ETHANOL, CALCIUM CHANNELS, AND PROTEIN KINASE C

该项目的总体目标是确定细胞适应酒精的分子事件，希望确定治疗酒精中毒的新药靶点。大量证据表明，电压依赖性钙通道与动物的酒精消耗和依赖有关。先前的工作主要集中在L型电压门控钙通道，其被乙醇抑制并且在体外被慢性乙醇暴露上调。此外，ω-芋螺毒素GVIA，一个特定的N通道拮抗剂，结合位点增加，在小鼠海马慢性暴露于乙醇。在PC 12细胞中，慢性乙醇暴露引起的N通道上调需要蛋白激酶C（PKC）介导。本项目的第一个目标是确定乙醇对N型通道的上调是否可逆并导致N通道功能的可逆变化。随后的研究将探讨乙醇是否通过增加通道亚基mRNA或通过PKC ε依赖性机制增加亚基蛋白的细胞内运输来增加N通道密度。使用一个新创建的PKC β基因敲除小鼠，PKC β在酒精偏好，耐受性和依赖性的作用将在两瓶偏好，翻正反射的损失，酒精戒断发作的行为研究中进行检查。最后，将使用这些PKC β基因敲除小鼠研究PKC β在体内乙醇诱导的N通道上调中的作用。这些研究将提供具体的信息，在介导乙醇诱导的功能性N通道的变化，在体外和体内的PKC β的作用。将获得关于PKC β在调节动物酒精偏好、耐受性和依赖性中的作用的重要信息。这些结果将推进我们对饮酒行为机制的认识，并可能将N型通道和PKC β作为开发治疗药物治疗酒精中毒的新靶点。

The overall goal of this project is to determine molecular events that underlie cellular adaptation to alcohol with the hope of identifying novel drug targets for treatment of alcoholism. Considerable evidence implicates voltage-dependent calcium channels in alcohol consumption and dependence in animals. Prior work has focused mainly on L-type, voltage- gated calcium channels, which are inhibited by ethanol and are up- regulated by chronic ethanol exposure in vitro. Moreover, binding sites for omega-conotoxin GVIA, a specific N channel antagonist, are increased in mouse hippocampus following chronic exposure to ethanol. In PC12 cells, up-regulation of N channels by chronic ethanol exposure requires protein kinase C (PKC)epsilon. The first goal of this project is to determine if up- regulation of N-type channels by ethanol is reversible and leads to reversible changes in N channel function. Subsequent studies will investigate whether ethanol increases N channel density by increasing channel subunit mRNA or intracellular trafficking of subunit proteins by PKCepsilon-dependent mechanisms. Using a newly created PKCepsilon knockout mouse, the role of PKCepsilon in alcohol preference, tolerance, and dependence will be examined in behavioral studies of two-bottle preference, loss of righting reflex, and alcohol withdrawal seizures. Finally, the role of PKCepsilon in ethanol-induced up-regulation of N channels in vivo will be examined using these PKCepsilon knockout mice. These studies will provide specific information about the role of PKCepsilon in mediating ethanol-induced changes in functional N channels in vitro and in vivo. Important information will be obtained regarding the role of PKCepsilon in regulating alcohol preference, tolerance, and dependance in animals. These results will advance our knowledge about mechanisms underlying drinking behavior and may identify N-type channels and PKCepsilon as new targets for development of therapeutic agents to treat alcoholism.