Preface ........................................................ xv
List of Contributors .......................................... xix
1 Introduction ............................................... 1
Mohammed A.A. Khalid and Ronald J. Clarke
1.1 History .................................................... 1
1.2 Energetics of Transport .................................... 6
1.3 Mechanistic Considerations ................................. 7
1.4 Ion Channels ............................................... 8
1.4.1 Voltage-Gated ....................................... 8
1.4.2 Ligand-Gated ........................................ 9
1.4.3 Mechanosensitive .................................... 9
1.4.4 Light-Gated ......................................... 9
1.5 Ion Pumps ................................................. 10
1.5.1 ATP-Activated ...................................... 10
1.5.2 Light-Activated .................................... 11
1.5.3 Redox-Linked ....................................... 12
1.6 Transporters .............................................. 13
1.6.1 Symporters and Antiporters ......................... 13
1.6.2 Na+-Linked and H+-Linked ........................... 14
1.7 Diseases of Ion Channels, Pumps, and Transporters ......... 15
1.7.1 Channelopathies .................................... 15
1.7.2 Pump Dysfunction ................................... 17
1.7.3 Transporter Dysfunction ............................ 18
1.8 Conclusion ................................................ 18
References ................................................ 19
2 Study of Ion Pump Activity Using Black Lipid Membranes .... 23
Hans-Jurgen Apell and Valerij S. Sokolov
2.1 Introduction .............................................. 23
2.2 Formation of Black Lipid Membranes ........................ 24
2.3 Reconstitution in Black Lipid Membranes ................... 25
2.3.1 Reconstitution of Na+, K+-ATPase in Black Lipid
Membranes .......................................... 25
2.3.2 Recording Transient Currents with Membrane
Fragments Adsorbed to a Black Lipid Membrane ....... 26
2.4 The Principles of Capacitive Coupling ..................... 28
2.4.1 Dielectric Coefficients ........................... 29
2.5 The Gated-Channel Concept ................................. 31
2.6 Relaxation Techniques ..................................... 34
2.6.1 Concentration-Jump Methods ......................... 34
2.6.2 Charge-Pulse Method ................................ 39
2.7 Admittance Measurements ................................... 39
2.8 The Investigation of Cytoplasmic and Extracellular
Ion Access Channels in the Na+, K+-ATPase ................. 42
2.9 Conclusions ............................................... 43
References ................................................ 45
3 Analyzing Ion Permeation in Channels and Pumps
Using Patch-Clamp Recording ............................... 51
Andrew J. Moorhouse, Trevor M. Lewis, and Peter H. Barry
3.1 Introduction .............................................. 51
3.2 Description of the Patch-Clamp Technique .................. 52
3.2.1 Development of Whole-Cell Dialysis with Voltage-
Clamp .............................................. 52
3.3 Patch-Clamp Measurement and Analysis of Single Channel
Conductance ............................................... 54
3.3.1 Conductance and Ohm's Law .......................... 54
3.3.2 Conductance of Channels versus Pumps ............... 56
3.3.3 Fluctuation Analysis ............................... 57
3.3.4 Single Channel Recordings .......................... 61
3.4 Determining Ion Selectivity and Relative Permeation in
Whole-Cell Recordings ..................................... 67
3.4.1 Dilution Potential Measurements .................... 67
3.4.2 Bi-Ionic Potential Measurements .................... 69
3.4.3 Voltage and Solution Control in Whole-Cell
Patch-Clamp Recordings ............................. 70
3.4.4 Ion Shift Effects During Whole-Cell Patch-Clamp
Experiments ........................................ 71
3.4.5 Liquid Junction Potential Corrections .............. 72
3.5 Influence of Voltage Corrections in Quantifying Ion
Selectivity in Channels ................................... 74
3.5.1 Analysis of Counterion Permeation in Glycine
Receptor Channels .................................. 74
3.5.2 Analysis of Anion-Cation Permeability in
Cation-Selective Mutant Glycine Receptor Channels .. 75
3.6 Ion Permeation Pathways through Channels and Pumps ........ 76
3.6.1 The Ion Permeation Pathway in Pentameric
Ligand-Gated Ion Channels .......................... 76
3.6.1.1 Extracellular and Intracellular Components of
the Permeation Pathway ......................... 78
3.6.1.2 The TM2 Pore is the Primary Ion Selectivity
Filter ......................................... 79
3.6.2 Ion Permeation Pathways in Pumps Identified Using
Patch-Clamp ........................................ 80
3.6.2.1 Palytoxin Uncouples the Occluded Gates of
the Na+, K+-ATPase ............................. 81
