The actions of ethanol on the central nervous system involve a number of biochemical sites, including the ligand-gated ion channels. Because GABAA and glycine receptors are the major inhibitory neurotransmitter receptors in the brain and brain stem/spinal cord, respectively, enhancement of the function of these receptors by ethanol may be responsible for some of the behavioral effects of ethanol observed in vivo. These is abundant biochemical and electrophysiological evidence implicating GABAA receptors as important sites of action of ethanol. More recently, the function of the phylogenetically-related glycine receptors has also been shown to be affected by ethanol. The work proposed involves the study of the interactions of ethanol with glycine and GABAA receptors on the molecular level. Homomeric glycine receptors composed of either alpha1 or alpha2 subunits are potentiated by concentrations of ethanol as low as 10 mM. In marked contrast, homomeric receptors composed of the related GABA rho1 subunit show strong inhibition by ethanol. Chimeric receptors made of glycine alpha1 and GABA rho1 subunits permitted the localization of ethanol action to a region of 63 amino acids on glycine receptors. Further work identified two amino acids which, when mutated, completely prevented ethanol enhancement of glycine and/or GABAA receptor function. This proposal aims to further characterize the molecular mechanisms of ethanol action on glycine and GABAA receptors. To obtain a clearer understanding of the nature of ethanol interactions with the glycine receptor, the amino acids implicated in ethanol action will be mutated to other amino acids, and the resulting receptors expressed in Xenopus oocytes and tested for ethanol sensitivity using the two-electrode voltage-clamp technique. Evidence suggests that amino acid residues in GABAA receptors equivalent to those shown to determine ethanol sensitivity of glycine receptors are also important for ethanol effects on the former. Unknown at present are the relative contributions of the alpha, beta and gamma GABAA subunits to this ethanol enhancement. Mutations of the glycine alpha1 subunit which yield interesting results will be tested at homologous positions in the different subunits of the GABAA receptor. Amino acid residues of both glycine and GABAA receptor subunits near these critical residues will also be studied. The manner in which ethanol interacts with mutant receptors will provide information on the molecular nature of the site at which these compounds interact with these receptors. An understanding of the mechanisms of ethanol actions will aid in the development of rational therapies for alcohol abuse and alcoholism.
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