on a much slower timescale In their case ion transport requires significant conformashytional changes to drive the ions or metabolites across the membrane Because these conformational changes involve a large number of amino acid side chains whose intermolecular interactions need to be broken and formed they typically have rate constants on the order of 100 sminus1 or slower The overall turnover of an ion pump or transporter then usually occurs on a timescale of milliseconds to seconds that is four to six orders of magnitude slower than that of ion channels This has important experimental consequences Because of the large ion fluxes that they produce the

8 INTRODUCTION

opening and closing of single channels can be observed via the patch‐clamp technique Typically the observed currents are in the picoampere range However single ion pumps or transporters produce only very small currents across the membranes that are exceedingly difficult if not impossible to measure by electrophysiological means An alternative approach for pumps is to use the whole‐cell patch‐clamp technique whereby the ion flux through many pumps or transporters is recorded simultaneously

The important point which we would like to make here is that because of these mechanistic and timescale differences experimental techniques designed for the investigation of ion channels can often not be applied to pumps or transporters In a similar fashion techniques devised for research on pumps or transporters cannot generally be directly applied to ion channels Therefore in the following chapters one will find some experimental techniques that have been specifically designed with ion channel investigations in mind and others with ion pumps or transporters in mind

14 ION CHANNeLS

Ion channels can be classified according to what it is that causes them to open that is their gating mechanism For a channel to allow ions to pass it must undergo a conformational change from one or more inactive closed states There are a number of mechanisms by which this conformational change might come about Below we consider the variety of possible mechanisms one by one

141 Voltage‐gated

First we consider voltage‐gated channels for example the Na+‐ and K+‐channels of nerve and muscle responsible for the action potential If a channel contains movshyable charged or dipolar amino acid residues then a change in voltage across the

Exterior

Pump Channel

OpenClosedCytosolATP ADP+Pi

FIgURe 12 Ion‐transporting membrane proteins Channels can exist in an open state in which ions move down an electrochemical potential gradient No energy is requiredmdashthe transport is termed facilitated diffusion Pumps transport ions against an electrochemical potential gradient The ion‐binding sites are open alternately to the cytosol and the exterior Energy is requiredmdashthe transport is termed active transport In the example shown the energy is derived from ATP hydrolysis Reproduced from Ref 26 with permission from Wiley

PUMPS CHANNELS AND TRANSPORTERS



EditorMARK F VITHA


STEPHEN G WEBER

VOLUME 183



Edited By











2015019124



10 9 8 7 6 5 4 3 2 1

1 2015

Contents

Preface xv








vi CoNTENTS
















CoNTENTS vii



















viii CoNTENTS
















CoNTENTS ix















x CoNTENTS













Q) Coupling 235



CoNTENTS xi













xii CoNTENTS















CoNTENTS xiii

















xiv CoNTENTS








Index 443





PREFACE



xvi PREFACE







PREFACE xvii




February 2015


February 2015




























11 HISTORY



INTRODUCTION



1

2 INTRODUCTION




Mndash

K+ K+

Clndash Clndash

Na+

Na+



ext = 4ndash5 mM

[Clminus]int

= 4 mM [Clminus]ext

= 120 mM [Na+]int

= 12 mM [Na+]ext



3minus across



HISTORY 3





4 INTRODUCTION





HISTORY 5




0F




6 INTRODUCTION






RT

c

czFVln in

outm (11)


RT

c

czFVln out

inm (12)


m









8 INTRODUCTION



14 ION CHANNeLS


141 Voltage‐gated


Exterior

Pump Channel





EditorMARK F VITHA


STEPHEN G WEBER

VOLUME 183



Edited By











2015019124



10 9 8 7 6 5 4 3 2 1

1 2015

Contents

Preface xv








vi CoNTENTS
















CoNTENTS vii



















viii CoNTENTS
















CoNTENTS ix















x CoNTENTS













Q) Coupling 235



CoNTENTS xi













xii CoNTENTS















CoNTENTS xiii

















xiv CoNTENTS








Index 443





PREFACE



xvi PREFACE







PREFACE xvii




February 2015


February 2015




























11 HISTORY



INTRODUCTION



1

2 INTRODUCTION




Mndash

K+ K+

Clndash Clndash

Na+

Na+



ext = 4ndash5 mM

[Clminus]int

= 4 mM [Clminus]ext

= 120 mM [Na+]int

= 12 mM [Na+]ext



3minus across



HISTORY 3





4 INTRODUCTION





HISTORY 5




0F




6 INTRODUCTION






RT

c

czFVln in

outm (11)