3.7 Conclusions ............................................... 82
References ................................................ 83
4 Probing Conformational Transitions of Membrane Proteins
with Voltage Clamp Fluorometry (VCF) ...................... 89
Thomas Friedrich
4.1 Introduction .............................................. 89
4.2 Description of The VCF Technique .......................... 90
4.2.1 Generation of Single-Cysteine Reporter
Constructs, Expression in Xenopus laevis Oocytes,
Site-Directed Fluorescence Labeling ................ 90
4.2.2 VCF Instrumentation ................................ 91
4.2.3 Technical Precautions and Controls ................. 93
4.3 Perspectives from Early Measurements on Voltage-Gated K+
Channels .................................................. 95
4.3.1 Early Results Obtained with VCF on Voltage-Gated
K+ Channels ........................................ 95
4.3.2 Probing the Environmental Changes: Fluorescence
Spectra, Anisotropy, and the Effects of Quenchers .. 98
4.4 VCF Applied to P-Type ATPases ............................ 100
4.4.1 Structural and Functional Aspects of Na+, K+-
and H+, K+-ATPase ................................. 100
4.4.2 The N790C Sensor Construct of Sheep Na+,K+-ATPase
ocl-Subunit ....................................... 102
4.4.2.1 Probing Voltage-Dependent Conformational
Changes of Na+, K+-ATPase ...................... 103
4.4.2.2 The Influence of Intracellular Na+
Concentrations ................................ 107
4.4.3 The Rat Gastric H+, K+-ATPase S806C Sensor
Construct ......................................... 108
4.4.3.1 Voltage-Dependent Conformational Shifts of
the H+, K+-ATPase Sensor Construct S806C
During the H+Transport Branch ................. 109
4.4.3.2 An Intra- or Extracellular Access Channel of
the Proton Pump? .............................. 110
4.4.3.3 Effects of Extracellular Ligands: K+ and Na+ .. 111
4.4.4 Probing Intramolecular Distances by Double
Labeling and FRET ................................. 113
4.5 Conclusions and Perspectives ............................. 116
References ............................................... 117
5 Patch Clamp Analysis of Transporters via Pre-Steady-
State Kinetic Methods .................................... 121
Christof Grewer
5.1 Introduction ............................................. 121
5.2 Patch Clamp Analysis of Secondary-Active Transporter
Function ................................................. 122
5.2.1 Patch Clamp Methods ............................... 122
5.2.2 Whole-Cell Recording .............................. 124
5.2.3 Recording from Excised Patches .................... 124
5.3 Perturbation Methods ..................................... 125
5.3.1 Concentration Jumps ............................... 126
5.3.2 Voltage Jumps ..................................... 129
5.4 Evaluation and Interpretation of Pre-Steady-State
Kinetic Data ............................................. 130
5.4.1 Integrating Rate Equations that Describe
Mechanistic Transport Models ...................... 131
5.4.2 Assigning Kinetic Components to Elementary
processes in the Transport Cycle .................. 131
5.5 Mechanistic Insight into Transporter Function ............ 133
5.5.1 Sequential Binding Mechanism ...................... 133
5.5.2 Electrostatics .................................... 134
5.5.3 Structure-Function Analysis ....................... 134
5.6 Case Studies ............................................. 136
5.6.1 Glutamate Transporter Mechanism ................... 136
5.6.2 Electrogenic Charge Movements Associated with
the Electroneutral Amino Acid Exchanger ASCT2 ..... 137
5.7 Conclusions .............................................. 139
References ............................................... 139
6 Recording of Pump and Transporter Activity Using
Solid-Supported Membranes (SSM-Based Electrophysiology) .. 147
Francesco Tadini-Buoninsegni and Klaus Fendler
6.1 Introduction ............................................. 147
6.2 The Instrument ........................................... 148
6.2.1 Rapid Solution Exchange Cuvette ................... 149
6.2.2 Setup and Flow Protocols .......................... 150
6.2.3 Protein Preparations .............................. 151
6.2.4 Commercial Instruments ............................ 152
6.3 Measurement Procedures, Data Analysis, and
Interpretation ........................................... 152
6.3.1 Current Measurement, Signal Analysis, and
Reconstruction of Pump Currents ................... 152
6.3.2 Voltage Measurement ............................... 156
6.3.3 Solution Exchange Artifacts ....................... 157
6.4 P-Type ATPases Investigated by SSM-Based
Electrophysiology ........................................ 159
6.4.1 Sarcoplasmic Reticulum Ca2+-ATPase ................ 159
6.4.2 Human Cu+-ATPases ATP7A and ATP7B ................. 163
6.5 Secondary Active Transporters ............................ 165
6.5.1 Antiport: Assessing the Forward and Reverse
