Potassium Channels: Methods and Protocols

300 Pages·2009·6.906 MB·English

by  Lezanne Ooi, Ian C. Wood (auth.), Jonathan D. Lippiat (eds.)

Potassium Channels
METHODS IN MOLECULAR BIOLOGY™
John M. Walker, SERIES EDITOR
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M M B ™
ETHODS IN OLECULAR IOLOGY
Potassium Channels
Methods and Protocols
Edited by
Jonathan D. Lippiat
Institute of Membrane and Systems Biology, Faculty of Biological Sciences
University of Leeds, Leeds, LS2 9JT, UK
Editor
Jonathan D. Lippiat
Institute of Membrane and Systems Biology
Faculty of Biological Sciences
University of Leeds
Leeds, LS2 9JT, UK
Series Editor
John M. Walker
School of life Sciences
University of Hertfordshire
Hatfield, Hertfordshire
AL10 9AB, UK
ISSN: 1064-3745 e-ISSN: 1940-6029
ISBN: 978-1-934115-65-7 e-ISBN: 978-1-59745-526-8
DOI: 10.1007/978-1-59745-526-8
Library of Congress Control Number: 2008931178
© Humana Press 2008, a part of Springer Science+Business Media, LLC
All rights reserved. This work may not be translated or copied in whole or in part without the written permission
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Cover illustration: Chapter 5, Figure 1
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Preface
Potassium channels are important regulators of membrane excitability, which in turn
determines cellular function. They form a super-family of ion channels that are regulated
by a diverse range of chemical and physical stimuli. In excess of 80 genes in the human
genome encode pore-forming potassium channel subunits. Diversity is further enhanced
by alternative splicing of subunit mRNA, heteromeric assembly between pore-forming
subunits, or by association with accessory proteins. The basis of several inherited diseases
lies in potassium channel gene mutations, and the pharmacological manipulation of
potassium channel function is increasingly important as a strategy in the treatment of
disease. This provides researchers with the challenge of developing tools and experimental
procedures to probe potassium channel function and identify chemicals that modulate
their behaviour.
We have progressed since the initial mid-twentieth century studies of delayed rectifier,
anomalous rectifier, and leak potassium currents, and now ask questions such as “What
does a potassium channel look like?”, “How do changes in membrane potential gate a
potassium channel?”, or “Which proteins assemble to form a potassium channel complex
in this particular cell?”. Many of these questions cannot be answered by electrophysi-
ological techniques alone, thus scientists have adopted techniques from other disciplines
to assist their studies.
This volume describes a range of experimental approaches that have been developed
to investigate potassium channel structure, function, pharmacology, cell biology, gene
expression, and their role in disease. Many of these techniques study potassium channels
as cellular proteins as well as the resultant membrane biophysics. They are proteins that are
synthesised according to mRNA sequence, trafficked to the correct location in the cell at the
appropriate time, interact with other protein components, change conformation following
stimulation, all in addition to providing a conduit for potassium ions to cross membranes.
Naturally, content covering the topic of ion channels would not be without electrophys-
iological techniques, but here they are focused on those that enable the study of intracellular
modulation, which is of particular importance to many potassium channels.
All investigators, including new researchers and discipline-hopping scientists, will
benefit from this volume. Within the various chapters you will also find protocols for
several standard laboratory techniques, such as cell culture, transfection, Xenopus oocyte
preparation, Western blotting, and whole-cell patch clamp recording. Indeed, the techniques
found in this book can be applied to the study of many other types of ion channel. There
is an increasing trend to answer scientific questions using a wide range of approaches and
it is hoped that the techniques outlined in this volume will help provide finishing touches
to research projects or provide new avenues of investigation.
