Expression and analysis of recombinant ion channels : from structural studies to pharmacological...
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作 者：edited by Jeffrey J. Clare and Derek J. Trezise.
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ISBN：9783527312092
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简介
Filling the gap created over the past five years, during which many new techniques have entered the market, this book is directed at both the new and the experienced ion channel researcher wishing to learn more about the considerations and methods for studying recombinant ion channels. These latest developments are covered here for the first time, contributed by editors and authors working for major pharmaceutical companies and who routinely apply these techniques in their daily work. The first three chapters cover the use of the Xenopus oocyte expression system for structure-function studies, from basic approaches for manipulating ion channel cDNAs to more specialized but powerful techniques. This is followed by reviews of strategies and methodologies available for expressing channels in mammalian cells and for their analysis by patch-clamp electrophysiology. Chapters 6 to 8 review the latest methodologies for ion channel drug discovery, including high throughput screening using fluorescence and luminescence, as well as automated planar array electrophysiology. The remaining two chapters focus on approaches for determining ion channel crystal structures and on computational approaches to understanding channel mechanisms at atomic resolution. Rather than provide detailed protocols, indicated by references in each chapter, the authors provide a comprehensive and easily accessible overview of the techniques involved, reviewing underlying principles and providing working guidelines as well as an understanding of the key theoretical and practical considerations associated with each topic. In each case, this practical advice is illustrated by real life examples, taken either from the author's own experience or from key examples in the literature, providing valuable practical hints not found elsewhere. The result is a compendium of practical ion channel information that will prove a valuable resource to academic and industrial workers alike.
目录
Contents 7
Preface 13
List of Contributors 15
Color Plates 19
1 Expression of Ion Channels in Xenopus Oocytes 33
1.1 Introduction 33
1.2 Advantages and Disadvantages of Xenopus Oocytes 34
1.3 Procedures for Using Oocytes 35
1.4 Types of Analyses 37
1.4.1 Electrophysiological Analysis 37
1.4.1.1 Two-electrode Whole Cell Voltage-clamp 37
1.4.1.2 Cut-open Oocyte Voltage-clamp 39
1.4.1.3 Macropatch Clamp 41
1.4.1.4 Single Channel Analysis 43
1.4.2 Biochemical Analysis 44
1.4.3 Compound Screening 45
1.4.3.1 Serial Recording Using the Roboocyte 46
1.4.3.2 Parallel Recording Using the OpusXpress 48
1.5 Examples of Use 49
1.5.1 Characterization of cDNA Clones for a Channel 49
1.5.2 Structure\u2013Function Correlations 50
1.5.3 Studies of Human Disease Mutations 51
1.6 Conclusions 53
Acknowledgments 53
References 53
2 Molecular Biology Techniques for Structure \u2013 Function Studies of Ion Channels 59
2.1 Introduction 59
2.2 Methods for cDNA Subcloning 60
