Patch clamp and molecular genetic techniques will be combined to study structure-function relationships in the Drosophila GABA-A receptor. The recent cloning of the GABA-A receptor of Drosophila provides an opportunity to carry out molecular genetic modifications of the receptor. The functional consequences of these structural alterations will be examined in expression systems such as Xenopus oocytes and transfected cells, and in a native cellular environment provided by cell culture of the Drosophila nervous system. In addition, Drosophila provides a powerful method of isolating mutants based on selection in terms of function, rather than site-directed mutagenesis. These advantages will be exploited to investigate the following basic problems relevant to the function of ligand-gated channels. We will use site-directed mutagenesis to determine the residues responsible for the anion selectivity of this channel. This information will then be exploited to elucidate the number of subunits in the native receptor and the number of copies of each cloned subunit in the native receptor. Ultimately, this work will lead to an understanding of the subunit stoichiometry of the GABA-A receptor of Drosophila. There is already some evidence to suggest that this receptor is a homomultimer, and if this is actually the case it would have considerable evolutionary and mechanistic significance. Proposed experiments will provide a clear test for this hypothesis. Along a different line, experiments will exploit mutants with reduced sensitivity to the classical GA BAA receptor antagonist picrotoxin. These mutants were isolated in fields sprayed with cyclodiene insecticides. Experiments proposed with these and other mutants will determine whether picrotoxin blocks by an allosteric mechanism, or by blockade of the open channel, or by both of these mechanisms. These experiments will also identify the residues of the GABA-A receptor that form the picrotoxin binding site. The information about the GABA-A receptor obtained in these studies will help researchers understand the nature of inhibition throughout the nervous system. Researchers will be able to develop better drugs in the treatment of epilepsy and many other neural disorders. The insight that will result from this study will be of general value in the development of drugs that act at other receptors in addition to the GABA-A receptor.
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