Leeds, UK Jonathan D. Lippiat
v
Contents
Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix
PART I. METHODS FOR STUDYING POTASSIUM CHANNEL
TRANSCRIPTION AND MRNA SPLICING
1. Identifying Transcriptional Regulatory Regions Using Reporter Genes
and DNA–Protein Interactions by Chromatin Immunoprecipitation . . . . . . . . . 3
Lezanne Ooi and Ian C. Wood
2. Quantitative RT-PCR Methods for Investigation of Low Copy Potassium
Channel Gene Expression in Native Murine Arteries . . . . . . . . . . . . . . . . . . . . . 19
Alex Cheong, Samuel J. Fountain, and David J. Beech
3. Cloning of Potassium Channel Splice Variants from Tissues and Cells . . . . . . . . 35
Lie Chen and Michael J. Shipston
PART II. METHODS FOR STUDYING THE CELL BIOLOGY
OF POTASSIUM CHANNEL PROTEINS
4. Chemiluminescence Assays to Investigate Membrane Expression
and Clathrin-Mediated Endocytosis of K Channels . . . . . . . . . . . . . . . . . . . . 63
ATP
Andrew J. Smith and Asipu Sivaprasadarao
5. Investigation of K Channel Endocytosis by Immunofluorescence. . . . . . . . . . 69
ATP
Andrew J. Smith and Asipu Sivaprasadarao
6. Investigation of K Channel Endocytosis and Cell Surface Density
ATP
by Biotinylation and Western Blotting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
Andrew J. Smith and Asipu Sivaprasadarao
7. Lipid Microdomains and K+ Channel Compartmentation: Detergent
and Non-Detergent-Based Methods for the Isolation and Characterisation
of Cholesterol-Enriched Lipid Rafts. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91
Laura J. Sampson and Caroline Dart
8. Determination of Phosphoinositide Binding to K+ Channel Subunits
Using a Protein–Lipid Overlay Assay. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103
Alison M. Thomas and Andrew Tinker
9. Protein Complex Analysis of Native Brain Potassium Channels
by Proteomics. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113
Guillaume Sandoz and Florian Lesage
PART III. ELECTROPHYSIOLOGICAL TECHNIQUES FOR THE STUDY
OF POTASSIUM CHANNEL FUNCTION
10. Xenopus Oocytes as a Heterologous Expression System for Studying
Ion Channels with the Patch-Clamp Technique. . . . . . . . . . . . . . . . . . . . . . . . . 127
Paolo Tammaro, Kenju Shimomura, and Peter Proks
vii
viii Contents
11. Whole-Cell Recording Using the Perforated Patch Clamp Technique . . . . . . . . 141
Jonathan D. Lippiat
12. Recording the Activity of ATP-Sensitive K+ Channels in Open-Cell
Cell-Attached Configuration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151
Andrei I. Tarasov
13. Planar Patch Clamp: Advances in Electrophysiology. . . . . . . . . . . . . . . . . . . . . . 165
Andrea Brüggemann, Cecilia Farre, Claudia Haarmann, Ali Haythornthwaite,
Mohamed Kreir, Sonja Stoelzle, Michael George, and Niels Fertig
14. Analysing Steroid Modulation of BK Channels Reconstituted
Ca
into Planar Lipid Bilayers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177
Heidi de Wet, Jonathan D. Lippiat, and Marcus Allen
PART IV. OPTICAL TECHNIQUES FOR THE STUDY OF POTASSIUM
CHANNEL FUNCTION AND INTERACTIONS
15. Using Bioluminescence Resonance Energy Transfer to Measure
Ion Channel Assembly. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189
Gina M. Whitaker and Eric A. Accili
16. The Use of FRET Microscopy to Elucidate Steady State Channel
Conformational Rearrangements and G Protein Interaction
with the GIRK Channels. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199
Adi Raveh, Inbal Riven, and Eitan Reuveny
17. The Voltage-Clamp Fluorometry Technique . . . . . . . . . . . . . . . . . . . . . . . . . . . 213
Chris S. Gandhi and Riccardo Olcese
PART V. METHODS FOR STUDYING POTASSIUM CHANNELOPATHIES
AND PHARMACOLOGICAL MODULATORS
18. Identification of Mutations in the Kir6.2 Subunit of the K Channel. . . . . . . . 235
ATP
Sarah E. Flanagan and Sian Ellard
19. Modulation of Potassium Ion Channel Proteins Utilising Antibodies. . . . . . . . . 247
Mark L. Dallas, Susan A. Deuchars, and Jim Deuchars
20. Fluorescence-Based Tl+-Influx Assays as a Novel Approach
for Characterization of Small-Conductance Ca2+-Activated
K+ Channel Modulators. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 257
Susanne Jørgensen, Tina H. Johansen, and Tino Dyhring
21. Rubidium Efflux as a Tool for the Pharmacological Characterisation
of Compounds with BK Channel Opening Properties . . . . . . . . . . . . . . . . . . . . 267
Neil G. McKay, Robert W. Kirby, and Kim Lawson
22. Recording hERG Potassium Currents and Assessing the Effects
of Compounds Using the Whole-Cell Patch-Clamp Technique . . . . . . . . . . . . . 279
Ray M. Helliwell
Index. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 297
Contributors
ERIC A. ACCILI (cid:127) Department of Cellular and Physiological Sciences, University
of British Columbia, Vancouver, British Columbia, BC, Canada
DAVID J. BEECH (cid:127) Institute of Membrane and Systems Biology, Faculty of Biological
Sciences, University of Leeds, Leeds, UK
