Pumps, Channels and Transporters

Ronald J. Clarke, Ph.D. is an Associate Professor in the School of Chemistry, University of Sydney, Australia. In 2010 he was awarded the McAulay-Hope Prize for Original Biophysics by the Australian Society for Biophysics. Mohammed A. A. Khalid, Ph.D. is an Associate Professor in the Department of Chemistry, College of Applied Medical and Sciences at Taif University, Turabah, Saudi Arabia.

"Overall Pumps, channels and transporters: methods of functional analysis is an excellent book full of useful, detailed information and well worth reading whether you are an experienced cellular biologist or just a curious science undergraduate." (Chemistry in Australia 2016)

• Preface xvList of Contributors xix1 Introduction 1Mohammed A. A. Khalid and Ronald J. Clarke1.1 History 11.2 Energetics of Transport 61.3 Mechanistic Considerations 71.4 Ion Channels 81.4.1 Voltage-Gated 81.4.2 Ligand-Gated 91.4.3 Mechanosensitive 91.4.4 Light-Gated 91.5 Ion Pumps 101.5.1 ATP-Activated 101.5.2 Light-Activated 111.5.3 Redox-Linked 121.6 Transporters 131.6.1 Symporters and Antiporters 131.6.2 Na+-Linked and H+-Linked 141.7 Diseases of Ion Channels, Pumps, and Transporters 151.7.1 Channelopathies 151.7.2 Pump Dysfunction 171.7.3 Transporter Dysfunction 181.8 Conclusion 18References 192 Study of Ion Pump Activity Using Black Lipid Membranes 23Hans-Jürgen Apell and Valerij S. Sokolov2.1 Introduction 232.2 Formation of Black Lipid Membranes 242.3 Reconstitution in Black Lipid Membranes 252.3.1 Reconstitution of Na+,K+-ATPase in Black Lipid Membranes 252.3.2 Recording Transient Currents with Membrane Fragments Adsorbed to a Black Lipid Membrane 262.4 The Principles of Capacitive Coupling 282.4.1 Dielectric Coefficients 292.5 The Gated-Channel Concept 312.6 Relaxation Techniques 342.6.1 Concentration-Jump Methods 342.6.2 Charge-Pulse Method 392.7 Admittance Measurements 392.8 The Investigation of Cytoplasmic and Extracellular Ion Access Channels in the Na+,K+-ATPase 422.9 Conclusions 43References 453 Analyzing Ion Permeation in Channels and Pumps Using Patch-Clamp Recording 51Andrew J. Moorhouse, Trevor M. Lewis, and Peter H. Barry3.1 Introduction 513.2 Description of the Patch-Clamp Technique 523.2.1 Development of Whole-Cell Dialysis with Voltage-Clamp 523.3 Patch-Clamp Measurement and Analysis of Single Channel Conductance 543.3.1 Conductance and Ohm’s Law 543.3.2 Conductance of Channels versus Pumps 563.3.3 Fluctuation Analysis 573.3.4 Single Channel Recordings 613.4 Determining Ion Selectivity and Relative Permeation in Whole-Cell Recordings 673.4.1 Dilution Potential Measurements 673.4.2 Bi-Ionic Potential Measurements 693.4.3 Voltage and Solution Control in Whole-Cell Patch-Clamp Recordings 703.4.4 Ion Shift Effects During Whole-Cell Patch-Clamp Experiments 713.4.5 Liquid Junction Potential Corrections 723.5 Influence of Voltage Corrections in Quantifying Ion Selectivity in Channels 743.5.1 Analysis of Counterion Permeation in Glycine Receptor Channels 743.5.2 Analysis of Anion-Cation Permeability inCation-Selective Mutant Glycine Receptor Channels 753.6 Ion Permeation Pathways through Channels and Pumps 763.6.1 The Ion Permeation Pathway in Pentameric Ligand-Gated Ion Channels 763.6.1.1 Extracellular and Intracellular Components of the Permeation Pathway 783.6.1.2 The TM2 Pore is the Primary Ion Selectivity Filter 793.6.2 Ion Permeation Pathways in Pumps Identified Using Patch-Clamp 803.6.2.1 Palytoxin Uncouples the Occluded Gates of the Na+,K+-ATPase 813.7 Conclusions 82References 834 Probing Conformational Transitions of Membrane Proteins with Voltage Clamp Fluorometry (VCF) 89Thomas Friedrich4.1 Introduction 894.2 Description of The Vcf Technique 904.2.1 Generation of Single-Cysteine Reporter Constructs, Expression