The multicomponental GABA-A receptor ionophore complex belongs to ligand- gated ion-channel gene family, and it is a site of action for a variety of centrally acting drugs. Recent cloning studies have also indicated heterogeneity of GABA-A receptors in term of subunit distribution in the CNS. This proposal seeks to test the hypothesis that chronic GABA agonist and barbiturate (which bind to two distinct sites) treatment produces downregulation and/or uncoupling of GABA benzodiazepine receptor ionophore complex. Additionally, we will examine the potential mechanism(s) involved in downregulation and uncoupling. This will be achieved by utilizing radioligand binding studies, GABA-gated 36Cl-influx studies and measuring mRNA levels of various subunits (alpha, beta, 2) in well characterized mammalian spinal cord neurons in culture. Parallel studies will also be conducted in mammalian cortical neurons in order to understand if different GABA-A receptor subtypes (spinal cord alpha 3 abundant v/s cortex alpha abundant) are regulated by GABA agonist and barbiturate treatment in a similar or different manner. We will determine if chronic GABA agonist and barbiturate treatments: a) alter the binding of benzodiazepine (BZ) agonists, antagonist and inverse antagonist; b) alter the sensitivity of the picrotoxin site; c) alter the to binding of GABA to receptor site; d) alter the coupling of GABA receptors with BZ and picrotoxin sites; e) decrease the efficacy of GABA-Aergic transmission by studying GABA-induced 36Cl-influx, and f) alter the levels of expression of different subunits (alpha, beta, 2) of GABA receptor complex by measuring mRNA levels, or involve other mechanisms like changes in receptor turnover or internalization of receptors. Preliminary studies indicate that both GABA and pentobarbital treatments produce downregulation of benzodiazepine binding sites and an uncoupling of GABA and benzodiazepine receptor sites. The proposed studies are aimed at increasing our understanding of GABA- Aergic transmission and its regulation. A clearer understanding of the GABA-Aergic transmission is a prerequisite to our understanding of normal biology and in the rational development of drugs for disorders involving a dysfunction of GABA-A receptor system.
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