2.2.1 Conventional Sub-cloning Using Restriction Enzymes and DNA Ligase 60
2.2.2 PCR-based cDNA Sub-cloning 63
2.2.3 Sub-cloning cDNA through Site-specific Recombination 65
2.3 Generation of Chimeric Channel cDNAs 68
2.3.1 Use of Restriction Enzymes to Generate Chimeric Channel cDNAs 68
2.3.2 PCR-mediated Overlap Extension for Chimera Generation 71
2.3.3 PCR-mediated Integration or Replacement of cDNA Fragments 75
2.4 Site-directed Mutagenesis 75
2.4.1 Examples of the Use of Site-directed Mutagenesis 77
2.4.2 Modification of the QuikChange Method for the Replacement of cDNA Fragments 82
2.5 Epitope-tagged Channels and Fusion Partners 82
2.6 Channel Subunit Concatamers 84
2.7 Concluding Remarks 85
References 86
3 Unnatural Amino Acids as Probes of Ion Channel Structure \u2013 Function and Pharmacology 91
3.1 Introduction 91
3.2 Unnatural Amino Acid Mutagenesis Methodology 92
3.3 Unnatural Amino Acid Mutagenesis for Ion Channel Studies 96
3.4 Structure\u2013Function Example Studies 97
3.4.1 Nicotinic Acetylcholine Receptor 97
3.4.2 Drug Interactions with the hERG Voltage-gated Potassium Ion Channel 99
3.5 Other Uses of Unnatural Amino Acids as Probes of Protein Structure and Function 104
3.6 Conclusions 105
Acknowledgements 106
References 106
4 Functional Expression of Ion Channels in Mammalian Systems 111
4.1 Introduction 111
4.2 cDNA Cloning and Manipulation 112
4.3 Choice of Host Cell Background 113
4.4 Post-translational Processing of Heterologous Expressed Ion Channels 117
4.5 Cytotoxicity 122
4.6 Transient Expression Systems 123
4.6.1 \u201cStandard\u201d Transient Expression 123
4.6.2 Viral Expression Systems 124
4.7 Stable Expression of Ion Channels 128
4.7.1 Bicistronic Expression Systems 128
4.7.2 Stable Expression of Multiple Subunits 132
4.7.3 Inducible Expression 133
4.8 Summary 135
Acknowledgements 135
References 136
5 Analysis of Electrophysiological Data 143
5.1 Overview 143
5.2 Introduction 143
5.3 Expression Systems and Related Recording Techniques 145
5.3.1 Expression in Xenopus Oocytes 145
5.3.2 Expression in Mammalian Cells 147
5.3.3 Leak and Capacitance Subtraction 148
5.4 Macroscopic Recordings 149
5.4.1 Analysis of Pore Properties \u2013 Permeation 150
5.4.2 Analysis of Fast Voltage-dependent Block \u2013 the Woodhull Model 153
5.4.3 Information on Gating Properties from Macroscopic Measurements 154
5.4.3.1 Equilibrium Properties \u2013 Voltage-gated Channels 156
5.4.3.2 Equilibrium Properties \u2013 Ligand Gated Channels 158
5.4.3.3 Macroscopic Kinetics 161
5.4.4 Channel Block 164
5.4.5 Nonstationary Noise Analysis 165
5.4.6 Gating Current Measurements in Voltage Gated Channels 167
5.5 Single Channel Analysis 168
5.5.1 Amplitude Histogram Analysis 168
5.5.2 Kinetic Single Channel Analysis 170
5.6 Summary 174
Acknowledgements 174
References 174
6 Automated Planar Array Electrophysiology for Ion Channel Research 177
6.1 Introduction 177
6.2 Overview of Planar Array Recording 177
6.3 Experimental Methods and Design 179
6.3.1 Cell Preparation 180
6.3.2 Cell Sealing and Recording 181
6.3.3 Drug Application 184
6.3.4 Experimental Design and Data Analysis 187
6.4 Overall Success Rates and Throughput 190
6.5 Population Patch Clamp 191
6.6 Summary and Perspective 194
Acknowledgments 194