RT

c

czFVln out

inm (12)


m









8 INTRODUCTION



14 ION CHANNeLS


141 Voltage‐gated


Exterior

Pump Channel




Edited By











2015019124



10 9 8 7 6 5 4 3 2 1

1 2015

Contents

Preface xv








vi CoNTENTS
















CoNTENTS vii



















viii CoNTENTS
















CoNTENTS ix















x CoNTENTS













Q) Coupling 235



CoNTENTS xi













xii CoNTENTS















CoNTENTS xiii

















xiv CoNTENTS








Index 443





PREFACE



xvi PREFACE







PREFACE xvii




February 2015


February 2015




























11 HISTORY



INTRODUCTION



1

2 INTRODUCTION




Mndash

K+ K+

Clndash Clndash

Na+

Na+



ext = 4ndash5 mM

[Clminus]int

= 4 mM [Clminus]ext

= 120 mM [Na+]int

= 12 mM [Na+]ext



3minus across



HISTORY 3





4 INTRODUCTION





HISTORY 5




0F




6 INTRODUCTION






RT

c

czFVln in

outm (11)


RT

c

czFVln out

inm (12)


m









8 INTRODUCTION



14 ION CHANNeLS


141 Voltage‐gated


Exterior

Pump Channel











2015019124



10 9 8 7 6 5 4 3 2 1

1 2015

Contents

Preface xv








vi CoNTENTS
















CoNTENTS vii



















viii CoNTENTS
















CoNTENTS ix















x CoNTENTS













Q) Coupling 235



CoNTENTS xi













xii CoNTENTS















CoNTENTS xiii

















xiv CoNTENTS








Index 443





PREFACE



xvi PREFACE







PREFACE xvii




February 2015


February 2015




























11 HISTORY



INTRODUCTION



1

2 INTRODUCTION




Mndash

K+ K+

Clndash Clndash

Na+

Na+



ext = 4ndash5 mM

[Clminus]int

= 4 mM [Clminus]ext

= 120 mM [Na+]int

= 12 mM [Na+]ext



3minus across



HISTORY 3





4 INTRODUCTION





HISTORY 5




0F




6 INTRODUCTION






RT

c

czFVln in

outm (11)


RT

c

czFVln out

inm (12)


m









8 INTRODUCTION



14 ION CHANNeLS


141 Voltage‐gated


Exterior

Pump Channel



Contents

Preface xv








vi CoNTENTS
















CoNTENTS vii



















viii CoNTENTS
















CoNTENTS ix















x CoNTENTS













Q) Coupling 235



CoNTENTS xi













xii CoNTENTS















CoNTENTS xiii

















xiv CoNTENTS








Index 443





PREFACE



xvi PREFACE







PREFACE xvii




February 2015


February 2015




























11 HISTORY



INTRODUCTION



1

2 INTRODUCTION




Mndash

K+ K+

Clndash Clndash

Na+

Na+



ext = 4ndash5 mM

[Clminus]int

= 4 mM [Clminus]ext

= 120 mM [Na+]int

= 12 mM [Na+]ext



3minus across



HISTORY 3





4 INTRODUCTION





HISTORY 5




0F




6 INTRODUCTION






RT

c

czFVln in

outm (11)


RT

c

czFVln out

inm (12)


m









8 INTRODUCTION



14 ION CHANNeLS


141 Voltage‐gated


Exterior

Pump Channel



vi CoNTENTS
















CoNTENTS vii



















viii CoNTENTS
















CoNTENTS ix















x CoNTENTS













Q) Coupling 235



CoNTENTS xi













xii CoNTENTS















CoNTENTS xiii

















xiv CoNTENTS








Index 443





PREFACE



xvi PREFACE







PREFACE xvii




February 2015


February 2015




























11 HISTORY



INTRODUCTION



1

2 INTRODUCTION




Mndash

K+ K+

Clndash Clndash

Na+

Na+



ext = 4ndash5 mM

[Clminus]int

= 4 mM [Clminus]ext

= 120 mM [Na+]int

= 12 mM [Na+]ext



3minus across



HISTORY 3





4 INTRODUCTION





HISTORY 5




0F




6 INTRODUCTION






RT

c

czFVln in

outm (11)


