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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.

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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.

Aus dem Klappentext

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.

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Expression and Analysis of Recombinant Ion Channels

John Wiley & Sons

Chapter One

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...