Research Component 3: Morrow

研究内容3-《依赖发病机制》慢性乙醇 导致乙醇依赖的消费改变了对GABA-A受体调节剂的敏感性，包括 乙醇，苯二氮卓类药物和神经类固醇。GABA-A受体改变的机制 功能似乎涉及突触GABA-A A1亚基受体的内化和升高 细胞表面含GABA-A4亚单位受体的表达。对人体的生理影响 GABA-A A1亚基受体表达降低和GABA-A A4亚基受体表达增加 包括中枢神经系统兴奋性增加、焦虑、失眠和震颤。然而，规范的机制(S) 这些受体亚型在细胞表面的表达尚不清楚。这项提案的总体目标是测试 特定的PKC同工酶调节特定GABA-A受体亚型的运输的假说。这个 恢复细胞表面GABA-A受体正常表达的能力将具有治疗意义 可能会促进酒精中毒的恢复。目标1将确定乙醇是否调节PKC(3，y和e同工酶 GABA-A受体亚型在体内和体外的表达及相互作用。PKC同工酶的表达 将通过使用特定抗体和PKC/GABA-A受体关联的蛋白质印迹分析来测量 将通过免疫共沉淀分析或双标记荧光免疫组织化学方法确定 通过共聚焦显微镜进行可视化。我们预测，特定的PKC同工酶将与特定的 乙醇对GABA-A受体亚型的影响目标2将确定PKCp、y和e亚型在 含有GABA-AA1和A4亚基的受体的运输。我们将确定特定的PKC是否 使用RNA抑制(RNAi)或特定的多肽抑制剂进行乙醇诱导的适应所必需的。表面 在生物素化试验、表面受体交联后，将通过蛋白质印迹来确定表达。 或亚细胞分馏。不同的PKC同工酶可能调节GABA-A的表面表达 A1与包含A4亚单位的受体。目的3研究PKCp、y和e亚型在 含有GABA-A和A4亚基的受体的磷酸化。受体将被变性为亚单位 用多肽和免疫共沉淀法测定慢性乙醇摄入量 改变GABA-A受体亚基的磷酸胺标记。PKC同工酶将被RNAi或 同工酶特异性拮抗剂。目标4将确定PKC同工酶在代谢营养效应中的作用 谷氨酸受体5型和肿瘤坏死因子-a对GABA-A的调节作用 感受器。与Hodge、Breese和Crews的合作研究可能将乙醇对多个受体的作用联系起来 到PKC信号转导，以确定一种新的策略来逆转慢性乙醇消费的有害影响。

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.