Expression and Analysis of Recombinant Ion Channels: From Structural Studies to Pharmacological Screening

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.

Table of Contents:
Preface xi List of Contributors xiii Color Plates xvii 1 Expression of Ion Channels in Xenopus Oocytes 1 Alan L. Goldin 1.1 Introduction 1 1.2 Advantages and Disadvantages of Xenopus Oocytes 2 1.3 Procedures for Using Oocytes 3 1.4 Types of Analyses 5 1.4.1 Electrophysiological Analysis 5 1.4.1.1 Two-electrode Whole Cell Voltage-clamp 5 1.4.1.2 Cut-open Oocyte Voltage-clamp 7 1.4.1.3 Macropatch Clamp 9 1.4.1.4 Single Channel Analysis 11 1.4.2 Biochemical Analysis 12 1.4.3 Compound Screening 13 1.4.3.1 Serial Recording Using the Roboocyte 14 1.4.3.2 Parallel Recording Using the OpusXpress 16 1.5 Examples of Use 17 1.5.1 Characterization of cDNA Clones for a Channel 17 1.5.2 Structure–Function Correlations 18 1.5.3 Studies of Human Disease Mutations 19 1.6 Conclusions 21 Acknowledgments 21 References 21 2 Molecular Biology Techniques for Structure – Function Studies of Ion Channels 27 Louisa Stevens, Andrew J. Powell, and Dennis Wray 2.1 Introduction 27 2.2 Methods for cDNA Subcloning 28 2.2.1 Conventional Sub-cloning Using Restriction Enzymes and DNA Ligase 28 2.2.2 PCR-based cDNA Sub-cloning 31 2.2.3 Sub-cloning cDNA through Site-specific Recombination 33 2.3 Generation of Chimeric Channel cDNAs 36 2.3.1 Use of Restriction Enzymes to Generate Chimeric Channel cDNAs 36 2.3.2 PCR-mediated Overlap Extension for Chimera Generation 39 2.3.3 PCR-mediated Integration or Replacement of cDNA Fragments 43 2.4 Site-directed Mutagenesis 43 2.4.1 Examples of the Use of Site-directed Mutagenesis 45 2.4.2 Modification of the QuikChange Method for the Replacement of cDNA Fragments 50 2.5 Epitope-tagged Channels and Fusion Partners 50 2.6 Channel Subunit Concatamers 52 2.7 Concluding Remarks 53 References 54 3 Unnatural Amino Acids as Probes of Ion Channel Structure – Function and Pharmacology 59 Paul B. Bennett, Niki Zacharias, John B. Nicholas, Sue Dee Sahba, Ashutosh Kulkarni, and Mark Nowak 3.1 Introduction 59 3.2 Unnatural Amino Acid Mutagenesis Methodology 60 3.3 Unnatural Amino Acid Mutagenesis for Ion Channel Studies 64 3.4 Structure–Function Example Studies 65 3.4.1 Nicotinic Acetylcholine Receptor 65 3.4.2 Drug Interactions with the hERG Voltage-gated Potassium Ion Channel 67 3.5 Other Uses of Unnatural Amino Acids as Probes of Protein Structure and Function 72 3.6 Conclusions 73 Acknowledgements 74 References 74 4 Functional Expression of Ion Channels in Mammalian Systems 79 Jeff J. Clare 4.1 Introduction 79 4.2 cDNA Cloning and Manipulation 80 4.3 Choice of Host Cell Background 81 4.4 Post-translational Processing of Heterologous Expressed Ion Channels 85 4.5 Cytotoxicity 90 4.6 Transient Expression Systems 91 4.6.1 “Standard” Transient Expression 91 4.6.2 Viral Expression Systems 92 4.7 Stable Expression of Ion Channels 96 4.7.1 Bicistronic Expression Systems 96 4.7.2 Stable Expression of Multiple Subunits 100 4.7.3 Inducible Expression 101 4.8 Summary 103 Acknowledgements 103 References 104 5 Analysis of Electrophysiological Data 111 Michael Pusch 5.1 Overview 111 5.2 Introduction 111 5.3 Expression Systems and Related Recording Techniques 113 5.3.1 Expression in Xenopus Oocytes 113 5.3.2 Expression in Mammalian Cells 115 5.3.3 Leak and Capacitance Subtraction 116 5.4 Macroscopic Recordings 117 5.4.1 Analysis of Pore Properties – Permeation 118 5.4.2 Analysis of Fast Voltage-dependent Block – the Woodhull Model 121 5.4.3 Information on Gating Properties from Macroscopic Measurements 122 5.4.3.1 Equilibrium Properties –Voltage-gated Channels 124 5.4.3.2 Equilibrium Properties – Ligand Gated Channels 126 5.4.3.3 Macroscopic Kinetics 129 5.4.4 Channel Block 132 5.4.5 Nonstationary Noise Analysis 133 5.4.6 Gating Current Measurements in Voltage Gated Channels 135 5.5 Single Channel Analysis 136 5.5.1 Amplitude Histogram Analysis 136 5.5.2 Kinetic Single Channel Analysis 138 5.6 Summary 142 Acknowledgements 142 References 142 6 Automated Planar Array Electrophysiology for Ion Channel Research 145 Derek J Trezise 6.1 Introduction 145 6.2 Overview of Planar Array Recording 145 6.3 Experimental Methods and Design 147 6.3.1 Cell Preparation 148 6.3.2 Cell Sealing and Recording 149 6.3.3 Drug Application 152 6.3.4 Experimental Design and Data Analysis 155 6.4 Overall Success Rates and