This project will study the structure and function of the receptor protein for the major brain inhibitory neurotransmitter gamma-aminobutyric acid (GABA). The GABA-A receptor complex is a ligand-gated chloride channel which is also the site of action of many important anticonvulsant, anxiolytic, and anesthetic drugs. The GABA-A receptor is actually a family of receptors, differing in gene sequence, subunit composition, pharmacological properties, and brain regional distribution. We can purify the receptor protein in large quantities by affinity chromatography. We will produce subunit-, subtype-, and functional domain-specific antibodies against synthetic peptides in the subunit sequences derived from cloning. Different subunits detected by gel electrophoresis of the purified receptor will be matched with different cDNA clones using clone-specific antibodies in Western blots and comparing regional location. The pharmacological specificity of ligand binding to GABA, benzodiazepine, barbiturate, and convulsant sites on the isolated subunits detected by photoaffinity labeling will be matched with brain regional ligand binding detected by autoradiography. This will allow us to determine the subunit composition, pharmacological properties, and cellular location of the receptor subtypes. The importance of receptor subtypes is that they vary in their function, susceptibility to disease processes, and sensitivity to pharmacological intervention. Domain-specific antibodies and active site sequencing of photolabeled protein will define functional domains within the protein structure. The purified protein will be reconstituted into membrane vesicles to study functional regulation, including phosphorylation by protein kinase C, which we have demonstrated in vitro on a specific A subunit. The information gained will be valuable in the understanding of normal and diseased brain function. Human disorders of particular relevance include epilepsy, anxiety, alcohol dependence, and sleep disorders.
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