Modes of the NhaA Na+/H+ Exchanger of E. coli ..... 166
6.5.2 Cotransport: A Sugar-Induced Electrogenic
Partial Reaction in the Lactose Permease Lac Y of
E. coli ........................................... 168
6.5.3 The Glutamate Transporter EAAC1: A Robust
Electrophysiological Assay with High Information
Content ........................................... 170
6.6 Conclusions .............................................. 172
References ............................................... 173
7 Stopped-FIow Fluorimetry Using Voltage-Sensitive
Fluorescent Membrane Probes .............................. 179
Ronald J. Clarke and Mohammed A.A. Khalid
7.1 Introduction ............................................. 179
7.2 Basics of the Stopped-FIow Technique ..................... 181
7.2.1 Flow Cell Design .................................. 181
7.2.2 Rapid Data Acquisition ............................ 181
7.2.3 Dead Time ......................................... 183
7.3 Covalent Versus Noncovalent Fluorescence Labeling ........ 184
7.3.1 Intrinsic Fluorescence ............................ 185
7.3.2 Covalently Bound Extrinsic Fluorescent Probes ..... 186
7.3.3 Noncovalently Bound Extrinsic Fluorescent Probes .. 187
7.4 Classes of Voltage-Sensitive Dyes ........................ 188
7.4.1 Slow Dyes ......................................... 188
7.4.2 Fast Dyes ......................................... 190
7.5 Measurement of the Kinetics of the Na+, K+-ATPase ........ 193
7.5.1 Dye Concentration ................................. 194
7.5.2 Excitation Wavelength and Light Source ............ 197
7.5.3 Monochromators and Filters ........................ 198
7.5.4 Photomultiplier and Voltage Supply ................ 199
7.5.5 Reactions Detected by RH421 ....................... 200
7.5.6 Origin of the RH421 Response ...................... 202
7.6 Conclusions .............................................. 204
References ............................................... 204
8 Nuclear Magnetic Resonance Spectroscopy .................. 211
Philip W. Kuchel
8.1 Introduction ............................................. 211
8.1.1 Definition of NMR ................................. 212
8.1.2 Why So Useful? .................................... 212
8.1.3 Magnetic Polarization ............................. 212
8.1.4 Larmor Equation ................................... 213
8.1.5 Chemical Shift .................................... 213
8.1.6 Free Induction Decay .............................. 214
8.1.7 Pulse Excitation .................................. 215
8.1.8 Relaxation Times .................................. 217
8.1.9 Splitting of Resonance Lines ...................... 217
8.1.10 Measuring Membrane Transport ...................... 217
8.2 Covalently-Induced Chemical Shift Differences ............ 218
8.2.1 Arginine Transport ................................ 218
8.2.2 Other Examples .................................... 220
8.3 Shift-Reagent-Induced Chemical Shift Differences ......... 220
8.3.1 DyPPP ............................................. 220
8.3.2 TmDTPA and TmDOTP ................................. 220
8.3.3 Fast Cation Exchange .............................. 220
8.4 pH-Induced Chemical Shift Differences .................... 223
8.4.1 Orthophosphate .................................... 223
8.4.2 Methylphosphonate ................................. 224
8.4.3 Triethylphosphate: 31P Shift Reference ............ 224
8.5 Hydrogen-Bond-Induced Chemical Shift Differences ......... 225
8.5.1 Phosphonates: DMMP ................................ 225
8.5.2 HPA ............................................... 225
8.5.3 Fluorides ......................................... 227
8.6 Ionic-Environment-Induced Chemical Shift Differences ..... 229
8.6.1 Cs+ Transport ..................................... 229
8.7 Relaxation Time Differences .............................. 229
8.7.1 Mn2+ Doping ....................................... 229
8.8 Diffusion Coefficient Differences ........................ 231
8.8.1 Stejskal-Tanner Plot .............................. 231
8.8.2 Andrasko's Method ................................. 231
8.9 Some Subtle Spectral Effects ............................. 233
8.9.1 Scalar (J) Coupling Differences ................... 233
8.9.2 Endogenous Magnetic Field Gradients ............... 233
8.9.2.1 Magnetic Induction and Magnetic Field
Strength ...................................... 234
8.9.2.2 Magnetic Field Gradients Across Cell
Membranes and CO Treatment of RBCs ............ 234
8.9.2.3 Exploiting Magnetic Field Gradients in
Membrane Transport Studies .................... 235
8.9.3 Residual Quadrupolar (νQ) Coupling ................ 235
8.10 A Case Study: The Stoichiometric Relationship Between
the Number of Na+ Ions Transported per Molecule of
Glucose Consumed in Human RBCs ........................... 236
8.11 Conclusions .............................................. 239
References ............................................... 239
