Expression and Analysis of Recombinant Ion Channels

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

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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 more

Product details

  • Digital | 302 pages
  • Wiley-VCH Verlag GmbH
  • Weinheim, Germany
  • 3527608095
  • 9783527608096

Author information

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 more

Table of contents

Preface.List of Contributors.Color Plates.1 Expression of Ion Channels in Xenopus Oocytes (Alan L. Goldin).1.1 Introduction.1.2 Advantages and Disadvantages of Xenopus Oocytes.1.3 Procedures for Using Oocytes.1.4 Types of Analyses.1.5 Examples of Use.1.6 Conclusions.Acknowledgments.References.2 Molecular Biology Techniques for Structure - Function Studies of Ion Channels (Louisa Stevens, Andrew J. Powell, and Dennis Wray).2.1 Introduction.2.2 Methods for cDNA Subcloning.2.3 Generation of Chimeric Channel cDNAs.2.4 Site-directed Mutagenesis.2.5 Epitope-tagged Channels and Fusion Partners.2.6 Channel Subunit Concatamers.2.7 Concluding Remarks.References.3 Unnatural Amino Acids as Probes of Ion Channel Structure - Function and Pharmacology (Paul B. Bennett, Niki Zacharias, John B. Nicholas, Sue Dee Sahba, Ashutosh Kulkarni, and Mark Nowak).3.1 Introduction.3.2 Unnatural Amino Acid Mutagenesis Methodology.3.3 Unnatural Amino Acid Mutagenesis for Ion Channel Studies.3.4 Structure-Function Example Studies.3.5 Other Uses of Unnatural Amino Acids as Probes of Protein Structure and Function.3.6 Conclusions.Acknowledgements.References.4 Functional Expression of Ion Channels in Mammalian Systems (Jeff J. Clare).4.1 Introduction.4.2 cDNA Cloning and Manipulation.4.3 Choice of Host Cell Background.4.4 Post-translational Processing of Heterologous Expressed Ion Channels.4.5 Cytotoxicity.4.6 Transient Expression Systems.4.7 Stable Expression of Ion Channels.4.8 Summary.Acknowledgements.References.5 Analysis of Electrophysiological Data (Michael Pusch).5.1 Overview.5.2 Introduction.5.3 Expression Systems and Related Recording Techniques.5.4 Macroscopic Recordings.5.5 Single Channel Analysis.5.6 Summary.Acknowledgements.References.6 Automated Planar Array Electrophysiology for Ion Channel Research (Derek J Trezise).6.1 Introduction.6.2 Overview of Planar Array Recording.6.3 Experimental Methods and Design.6.4 Overall Success Rates and Throughput.6.5 Population Patch Clamp.6.6 Summary and Perspective.Acknowledgments.References.7 Ion Flux and Ligand Binding Assays for Analysisof Ion Channels (Georg C. Terstappen).7.1 Introduction.7.2 Ion Flux Assays.7.3 Ligand Binding Assays.Acknowledgements.References.8 Ion Channel Assays Based on Ion and Voltage-sensitive Fluorescent Probes (Jesus E. Gonzalez, Jennings Worley, and Fredrick Van Goor).8.1 Introduction.8.2 Membrane Potential Probes.8.3 Ion-sensitive Fluorescent Probes.8.4 Fluorescence Assays for Ion Channels.8.5 Assays for Monitoring Channel Trafficking.8.6 Summary.References.9 Approaches for Ion Channel Structural Studies (Randal B. Bass and Robert H. Spencer).9.1 Introduction.9.2 Expression of Membrane Proteins for Structural Studies.9.3 The Detergent Factor.9.4 Purification.9.5 Crystallization.9.6 Use of Antibody Fragments.9.7 Generation of First Diffraction Datasets.9.8 Selenomethionine Phasing of Membrane Proteins.9.9 MAD Phasing and Edge Scanning.9.10 Negative B- factor Application (Structure Factor Sharpening).9.11 Conclusions.References.10 Molecular Modeling and Simulations of Ion Channels: Applications to Potassium Channels (Daniele Bemporad, Alessandro Grottesi, Shozeb Haider, Zara A. Sands, and Mark S.P. Sansom).10.1 Introduction.10.2 Computational Methods.10.3 Kir Channels.10.4 Kv Channels.10.5 Summary and Future Directions.Acknowledgements.References.Subject more