Expression and Analysis of Recombinant Ion Channels: From Structural Studies to Pharmacological Scre重组细胞离子通道表达与分析：从结构研究到药理解析

作者： 本社 编

出 版 社：

出版时间： 2006-3-1字数：版次： 1页数： 273印刷时间： 2006/03/01开本： 16开印次： 1纸张： 胶版纸I S B N ： 9783527312092包装： 精装内容简介

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

Preface

List of Contributors

Color Plates

1 Expression of Ion Channels in Xenopus Oocytes （Alan LGoldin）

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 JPowell, 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 BBennett, Niki Zacharias, John BNicholas, 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 JClare）

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）

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

7 Ion Flux and Ligand Binding Assays for Analysisof Ion Channels （Georg CTerstappen）

8 Ion Channel Assays Based on Ion and Voltage-sensitive Fluorescent Probes

9 Approaches for Ion Channel Structural Studies （Randal BBass and Robert HSpencer）0 Molecular 10 Modeling and Simulations of Ion Channels: Applications to Potassium Channels

Acknowledgements

References

Subject Index