The mRNAs for the various gamma-aminobutyric acid type A (GABA-A) receptor subunits and their subtypes show significantly different distribution patterns within the brain, a finding that is consistent with pharmacological and biochemical evidence for polymorphism in the allosteric modulatory sites of this receptor. The mammalian brain, including that of human, has the ability to synthesize steroids--herein called neurosteroids--that function as positive or negative endogenous allosteric modulators of GABA-A receptor function; these steroids appear to be synthesized on glial cells. Thus, modulation of GABA-A receptors by drugs is determined by both the polymorphism of GABA-A receptor subtypes and the ability of the central nervous system (CNS) to produce neurosteroids. Studies with recombinant GABA-A receptors expressed in the 293 kidney tumor cell line have indicated that structural diversity of the allosteric modulatory sites associated with the extracellular domain of the receptor determines the pharmacological profiles of several classes of benzodiazepine (BZD) ligands. However, the pharmacological specificity of certain BZDs and their congeners may be attributable to their ability to bind to BZD recognition sites located in the outer mitochondrial membrane of glial cells and to modulate the rate or quality of mitochondrial neurosteroidogenesis. In turn, neurosteroids may modulate GABA-A receptor function by acting at the allosteric center associated with the gating mechanisms of the receptor-operated Cl- channel. The first objective of our proposal is to examine the functional and structural heterogeneity of native GABA-A receptors in different areas of the CNS. The second objective is to determine the pharmacological profiles of BZDs and their congeners that act on the extracellular domain of various recombinant GABA-A receptors as reconstituted in transfected tumor cell lines. The third goal will be to relate the data pertaining to objectives 1 and 2 to the acute in vivo pharmacological effects of BZDs after injection into rats. In the fourth objective, we will address the role of neurosteroids in drug-induced positive and negative modulation of GABA-A receptor function. Together, these studies will help to develop both new criteria for drug classification and new strategies for the development of drugs with preferential action at distinct allosteric modulatory sites of GABA-A receptor subtypes. This approach may help to identify drugs that are both devoid of side effects and highly selective for the treatment of anxiety disorders--such as generalized anxiety or panic disorders--sleep disorders, and epilepsy.
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