The long term goal of this research program continues to be the understanding of mechanisms by which GABA-A receptor expression is regulated and the contribution of individual subunit proteins to receptor function. This includes analysis of the role of the receptor subunits in mediating the physiologic effects of benzodiazepines and barbiturates. Gamma-Aminobutyric acid (GABA) is the principal inhibitory neurotransmitter in the central nervous system. A substantial portion of the inhibitory actions of GABA are achieved via the GABA-A receptors. The GABA-A receptors appear to be constituted of 5 subunit proteins that are the products of transcription, alternative splicing and translation of at least 16 different genes. Each specific combination of these subunit proteins results in a receptor complex with subtly different characteristics in terms of the chloride conductance which GABA elicits and the modulatory influences of benzodiazepines, barbiturates, and steroids on these receptors. During years 17 through 20 of this research program, we propose to focus on four major hypotheses related to the regulation of GABA-A receptor structure and function, as well as its role in development. First, we will test the hypothesis that GABA-A subunit expression is regulated by metabotropic acidic amino acid/peptide receptors. Based on data obtained during the previous support interval, we propose that activation of metabotropic receptors by NAAG and other agonists influences alpha6 GABA-A receptor subunit expression in cerebellar granule cells. Subunit mRNA and protein expression will be examined in granule cell cultures using PCR and immunoblot methods. We will continue to develop subunit specific (alpha6, gamma2, beta2) antibodies which will be used to confirm subunit protein expression. Second, we will test the hypothesis that phosphorylation of the GABA-A receptors is influenced by the balance of excitatory and inhibitory receptor activation. We will use cerebellar granule cells in culture to define the extent of subunit phosphorylation under conditions of activation and blockade of GABA-A and 'glutamate' receptors. Third, we will test the hypothesis that individual neurons with similar structure and position in the CNS express different subsets of GABA-A receptor subunit mRNA and that these differences result in distinguishable electrophysiological responses to GABA, benzodiazepines and barbiturates. We have succeeded in identifying the mRNA for two different GABA-A subunit proteins in single cells in which the GABA-A receptor responses have been characterized under patch clamp conditions. We propose to extend this approach to the preparation of mRNA libraries from individual, electrophysiologically characterized cells. This technically demanding approach will permit us to test in primary cell culture and in brain slices, a series of hypotheses, derived from transfection studies, on the function of GABA-A subunit proteins in situ. Additionally, this work may confirm and perhaps extend hypotheses on preferential subunit association in GABA-A receptors that have been derived from in situ hybridization and transfection studies. Fourth, we will test the hypothesis that GABA-A receptors contribute to the establishing patterns of synaptic connectivity in the developing central nervous system. Rapidly, evolving transgenic methods will be applied to produce null mutations of the alpha6 and gamma2 subunit genes and conditional deletions of the alpha1 and beta2 subunit expression via the controlled expressions of ribozymes targeted for subunit transcripts.
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