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

Hardcover

ISBN 10: 3527312099 ISBN 13: 9783527312092

Publisher: Wiley-VCH, 2006

Synopsis

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.

About this title

About the Author

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.

From the Back Cover

Aimed at filling the gap created by recent technical development sin several key areas, this book is directed at both the new and the experienced researcher wishing to learn more about the considerations and methods for studying recombinant ion channels. Covering many of these new techniques for the first time, the book contains contributions from authors who routinely apply these approaches in key academic labs or major pharmaceutical companies.

Rather than describing detailed protocols, to which the reader is directed by key references in each chapter, the authors instead provide a comprehensive and easily accessible overview of the techniques involved, reviewing underlying principles and 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 results is a compendium of practical ion channel information that will prove a valuable resource to academic and industrial researchers alike.

Rather than describing detailed protocols, to which the reader is directed by key references in each chapter, the authors instead provide a comprehensive and easily accessible overview of the techniques involved, reviewing underlying principles and 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 results is a compendium of practical ion channel information that will prove a valuable resource to academic and industrial researchers alike.

Excerpt. © Reprinted by permission. All rights reserved.

Expression and Analysis of Recombinant Ion Channels

John Wiley & Sons

Copyright © 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
All right reserved.
ISBN: 978-3-527-31209-2

Chapter One

Expression of Ion Channels in Xenopus Oocytes

Alan L. Goldin

1.1 Introduction

Xenopus oocytes have been widely used for studying ion channels in a controlled in vivo environment since the system was initially developed for this purpose by Miledi and coworkers. There have been at least five major types of studies using oocytes to examine ion channel expression. The earliest use was to examine the properties of specific ion channels in a living cell free from other responses. The oocytes were injected with RNA isolated from whole brains, and the responses were analyzed using the two-microelectrode whole cell voltage-clamp, the patch clamp, or a variety of biochemical techniques [5, 6]. Once the responses were isolated, Xenopus oocytes were then used in a second type of study as an assay system to isolate cDNA clones encoding the proteins involved. For example, cDNA clones encoding the 5-HT_1C receptor were isolated using electrophysiological assays, both by hybrid depletion and by directly transcribing RNA from a cDNA library and injecting the transcripts into oocytes. These types of studies are much less commonly used now because of the large number of available heterologous expression systems and cDNA clones encoding ion channels.

The third major type of study for which the Xenopus oocyte expression system has been, and continues to be, particularly useful is the correlation of molecular structure with electrophysiological function of a specific channel. The two basic approaches have been to construct defined mutations whose effects are determined by expression in oocytes, and to construct chimeric molecules between two closely related channels or receptors followed by expression in oocytes and electrophysiological analysis. These types of approach are still commonly used but with more sophisticated structural alterations and functional analyses.

The fourth general approach utilizing expression in oocytes is to determine the functional effects of mutations that cause human diseases. These types of studies are also performed using expression in other heterologous systems such as mammalian cells, and the advantages and disadvantages of each approach will be discussed in Section 1.5.3.

The final approach that takes advantage of expression in Xenopus oocytes is to screen potential drugs to determine their relative efficacies against specific types of ion channels. These studies have been made feasible by the development of automated voltage-clamp devices. Two such devices are the Roboocyte from Multichannel Systems and the OpusXpress from Axon Instruments, which is now part of Molecular Devices. The features of these instruments are described in Section 1.4.3.

1.2 Advantages and Disadvantages of Xenopus Oocytes

While the Xenopus oocyte system is a valuable tool for the study of ion channel function, there are a number of important factors to consider in deciding whether oocytes are the most appropriate system to use. One of the primary advantages of oocytes is that these cells do not express a large number of ion channels and receptors, so that the exogenous protein can be studied without contamination from endogenous channels. This advantage is not true in all cases, however, as oocytes do express some channels and receptors. These responses are not usually a problem for two reasons. First, only some oocytes express the channels, so that it is frequently possible to obtain oocytes that do not have endogenous current. Second, current from the injected RNA is usually much larger than that through the endogenous channels, so that it is generally possible to record the expressed current without significant contamination from native oocyte currents. In some cases, the presence of an endogenous response can be used to advantage as a second messenger system that is coupled to the initial response that is being studied.

Another advantage is that some channels can only be expressed in oocytes and not in mammalian cells. It is not possible to predict which channels fall into this category, and success using mammalian cells often depends very strongly on choosing the appropriate cell type (see Chapter 4). Even when the channels can be expressed in other systems, oocytes may still be advantageous for examination of the roles of different subunits. Since expression in oocytes involves injection of RNA, it is possible to adjust the ratio of RNA encoding each subunit and thus examine channels with a relatively well controlled composition. In contrast, it is more difficult to control the ratios of different subunits expressed by transfection in mammalian cells.

