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

by Clare, Jeffrey J.; Trezise, Derek J.

• ISBN13: 9783527312092
• ISBN10: 3527312099
• Edition: 1st
• Format: Hardcover
• Copyright: 2006-03-10
• Publisher: Wiley-VCH

Summary

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.

Author Biography

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 receptors.

Table of Contents

Preface	xi
List of Contributors	xiii
Color Plates	xvii
Expression of Ion Channels in Xenopus Oocytes	1	(26)
Alan L. Goldin
Introduction	1	(1)
Advantages and Disadvantages of Xenopus Oocytes	2	(1)
Procedures for Using Oocytes	3	(2)
Types of Analyses	5	(12)
Electrophysiological Analysis	5	(1)
Two-electrode Whole Cell Voltage-clamp	5	(2)
Cut-open Oocyte Voltage-clamp	7	(2)
Macropatch Clamp	9	(2)
Single Channel Analysis	11	(1)
Biochemical Analysis	12	(1)
Compound Screening	13	(1)
Serial Recording Using the Roboocyte	14	(2)
Parallel Recording Using the OpusXpress	16	(1)
Examples of Use	17	(4)
Characterization of cDNA Clones for a Channel	17	(1)
Structure-Function Correlations	18	(1)
Studies of Human Disease Mutations	19	(2)
Conclusions	21	(6)
Acknowledgments	21	(1)
References	21	(6)
Molecular Biology Techniques for Structure -- Function Studies of Ion Channels	27	(32)
Louisa Stevens
Andrew J. Powell
Dennis Wray
Introduction	27	(1)
Methods for cDNA Subcloning	28	(8)
Conventional Sub-cloning Using Restriction Enzymes and DNA Ligase	28	(3)
PCR-based cDNA Sub-cloning	31	(2)
Sub-cloning cDNA through Site-specific Recombination	33	(3)
Generation of Chimeric Channel cDNAs	36	(7)
Use of Restriction Enzymes to Generate Chimeric Channel cDNAs	36	(3)
PCR-mediated Overlap Extension for Chimera Generation	39	(4)
PCR-mediated Integration or Replacement of cDNA Fragments	43	(1)
Site-directed Mutagenesis	43	(7)
Examples of the Use of Site-directed Mutagenesis	45	(5)
Modification of the QuikChange Method for the Replacement of cDNA Fragments	50	(1)
Epitope-tagged Channels and Fusion Partners	50	(2)
Channel Subunit Concatamers	52	(1)
Concluding Remarks	53	(6)
References	54	(5)
Unnatural Amino Acids as Probes of Ion Channel Structure -- Function and Pharmacology	59	(20)
Paul B. Bennett
Niki Zacharias
John B. Nicholas
Sue Dee Sahba
Ashutosh Kulkarni
Mark Nowak
Introduction	59	(1)
Unnatural Amino Acid Mutagenesis Methodology	60	(4)
Unnatural Amino Acid Mutagenesis for Ion Channel Studies	64	(1)
Structure-Function Example Studies	65	(7)
Nicotinic Acetylcholine Receptor	65	(2)
Drug Interactions with the hERG Voltage-gated Potassium Ion Channel	67	(5)
Other Uses of Unnatural Amino Acids as Probes of Protein Structure and Function	72	(1)
Conclusions	73	(6)
Acknowledgements	74	(1)
References	74	(5)
Functional Expression of Ion Channels in Mammalian Systems	79	(32)
Jeff J. Clare
Introduction	79	(1)
cDNA Cloning and Manipulation	80	(1)
Choice of Host Cell Background	81	(4)
Post-translational Processing of Heterologous Expressed Ion Channels	85	(5)
Cytotoxicity	90	(1)
Transient Expression Systems	91	(5)
``Standard'' Transient Expression	91	(1)
Viral Expression Systems	92	(4)
Stable Expression of Ion Channels	96	(7)
Bicistronic Expression Systems	96	(4)
Stable Expression of Multiple Subunits	100	(1)
Inducible Expression	101	(2)
Summary	103	(8)
Acknowledgements	103	(1)
References	104	(7)
Analysis of Electrophysiological Data	111	(34)
Michael Pusch
Overview	111	(1)
Introduction	111	(2)
Expression Systems and Related Recording Techniques	113	(4)
Expression in Xenopus Oocytes	113	(2)
Expression in Mammalian Cells	115	(1)
Leak and Capacitance Subtraction	116	(1)
Macroscopic Recordings	117	(19)
Analysis of Pore Properties -- Permeation	118	(3)
Analysis of Fast Voltage-dependent Block -- the Woodhull Model	121	(1)
Information on Gating Properties from Macroscopic Measurements	122	(2)
