Protein Kinase C Modulation of NMDAR Ethanol Sensitivity

描述(由申请人提供)：本研究计划的目的 是阐明蛋白激酶C(PKC)如何调节乙醇(Etoh)抑制 N-甲基-D-天冬氨酸受体(NMDAR)。NMDAR据信是 参与调节学习和记忆的神经元过程，兴奋性毒性 和细胞死亡，以及许多神经和行为的变化归因于 滥用Etoh。我们的初步结果提示PKC可能参与了乙醇脱氢反应。 抑制原代培养小脑颗粒细胞NMDARs的表达 (CGC)S体外培养6-14天(DIV)。既使用全细胞也使用穿孔 我们观察到的膜片钳电生理技术具有重要意义 An对乙醇抑制NMDA诱导的稳态电流的减弱作用 PKC抑制剂，100 NM的Calphostin C作用8分钟。这一逆转 乙醇浓度为10、50、100 mM时，抑制作用显著。 免疫印迹结果显示，PKCsα、β1、βII、epsilon、γ、 Delta、Mu、theta、lambda/iota和Zeta包含在我们的文化中 6-14 DIV。这些初步数据支持急性酒精中毒的假设 暴露可激活这些PKC亚型中的一个(或多个)。激活会导致 将酶转移到其底物的位置，在那里 磷酸化介导的事件发生。这种磷酸化介导的事件 调节对本地NMDAR的乙醇抑制。这一假设将在#年进行检验。 接下来的实验。CGCs将暴露在乙醇和PKC的激活下 将通过测量从胞浆到胞浆的酶活性增加来确认 用酶活性测定法测定细胞膜的活性。免疫印迹分析与PKC 异构体特异性抗体将被用来识别哪些异构体 转移到膜部分。以确定乙醇介导的激活 PKC的突变导致NMDAR NRI亚基的磷酸化， 将使用NR1抗体进行免疫沉淀研究，然后 识别磷酸化位点特异性抗体的免疫印迹 PKC介导的NRI的磷酸化。易位、激酶活性和 磷酸化实验将在不同生理条件下进行 确定乙醇是否直接或间接激活PKC的条件。 电生理实验将确定我们观察到的Calphostin C NMDARs的乙醇抑制的减弱是由PKC介导的，通过评估 NMDAR对PKC激活剂和催化作用下乙醇敏感性的研究 在存在和不存在PKC抑制剂的情况下激活PKC。PKC异构体特异性 抑制剂将确定哪个PKC亚型正在调节Etoh抑制 NMDAR函数。其他电生理实验将检验 PKC与其他细胞内信号级联的作用以及如何融合 这些级联改变了NMDAR的乙醇灵敏度。总的来说，这些 研究将为急性乙醇中毒的分子机制提供洞察力 暴露会改变NMDAR的功能。从而为小说未来的设计提供了便利 在酒精中毒和酒精中毒治疗中使用的治疗策略 虐待。

DESCRIPTION (provided by applicant): The aim of this proposed research project is to elucidate how protein kinase C (PKC) modulates ethanol (EtOH) inhibition of the N-methyl-D-aspartate receptor (NMDAR). The NMDAR is believed to be involved in neuronal processes that mediate learning and memory, excitotoxicity and cell death, as well as many neural and behavioral changes attributed to the abuse of EtOH. Our preliminary results suggest PKC may be involved in EtOH inhibition of NMDARs expressed in primary cultured cerebellar granule cells (CGC)s 6-14 days in vitro (DIV). Using both whole-cell and perforated patch-clamp electrophysiological techniques we have observed significant attenuation of EtOH inhibition of NMDA-induced steady-state currents by an eight minute exposure to 100 nM calphostin C, a PKC inhibitor. This reversal of inhibition was significant for three concentrations of EtOH: 10, 50 and 100 mM. Immunoblot results indicate that PKCs alpha, betaI, betaII, epsilon, gamma, delta, mu, theta, lambda/iota and zeta are contained in our cultures between 6-14 DIV. These preliminary data support the hypothesis that acute EtOH exposure activates one (or more) of these PKC isoforms. Activation results in a translocation of the enzyme to the location of its substrate where a phosphorylation-mediated event occurs. This phosphorylation-mediated event modulates EtOH inhibition of native NMDARs. This hypothesis will be tested in the following experiments. CGCs will be exposed to EtOH and activation of PKC will be confirmed by measuring increases in enzyme activity from the cytosol to the membrane by kinase activity assays. Immunoblot analysis and PKC isoform-specific antibodies will be used to identify which isoforms have translocated to the membrane fraction. To determine if EtOH-mediated activation of PKC results in the phosphorylation of the NMDAR NRI subunit, immunoprecipitation studies will be performed with an NR1 antibody followed by immunoblotting with a phosphorylation site-specific antibody that recognizes PKC-mediated phosphorylation of the NRI. The translocation, kinase activity and phosphorylation experiments will be conducted under various physiological conditions to determine if PKC is directly or indirectly activated by EtOH. Electrophysiological experiments will establish that our observed calphostin C attenuation of EtOH inhibition of NMDARs is mediated by PKC, by assessing the EtOH sensitivity of NMDARs after exposure to PKC activators and catalytically active PKC in the presence and absence of PKC inhibitors. PKC isoform-specific inhibitors will identify which PKC isoform is modulating EtOH inhibition of NMDAR function. Additional electrophysiological experiments will examine the role of PKC with other intracellular signaling cascades and how the convergence of these cascades alters the EtOH sensitivity of the NMDAR. Overall, these studies will provide insights into the molecular mechanisms by which acute EtOH exposure alters NMDAR function. Thus, facilitating the future design of novel therapeutic strategies to be used in the treatment of alcoholism and alcohol abuse.