Research Component 3: Morrow

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Research Component 3 - Morrow "Mechanisms of Dependence Pathogenesis" Chronic ETOH consumption that induces ETOH dependence alters sensitivity to GABA-A receptor modulators, including ethanol, benzodiazepines and neurosteroids. The mechanisms that underlie alterations in GABA-A receptor function appear to involve internalization of synaptic GABA-A a1 subunit-containing receptors and elevated cell surface expression of GABA-A a4 subunit-containing receptors. The physiological consequences of diminished GABA-A a1 subunit receptor expression and elevated GABA-A a4 subunit-containing receptors include increased CNS excitability, anxiety, insomnia and tremor. However, the mechanism(s) that regulate cell surface expression of these receptor subtypes remain unclear. The overall goal of this proposal is to test the hypothesis that specific PKC isozvmes regulate trafficking of specific GABA-A receptor subtypes. The ability to restore normal cell surface expression of GABA-A receptors would have therapeutic relevance that may enhance recovery from alcoholism. Aim 1 will determine if ETOH regulates PKC(3, y and e isozyme expression and interactions with GABA-A receptor subtypes both in vivo and in vitro. PKC isozyme expression will be measured by western blot analysis using specific antibodies and PKC/ GABA-A receptor association will be determined by co-immunoprecipitation analysis or dual-label fluorescent immunohistochemistry with visualization by confocal microscopy. We predict that specific PKC isozymes will associate with specific GABA-A receptor subtypes in response to ETOH. Aim 2 will determine the role of PKCp, y and e isoforms in the trafficking of GABA-A a1 and a4 subunit-containing receptors. We will determine if specific PKCs are required for ETOH-induced adaptations using RNA inhibition (RNAi) or specific peptide inhibitors. Surface expression will be determined by western blotting following biotinylation assays, surface receptor crosslinking or subcellular fractionation. Distinct PKC isozymes may regulate the surface expression of GABA-A a1 vs. a4 subunit-containing receptors. Aim 3 investigates the role of PKCp, y and e isoforms in phosphorylation of GABA-A <x1 and a4 subunit-containing receptors. Receptors will be denatured into subunit peptides and immunoprecipitated using phosphor antibodies to determine if chronic ETOH consumption alters phosphoramine labeling of GABA-A receptor subunits. PKC isozymes will be inhibited by RNAi or isozyme-specific antagonists. Aim 4 will define the role of PKC isozymes in the effects of metabotrophic glutamate receptor (mGluR) type 5 and tumor necrosis factor-a (TNF-a)-mediated regulation of GABA-A receptors. Collaborative studies with Hodge, Breese and Crews may link ETOH actions on multiple receptors to PKC signaling to identify a new strategy to reverse the detrimental effects of chronic ETOH consumption.