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

描述（由申请人提供）：对蛋白激酶C δ敲除（PKC4-/-）小鼠的研究表明PKC4调节乙醇中毒和自我给药。这些影响可能与PKC4作用于突触外GABAA受体有关，因为PKC4-/-小鼠的丘脑和海马神经元缺乏乙醇增强强直性抑制性GABA电流。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，这些蛋白质改变了控制这些行为的脑区突触外GABAA受体的功能。这一假设将通过一种新的小鼠模型进行验证，该模型是一种表达ATP类似物敏感的PKC4突变体（AS-PKC4）的敲入小鼠。AS-PKC4可以被一般激酶抑制剂PP1的类似物选择性和有效地抑制，PP1可以穿过血脑屏障，可以口服或通过腹腔注射给药。研究将确定AS-PKC4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能力。脑切片电生理研究将探讨抑制AS-PKC4是否会阻断乙醇增强海马、丘脑和杏仁核神经元的强直性GABA电流。体外激酶测定将确定22、23和4亚基是否可能是PKC4的底物。新的ATP类似物将用于AS-PKC4小鼠的组织，以鉴定GABAA受体相关蛋白上的PKC4磷酸化位点。对异源表达1422/34受体和PKC4磷酸化位点丙氨酸取代受体相关蛋白的细胞进行研究，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PKC4信号通路，该通路调节突触外GABAA受体的乙醇敏感性，并可能包含开发治疗酒精使用障碍的新疗法的靶点。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类似物敏感形式，可以高选择性和特异性地抑制，并可用于鉴定PKC4底物。这些研究结果将揭示调节GABA信号、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.