Alcohol abuse and alcoholism are serious health problems in the United States. However, the molecular bases of alcohol's actions and alcoholism are unknown. The overall goal of this proposal is to study the molecular mechanism(s) of action of alcohol at the neurotransmitter-gated ion channel level. The effects of ethanol on GABA-A and NMDA receptors, the major inhibitory and excitatory neurotransmitter-gated ion channels in the mammalian central nervous system, respectively, will be studied. The function of these ion channels is regulated by protein phosphorylation/dephosphorylation. This regulatory process is mediated by multiple kinases and phosphatases, and appears to be important for ethanol's actions. A combination of electrophysiological and biochemical techniques in cultured transfected cell lines is proposed to be used to study the role of protein phosphorylation in the ethanol-induced effects on these channels. Electropysiological experiments will determine the effect of ethanol on receptor function. Immunoprecipitation experiments in the same batch of cells will directly examine the phosphorylation state of GABA-A and NMDA receptor subunits in the presence of ethanol. If ethanol changes the phosphorylation state of receptor subunits, then phosphoamino acid and phosphopeptide analyses, and studies with kinase & phosphatase inhibitors, will determine which kinases or phosphatases are required for ethanol's actions. Correlation of electrophysiological and site-directed mutagenesis experiments will determine whether phosphorylation of specific amino acids in GABA-A AND NMDA receptor subunits is required for ethanol's actions. The findings of the proposed experiments will contribute toward a better understanding of the actions of ethanol in the central nervous system.
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