Andrea Brüggemann (cid:127) Nanion Technologies GmbH, Munich, Germany
LIE CHEN (cid:127) Centre for Integrative Physiology, College of Medicine and Veterinary
Medicine, University of Edinburgh, Edinburgh, UK
ALEX CHEONG (cid:127) Institute of Membrane and Systems Biology, Faculty of Biological
Sciences, University of Leeds, Leeds, UK
MARK L. DALLAS (cid:127) School of Medicine, University of Leeds, Leeds, UK
CAROLINE DART (cid:127) School of Biological Sciences, University of Liverpool, Liverpool, UK
JIM DEUCHARS (cid:127) Institute of Membrane and Systems Biology, Faculty of Biological
Sciences, University of Leeds, Leeds, UK
SUSAN A. DEUCHARS (cid:127) Institute of Membrane and Systems Biology, Faculty of Biological
Sciences, University of Leeds, Leeds, UK
TINO DYHRING (cid:127) NeuroSearch A/S, Ballerup, Denmark
SIAN ELLARD (cid:127) Institute of Biomedical and Clinical Science, Peninsula Medical
School, Exeter, UK
CECILIA FARRE (cid:127) Nanion Technologies GmbH, Munich, Germany
NIELS FERTIG (cid:127) Nanion Technologies GmbH, Munich, Germany
SARAH E. FLANAGAN (cid:127) Institute of Biomedical and Clinical Science, Peninsula Medical
School, Exeter, UK
SAMUEL J. FOUNTAIN (cid:127) Institute of Membrane and Systems Biology, Faculty of Biological
Sciences, University of Leeds, Leeds, UK
CHRIS S. GANDHI (cid:127) Division of Chemistry and Chemical Engineering, Howard Hughes
Medical Institute, California Institute of Technology, Pasadena, CA, USA
MICHAEL GEORGE (cid:127) Nanion Technologies GmbH, Munich, Germany
CLAUDIA HAARMANN (cid:127) Nanion Technologies GmbH, Munich, Germany
ALI HAYTHORNTHWAITE (cid:127) Nanion Technologies GmbH, Munich, Germany
RAY M. HELLIWELL (cid:127) Millipore UK, Cambridge, UK
TINA H. JOHANSEN (cid:127) NeuroSearch A/S, DK-2750 Ballerup, Denmark
SUSANNE JØRGENSEN (cid:127) NeuroSearch A/S, DK-2750 Ballerup, Denmark
ROBERT W. KIRBY (cid:127) Biomedical Research Centre, Faculty of Health and Wellbeing,
Sheffi eld Hallam University, Sheffi eld, UK
MOHAMED KREIR (cid:127) Nanion Technologies GmbH, Munich, Germany
ix
x Contributors
KIM LAWSON (cid:127) Biomedical Research Centre, Faculty of Health and Wellbeing,
Sheffi eld Hallam University, Sheffi eld, UK
FLORIAN LESAGE (cid:127) Institut de Pharmacologie Moléculaire et Cellulaire, CNRS,
06560 Valbonne Sophia-Antipolis, France
JONATHAN D. LIPPIAT (cid:127) Institute of Membrane and Systems Biology, Faculty
of Biological Sciences, University of Leeds, Leeds, UK
NEIL G. MCKAY (cid:127) Biomedical Research Centre, Faculty of Health and Wellbeing,
Sheffi eld Hallam University, Sheffi eld, UK
PETER PROKS (cid:127) Department of Physiology, Anatomy and Genetics, University
of Oxford, Oxford, UK
RICCARDO OLCESE (cid:127) Division of Molecular Medicine, Department of Anesthesiology,
David Geffen School of Medicine at University of California, Los Angeles, CA, USA
LEZANNE OOI (cid:127) Institute of Membrane and Systems Biology, Faculty of Biological
Sciences, University of Leeds, Leeds, UK
ADI RAVEH (cid:127) Department of Biological Chemistry, Weizmann Institute of Science,
Rehovot, Israel
EITAN REUVENY (cid:127) Department of Biological Chemistry, Weizmann Institute
of Science, Rehovot, Israel
INBAL RIVEN (cid:127) Department of Physiology and Pharmacology, Sackler School
of Medicine, Tel Aviv University, Tel Aviv, Israel
LAURA J. SAMPSON (cid:127) Department of Cell Physiology and Pharmacology,
University of Leicester, UK
GUILLAUME SANDOZ (cid:127) Institut de Pharmacologie Moléculaire et Cellulaire,
CNRS, 06560 Valbonne Sophia-Antipolis, France
KENJU SHIMOMURA (cid:127) Department of Physiology, Anatomy and Genetics,
University of Oxford, Oxford, UK
MICHAEL J. SHIPSTON (cid:127) Centre for Integrative Physiology, College of Medicine
and Veterinary Medicine, University of Edinburgh, Edinburgh, UK
ASIPU SIVAPRASADARAO (cid:127) Institute of Membrane and Systems Biology, Faculty
of Biological Sciences, University of Leeds, Leeds, UK
ANDREW J. SMITH (cid:127) Institute of Membrane and Systems Biology, Faculty of Biological
Sciences, University of Leeds, Leeds, UK
SONJA STOELZLE (cid:127) Nanion Technologies GmbH, Munich, Germany
PAOLO TAMMARO (cid:127) Department of Physiology, Anatomy and Genetics,
University of Oxford, Oxford, UK
ANDREI I. TARASOV (cid:127) Section of Cell Biology, Division of Medicine, Faculty
of Medicine, Imperial College London, London, UK
ALISON M. THOMAS (cid:127) Department of Medicine, University College London, London, UK
ANDREW TINKER (cid:127) Department of Medicine, University College London, London, UK
GINA M. WHITAKER (cid:127) Department of Cellular and Physiological Sciences, University
of British Columbia, Vancouver, British Columbia, BC, Canada
IAN C. WOOD (cid:127) Institute of Membrane and Systems Biology, Faculty of Biological
Sciences, University of Leeds, Leeds, UK
Part I
Methods for Studying Potassium Channel Transcription
and mRNA Splicing