RT

c

czFVln out

inm (12)


m









8 INTRODUCTION



14 ION CHANNeLS


141 Voltage‐gated


Exterior

Pump Channel



CoNTENTS vii



















viii CoNTENTS
















CoNTENTS ix















x CoNTENTS













Q) Coupling 235



CoNTENTS xi













xii CoNTENTS















CoNTENTS xiii

















xiv CoNTENTS








Index 443





PREFACE



xvi PREFACE







PREFACE xvii




February 2015


February 2015




























11 HISTORY



INTRODUCTION



1

2 INTRODUCTION




Mndash

K+ K+

Clndash Clndash

Na+

Na+



ext = 4ndash5 mM

[Clminus]int

= 4 mM [Clminus]ext

= 120 mM [Na+]int

= 12 mM [Na+]ext



3minus across



HISTORY 3





4 INTRODUCTION





HISTORY 5




0F




6 INTRODUCTION






RT

c

czFVln in

outm (11)


RT

c

czFVln out

inm (12)


m









8 INTRODUCTION



14 ION CHANNeLS


141 Voltage‐gated


Exterior

Pump Channel



viii CoNTENTS
















CoNTENTS ix















x CoNTENTS













Q) Coupling 235



CoNTENTS xi













xii CoNTENTS















CoNTENTS xiii

















xiv CoNTENTS








Index 443





PREFACE



xvi PREFACE







PREFACE xvii




February 2015


February 2015




























11 HISTORY



INTRODUCTION



1

2 INTRODUCTION




Mndash

K+ K+

Clndash Clndash

Na+

Na+



ext = 4ndash5 mM

[Clminus]int

= 4 mM [Clminus]ext

= 120 mM [Na+]int

= 12 mM [Na+]ext



3minus across



HISTORY 3





4 INTRODUCTION





HISTORY 5




0F




6 INTRODUCTION






RT

c

czFVln in

outm (11)


RT

c

czFVln out

inm (12)


m









8 INTRODUCTION



14 ION CHANNeLS


141 Voltage‐gated


Exterior

Pump Channel



CoNTENTS ix















x CoNTENTS













Q) Coupling 235



CoNTENTS xi













xii CoNTENTS















CoNTENTS xiii

















xiv CoNTENTS








Index 443





PREFACE



xvi PREFACE







PREFACE xvii




February 2015


February 2015




























11 HISTORY



INTRODUCTION



1

2 INTRODUCTION




Mndash

K+ K+

Clndash Clndash

Na+

Na+



ext = 4ndash5 mM

[Clminus]int

= 4 mM [Clminus]ext

= 120 mM [Na+]int

= 12 mM [Na+]ext



3minus across



HISTORY 3





4 INTRODUCTION





HISTORY 5




0F




6 INTRODUCTION






RT

c

czFVln in

outm (11)


RT

c

czFVln out

inm (12)


m









8 INTRODUCTION



14 ION CHANNeLS


141 Voltage‐gated


Exterior

Pump Channel



x CoNTENTS













Q) Coupling 235



CoNTENTS xi













xii CoNTENTS















CoNTENTS xiii

















xiv CoNTENTS








Index 443





PREFACE



xvi PREFACE







PREFACE xvii




February 2015


February 2015




























11 HISTORY



INTRODUCTION



1

2 INTRODUCTION




Mndash

K+ K+

Clndash Clndash

Na+

Na+



ext = 4ndash5 mM

[Clminus]int

= 4 mM [Clminus]ext

= 120 mM [Na+]int

= 12 mM [Na+]ext



3minus across



HISTORY 3





4 INTRODUCTION





HISTORY 5




0F




6 INTRODUCTION






RT

c

czFVln in

outm (11)


RT

c

czFVln out

inm (12)


m









8 INTRODUCTION



14 ION CHANNeLS


141 Voltage‐gated


Exterior

Pump Channel



CoNTENTS xi













xii CoNTENTS















CoNTENTS xiii

















xiv CoNTENTS








Index 443





PREFACE



xvi PREFACE







PREFACE xvii




February 2015


February 2015




























11 HISTORY



INTRODUCTION



1

2 INTRODUCTION




Mndash

K+ K+

Clndash Clndash

Na+

Na+



ext = 4ndash5 mM

[Clminus]int

= 4 mM [Clminus]ext

= 120 mM [Na+]int

= 12 mM [Na+]ext



3minus across



HISTORY 3





4 INTRODUCTION





HISTORY 5




0F




6 INTRODUCTION






RT

c

czFVln in

outm (11)


