Picrotoxin is a non-competitive antagonist of the GABAa receptor. The use dependent nature of this compound suggests that the binding site is located within the pore region. Mutagenesis studies have identified a number of residues, all within TM2, that when mutated affect picrotoxin inhibition. In particular Val at the 2’ position (Xu et a l, 1995), Thr at the 6’ position (Gurley et a l, 1995; Xu et a l, 1995) and Leu at the 9’ position (Chang & Weiss, 1999).
1.7. Native verses recombinant GABAa receptors.
Single channel characteristics of native GABAa receptors have revealed that these channels exhibit multiple conductance levels including 12, 17-20 and 27-30 pS (Bormann et a l, 1987; Macdonald et a l, 1989; Macdonald & Olsen, 1994). The major level, accounting for approximately 95% of the current, is the 27-30 pS conductance level. Similarly the single channel characteristics of a number of recombinant receptors expressed in a variety of cells have been reported (Levitan et a l, 1988a; Verdoom et a l, 1990; Herb et a l, 1992; Angelotti et a l, 1993; Saxena & Macdonald, 1994). In general, recombinant receptors display similar conductance levels to native receptors, however differences in the most abundant conductance level are reported.
Studies with G A BAareceptor modulators have demonstrated clear differences in their mechanisms of action at the single channel level. For example the competitive antagonist bicuculline reduces the frequency and mean duration of GABA-induced single channel events (Macdonald et a l, 1989). Picrotoxin and benzodiazepine inverse agonists reduce the channel opening frequency (Newland & Cull-Candy, 1992; Porter et a l, 1992; Rogers et a l, 1994), whereas benzodiazepine agonists
Chapter 1 General Introduction
increase the channel opening frequency (Macdonald & Twyman, 1992; Macdonald & Olsen, 1994). Unlike benzodiazepines, barbiturates increase the mean open time to exogenously applied GABA with no effect on the single-channel conductances or frequency of openings (Study & Barker, 1981; Macdonald et al., 1989; Twyman et a l, 1989). More recent studies however have shown that in addition to increasing channel activity benzodiazepines and pentobarbitone also increase channel conductance while bicuculline has been demonstrated to reduce channel conductance (Eghbali et at., 1997; Bimir et a l, 2000a; Eghbali et a l, 2000).
Recombinant receptors display very similar pharmacological characteristics to native GAB A A receptors and therefore represent a powerful tool with which to characterise the pharmacological properties of GABAareceptors. Significant progress has been made over the last 25 years in understanding the pharmacological properties and the structure of recombinant G A BAareceptors. The challenge for the future however is to link receptor subtype with physiological function and to the develop therapeutic molecules which target specific receptor subtypes. This goal will require the input of many disciplines including electrophysiology, development of transgenic animals, single cell PCR etc. Two recent reports by Rudolph et al. (1999) and McKeman et al. (2000) illustrate that these goals are achievable. Both groups developed genetically modified mice in which histidine 101 had been mutated to arginine within the a l subunit resulting in a mouse in which the a l receptor subtype was insensitive to benzodiazepines. In addition, McKeman et al. (2000) synthesised a benzodiazepine site ligand that selectively potentiated a2P3y2s, a3P3y2s and a5P3y2s receptors but not aip3y2s receptors. Given these tools both groups were able to conclude that the a l subtype was responsible for the sedative but not the anxiolytic effects of benzodiazepines.
1.8. The Xenopus oocyte expression system.
The Xenopus laevis oocyte was first introduced as an in vitro system for the transient expression of foreign mRNA for haemoglobin and mouse myeloma protein by
Chapter 1 General Introduction
Advantages of using cDNA over mRNA are that it is less labile than mRNA and easier to handle. Other advantages with Xenopus oocytes are that they are large robust cells (up to 1.5mm in diameter) which makes isolation and penetration of the oocyte with fine injection needles and microelectrodes relatively easy. A disadvantage of the large size of the cell is that drug application times are slow. A second disadvantage of the Xenopus oocyte is that the cell originates from an amphibian and consequently may perform different post-translational modifications to proteins as well as containing different phosphorylase / kinase enzymes to a mammalian cell. It must also be remembered that although Xenopus oocytes do not contain genes encoding GABAa receptors, they do express endogenous cell surface receptors such as muscarinic acetylcholine receptors (Kusano et al., 1977), voltage-gated ion channels such as the Ca^^-dependent chloride channel (Miledi, 1982) and stretch-activated channels (Yang & Sachs, 1989). Overall however the Xenopus oocyte expression system represents a very useful robust technique which has been used throughout this study.
1.9. Aims and Objectives.
A broad aim for this study is to enhance our pharmacological knowledge of recombinant GABAa receptors. The studies within this thesis will either examine the pharmacological properties of novel GABAa receptor subunits and mutated subunits, or characterise, in detail, the pharmacology of specific compounds that interact with GABAa receptors. This will be achieved by implementing the two-electrode voltage- dam p technique on Xenopus oocytes expressing wild type or mutated human recombinant GABAa receptors.
Chapter 2
Chapter 2 Methods
2.1. M olecular biology.
The wild type and mutant cDNAs that were used in this project were all generated by m em bers o f the M olecular Biology Departm ent, headed by Dr. P. J. W hiting, at M erck Sharp & Dohme. Standard cloning and site directed m utagenesis techniques were em ployed (W hiting et a l, 1995b; Sambrook & Russell, 2001) and the resulting cDNAs cloned into the eukaryotic expression vectors pCDMS or p cD N A l.lA m p , which when introduced into Xenopus oocytes behave essentially the same, how ever see C hapter 3.
Figure 2.1. Pictorial representation of a GABAa receptor cDNA inserted in a eukaryotic expression vector.