Throughput 158 6.5 Population Patch Clamp 159 6.6 Summary and Perspective 162 Acknowledgments 162 References 162 7 Ion Flux and Ligand Binding Assays for Analysis of Ion Channels 165 Georg C. Terstappen 7.1 Introduction 165 7.2 Ion Flux Assays 166 7.2.1 Radioactive Ion Flux Assays 167 7.2.2 Nonradioactive Ion Flux Assays based on Atomic Absorption Spectrometry 168 7.2.2.1 Nonradioactive Rubidium Efflux Assay 168 7.2.2.2 Nonradioactive Lithium Influx Assay 174 7.2.2.3 Nonradioactive Chloride Influx Assay 174 7.2.2.4 Conclusions 174 7.3 Ligand Binding Assays 175 7.3.1 Heterogeneous Binding Assays Employing Radioligands 177 7.3.2 Homogeneous Binding Assays Employing Radioligands 178 7.3.3 Homogeneous Binding Assays Employing Fluorescent-Labeled Ligands and Fluorescence Polarization 180 7.3.4 Conclusions 181 Acknowledgements 182 References 182 8 Ion Channel Assays Based on Ion and Voltage-sensitive Fluorescent Probes 187 Jesús E. González, Jennings Worley, and Fredrick Van Goor 8.1 Introduction 187 8.2 Membrane Potential Probes 188 8.2.1 Redistribution Probes 188 8.2.2 FRET Probes 190 8.2.3 Advantages and Limitations of Membrane Potential Probes 192 8.3 Ion-sensitive Fluorescent Probes 194 8.3.1 Calcium Dyes 194 8.3.2 Indicators of Other Ions 195 8.4 Fluorescence Assays for Ion Channels 196 8.4.1 Calcium Channels 196 8.4.2 Non-voltage-gated Calcium Permeable Channels 197 8.4.3 Sodium Channels 200 8.4.4 Potassium Channels 201 8.4.5 Chloride Channels 203 8.5 Assays for Monitoring Channel Trafficking 205 8.6 Summary 207 References 208 9 Approaches for Ion Channel Structural Studies 213 Randal B. Bass and Robert H. Spencer 9.1 Introduction 213 9.2 Expression of Membrane Proteins for Structural Studies 216 9.2.1 Mammalian Expression 216 9.2.2 Insect Expression 217 9.2.3 Yeast Expression 217 9.2.4 Bacterial Expression 218 9.3 The Detergent Factor 219 9.4 Purification 223 9.5 Crystallization 227 9.6 Use of Antibody Fragments 229 9.7 Generation of First Diffraction Datasets 230 9.8 Selenomethionine Phasing of Membrane Proteins 232 9.9 MAD Phasing and Edge Scanning 233 9.10 Negative B- factor Application (Structure Factor Sharpening) 234 9.11 Conclusions 235 References 235 10 Molecular Modeling and Simulations of Ion Channels: Applications to Potassium Channels 241 Daniele Bemporad, Alessandro Grottesi, Shozeb Haider, Zara A. Sands, and Mark S.P. Sansom 10.1 Introduction 241 10.2 Computational Methods 242 10.3 Kir Channels 246 10.3.1 Structures 246 10.3.2 Molecular Modeling 247 10.3.3 Simulations 248 10.3.4 Filter Flexibility 248 10.3.5 M2 Helices and Hinge Motion 250 10.3.6 Intracellular Domain Dynamics 251 10.3.7 Interactions with Ligands 251 10.3.8 Towards an Integrated Gating Model 253 10.4 Kv Channels 254 10.4.1 Structures 254 10.4.2 S6 Helices, Hinges and Gating 256 10.4.3 The Barrier at the Gate 257 10.4.4 The Nature of the Voltage Sensor 258 10.4.5 A Possible Gating Model 260 10.5 Summary and Future Directions 261 Acknowledgements 262 References 262 Subject Index 269

About the Author :
Jeff Clare has worked in ion channel drug discovery for the last 13 years and is currently Head of Gene Expression for ion channels at GlaxoSmithKline. Having obtained his PhD from Kent University, he spent 5 years studying retrotransposable elements at UMIST and University of Connecticut. He then entered the pharmaceutical industry first joining Wellcome BioTech, to develop expression systems for vaccine antigen production, before transferring to Wellcome Research to initiate efforts in ion channel molecular cloning and expression. Subsequently, at GlaxoWellcome and then GlaxoSmithKline, he has been engaged in drug discovery and research projects for a range of ion channels, particularly voltage-gated sodium channels. Derek Trezise has over fifteen years experience in the functional analysis of ion channels, in both academic and industrial environments. After completing his PhD on smooth muscle K+ channels at the Victoria University of Manchester, UK he spent 5 years as a postdoctoral researcher at the Glaxo Institute of Applied Pharmacology at Cambridge, studying P2X channels. He then moved to GlaxoSmithKline R&D, Stevenage where he has held positions of increasing responsibility in Neuroscience, Molecular Pharmacology and Assay Development and Compound Profiling departments. His current role is Head of Assay Development for ion channels and G-protein coupled receptors.