Regulation of GABA Alpha Receptors by PKC epsilon.

在前期支持期间的发现表明，PKCe是一种下调 GABAA受体通过三种机制发挥功能：（1）通过调节变构敏感性， 受体亚单位的磷酸化;（2）下调细胞表面丰度，可能通过 N-乙基马来酰亚胺敏感因子（NSF）的磷酸化，和（3）GABAA受体的下调 功能在这一延续中，我们将通过检测32和y2 S的PKCe磷酸化来扩展这项工作。 GABAA受体亚基，PKCe对NSF的调节，cofilin-1和亲环素A的作用， 调节GABAA受体对变构药物敏感性的PKCe底物，以及增加α 2 介导PKCe无效表型的纹状体中的亚基丰度。研究将使用转染的细胞系 表达特异性受体亚基组合和PKC类似物敏感突变体。其他研究 将使用从PKCe缺失和野生型小鼠培养的原代神经元。将确定磷酸化位点 通过质谱和磷酸化位点丙氨酸突变体将被构建以确定这些突变体是否 突变阻止PKCe的磷酸化。完整细胞中的GABAA受体电流将通过以下方法检查： 全细胞膜片钳a2亚单位将在纹状体中过表达，以观察过表达是否减少 野生型小鼠的酒精消耗。将开发一种RNAi载体来敲低纹状体中的α 2， 确定降低α 2亚基的表达是否增加PKCe缺失小鼠中的酒精消耗。这些 这些研究将提供关于PKCe如何调节GABAA敏感性的新的分子信息， 乙醇受体，并将提供有关PKCe如何调节GABAA受体细胞表面的新信息 S1能力和功能。

Discoveries during the prior period of support demonstrated that PKCe is a key enzyme that down-regulates GABAA receptor function through three mechanisms: (1) modulation of allosteric sensitivity via phosphorylation of receptor subunits; (2) down-regulation of cell surface abundance, possibly through phosphorylation of the N-ethylmaleimide-sensitive factor (NSF), and (3) down-regulation of GABAA receptor function. In this continuation, we will extend this work by examining PKCe phosphorylation of 32 and y2S GABAA receptor subunits, the regulation of NSF by PKCe, the role of cofilin-1 and cyclophilin A as possible PKCe substrates that regulate GABAA receptor sensitivity to allosteric drugs, and the role of increased a2 subunit abundance in the striatum in mediating PKCe null phenotypes. Studies will use transfected cell lines expressing specific receptor subunit combinations and an analog-sensitive mutant of PKCs. Other studies will use primary neurons cultured from PKCe null and wild type mice. Phosphorylation sites will be identified by mass spectrometry and phosphorylation site alanine mutants will be constructed to determine if these mutations prevent phosphorylation by PKCe. GABAA receptor currents in intact cells will be examined by whole cell patch clamp. a2 subunits will be overexpressed in the striatum to see if overexpression reduces alcohol consumption in wild type mice. An RNAi vector will be developed to knock down ot2in the striatum to determine if decreasing expressing of a2 subunits increases alcohol consumption in PKCe null mice. These studies will provide novel molecular information about how PKCe regulates the sensitivity of GABAA receptors to ethanol and will provide new information about how PKCe regulates GABAA receptor cell surface s1 ability and function.