Expression and Analysis ofRecombinant Ion Channels

From Structural Studies to Pharmacological Screening

Edited byJeffrey J. Clare and Derek J.Trezise

InnodataFile Attachment3527607935.jpg

Expression and Analysis ofRecombinant Ion Channels

Edited byJeffrey J. Clare and Derek J.Trezise

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Expression and Analysis ofRecombinant Ion Channels

From Structural Studies to Pharmacological Screening

Edited byJeffrey J. Clare and Derek J.Trezise

The Editors

Dr. Jeffrey J. ClareGlaxoSmithKlineDepartment of Gene Expressionand Protein BiochemistryGunnels Wood RoadStevenage, SG1 2NYGreat Britain

Dr. Derek J. TreziseGlaxoSmithKlineDepartment of Assay DevelopmentGunnels Wood RoadStevenage SG1 2NYGreat Britain

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2006 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim

All rights reserved (including those of translationinto other languages). No part of this book may bereproduced in any form by photoprinting, micro-film, or any other means nor transmitted ortranslated into a machine language without writ-ten permission from the publishers. Registerednames, trademarks, etc. used in this book, evenwhen not specifically marked as such, are not tobe considered unprotected by law.

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Printed in the Federal Republic of GermanyPrinted on acid-free paper

ISBN-13: 978-3-527-31209-2ISBN-10: 3-527-31209-9

Contents

Preface XIList of Contributors XIIIColor Plates XVII

1 Expression of Ion Channels in Xenopus Oocytes 1Alan L. Goldin

1.1 Introduction 11.2 Advantages and Disadvantages of Xenopus Oocytes 21.3 Procedures for Using Oocytes 31.4 Types of Analyses 51.4.1 Electrophysiological Analysis 51.4.1.1 Two-electrode Whole Cell Voltage-clamp 51.4.1.2 Cut-open Oocyte Voltage-clamp 71.4.1.3 Macropatch Clamp 91.4.1.4 Single Channel Analysis 111.4.2 Biochemical Analysis 121.4.3 Compound Screening 131.4.3.1 Serial Recording Using the Roboocyte 141.4.3.2 Parallel Recording Using the OpusXpress 161.5 Examples of Use 171.5.1 Characterization of cDNA Clones for a Channel 171.5.2 StructureFunction Correlations 181.5.3 Studies of Human Disease Mutations 191.6 Conclusions 21

Acknowledgments 21References 21

2 Molecular Biology Techniques for Structure Function Studies of Ion Channels 27Louisa Stevens, Andrew J. Powell, and Dennis Wray

2.1 Introduction 272.2 Methods for cDNA Subcloning 28

V

2.2.1 Conventional Sub-cloning Using Restriction Enzymesand DNA Ligase 28

2.2.2 PCR-based cDNA Sub-cloning 312.2.3 Sub-cloning cDNA through Site-specific Recombination 332.3 Generation of Chimeric Channel cDNAs 362.3.1 Use of Restriction Enzymes to Generate Chimeric Channel cDNAs 362.3.2 PCR-mediated Overlap Extension for Chimera Generation 392.3.3 PCR-mediated Integration or Replacement of cDNA Fragments 432.4 Site-directed Mutagenesis 432.4.1 Examples of the Use of Site-directed Mutagenesis 452.4.2 Modification of the QuikChange Method for the Replacement of cDNA

Fragments 502.5 Epitope-tagged Channels and Fusion Partners 502.6 Channel Subunit Concatamers 522.7 Concluding Remarks 53

References 54

3 Unnatural Amino Acids as Probes of Ion Channel Structure Functionand Pharmacology 59Paul B. Bennett, Niki Zacharias, John B. Nicholas, Sue Dee Sahba,Ashutosh Kulkarni, and Mark Nowak

3.1 Introduction 593.2 Unnatural Amino Acid Mutagenesis Methodology 603.3 Unnatural Amino Acid Mutagenesis for Ion Channel Studies 643.4 StructureFunction Example Studies 653.4.1 Nicotinic Acetylcholine Receptor 653.4.2 Drug Interactions with the hERG Voltage-gated Potassium

Ion Channel 673.5 Other Uses of Unnatural Amino Acids as Probes of Protein Structure

and Function 723.6 Conclusions 73

Acknowledgements 74References 74

4 Functional Expression of Ion Channels in Mammalian Systems 79Jeff J. Clare

4.1 Introduction 794.2 cDNA Cloning and Manipulation 804.3 Choice of Host Cell Background 814.4 Post-translational Processing of Heterologous Expressed

Ion Channels 854.5 Cytotoxicity 904.6 Transient Expression Systems 914.6.1 Standard Transient Expression 914.6.2 Viral Expression Systems 92