References 194
7 Ion Flux and Ligand Binding Assays for Analysis of Ion Channels 197
7.1 Introduction 197
7.2 Ion Flux Assays 198
7.2.1 Radioactive Ion Flux Assays 199
7.2.2 Nonradioactive Ion Flux Assays based on Atomic Absorption Spectrometry 200
7.2.2.1 Nonradioactive Rubidium Efflux Assay 200
7.2.2.2 Nonradioactive Lithium Influx Assay 206
7.2.2.3 Nonradioactive Chloride Influx Assay 206
7.2.2.4 Conclusions 206
7.3 Ligand Binding Assays 207
7.3.1 Heterogeneous Binding Assays Employing Radioligands 209
7.3.2 Homogeneous Binding Assays Employing Radioligands 210
7.3.3 Homogeneous Binding Assays Employing Fluorescent-Labeled Ligands and Fluorescence Polarization 212
7.3.4 Conclusions 213
Acknowledgements 214
References 214
8 Ion Channel Assays Based on Ion and Voltage-sensitive Fluorescent Probes 219
8.1 Introduction 219
8.2 Membrane Potential Probes 220
8.2.1 Redistribution Probes 220
8.2.2 FRET Probes 222
8.2.3 Advantages and Limitations of Membrane Potential Probes 224
8.3 Ion-sensitive Fluorescent Probes 226
8.3.1 Calcium Dyes 226
8.3.2 Indicators of Other Ions 227
8.4 Fluorescence Assays for Ion Channels 228
8.4.1 Calcium Channels 228
8.4.2 Non-voltage-gated Cation Permeable Channels 229
8.4.3 Sodium Channels 232
8.4.4 Potassium Channels 233
8.4.5 Chloride Channels 235
8.5 Assays for Monitoring Channel Trafficking 237
8.6 Summary 239
References 240
9 Approaches for Ion Channel Structural Studies 245
9.1 Introduction 245
9.2 Expression of Membrane Proteins for Structural Studies 248
9.2.1 Mammalian Expression 248
9.2.2 Insect Expression 249
9.2.3 Yeast Expression 249
9.2.4 Bacterial Expression 250
9.3 The Detergent Factor 251
9.4 Purification 255
9.5 Crystallization 259
9.6 Use of Antibody Fragments 261
9.7 Generation of First Diffraction Datasets 262
9.8 Selenomethionine Phasing of Membrane Proteins 264
9.9 MAD Phasing and Edge Scanning 265
9.10 Negative B- factor Application (Structure Factor Sharpening) 266
9.11 Conclusions 267
References 267
10 Molecular Modeling and Simulations of Ion Channels: Applications to Potassium Channels 273
10.1 Introduction 273
10.2 Computational Methods 274
10.3 Kir Channels 278
10.3.1 Structures 278
10.3.2 Molecular Modeling 279
10.3.3 Simulations 280
10.3.4 Filter Flexibility 280
10.3.5 M2 Helices and Hinge Motion 282
10.3.6 Intracellular Domain Dynamics 283
10.3.7 Interactions with Ligands 283
10.3.8 Towards an Integrated Gating Model 285
10.4 Kv Channels 286
10.4.1 Structures 286
10.4.2 S6 Helices, Hinges and Gating 288
10.4.3 The Barrier at the Gate 289
10.4.4 The Nature of the Voltage Sensor 290
10.4.5 A Possible Gating Model 292
10.5 Summary and Future Directions 293
Acknowledgements 294
References 294
Subject Index 301
Preface 13
List of Contributors 15
Color Plates 19
1 Expression of Ion Channels in Xenopus Oocytes 33
1.1 Introduction 33
1.2 Advantages and Disadvantages of Xenopus Oocytes 34
1.3 Procedures for Using Oocytes 35
1.4 Types of Analyses 37
1.4.1 Electrophysiological Analysis 37
1.4.1.1 Two-electrode Whole Cell Voltage-clamp 37
1.4.1.2 Cut-open Oocyte Voltage-clamp 39
1.4.1.3 Macropatch Clamp 41
1.4.1.4 Single Channel Analysis 43
1.4.2 Biochemical Analysis 44
1.4.3 Compound Screening 45
1.4.3.1 Serial Recording Using the Roboocyte 46
