This application focuses upon the purification and characterization of the complex of proteins involved in the psychopharmacological actions of the benzodiazepines. Using a benzodiazepine (3-aminoclonazepam), which can be coupled to a solid matrix, a method for the purification of the benzodiazepine receptor protein is being developed. Along with the benzodiazepine receptor, it is possible to copurify both high and intermediate affinity binding sites for Gamma-aminobutyric acid. The kinetics, stoichiometry and molecular properties of these sites are investigated. Monoclonal antibodies against the purified complex are being prepared and several methods for detecting different properties of these antibodies are being developed. The physical locus of action of these antibodies will be compared to their pharmacological activities with the goal of defining the proximity of the sites of action of various drugs. The purified proteins will be reconstituted into a model system to directly study their action. It is expected that by a careful examination of the biochemical parameters of receptor function, greater reliability in predicting the actions of drugs which act at these sites can be made at a preclinical level. Changes (desensitization) in the complex due to chronic benzodiazepine administration or overactivity of GABA will also be studied in the model membrane system. By studying the molecular structure of the GABA-benzodiazepine receptor complex, we expect to gain greater insight into the pharmacology of psychoactive compounds such as diazepam, chlodiazepoxide and flurazepam. These compounds are among the most widely prescribed drugs in the world for the treatment of generalized anxiety and sleep disorders. Our studies will provide insights into the etiology of these conditions and the development of more effective therapy.