There are also some advantages to the use of oocytes with respect to electrophysiological recording. The oocyte system is particularly well suited for the study of many different mutations because injection and two-electrode voltage-clamping can be carried out rapidly and in a semi-automated fashion. In addition, studies involving modulation by second messenger systems such as phosphorylation are particularly well suited to oocytes because it is possible to express multiple proteins in the same cell, and modulators can be injected while recording with the two-electrode voltage-clamp. Finally, some analysis techniques are unique to oocytes. For example, the cut-open oocyte voltage-clamp was developed specifically for high resolution electrophysiological recording from oocytes and it is particularly well-suited for analyzing fast ionic and gating currents.

There are a number of disadvantages to using oocytes for expression of ion channels. First, as pointed out above, oocytes do express some endogenous channels and receptors. A second major disadvantage is that it has not been possible to express every channel in oocytes. On the other hand, most channels that have not been expressed in oocytes have not been expressed in other heterologous systems either, so that this problem is not unique to the use of oocytes.

Another disadvantage of oocytes is that many pharmacological agents are less potent on channels in oocytes compared to the channels in mammalian cells or native tissues. This difference in potency most likely reflects decreased accessibility of the drug because of the large number of invaginations in the oocyte membrane, the vitelline membrane surrounding the oocyte surface, or the follicle cells around the oocyte. However, although the absolute concentration of drug that is required for block is often higher than that required in native tissues, the relative efficacies of drugs against different channels are generally representative of those in native tissues. Other potential disadvantages with the use of oocytes include the need for procedures and equipment beyond that usually found in a standard research laboratory, occasional wide variations in quality due to seasonal and other factors, and the fact that they are best maintained at ambient temperature which may lead to altered synthesis and processing of mammalian channels compared to physiological conditions.

The most serious disadvantage of using expression in oocytes as an assay system is that the cells are not the native cells in which the channels are normally expressed. This can be reflected in two major ways. First, the functional properties that are observed may not be identical to those characterized in native tissues, although it is often difficult to make a direct comparison because the native cell usually contains multiple different types of channels. In addition, the functional properties may depend on the subunit composition, in which case the oocyte system can be used to determine which subunits are required for properties similar to those observed in vivo. The second consequence of oocytes not being the native tissue is that cellular trafficking is different, particularly in comparison to neurons. Because of this difference, some channels may not be expressed on the cell surface in oocytes and the effects of mutations that affect trafficking cannot be studied at all.

1.3 Procedures for Using Oocytes

The procedures for maintenance of Xenopus laevis, preparation of oocytes and injection with mRNA have been previously described. In addition, details concerning the maintenance of Xenopus laevis and the use of oocytes can be obtained from the Xenopus Express website (http://www.xenopus.com/links.htm).

A frog colony does not require elaborate equipment, although there are two important considerations. First, amphibians are sensitive to both chlorine and chloramine, so the water must be purified to remove both compounds. Second, although frogs can tolerate a wide range of temperature fluctuations, inconsistent temperature, and particularly elevated temperatures above 20 °C, greatly diminish oocyte viability.

Surgery to remove oocytes is a relatively simple procedure that can be carried out on a bench top in a clean room. After preparation, the follicle cells are usually removed by treatment with collagenase, although oocytes can be injected and voltage-clamped with intact follicle cells around them. Some electrophysiological responses in oocytes either depend on the presence of follicle cells or occur in the follicle cells, which would be a reason to maintain the cells. However, all procedures are technically more difficult because the follicle cells are harder to pierce and it is time-consuming to separate out individual oocytes, so it is usually best to use defolliculated oocytes.

It is possible to obtain oocytes that have been surgically removed and prepared for injection by commercial vendors. The advantages of this approach are that there is no need to maintain a frog colony, an animal use protocol is not required because no vertebrate animals will be used, and there is a considerable saving in time. The disadvantages are that the oocytes are significantly more expensive and they are only available in specific geographical areas, although the number of vendors may increase, depending on demand. One commercial source for oocytes is EcoCyte Bioscience in Germany (http://www.ecocyte.de).

For most studies, oocytes are injected with RNA that has been transcribed in vitro from a cDNA clone using either a T7 or T3 promoter. Transcription is easily performed using commercially available kits, although there are some important considerations that affect expression levels. First, the RNA needs to be capped for optimal efficiency, which is part of the procedure in most kits. Second, inclusion of Xenopus ß-globin 5' and 3' untranslated mRNA regions and a poly(A) tail at the 3' end usually enhances stability and translatability. Third, the length of the untranslated 5' region can have a dramatic effect on the level of current, with shorter regions generally resulting in greater efficiency of expression.

It is also possible to inject DNA into the nucleus of oocytes. In this case, the cDNA should be cloned downstream of a eukaryotic promoter such as the commonly used Cytomegalovirus (CMV) promoter. The advantage of injecting DNA is that there is no need to perform in vitro transcription reactions, which saves both time and money. The disadvantage is that the procedure is more difficult and requires a more sophisticated injection apparatus.