VI Contents

4.7 Stable Expression of Ion Channels 964.7.1 Bicistronic Expression Systems 964.7.2 Stable Expression of Multiple Subunits 1004.7.3 Inducible Expression 1014.8 Summary 103

Acknowledgements 103References 104

5 Analysis of Electrophysiological Data 111Michael Pusch

5.1 Overview 1115.2 Introduction 1115.3 Expression Systems and Related Recording Techniques 1135.3.1 Expression in Xenopus Oocytes 1135.3.2 Expression in Mammalian Cells 1155.3.3 Leak and Capacitance Subtraction 1165.4 Macroscopic Recordings 1175.4.1 Analysis of Pore Properties Permeation 1185.4.2 Analysis of Fast Voltage-dependent Block the Woodhull Model 1215.4.3 Information on Gating Properties from Macroscopic Measurements 1225.4.3.1 Equilibrium Properties Voltage-gated Channels 1245.4.3.2 Equilibrium Properties Ligand Gated Channels 1265.4.3.3 Macroscopic Kinetics 1295.4.4 Channel Block 1325.4.5 Nonstationary Noise Analysis 1335.4.6 Gating Current Measurements in Voltage Gated Channels 1355.5 Single Channel Analysis 1365.5.1 Amplitude Histogram Analysis 1365.5.2 Kinetic Single Channel Analysis 1385.6 Summary 142

Acknowledgements 142References 142

6 Automated Planar Array Electrophysiology for Ion Channel Research 145Derek J Trezise

6.1 Introduction 1456.2 Overview of Planar Array Recording 1456.3 Experimental Methods and Design 1476.3.1 Cell Preparation 1486.3.2 Cell Sealing and Recording 1496.3.3 Drug Application 1526.3.4 Experimental Design and Data Analysis 1556.4 Overall Success Rates and Throughput 1586.5 Population Patch Clamp 1596.6 Summary and Perspective 162

VIIContents

Acknowledgments 162References 162

7 Ion Flux and Ligand Binding Assays for Analysisof Ion Channels 165Georg C. Terstappen

7.1 Introduction 1657.2 Ion Flux Assays 1667.2.1 Radioactive Ion Flux Assays 1677.2.2 Nonradioactive Ion Flux Assays based on Atomic Absorption

Spectrometry 1687.2.2.1 Nonradioactive Rubidium Efflux Assay 1687.2.2.2 Nonradioactive Lithium Influx Assay 1747.2.2.3 Nonradioactive Chloride Influx Assay 1747.2.2.4 Conclusions 1747.3 Ligand Binding Assays 1757.3.1 Heterogeneous Binding Assays Employing Radioligands 1777.3.2 Homogeneous Binding Assays Employing Radioligands 1787.3.3 Homogeneous Binding Assays Employing Fluorescent-Labeled Ligands

and Fluorescence Polarization 1807.3.4 Conclusions 181

Acknowledgements 182References 182

8 Ion Channel Assays Based on Ion and Voltage-sensitiveFluorescent Probes 187Jess E. Gonzlez, Jennings Worley, and Fredrick Van Goor

8.1 Introduction 1878.2 Membrane Potential Probes 1888.2.1 Redistribution Probes 1888.2.2 FRET Probes 1908.2.3 Advantages and Limitations of Membrane Potential Probes 1928.3 Ion-sensitive Fluorescent Probes 1948.3.1 Calcium Dyes 1948.3.2 Indicators of Other Ions 1958.4 Fluorescence Assays for Ion Channels 1968.4.1 Calcium Channels 1968.4.2 Non-voltage-gated Calcium Permeable Channels 1978.4.3 Sodium Channels 2008.4.4 Potassium Channels 2018.4.5 Chloride Channels 2038.5 Assays for Monitoring Channel Trafficking 2058.6 Summary 207

References 208

VIII Contents

9 Approaches for Ion Channel Structural Studies 213Randal B. Bass and Robert H. Spencer

9.1 Introduction 2139.2 Expression of Membrane Proteins for Structural Studies 2169.2.1 Mammalian Expression 2169.2.2 Insect Expression 2179.2.3 Yeast Expression 2179.2.4 Bacterial Expression 2189.3 The Detergent Factor 2199.4 Purification 2239.5 Crystallization 2279.6 Use of Antibody Fragments 2299.7 Generation of First Diffraction Datasets 2309.8 Selenomethionine Phasing of Membrane Proteins 2329.9 MAD Phasing and Edge Scanning 2339.10 Negative B- factor Application (Structure Factor Sharpening) 2349.11 Conclusions 235

References 235

10 Molecular Modeling and Simulations of Ion Channels:Applications to Potassium Channels 241Daniele Bemporad, Alessandro Grottesi, Shozeb Haider, Zara A. Sands,and Mark S.P. Sanso