1.4.3.2 Parallel Recording Using the OpusXpress 48
1.5 Examples of Use 49
1.5.1 Characterization of cDNA Clones for a Channel 49
1.5.2 Structure\u2013Function Correlations 50
1.5.3 Studies of Human Disease Mutations 51
1.6 Conclusions 53
Acknowledgments 53
References 53
2 Molecular Biology Techniques for Structure \u2013 Function Studies of Ion Channels 59
2.1 Introduction 59
2.2 Methods for cDNA Subcloning 60
2.2.1 Conventional Sub-cloning Using Restriction Enzymes and DNA Ligase 60
2.2.2 PCR-based cDNA Sub-cloning 63
2.2.3 Sub-cloning cDNA through Site-specific Recombination 65
2.3 Generation of Chimeric Channel cDNAs 68
2.3.1 Use of Restriction Enzymes to Generate Chimeric Channel cDNAs 68
2.3.2 PCR-mediated Overlap Extension for Chimera Generation 71
2.3.3 PCR-mediated Integration or Replacement of cDNA Fragments 75
2.4 Site-directed Mutagenesis 75
2.4.1 Examples of the Use of Site-directed Mutagenesis 77
2.4.2 Modification of the QuikChange Method for the Replacement of cDNA Fragments 82
2.5 Epitope-tagged Channels and Fusion Partners 82
2.6 Channel Subunit Concatamers 84
2.7 Concluding Remarks 85
References 86
3 Unnatural Amino Acids as Probes of Ion Channel Structure \u2013 Function and Pharmacology 91
3.1 Introduction 91
3.2 Unnatural Amino Acid Mutagenesis Methodology 92
3.3 Unnatural Amino Acid Mutagenesis for Ion Channel Studies 96
3.4 Structure\u2013Function Example Studies 97
3.4.1 Nicotinic Acetylcholine Receptor 97
3.4.2 Drug Interactions with the hERG Voltage-gated Potassium Ion Channel 99
3.5 Other Uses of Unnatural Amino Acids as Probes of Protein Structure and Function 104
3.6 Conclusions 105
Acknowledgements 106
References 106
4 Functional Expression of Ion Channels in Mammalian Systems 111
4.1 Introduction 111
4.2 cDNA Cloning and Manipulation 112
4.3 Choice of Host Cell Background 113
4.4 Post-translational Processing of Heterologous Expressed Ion Channels 117
4.5 Cytotoxicity 122
4.6 Transient Expression Systems 123
4.6.1 \u201cStandard\u201d Transient Expression 123
4.6.2 Viral Expression Systems 124
4.7 Stable Expression of Ion Channels 128
4.7.1 Bicistronic Expression Systems 128
4.7.2 Stable Expression of Multiple Subunits 132
4.7.3 Inducible Expression 133
4.8 Summary 135
Acknowledgements 135
References 136
5 Analysis of Electrophysiological Data 143
5.1 Overview 143
5.2 Introduction 143
5.3 Expression Systems and Related Recording Techniques 145
5.3.1 Expression in Xenopus Oocytes 145
5.3.2 Expression in Mammalian Cells 147
5.3.3 Leak and Capacitance Subtraction 148
5.4 Macroscopic Recordings 149
5.4.1 Analysis of Pore Properties \u2013 Permeation 150
5.4.2 Analysis of Fast Voltage-dependent Block \u2013 the Woodhull Model 153
5.4.3 Information on Gating Properties from Macroscopic Measurements 154
5.4.3.1 Equilibrium Properties \u2013 Voltage-gated Channels 156
5.4.3.2 Equilibrium Properties \u2013 Ligand Gated Channels 158
5.4.3.3 Macroscopic Kinetics 161
5.4.4 Channel Block 164
5.4.5 Nonstationary Noise Analysis 165
5.4.6 Gating Current Measurements in Voltage Gated Channels 167
5.5 Single Channel Analysis 168
5.5.1 Amplitude Histogram Analysis 168
5.5.2 Kinetic Single Channel Analysis 170
5.6 Summary 174
Acknowledgements 174
References 174
6 Automated Planar Array Electrophysiology for Ion Channel Research 177