in Xenopus laevis Oocytes, Site-Directed Fluorescence Labeling 904.2.2 VCF Instrumentation 914.2.3 Technical Precautions and Controls 934.3 Perspectives from Early Measurements on Voltage-Gated K+ Channels 954.3.1 Early Results Obtained with VCF on Voltage-Gated K+ Channels 954.3.2 Probing the Environmental Changes: Fluorescence Spectra, Anisotropy, and the Effects of Quenchers 984.4 Vcf Applied to P-Type Atpases 1004.4.1 Structural and Functional Aspects of Na+, K+- and H+,K+-ATPase 1004.4.2 The N790C Sensor Construct of Sheep Na+,K+-ATPase α1-Subunit 1024.4.2.1 Probing Voltage-Dependent Conformational Changes of Na+,K+-ATPase 1034.4.2.2 The Influence of Intracellular Na+ Concentrations 1074.4.3 The Rat Gastric H+,K+-ATPase S806C Sensor Construct 1084.4.3.1 Voltage-Dependent Conformational Shifts of the H+,K+-ATPase Sensor Construct S806CDuring the H+ Transport Branch 1094.4.3.2 An Intra- or Extracellular Access Channel of the Proton Pump? 1104.4.3.3 Effects of Extracellular Ligands: K+ and Na+ 1114.4.4 Probing Intramolecular Distances by Double Labeling and FRET 1134.5 Conclusions and Perspectives 116References 1175 Patch Clamp Analysis of Transporters via Pre-Steady-State Kinetic Methods 121Christof Grewer5.1 Introduction 1215.2 Patch Clamp Analysis of Secondary-Active Transporter Function 1225.2.1 Patch Clamp Methods 1225.2.2 Whole-Cell Recording 1245.2.3 Recording from Excised Patches 1245.3 Perturbation Methods 1255.3.1 Concentration Jumps 1265.3.2 Voltage Jumps 1295.4 Evaluation and Interpretation of Pre-Steady-State Kinetic Data 1305.4.1 Integrating Rate Equations that Describe Mechanistic Transport Models 1315.4.2 Assigning Kinetic Components to Elementary processes in the Transport Cycle 1315.5 Mechanistic Insight into Transporter Function 1335.5.1 Sequential Binding Mechanism 1335.5.2 Electrostatics 1345.5.3 Structure-Function Analysis 1345.6 Case Studies 1365.6.1 Glutamate Transporter Mechanism 1365.6.2 Electrogenic Charge Movements Associated with the Electroneutral Amino Acid Exchanger ASCT2 1375.7 Conclusions 139References 1396 Recording of Pump and Transporter Activity Using Solid-Supported Membranes (SSM-Based Electrophysiology) 147Francesco Tadini-Buoninsegni and Klaus Fendler6.1 Introduction 1476.2 The Instrument 1486.2.1 Rapid Solution Exchange Cuvette 1496.2.2 Setup and Flow Protocols 1506.2.3 Protein Preparations 1516.2.4 Commercial Instruments 1526.3 Measurement Procedures, Data Analysis, and Interpretation 1526.3.1 Current Measurement, Signal Analysis, and Reconstruction of Pump Currents 1526.3.2 Voltage Measurement 1566.3.3 Solution Exchange Artifacts 1576.4 P-Type Atp ases Investigated by Ssm-Based Electrophysiology 1596.4.1 Sarcoplasmic Reticulum Ca2+-ATPase 1596.4.2 Human Cu+-ATPases ATP7A and ATP7B 1636.5 Secondary Active Transporters 1656.5.1 Antiport: Assessing the Forward and Reverse Modes of the NhaA Na+/H+ Exchanger of E. coli 1666.5.2 Cotransport: A Sugar-Induced Electrogenic Partial Reaction in the Lactose Permease LacY of E. coli 1686.5.3 The Glutamate Transporter EAAC1: A Robust Electrophysiological Assay with High Information Content 1706.6 Conclusions 172References 1737 Stopped-Flow Fluorimetry Using Voltage-Sensitive Fluorescent Membrane Probes 179Ronald J. Clarke and Mohammed A. A. Khalid7.1 Introduction 1797.2 Basics of the Stopped-Flow Technique 1817.2.1 Flow Cell Design 1817.2.2 Rapid Data Acquisition 1817.2.3 Dead Time 1837.3 Covalent Versus Noncovalent Fluorescence Labeling 1847.3.1 Intrinsic Fluorescence 1857.3.2 Covalently Bound Extrinsic Fluorescent Probes 1867.3.3 Noncovalently Bound Extrinsic Fluorescent Probes 1877.4 Classes of Voltage-Sensitive Dyes 1887.4.1 Slow Dyes 1887.4.2 Fast Dyes 1907.5 Measurement of the Kinetics of the Na+,K+-Atpase 1937.5.1 Dye Concentration 1947.5.2 Excitation Wavelength and Light Source 1977.5.3 Monochromators and Filters 1987.5.4 Photomultiplier and Voltage Supply 1997.5.5 Reactions Detected by RH421 2007.5.6 Origin of the RH421 Response 2027.6 Conclusions 204References 2048 Nuclear Magnetic Resonance Spectroscopy 211Philip W. Kuchel8.1 Introduction 2118.1.1 Definition of NMR 2128.1.2 Why So Useful? 