Research Component 3 - Morrow 'Mechanisms of Dependence Pathogenesis' Chronic ETOHconsumption that induces ETOH dependence alters sensitivity to GABA-A receptor modulators, includingethanol, benzodiazepines and neurosteroids. The mechanisms that underlie alterations in GABA-A receptorfunction appear to involve internalization of synaptic GABA-A a1 subunit-containing receptors and elevatedcell surface expression of GABA-A a4 subunit-containing receptors. The physiological consequences ofdiminished GABA-A a1 subunit receptor expression and elevated GABA-A a4 subunit-containing receptorsinclude increased CNS excitability, anxiety, insomnia and tremor. However, the mechanism(s) that regulatecell surface expression of these receptor subtypes remain unclear. The overall goal of this proposal is to testthe hypothesis that specific PKC isozvmes regulate trafficking of specific GABA-A receptor subtypes. Theability to restore normal cell surface expression of GABA-A receptors would have therapeutic relevance thatmay enhance recovery from alcoholism.
Aim 1 will determine if ETOH regulates PKC(3, y and e isozymeexpression and interactions with GABA-A receptor subtypes both in vivo and in vitro. PKC isozyme expressionwill be measured by western blot analysis using specific antibodies and PKC/ GABA-A receptor associationwill be determined by co-immunoprecipitation analysis or dual-label fluorescent immunohistochemistrywith visualization by confocal microscopy. We predict that specific PKC isozymes will associate with specificGABA-A receptor subtypes in response to ETOH.
Aim 2 will determine the role of PKCp, y and e isoforms inthe trafficking of GABA-A a1 and a4 subunit-containing receptors. We will determine if specific PKCs arerequired for ETOH-induced adaptations using RNA inhibition (RNAi) or specific peptide inhibitors. Surfaceexpression will be determined by western blotting following biotinylation assays, surface receptor crosslinkingor subcellular fractionation. Distinct PKC isozymes may regulate the surface expression of GABA-Aa1 vs. a4 subunit-containing receptors.
Aim 3 investigates the role of PKCp, y and e isoforms inphosphorylation of GABA-A <x1 and a4 subunit-containing receptors. Receptors will be denatured into subunitpeptides and immunoprecipitated using phosphor antibodies to determine if chronic ETOH consumptionalters phosphoramine labeling of GABA-A receptor subunits. PKC isozymes will be inhibited by RNAi orisozyme-specific antagonists.
Aim 4 will define the role of PKC isozymes in the effects of metabotrophicglutamate receptor (mGluR) type 5 and tumor necrosis factor-a (TNF-a)-mediated regulation of GABA-Areceptors. Collaborative studies with Hodge, Breese and Crews may link ETOH actions on multiple receptorsto PKC signaling to identify a new strategy to reverse the detrimental effects of chronic ETOH consumption.
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