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

描述（由申请方提供）：蛋白激酶C δ敲除（PKC 4-/-）小鼠的研究表明，PKC 4调节乙醇中毒和自我给药。这些影响可能与PKC 4在突触外GABAA受体的作用有关，因为PKC 4-/-小鼠的丘脑和海马神经元缺乏乙醇增强的强直抑制性GABA电流。该项目的主要假设是PKC 4通过磷酸化蛋白质来调节乙醇中毒和自我给药，磷酸化蛋白质改变了控制这些行为的脑区域中突触外GABAA受体的功能。这一假设将使用一种新的小鼠模型进行测试，该模型是一种表达PKC 4的ATP类似物敏感突变体（AS-PKC 4）的基因敲入小鼠。AS-PKC 4可被穿过血脑屏障的一般激酶抑制剂PP 1的类似物选择性和有效地抑制，并可口服或通过腹膜内注射给药。研究将确定AS-PKC 4小鼠的抑制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。脑切片的电生理学研究将研究抑制AS-PKC 4是否阻断海马、丘脑和杏仁核神经元中的强直性GABA电流的乙醇增强。体外激酶试验将确定22、23和4亚基是否是PKC 4的可能底物。新的ATP类似物将与AS-PKC 4小鼠的组织一起使用，以鉴定GABAA受体相关蛋白上的PKC 4磷酸化位点。在异源表达1422/34受体和在PKC 4磷酸化位点具有丙氨酸取代的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的PKC 4信号通路，该通路调节突触外GABAA受体的乙醇敏感性，并可能包含用于开发治疗酒精使用障碍的新疗法的靶点。我们将研究PKC 4在乙醇对行为和GABAA受体调节的影响中的作用。这些研究将利用一种ATP类似物敏感形式的PKC 4，它可以被高选择性和特异性抑制，并可用于鉴定PKC 4底物。这些研究的结果将揭示调节GABA信号传导、对乙醇的行为敏感性和乙醇消耗的新分子机制。

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.