2128.1.3 Magnetic Polarization 2128.1.4 Larmor Equation 2138.1.5 Chemical Shift 2138.1.6 Free Induction Decay 2148.1.7 Pulse Excitation 2158.1.8 Relaxation Times 2178.1.9 Splitting of Resonance Lines 2178.1.10 Measuring Membrane Transport 2178.2 Covalently-Induced Chemical Shift Differences 2188.2.1 Arginine Transport 2188.2.2 Other Examples 2208.3 Shift-Reagent-Induced Chemical Shift Differences 2208.3.1 DyPPP 2208.3.2 TmDTPA and TmDOTP 2208.3.3 Fast Cation Exchange 2208.4 pH-Induced Chemical Shift Differences 2238.4.1 Orthophosphate 2238.4.2 Methylphosphonate 2248.4.3 Triethylphosphate: 31P Shift Reference 2248.5 Hydrogen-Bond-Induced Chemical Shift Differences 2258.5.1 Phosphonates: DMMP 2258.5.2 HPA 2258.5.3 Fluorides 2278.6 Ionic-Environment-Induced Chemical Shift Differences 2298.6.1 Cs+ Transport 2298.7 Relaxation Time Differences 2298.7.1 Mn2+ Doping 2298.8 Diffusion Coefficient Differences 2318.8.1 Stejskal-Tanner Plot 2318.8.2 Andrasko’s Method 2318.9 Some Subtle Spectral Effects 2338.9.1 Scalar (J) Coupling Differences 2338.9.2 Endogenous Magnetic Field Gradients 2338.9.2.1 Magnetic Induction and Magnetic Field Strength 2348.9.2.2 Magnetic Field Gradients Across Cell Membranes and CO Treatment of RBCs 2348.9.2.3 Exploiting Magnetic Field Gradients in Membrane Transport Studies 2358.9.3 Residual Quadrupolar (νQ) Coupling 2358.10 A Case Study: The Stoichiometric Relationship Between the Number of Na+ Ions Transported per Molecule of Glucose Consumed in Human Rbcs 2368.11 Conclusions 239References 2399 Time-Resolved and Surface-Enhanced Infrared Spectroscopy 245Joachim Heberle9.1 Introduction 2459.2 Basics of Ir Spectroscopy 2469.2.1 Vibrational Spectroscopy 2469.2.2 FTIR Spectroscopy 2479.2.3 IR Spectra of Biological Compounds 2489.2.4 Difference Spectroscopy 2509.3 Reflection Techniques 2509.3.1 Attenuated Total Reflection 2509.3.2 Surface-Enhanced IR Absorption 2519.4 Application to Electron-Transferring Proteins 2529.4.1 Cytochrome c 2529.4.2 Cytochrome c Oxidase 2539.5 Time-Resolved ir Spectroscopy 2549.5.1 The Rapid-Scan Technique 2549.5.2 The Step-Scan Technique 2559.5.3 Tunable QCLs 2559.6 Applications to Retinal Proteins 2569.6.1 Bacteriorhodopsin 2569.6.2 Channelrhodopsin 2609.7 Conclusions 263References 26410 Analysis of Membrane-Protein Complexes by Single-Molecule Methods 269Katia Cosentino, Stephanie Bleicken, and Ana J. García-Sáez10.1 Introduction 26910.2 Fluorophores for Single Particle Labeling 27010.3 Principles of Fluorescence Correlation Spectroscopy 27110.3.1 Analysis of Molecular Complexes by Two-Color FCS 27510.3.2 FCS Variants to Study Lipid Membranes 27510.3.3 FCS Applications to Membranes 27810.4 Principle and Analysis of Single-Molecule Imaging 27910.4.1 TIRF Microscopy 28010.4.2 Single-Molecule Detection 28210.4.3 Single Particle Tracking and Trajectory Analysis 28410.5 Complex Dynamics and Stoichiometry by Single-Molecule Microscopy 28510.5.1 Application to Single-Molecule Stoichiometry Analysis 28510.5.2 Application to Kinetics Processes in Cell Membranes 29010.6 Fcs Versus Spt 291References 29111 Probing Channel, Pump, and Transporter Function Using Single-Molecule Fluorescence 299Eve E. Weatherill, John S. H. Danial, and Mark I. Wallace11.1 Introduction 29911.1.1 Basic Principles 30011.2 Practical Considerations 30011.2.1 Observables 30111.2.2 Apparatus 30111.2.3 Labels 30211.2.4 Bilayers 30311.3 smf Imaging 30311.3.1 