Cytoplasmic injection is a relatively rapid and easy procedure. The basic requirements are a dissecting microscope, a micromanipulator, and an injector that can be a simple manual or motor driven dispenser (Fig. 1.1). Alternatively, oocytes can be injected using an automated device such as the Roboocyte from Multichannel Systems, as described in Section 1.4.3.1. This instrument was developed for automated two-electrode voltage-clamping of oocytes, but it can also function for both cytoplasmic and nuclear injection of oocytes.

1.4 Types of Analyses

1.4.1 Electrophysiological Analysis

The most sensitive approach for analyzing ion channel function in Xenopus oocytes is the use of electrophysiology. Essentially all of the standard electrophysiological techniques can be performed on oocytes, including whole cell recording and patch-clamp recording of both macroscopic and single channel currents. In addition, techniques such as cut-open oocyte voltage-clamp recording have been developed specifically for analyzing currents in Xenopus oocytes. The goal of this chapter is to present the general considerations involved in using the various approaches, as detailed procedures have been previously described.

1.4.1.1 Two-electrode Whole Cell Voltage-clamp

Whole cell voltage-clamping of oocytes involves using two electrodes inserted into the oocyte, rather than using one electrode to make a patch on the surface followed by rupturing the membrane, as is done in mammalian cells. One electrode is used to measure the internal potential of the oocyte and the other electrode is used to inject current (Fig. 1.2). The large size of the oocyte (about 1 mm in diameter and 0.5–1 µl in volume for stage V oocytes) makes this feasible, and is both the major advantage and disadvantage of the system. One advantage is that the procedure is easy to learn and fast to perform. The electrodes are simple to prepare and it is generally possible to obtain records from all oocytes if they are healthy, so that there is very little time lost in preparation. In addition, perfusion of the external medium can be easily changed multiple times. These features make the two-electrode voltage-clamp ideal for screening purposes. A second advantage is that the recordings can be stable over long periods of time, which makes it particularly useful for analyzing properties that require long protocols, such as slow inactivation. A third advantage is that it is possible to insert multiple electrodes and injection needles into the same oocyte. Therefore, modulators of channel function can be injected inside the cell while recording, so that a rapid and direct response to an intracellular signal can be observed. The final advantage of the two-electrode voltage-clamp is that it records currents through channels present in the whole cell, so that it is very sensitive. For example, currents as small as 50 nA can be detected, which corresponds to only 5 x 10⁴ molecules if the single channel current is 1 pA. The two-electrode voltage-clamp can be used to record currents over a wide range of amplitudes from about 10 nA to over 100 µA, depending on the amount of RNA that is injected. However, it is important to adjust the amount of RNA to obtain currents that can be reliably clamped because it is difficult to accurately clamp the membrane potential of the oocyte if the currents are larger than 5 µA.

The major disadvantage of recording from the entire oocyte is that the large size and extensive membrane invaginations result in an extremely large membrane capacitance, approximately 150200 nF. The large capacitance causes a slow clamp settling time when the membrane potential is changed. The capacity transient can be minimized by using electrodes with low impedances of 500 kΩ or less. This can be accomplished by filling the electrode tips with low-melting temperature agarose, making it possible to have a large tip opening without significant leakage of KCl into the oocyte. However, even with the best electrodes it is difficult to obtain data during the first 1–2 ms of a depolarization, which is the time during which rapidly activating voltage-gated channels such as sodium channels are activated (Fig. 1.3). The large capacity transient is not a problem when recording slow responses or ligand-gated currents that do not require changes in voltage. A second major disadvantage is that there is no control of the internal cellular environment, so that it is difficult to perform quantitative studies, for example examining selective permeability.

1.4.1.2 Cut-open Oocyte Voltage-clamp

The cut-open oocyte voltage-clamp was developed to circumvent many of the disadvantages involved in using the two-electrode whole cell voltage-clamp. In this procedure, the oocyte is inserted in a chamber that separates the surface into three regions (Fig. 1.4). The top portion of the oocyte membrane is the region that is clamped and is the section from which currents are actually recorded. The middle portion is a guard that is clamped to the same potential as the top to null leakage currents though the seals. The bottom portion is the region of the oocyte that is "cut-open", either by permeabilization with saponin or by insertion of a cannula, thus making it possible to perfuse the internal environment and to inject current intracellularly through a low resistance pathway. The internal environment is clamped to ground, as measured by a low resistance electrode inserted through the top of the oocyte, which ensures that the region of the oocyte near the top portion is accurately held at ground. The bath surrounding the top portion of the oocyte is clamped to the command potential, which can be rapidly changed with minimal series resistance. Currents are recorded through low resistance electrodes in the top chamber.

(Continues...)

Excerpted from Expression and Analysis of Recombinant Ion Channels Copyright © 2006 by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. Excerpted by permission of John Wiley & Sons. All rights reserved. No part of this excerpt may be reproduced or reprinted without permission in writing from the publisher.
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Edition details

• Publisher: Wiley-VCH
• Publication date: 2006
• Language: English
• ISBN 10: 3527312099
• ISBN 13: 9783527312092
• Binding: Hardcover
• Edition number: 1
• Number of pages: 302
• Editor: Clare Jeffrey J., Trezise Derek J.