The objectives of this proposal are to study the molecular mechanisms of action of benzodiazepines (BZs) and mechanisms of regulation of BZ receptors; to purify and characterize BZ receptors and endogenous modulator; and to quantitate the relationship between receptor occupancy and pharmacological effects. Our earlier kinetic studies on the binding of BZs to cerebral cortices provided the first evidence that BZ receptors exist as two interconvertible conformations. We will further pursue the binding kinetics, under a variety of conditions that are known to affect BZ binding, in different brain regions using three classes of radiolabeled ligands (i.e., BZ agonists and antagonists, and Beta-carboline convulsants). Detailed analysis of binding kinetics may yield information not available from equilibrium binding measurements. The effects of BZs on the binding of GABA to a synaptosomal membrane prepared by a novel """"""""gentle"""""""" method (i.e., treating the membrane with two intrinsic cellular enzymes, GABA transaminase and succinic semialdehyde dehydrogenase, to remove endogenous GABA), will be studied. We will explore the relationship and inter-regulation between BZ and GABA receptors by studying the binding of GABA to different brain regions after chronic BZ treatment which is known to cause BZ receptor down-regulation. The effects of chronic treatment with BZ antagonists on the binding of BZ and GABA will also be studied. The turnover rate of BZ receptors will be studied using irreversible BZ ligands, protein synthesis inhibitors, and an axoplasmic flow inhibitor. The occupancy of BZ receptors measured by co-administered BZs and their corresponding (3H)BZs will be correlated with their anticonvulsant and anticonflict activity. Such study may also reveal the presence of spare receptors. BZ receptors and endogenous modulator will be solubilized with the nonionic detergent, n-octyl glucoside, purified with affinity chromatography, high performance liquid chromatography (HPLC) and other separation techniques, and their biochemical and physical properties characterized. Understanding the mechanisms of action of BZs, the regulation of BZ receptors, and their properties will help us understand this important class of drugs and the underlying mechanisms of anxiety and seizure disorders.
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