A major challenge in alcohol research is to determine the molecular events that underlie alcohol abuse. Much evidence implicates ethanol- induced increases n voltage-dependent calcium channels in alcohol dependence and manifestations of alcohol withdrawal in animals. Studies in our laboratory, using the neural cell line PC12, demonstrated that exposure to 25-200 mM ethanol for 2-6 days increases depolarization- stimulated calcium uptake, which remains elevated for several hours following removal of ethanol. This is associated with an increase in the number of binding sites for dihydropyridine calcium channel antagonists, indicating that ethanol increases the number of functional L-type calcium channels. The process by which this occurs requires protein kinase C (PKC) and chronic ethanol exposure appears to activate PKC by increasing levels of two isozymes, deltaPKC and epsilon PKC. The first goal of this project is to determine which of these isozymes mediates up-regulation of L-channels by ethanol. Since L-channels are composed of multiple subunits, a second goal is to identify which subunits are regulated by ethanol. The final goal is to determine whether similar PKC isozymes and L-channel subunits are regulated by ethanol in experimental animals. Studies are planned to use antisense DNA and RNA to inhibit PKC isozyme expression and block ethanol's effects on L-channels, and to make stable transfectants of PC12 cells that over-express PKC isozymes to mimic or enhance ethanol's effects. Antibodies will be made against L-channel subunits which will be analyzed for ethanol-induced changes in abundance by Northern analysis, Western analysis, and immunoassay. Finally, strains of mice bred for susceptibility or resistance to alcohol withdrawal seizures will be provide specific information about which PKC isozymes and L-channel subunits are involved in up-regulation of calcium channels by ethanol. In addition, they may uncover differences in regulations of L-channels by PCK that underlie genetic differences in the development of alcohol dependence and susceptibility to withdrawal seizures. This will advance the search for genes involved in the development of dependence on alcohol.
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