9 Time-Resolved and Surface-Enhanced Infrared
Spectroscopy ............................................. 245
Joachim Heberle
9.1 Introduction ............................................. 245
9.2 Basics of IR Spectroscopy ................................ 246
9.2.1 Vibrational Spectroscopy .......................... 246
9.2.2 FTIR Spectroscopy ................................. 247
9.2.3 IR Spectra of Biological Compounds ................ 248
9.2.4 Difference Spectroscopy ........................... 250
9.3 Reflection Techniques .................................... 250
9.3.1 Attenuated Total Reflection ....................... 250
9.3.2 Surface-Enhanced IR Absorption .................... 251
9.4 Application to Electron-Transferring Proteins ............ 252
9.4.1 Cytochrome с ...................................... 252
9.4.2 Cytochrome с Oxidase .............................. 253
9.5 Time-Resolved IR Spectroscopy ............................ 254
9.5.1 The Rapid-Scan Technique .......................... 254
9.5.2 The Step-Scan Technique ........................... 255
9.5.3 Tunable QCLs ...................................... 255
9.6 Applications to Retinal Proteins ......................... 256
9.6.1 Bacteriorhodopsin ................................. 256
9.6.2 Channelrhodopsin .................................. 260
9.7 Conclusions .............................................. 263
References ............................................... 264
10 Analysis of Membrane-Protein Complexes by Single-
Molecule Methods ......................................... 269
Katia Cosentino, Stephanie Bleicken, and Ana J.
García-Sáez
10.1 Introduction ............................................. 269
10.2 Fluorophores for Single Particle Labeling ................ 270
10.3 Principles of Fluorescence Correlation Spectroscopy ...... 271
10.3.1 Analysis of Molecular Complexes by Two-Color FCS .. 275
10.3.2 FCS Variants to Study Lipid Membranes ............. 275
10.3.3 FCS Applications to Membranes ..................... 278
10.4 Principle and Analysis of Single-Molecule Imaging ........ 279
10.4.1 TIRF Microscopy ................................... 280
10.4.2 Single-Molecule Detection ......................... 282
10.4.3 Single Particle Tracking and Trajectory Analysis .. 284
10.5 Complex Dynamics and Stoichiometry by Single-Molecule
Microscopy ............................................... 285
10.5.1 Application to Single-Molecule Stoichiometry
Analysis .......................................... 285
10.5.2 Application to Kinetics Processes in Cell
Membranes ......................................... 290
10.6 FCS Versus SPT ........................................... 291
References ............................................... 291
11 Probing Channel, Pump, and Transporter Function Using
Single-Molecule Fluorescence ............................. 299
Eve E. Weatherill, John S.H. Danial, and Mark I. Wallace
11.1 Introduction ............................................. 299
11.1.1 Basic Principles .................................. 300
11.2 Practical Considerations ................................. 300
11.2.1 Observables ....................................... 301
11.2.2 Apparatus ......................................... 301
11.2.3 Labels ............................................ 302
11.2.4 Bilayers .......................................... 303
11.3 SMF Imaging .............................................. 303
11.3.1 Fluorescence Colocalization ....................... 304
11.3.2 Conformational Changes ............................ 306
11.3.3 Superresolution Microscopy ........................ 307
11.4 Single Molecule Forster Resonance Energy Transfer ........ 308
11.4.1 Interactions/Stoichiometry ........................ 308
11.4.2 Conformational Changes ............................ 309
11.5 Single-Molecule Counting by Photobleaching ............... 312
11.6 Optical Channel Recording ................................ 314
11.7 Simultaneous Techniques .................................. 315
11.8 Summary .................................................. 318
References ............................................... 318
12 Electron Paramagnetic Resonance: Site-Directed Spin
Labeling ................................................. 327
Louise J. Brown and Joanna E. Hare
12.1 Introduction ............................................. 327
12.1.1 Development of EPR as a Tool for Structural
Biology ........................................... 329
12.1.2 SDSL-EPR: A Complementary Approach to Determine
Structure-Function Relationships .................. 330
12.2 Basics of the EPR Method ................................. 331
12.2.1 Physical Basis of the EPR Signal .................. 331
12.2.2 Spin Labeling ..................................... 333
12.2.3 EPR Instrumentation ............................... 336
12.3 Structural and Dynamic Information from SDSL-EPR ......... 336
12.3.1 Mobility Measurements ............................. 336
12.3.2 Solvent Accessibility ............................. 341
