Expression and Analysis of Recombinant Ion Channels Edited by Jeffrey J. Clare and Derek J.Trezise Related Titles Daunert, S., Deo, S. K. (eds.) Sibley, D. R. Photoproteins in Bioanalysis Receptor Biochemistry and approx. 300 pages Methodology Series Hardcover 6 Volume Set ISBN 3-527-31016-9 approx. 2500 pages 2002 Dingermann, T., Steinhilber, D., Hardcover ISBN 0-471-32258-X Folkers, G. (eds.) Molecular Biology in Medicinal Chemistry Novartis 435 pages with 113 figures and 32 tables Novartis Foundation 2004 Symposium 245 – Ion Channels – Hardcover From Atomic Resolution Physiology ISBN 3-527-30431-2 to Functional Genomics 284 pages Babine, R. E., Abdel-Meguid, S. S. (eds.) 2002 Protein Crystallography in Hardcover ISBN 0-470-84375-6 Drug Discovery 278 pages with 131 figures and 11 tables 2004 Hardcover ISBN 3-527-30678-1 Expression and Analysis of Recombinant Ion Channels From Structural Studies to Pharmacological Screening Edited by Jeffrey J. Clare and Derek J.Trezise The Editors & All books published by Wiley-VCH are carefully produced. Nevertheless, authors, editors, and Dr. Jeffrey J. Clare publisher do not warrant the information con- GlaxoSmithKline tained in these books, including this book, to be Department of Gene Expression free of errors. Readers are advised to keep in mind and Protein Biochemistry that statements, data, illustrations, procedural Gunnels Wood Road details or other items may inadvertently be inaccu- Stevenage, SG1 2NY rate. Great Britain Library of Congress Card No.: Dr. Derek J. Trezise applied for GlaxoSmithKline Department of Assay Development British Library Cataloguing-in-Publication Data Gunnels Wood Road A catalogue record for this book is available from Stevenage SG1 2NY the British Library. Great Britain Bibliographic information published by Die Deutsche Bibliothek Die Deutsche Bibliothek lists this publication in the Deutsche Nationalbibliografie; detailed biblio- graphic data is available in the Internet at <http://dnb.ddb.de>. 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim All rights reserved (including those of translation into other languages). No part of this book may be reproduced in any form – by photoprinting, micro- film, or any other means – nor transmitted or translated into a machine language without writ- ten permission from the publishers. Registered names, trademarks, etc. used in this book, even when not specifically marked as such, are not to be considered unprotected by law. Typesetting ProSatz Unger,Weinheim Printing Strauss GmbH, Mörlenbach Binding J. Schäffer GmbH i.G., Grünstadt Printed in the Federal Republic of Germany Printed on acid-free paper ISBN-13: 978-3-527-31209-2 ISBN-10: 3-527-31209-9 V 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 Expression and Analysis of Recombinant Ion Channels. Edited by Jeffrey J. Clare and Derek J. Trezise Copyright # 2006 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim ISBN: 3-527-31209-9 VI Contents 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 Contents VII 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 VIII Contents Acknowledgments 162 References 162 7 Ion Flux and Ligand Binding Assays for Analysisof 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 Contents IX 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.