PKC Delta in Ethanol Regulation of GABA-A Receptors and Behavior

GABA-A 受体和行为的乙醇调节中的 PKC Delta

• 批准号: 8643855
• 负责人: ROBERT O. MESSING
• 金额: $ 27.3万
• 依托单位: UNIVERSITY OF TEXAS AT AUSTIN
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-20 至 2014-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8643855
• 关键词: PKC; Delta; Ethanol; Regulation; GABA

DESCRIPTION (provided by applicant): Studies with protein kinase C delta knockout (PKC4-/-) mice indicate that PKC4 regulates ethanol intoxication and self-administration. These effects may be related to PKC4 actions at extrasynaptic GABAA receptors, since thalamic and hippocampal neurons from PKC4-/- mice lack ethanol enhancement of tonic inhibitory GABA currents. The main hypothesis of this project is that PKC4 regulates ethanol intoxication and self-administration by phosphorylating proteins that alter the function of extrasynaptic GABAA receptors in brain regions controlling these behaviors. This hypothesis will be tested using a novel mouse model, a knock-in mouse that expresses an ATP analog-sensitive mutant of PKC4 (AS-PKC4). AS-PKC4 can be selectively and potently inhibited by analogs of the general kinase inhibitor PP1 that cross the blood brain barrier and can be administered orally or by i.p. injection. Studies will determine if inhibition of AS-PKC4 mice increases signs of ethanol intoxication and enhances ethanol self-administration in adult mice. Electrophysiological studies in brain slices will investigate whether inhibiting AS-PKC4 blocks ethanol potentiation of tonic GABA currents in hippocampal, thalamic, and amygdala neurons. In vitro kinase assays will determine if 22, 23, and 4 subunits are possible substrates of PKC4. Novel ATP analogs will be used with tissues from AS-PKC4 mice to identify PKC4 phosphorylation sites on GABAA receptor-associated proteins. Studies in cells that heterologously express 1422/34 receptors and receptor-associated proteins with alanine substitutions at PKC4 phosphorylation sites, will determine if these sites regulate the ethanol sensitivity of 1422/34 receptors. The overall goal of this project is to identify a novel PKC4 signaling pathway that regulates the ethanol sensitivity of extrasynaptic GABAA receptors and may contain targets for the development of new therapeutics to treat alcohol use disorders. PUBLIC HEALTH RELEVANCE We will investigate the role of PKC4 in ethanol's effects on behavior and on regulation of GABAA receptors. These studies will utilize an ATP-analog sensitive form of PKC4 that can be inhibited with high selectivity and specificity, and can be used to identify PKC4 substrates. The results of these studies will reveal new molecular mechanisms that regulate GABA signaling, behavioral sensitivity to ethanol, and ethanol consumption.

描述（由申请人提供）：蛋白激酶C Delta敲除（PKC4 - / - ）的研究表明，PKC4调节乙醇中毒和自我给药。这些作用可能与PKC4在外部GABAA受体上的作用有关，因为来自PKC4 - / - 小鼠的丘脑和海马神经元缺乏补品抑制性GABA电流的乙醇增强。该项目的主要假设是，PKC4通过磷酸化蛋白质来调节乙醇中毒和自我给药，从而改变控制这些行为的大脑区域中外部挑战GABAA受体的功能。该假设将使用新型小鼠模型进行测试，这是一种表达PKC4的ATP模拟敏感突变体（AS-PKC4）的敲门小鼠。 AS-PKC4可以通过跨越血脑屏障的一般激酶抑制剂PP1的类似物进行选择性和有效的抑制，并且可以口服或i.p.施用。注射。研究将确定抑制AS-PKC4小鼠是否会增加乙醇中毒的迹象，并增强成年小鼠的乙醇自我给药。脑切片中的电生理研究将研究抑制AS-PKC4是否阻止海马，丘脑和杏仁核神经元中补品GABA电流的乙醇增强。体外激酶测定将确定PKC4的底物是否可能是22、23和4个亚基。新型的ATP类似物将与来自AS-PKC4小鼠的组织一起使用，以鉴定与GABAA受体相关蛋白上的PKC4磷酸化位点。在PKC4磷酸化位点，在异源表达1422/34受体和受体相关蛋白的细胞中的研究将确定这些位点是否调节1422/34受体的乙醇敏感性。该项目的总体目标是确定一种新型的PKC4信号传导途径，该途径可以调节外部挑战GABAA受体的乙醇敏感性，并可能包含用于开发新的治疗酒精使用障碍的新疗法的靶标。公共卫生相关性，我们将研究PKC4在乙醇对行为和GABAA受体调节的影响中的作用。这些研究将利用PKC4的ATP-Analog敏感形式，可以以高选择性和特异性抑制，并可用于识别PKC4底物。这些研究的结果将揭示调节GABA信号传导，行为敏感性和乙醇消耗的新分子机制。