Equilibrium Properties -- Voltage-gated Channels	124	(2)
Equilibrium Properties -- Ligand Gated Channels	126	(3)
Macroscopic Kinetics	129	(3)
Channel Block	132	(1)
Nonstationary Noise Analysis	133	(2)
Gating Current Measurements in Voltage Gated Channels	135	(1)
Single Channel Analysis	136	(6)
Amplitude Histogram Analysis	136	(2)
Kinetic Single Channel Analysis	138	(4)
Summary	142	(3)
Acknowledgements	142	(1)
References	142	(3)
Automated Planar Array Electrophysiology for Ion Channel Research	145	(20)
Derek J Trezise
Introduction	145	(1)
Overview of Planar Array Recording	145	(2)
Experimental Methods and Design	147	(11)
Cell Preparation	148	(1)
Cell Sealing and Recording	149	(3)
Drug Application	152	(3)
Experimental Design and Data Analysis	155	(3)
Overall Success Rates and Throughput	158	(1)
Population Patch Clamp	159	(3)
Summary and Perspective	162	(3)
Acknowledgements	162	(1)
References	162	(3)
Ion Flux and Ligand Binding Assays for Analysisof Ion Channels	165	(22)
Georg C. Terstappen
Introduction	165	(1)
Ion Flux Assays	166	(9)
Radioactive Ion Flux Assays	167	(1)
Nonradioactive Ion Flux Assays based on Atomic Absorption Spectrometry	168	(1)
Nonradioactive Rubidium Efflux Assay	168	(6)
Nonradioactive Lithium Influx Assay	174	(1)
Nonradioactive Chloride Influx Assay	174	(1)
Conclusions	174	(1)
Ligand Binding Assays	175	(12)
Heterogeneous Binding Assays Employing Radioligands	177	(1)
Homogeneous Binding Assays Employing Radioligands	178	(2)
Homogeneous Binding Assays Employing Fluorescent-Labeled Ligands and Fluorescence Polarization	180	(1)
Conclusions	181	(1)
Acknowledgements	182	(1)
References	182	(5)
Ion Channel Assays Based on Ion and Voltage-sensitive Fluorescent Probes	187	(26)
Jesus E. Gonzalez
Jennings Worley
Fredrick Van Goor
Introduction	187	(1)
Membrane Potential Probes	188	(6)
Redistribution Probes	188	(2)
FRET Probes	190	(2)
Advantages and Limitations of Membrane Potential Probes	192	(2)
Ion-sensitive Fluorescent Probes	194	(2)
Calcium Dyes	194	(1)
Indicators of Other Ions	195	(1)
Fluorescence Assays for Ion Channels	196	(9)
Calcium Channels	196	(1)
Non-voltage-gated Calcium Permeable Channels	197	(3)
Sodium Channels	200	(1)
Potassium Channels	201	(2)
Chloride Channels	203	(2)
Assays for Monitoring Channel Trafficking	205	(2)
Summary	207	(6)
References	208	(5)
Approaches for Ion Channel Structural Studies	213	(28)
Randal B. Bass
Robert H. Spencer
Introduction	213	(3)
Expression of Membrane Proteins for Structural Studies	216	(3)
Mammalian Expression	216	(1)
Insect Expression	217	(1)
Yeast Expression	217	(1)
Bacterial Expression	218	(1)
The Detergent Factor	219	(4)
Purification	223	(4)
Crystallization	227	(2)
Use of Antibody Fragments	229	(1)
Generation of First Diffraction Datasets	230	(2)
Selenomethionine Phasing of Membrane Proteins	232	(1)
MAD Phasing and Edge Scanning	233	(1)
Negative B- factor Application (Structure Factor Sharpening)	234	(1)
Conclusions	235	(6)
References	235	(6)
Molecular Modeling and Simulations of Ion Channels: Applications to Potassium Channels	241	(28)
Daniele Bemporad
Alessandro Grottesi
Shozeb Haider
Zara A. Sands
Mark S.P. Sansom
Introduction	241	(1)
Computational Methods	242	(4)
Kir Channels	246	(8)
Structures	246	(1)
Molecular Modeling	247	(1)
Simulations	248	(1)
Filter Flexibility	248	(2)
M2 Helices and Hinge Motion	250	(1)
Intracellular Domain Dynamics	251	(1)
Interactions with Ligands	251	(2)
Towards an Integrated Gating Model	253	(1)
Kv Channels	254	(7)
Structures	254	(2)
S6 Helices, Hinges and Gating	256	(1)
The Barrier at the Gate	257	(1)
The Nature of the Voltage Sensor	258	(2)
A Possible Gating Model	260	(1)
Summary and Future Directions	261	(8)
Acknowledgements	262	(1)
References	262	(7)
Subject Index	269