Protein Kinase C Modulation of NMDAR Ethanol Sensitivity

描述（由申请人提供）：本研究项目的目的 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被认为是 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变化归因于 滥用乙醇。我们的初步结果表明，PKC可能参与EtOH 抑制原代培养的小脑颗粒细胞中NMDAR的表达 （CGC）6-14天体外（DIV）。使用全细胞和穿孔 膜片钳电生理技术，我们已经观察到显着 用一种抑制剂减弱EtOH对NMDA诱导稳态电流的抑制作用 暴露于100 nM calphostin C（一种PKC抑制剂）8分钟。的反转 对于三种浓度的EtOH：10、50和100 mM，抑制是显著的。 免疫印迹结果表明，PKC α，β I，β II，γ， δ，μ，θ，λ/i和ζ包含在我们的文化中， 6-14 DIV.这些初步数据支持急性EtOH 暴露激活这些PKC同种型中的一种（或多种）。激活导致 转移酶到其底物的位置， 磷酸化介导的事件发生。这种磷酸化介导的事件 调节天然NMDAR的EtOH抑制。这一假设将在 以下实验。CGCs将暴露于EtOH并激活PKC 将通过测量从细胞质到细胞质的酶活性的增加来证实。 通过激酶活性测定来测定膜。免疫印迹分析和PKC 同种型特异性抗体将用于鉴定哪些同种型具有 转移到膜部分。为了确定EtOH介导的活化是否 PKC的激活导致NMDAR NRI亚基的磷酸化， 将用NR 1抗体进行免疫沉淀研究， 用磷酸化位点特异性抗体进行免疫印迹， PKC介导的NRI磷酸化。易位，激酶活性和 磷酸化实验将在各种生理条件下进行。 条件以确定PKC是否被EtOH直接或间接激活。 电生理实验将证实我们观察到的钙磷蛋白C NMDAR的EtOH抑制的减弱是由PKC介导的，通过评估 暴露于PKC激活剂和催化剂后NMDAR的EtOH敏感性 在存在和不存在PKC抑制剂的情况下的活性PKC。PKC亚型特异性 抑制剂将鉴定哪种PKC亚型调节EtOH抑制 NMDAR功能。额外的电生理学实验将检查 PKC与其他细胞内信号级联的作用以及如何收敛 这些级联改变了NMDAR的EtOH灵敏度。总的来说，这些 研究将提供深入了解急性乙醇 暴露改变NMDAR功能。从而为今后的小说设计提供便利 用于治疗酒精中毒和酒精的治疗策略 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.