Expression and Analysis of Recombinant Ion Channels

Abstract

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 Index.