Fluorescence Colocalization 30411.3.2 Conformational Changes 30611.3.3 Superresolution Microscopy 30711.4 Single Molecule Förster Resonance Energy Transfer 30811.4.1 Interactions/Stoichiometry 30811.4.2 Conformational Changes 30911.5 Single-Molecule Counting by Photobleaching 31211.6 Optical Channel Recording 31411.7 Simultaneous Techniques 31511.8 Summary 318References 31812 Electron Paramagnetic Resonance: Site-Directed Spin Labeling 327Louise J. Brown and Joanna E. Hare12.1 Introduction 32712.1.1 Development of EPR as a Tool for Structural Biology 32912.1.2 SDSL-EPR: A Complementary Approach to Determine Structure-Function Relationships 33012.2 Basics of the Epr Method 33112.2.1 Physical Basis of the EPR Signal 33112.2.2 Spin Labeling 33312.2.3 EPR Instrumentation 33612.3 Structural and Dynamic Information from Sdsl-Epr 33612.3.1 Mobility Measurements 33612.3.2 Solvent Accessibility 34112.4 Distance Measurements 34512.4.1 Interspin Distance Measurements 34512.4.2 Continuous Wave 34712.4.3 Pulsed Methods: DEER 34912.5 Challenges 35312.5.1 New Labels 35312.5.2 Spin-Label Flexibility 35512.5.3 Production and Reconstitution Challenges: Nanodiscs 35512.6 Conclusions 356References 35713 Radioactivity-Based Analysis of Ion Transport 367Ingolf Bernhardt and J. Clive Ellory13.1 Introduction 36713.2 Membrane Permeability for Electroneutral Substances and Ions 36813.3 Kinetic Considerations 37013.4 Techniques for Ion Flux Measurements 37113.4.1 Conventional Methods 37113.4.2 Alternative Method 37313.5 Kinetic Analysis of Ion Transporter Properties 37513.6 Selected Cation Transporter Studies on Red Blood Cells 37613.6.1 K+,Cl− Cotransport (KCC) 37813.6.2 Residual Transport 37813.7 Combination of Radioactive Isotope Studies with Methods using Fluorescent Dyes 37913.8 Conclusions 382References 38314 Cation Uptake Studies with Atomic Absorption Spectrophotometry (Aas) 387Thomas Friedrich14.1 Introduction 38714.2 Overview of the Technique of Aas 38914.2.1 Historical Account of AAS with Flame Atomization 39014.2.2 Element-Specific Radiation Sources 39114.2.3 Electrothermal Atomization in Heated Graphite Tubes 39214.2.4 Correction for Background Absorption 39414.3 The Expression System of Xenopus laevis Oocytes for Cation Flux Studies: Practical Considerations 39514.4 Experimental Outline of the Aas Flux Quantification Technique 39514.5 Representative Results Obtained with the Aas Flux Quantification Technique 39714.5.1 Reaction Cycle of P-Type ATPases 39814.5.2 Rb+ Uptake Kinetics: Inhibitor Sensitivity 39814.5.3 Dependence of Rb+ Transport of Gastric H+,K+-ATPase on Extra- and Intracellular pH 40014.5.4 Determination of Na+,K+-ATPase Transport Stoichiometry and Voltage Dependence of H+,K+-ATPase Rb+ Transport 40314.5.5 Effects of C-Terminal Deletions of the H+,K+-ATPase α-Subunit 40414.5.6 Li+ and Cs+ Uptake Studies 40514.6 Concluding Remarks 407References 40815 Long Timescale Molecular Simulations for Understanding Ion Channel Function 411Ben Corry15.1 Introduction 41115.2 Fundamentals of Md Simulation 41215.2.1 The Main Idea 41215.2.2 Force Fields 41415.2.3 O ther Simulation Considerations 41615.2.4 Why Do MD Simulations Take So Much Computational Power? 41615.2.4.1 Force Calculations 41715.2.4.2 Time Step 41715.3 Simulation Duration and Simulation Size 41815.4 Historical Development of Long Md Simulations 42115.5 Limitations and Challenges Facing Md Simulations 42315.5.1 Force Field and Algorithm Accuracy 42315.5.2 Sampling Problems 42415.6 Example Simulations of Ion Channels 42515.6.1 Simulations of Ion Permeation 42515.6.2 Simulations of Ion Selectivity 42815.6.3 Simulations of Channel Gating 43215.7 Conclusions 433References 436Index 443Chemical Analysis: A Series of Monographs on AnalyticalChemistry and its Applications 461