RT

c

czFVln out

inm (12)


m









8 INTRODUCTION



14 ION CHANNeLS


141 Voltage‐gated


Exterior

Pump Channel



xii CoNTENTS















CoNTENTS xiii

















xiv CoNTENTS








Index 443





PREFACE



xvi PREFACE







PREFACE xvii




February 2015


February 2015




























11 HISTORY



INTRODUCTION



1

2 INTRODUCTION




Mndash

K+ K+

Clndash Clndash

Na+

Na+



ext = 4ndash5 mM

[Clminus]int

= 4 mM [Clminus]ext

= 120 mM [Na+]int

= 12 mM [Na+]ext



3minus across



HISTORY 3





4 INTRODUCTION





HISTORY 5




0F




6 INTRODUCTION






RT

c

czFVln in

outm (11)


RT

c

czFVln out

inm (12)


m









8 INTRODUCTION



14 ION CHANNeLS


141 Voltage‐gated


Exterior

Pump Channel



CoNTENTS xiii

















xiv CoNTENTS








Index 443





PREFACE



xvi PREFACE







PREFACE xvii




February 2015


February 2015




























11 HISTORY



INTRODUCTION



1

2 INTRODUCTION




Mndash

K+ K+

Clndash Clndash

Na+

Na+



ext = 4ndash5 mM

[Clminus]int

= 4 mM [Clminus]ext

= 120 mM [Na+]int

= 12 mM [Na+]ext



3minus across



HISTORY 3





4 INTRODUCTION





HISTORY 5




0F




6 INTRODUCTION






RT

c

czFVln in

outm (11)


RT

c

czFVln out

inm (12)


m









8 INTRODUCTION



14 ION CHANNeLS


141 Voltage‐gated


Exterior

Pump Channel



xiv CoNTENTS








Index 443





PREFACE



xvi PREFACE







PREFACE xvii




February 2015


February 2015




























11 HISTORY



INTRODUCTION



1

2 INTRODUCTION




Mndash

K+ K+

Clndash Clndash

Na+

Na+



ext = 4ndash5 mM

[Clminus]int

= 4 mM [Clminus]ext

= 120 mM [Na+]int

= 12 mM [Na+]ext



3minus across



HISTORY 3





4 INTRODUCTION





HISTORY 5




0F




6 INTRODUCTION






RT

c

czFVln in

outm (11)


RT

c

czFVln out

inm (12)


m









8 INTRODUCTION



14 ION CHANNeLS


141 Voltage‐gated


Exterior

Pump Channel






PREFACE



xvi PREFACE







PREFACE xvii




February 2015


February 2015




























11 HISTORY



INTRODUCTION



1

2 INTRODUCTION




Mndash

K+ K+

Clndash Clndash

Na+

Na+



ext = 4ndash5 mM

[Clminus]int

= 4 mM [Clminus]ext

= 120 mM [Na+]int

= 12 mM [Na+]ext



3minus across



HISTORY 3





4 INTRODUCTION





HISTORY 5




0F




6 INTRODUCTION






RT

c

czFVln in

outm (11)


RT

c

czFVln out

inm (12)


m









8 INTRODUCTION



14 ION CHANNeLS


141 Voltage‐gated


Exterior

Pump Channel



xvi PREFACE







PREFACE xvii




February 2015


February 2015




























11 HISTORY



INTRODUCTION



1

2 INTRODUCTION




Mndash

K+ K+

Clndash Clndash

Na+

Na+



ext = 4ndash5 mM

[Clminus]int

= 4 mM [Clminus]ext

= 120 mM [Na+]int

= 12 mM [Na+]ext



3minus across



HISTORY 3





4 INTRODUCTION





HISTORY 5




0F




6 INTRODUCTION






RT

c

czFVln in

outm (11)


