The actions of ethanol on the central nervous system involve a number of biochemical sites, including the ligand-gated ion channels. Because GABA-A 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. There is abundant biochemical and electrophysiological evidence implicating GABA-A receptors as important sites of action of ethanol. More recently, the functioning of 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 GABA-A receptors on the molecular level. Chimeragenesis studies permitted the localization of ethanol action to a domain on glycine receptors encompassing transmembrane domains two (2) and three (3). Subsequent work identified two (2) amino acids that, when mutated, completely prevented ethanol enhancement of glycine and GABA-A receptor function. This proposal aims to characterize the molecular mechanisms of ethanol action on glycine and GABA-A receptors. To obtain a clearer understanding of the nature of ethanol interactions with these receptors, the amino acids implicated in ethanol action will be mutated, and the resulting receptors expressed in a mammalian cell line and tested for ethanol sensitivity using whole-cell and single-channel electrophysiological techniques.
The aims of the proposed research are: (1) To determine how mutations of amino acids in transmembrane domains of glycine and GABA-A receptor subunits render the resulting receptors to ethanol, or in some cases yield receptors displaying inhibition of receptor function by ethanol; (2) To determine if amino acids in transmembrane domains two (2) and three (3) of glycine and GABA-A receptors constitute a portion of the ethanol binding site on these receptors; and (3) To clarify the molecular mechanisms underlying the enhancing actions of ethanol on glycine and GABA-A receptor function, using the tonic opening and desensitization that occur spontaneously in some glycine and GABA-A receptor mutants.
Showing the most recent 10 out of 19 publications
