This research is aimed at understanding the subunit composition of the GABA(A)/benzodiazepine receptor complexes in different areas of the brain as well as in different cell types and domains within the same cell. Recent cloning studies have revealed a very high degree of heterogeneity in the GABA(A)/benzodiazepine receptor subunits. We will address the study of the subunit heterogeneity and subunit composition of the GABA(A)/benzodiazepine receptors at the peptide level. Our strategy is based on making and using subunit-specific monoclonal antibodies for the purification and characterization of various types of GABA(A)/ benzodiazepine receptors. Considerable effort will be invested in making subunit-specific monoclonal antibodies. The large intracellular loop of individual receptor subunits will be expressed in bacteria as fusion proteins which will be purified and used for immunizations. If necessary, subunit-specific synthetic peptides will also be used for immunizations. The purification of the various types of GABA(A)/ benzodiazepine receptors from several brain regions will be accomplished by immunoaffinity chromatography on immobilized subunit-specific antibodies. The subunit composition of the immunopurified receptors will be determined by immunoblotting with other subunit-specific antibodies as well as by biochemical methods. The radioligand binding specificities of the immunopurified receptors will also be determined aiming to establish relationships between subunit composition and ligand binding affinities. Immunocytochemistry with subunit-specific monoclonal antibodies will be used for studying the distribution of the GABA(A)/ benzodiazepine receptor subunits throughout the brain as well as for revealing their cellular and subcellular localization. Comparisons with receptor distribution in the brain revealed by radioligand autoradiography will also establish relationships between subunit composition and ligand binding affinities. These studies should be relevant for developing new benzodiazepines and other drugs that selectively interact with certain benzodiazepine receptor types and, therefore, have fewer side effects. In addition, they will help to understand in molecular terms the functional roles of GABA(A)/ benzodiazepine receptors both in mental health and disease including anxiety, epilepsy, sleep disorders and amnesia among others.
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