12.4 Distance Measurements .................................... 345
12.4.1 Interspin Distance Measurements ................... 345
12.4.2 Continuous Wave ................................... 347
12.4.3 Pulsed Methods: DEER .............................. 349
12.5 Challenges ............................................... 353
12.5.1 New Labels ........................................ 353
12.5.2 Spin-Label Flexibility ............................ 355
12.5.3 Production and Reconstitution Challenges:
Nanodiscs ......................................... 355
12.6 Conclusions .............................................. 356
References ............................................... 357
13 Radioactivity-Based Analysis of Ion Transport ............ 367
Ingolf Bernhardt and J. Clive Ellory
13.1 Introduction ............................................. 367
13.2 Membrane Permeability for Electroneutral Substances
and Ions ................................................. 368
13.3 Kinetic Considerations ................................... 370
13.4 Techniques for Ion Flux Measurements ..................... 371
13.4.1 Conventional Methods .............................. 371
13.4.2 Alternative Method ................................ 373
13.5 Kinetic Analysis of Ion Transporter Properties ........... 375
13.6 Selected Cation Transporter Studies on Red Blood Cells ... 376
13.6.1 K+,C1" Cotransport (KCC) .......................... 378
13.6.2 Residual Transport ................................ 378
13.7 Combination of Radioactive Isotope Studies with Methods
using Fluorescent Dyes ................................... 379
13.8 Conclusions .............................................. 382
References ............................................... 383
14 Cation Uptake Studies with Atomic Absorption
Spectrophotometry (AAS) .................................. 387
Thomas Friedrich
14.1 Introduction ............................................. 387
14.2 Overview of the Technique of AAS ......................... 389
14.2.1 Historical Account of AAS with Flame Atomization .. 390
14.2.2 Element-Specific Radiation Sources ................ 391
14.2.3 Electrothermal Atomization in Heated Graphite
Tubes ............................................. 392
14.2.4 Correction for Background Absorption .............. 394
14.3 The Expression System of Xenopus laevis Oocytes
for Cation Flux Studies: Practical Considerations ........ 395
14.4 Experimental Outline of the AAS Flux Quantification
Technique ................................................ 395
14.5 Representative Results Obtained with the AAS Flux
Quantification Technique ................................. 397
14.5.1 Reaction Cycle of P-Type ATPases .................. 398
14.5.2 Rb+Uptake Kinetics: Inhibitor Sensitivity ......... 398
14.5.3 Dependence of Rb+ Transport of Gastric
H+, K+-ATPase on Extra- and Intracellular pH ...... 400
14.5.4 Determination of Na+, K+-ATPase Transport
Stoichiometry and Voltage Dependence of H+, K+-
ATPase Rb+ Transport .............................. 403
14.5.5 Effects of C-Terminal Deletions of the
H+, K+-ATPase α-Subunit ........................... 404
14.5.6 Li+ and Cs+ Uptake Studies ........................ 405
14.6 Concluding Remarks ....................................... 407
References ............................................... 408
15 Long Timescale Molecular Simulations for Understanding
Ion Channel Function ..................................... 411
Ben Corry
15.1 Introduction ............................................. 411
15.2 Fundamentals of MD Simulation ............................ 412
15.2.1 The Main Idea ..................................... 412
15.2.2 Force Fields ...................................... 414
15.2.3 Other Simulation Considerations ................... 416
15.2.4 Why Do MD Simulations Take So Much Computational
Power? ............................................ 416
15.2.4.1 Force Calculations ............................ 417
15.2.4.2 Time Step ..................................... 417
15.3 Simulation Duration and Simulation Size .................. 418
15.4 Historical Development of Long MD Simulations ............ 421
15.5 Limitations and Challenges Facing MD Simulations ......... 423
15.5.1 Force Field and Algorithm Accuracy ................ 423
15.5.2 Sampling Problems ................................. 424
15.6 Example Simulations of Ion Channels ...................... 425
15.6.1 Simulations of Ion Permeation ..................... 425
15.6.2 Simulations of Ion Selectivity .................... 428
15.6.3 Simulations of Channel Gating ..................... 432
15.7 Conclusions .............................................. 433
References ............................................... 436
Index ......................................................... 443
Chemical Analysis: A Series of Monographs on Analytical
Chemistry and its Applications ................................ 461
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