A critical role for zinc in ethanol action at the glycine receptor



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Ethanol is a widely used drug, yet an understanding of its sites and mechanisms of action remains incomplete. Among the protein targets of ethanol are glycine receptors (GlyRs). In addition to ethanol, zinc also modulates GlyR function. Although the individual effects of zinc and alcohols on GlyR function have been well studied, the combined effects of these agents have not been thoroughly examined. This project investigated the effects of zinc on alcohol action at the glycine receptor (GlyR). In Aim 1, the effects of zinc on ethanol modulation of GlyR function were tested and characterized in three GlyR [alpha] subunits ([alpha]1-3). Aim 2 explored a site of action for the augmenting effects of zinc on ethanol action at the GlyR. Mutant D80A GlyRs, which lack a zinc binding site (D80), were constructed and allowed us determine if this zinc binding site is important for the zinc/ethanol interactions that were observed in Aim 1. The effects of ethanol were reduced in mutant D80A GlyRs compared to wild type (WT). In addition, manipulating zinc levels in our buffers either by adding or chelating zinc did not change the magnitude of ethanol enhancement of mutant D80A GlyRs as it did in WT GlyRs suggesting that the D80 position is important for zinc modulation of ethanol action. Finally, Aim 3 extended the findings from Aims 1 and 2 by evaluating the effects of a GlyR point-mutation on alcohol consumption and other behavioral tests in mice. Glra1(D80A) knock-in mice provided an animal model for behavioral studies of zinc/ethanol interactions at the GlyR and showed decreased alcohol consumption and preference compared to their WT littermates. In addition, D80A KI mice had increased startle responses compared to their WT littermates. Other behavioral tests were also conducted including tests of ethanol motor incoordination and strychnine induced convulsions; there were no differences detected between KI and WT mice in these assays. Overall, our findings demonstrate that zinc is critical in determining the effects of ethanol at GlyRs and suggest that zinc signaling at the D80 position may be important for mediating the behavioral effects of ethanol action at GlyRs.