6.1 Introduction 177
6.2 Overview of Planar Array Recording 177
6.3 Experimental Methods and Design 179
6.3.1 Cell Preparation 180
6.3.2 Cell Sealing and Recording 181
6.3.3 Drug Application 184
6.3.4 Experimental Design and Data Analysis 187
6.4 Overall Success Rates and Throughput 190
6.5 Population Patch Clamp 191
6.6 Summary and Perspective 194
Acknowledgments 194
References 194
7 Ion Flux and Ligand Binding Assays for Analysis of Ion Channels 197
7.1 Introduction 197
7.2 Ion Flux Assays 198
7.2.1 Radioactive Ion Flux Assays 199
7.2.2 Nonradioactive Ion Flux Assays based on Atomic Absorption Spectrometry 200
7.2.2.1 Nonradioactive Rubidium Efflux Assay 200
7.2.2.2 Nonradioactive Lithium Influx Assay 206
7.2.2.3 Nonradioactive Chloride Influx Assay 206
7.2.2.4 Conclusions 206
7.3 Ligand Binding Assays 207
7.3.1 Heterogeneous Binding Assays Employing Radioligands 209
7.3.2 Homogeneous Binding Assays Employing Radioligands 210
7.3.3 Homogeneous Binding Assays Employing Fluorescent-Labeled Ligands and Fluorescence Polarization 212
7.3.4 Conclusions 213
Acknowledgements 214
References 214
8 Ion Channel Assays Based on Ion and Voltage-sensitive Fluorescent Probes 219
8.1 Introduction 219
8.2 Membrane Potential Probes 220
8.2.1 Redistribution Probes 220
8.2.2 FRET Probes 222
8.2.3 Advantages and Limitations of Membrane Potential Probes 224
8.3 Ion-sensitive Fluorescent Probes 226
8.3.1 Calcium Dyes 226
8.3.2 Indicators of Other Ions 227
8.4 Fluorescence Assays for Ion Channels 228
8.4.1 Calcium Channels 228
8.4.2 Non-voltage-gated Cation Permeable Channels 229
8.4.3 Sodium Channels 232
8.4.4 Potassium Channels 233
8.4.5 Chloride Channels 235
8.5 Assays for Monitoring Channel Trafficking 237
8.6 Summary 239
References 240
9 Approaches for Ion Channel Structural Studies 245
9.1 Introduction 245
9.2 Expression of Membrane Proteins for Structural Studies 248
9.2.1 Mammalian Expression 248
9.2.2 Insect Expression 249
9.2.3 Yeast Expression 249
9.2.4 Bacterial Expression 250
9.3 The Detergent Factor 251
9.4 Purification 255
9.5 Crystallization 259
9.6 Use of Antibody Fragments 261
9.7 Generation of First Diffraction Datasets 262
9.8 Selenomethionine Phasing of Membrane Proteins 264
9.9 MAD Phasing and Edge Scanning 265
9.10 Negative B- factor Application (Structure Factor Sharpening) 266
9.11 Conclusions 267
References 267
10 Molecular Modeling and Simulations of Ion Channels: Applications to Potassium Channels 273
10.1 Introduction 273
10.2 Computational Methods 274
10.3 Kir Channels 278
10.3.1 Structures 278
10.3.2 Molecular Modeling 279
10.3.3 Simulations 280
10.3.4 Filter Flexibility 280
10.3.5 M2 Helices and Hinge Motion 282
10.3.6 Intracellular Domain Dynamics 283
10.3.7 Interactions with Ligands 283
10.3.8 Towards an Integrated Gating Model 285
10.4 Kv Channels 286
10.4.1 Structures 286
10.4.2 S6 Helices, Hinges and Gating 288
10.4.3 The Barrier at the Gate 289
10.4.4 The Nature of the Voltage Sensor 290
10.4.5 A Possible Gating Model 292
10.5 Summary and Future Directions 293
Acknowledgements 294
References 294
Subject Index 301
Expression and analysis of recombinant ion channels : from structural studies to pharmacological...
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