RT

c

czFVln out

inm (12)


m









8 INTRODUCTION



14 ION CHANNeLS


141 Voltage‐gated


Exterior

Pump Channel



PREFACE xvii




February 2015


February 2015




























11 HISTORY



INTRODUCTION



1

2 INTRODUCTION




Mndash

K+ K+

Clndash Clndash

Na+

Na+



ext = 4ndash5 mM

[Clminus]int

= 4 mM [Clminus]ext

= 120 mM [Na+]int

= 12 mM [Na+]ext



3minus across



HISTORY 3





4 INTRODUCTION





HISTORY 5




0F




6 INTRODUCTION






RT

c

czFVln in

outm (11)


RT

c

czFVln out

inm (12)


m









8 INTRODUCTION



14 ION CHANNeLS


141 Voltage‐gated


Exterior

Pump Channel






























11 HISTORY



INTRODUCTION



1

2 INTRODUCTION




Mndash

K+ K+

Clndash Clndash

Na+

Na+



ext = 4ndash5 mM

[Clminus]int

= 4 mM [Clminus]ext

= 120 mM [Na+]int

= 12 mM [Na+]ext



3minus across



HISTORY 3





4 INTRODUCTION





HISTORY 5




0F




6 INTRODUCTION






RT

c

czFVln in

outm (11)


RT

c

czFVln out

inm (12)


m









8 INTRODUCTION



14 ION CHANNeLS


141 Voltage‐gated


Exterior

Pump Channel



















11 HISTORY



INTRODUCTION



1

2 INTRODUCTION




Mndash

K+ K+

Clndash Clndash

Na+

Na+



ext = 4ndash5 mM

[Clminus]int

= 4 mM [Clminus]ext

= 120 mM [Na+]int

= 12 mM [Na+]ext



3minus across



HISTORY 3





4 INTRODUCTION





HISTORY 5




0F




6 INTRODUCTION






RT

c

czFVln in

outm (11)


RT

c

czFVln out

inm (12)


m









8 INTRODUCTION



14 ION CHANNeLS


141 Voltage‐gated


Exterior

Pump Channel




11 HISTORY



INTRODUCTION



1

2 INTRODUCTION




Mndash

K+ K+

Clndash Clndash

Na+

Na+



ext = 4ndash5 mM

[Clminus]int

= 4 mM [Clminus]ext

= 120 mM [Na+]int

= 12 mM [Na+]ext



3minus across



HISTORY 3





4 INTRODUCTION





HISTORY 5




0F




6 INTRODUCTION






RT

c

czFVln in

outm (11)


RT

c

czFVln out

inm (12)


m









8 INTRODUCTION



14 ION CHANNeLS


141 Voltage‐gated


Exterior

Pump Channel



2 INTRODUCTION




Mndash

K+ K+

Clndash Clndash

Na+

Na+



ext = 4ndash5 mM

[Clminus]int

= 4 mM [Clminus]ext

= 120 mM [Na+]int

= 12 mM [Na+]ext



3minus across



HISTORY 3





4 INTRODUCTION





HISTORY 5




0F




6 INTRODUCTION






RT

c

czFVln in

outm (11)


RT

c

czFVln out

inm (12)


m









8 INTRODUCTION



14 ION CHANNeLS


141 Voltage‐gated


Exterior

Pump Channel



HISTORY 3





4 INTRODUCTION





HISTORY 5




0F




6 INTRODUCTION






RT

c

czFVln in

outm (11)


RT

c

czFVln out

inm (12)


m









8 INTRODUCTION



14 ION CHANNeLS


141 Voltage‐gated


Exterior

Pump Channel



4 INTRODUCTION





HISTORY 5




0F




6 INTRODUCTION






RT

c

czFVln in

outm (11)


RT

c

czFVln out

inm (12)


m









8 INTRODUCTION



14 ION CHANNeLS


141 Voltage‐gated


Exterior

Pump Channel



HISTORY 5




0F




6 INTRODUCTION






RT

c

czFVln in

outm (11)


RT

c

czFVln out

inm (12)


m









8 INTRODUCTION



14 ION CHANNeLS


141 Voltage‐gated


Exterior

Pump Channel



6 INTRODUCTION






RT

c

czFVln in

outm (11)


RT

c

czFVln out

inm (12)


m









8 INTRODUCTION



14 ION CHANNeLS


141 Voltage‐gated


Exterior

Pump Channel









8 INTRODUCTION



14 ION CHANNeLS


141 Voltage‐gated


Exterior

Pump Channel



8 INTRODUCTION



14 ION CHANNeLS


141 Voltage‐gated


Exterior

Pump Channel



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