Position 52 in alpha1 glycine receptors is important for the action of ethanol

UnrestrictedGlycine receptors (GlyRs) have received considerable attention as putative sites of action causing the behavioral effects of ethanol. Studies over the last decade have identified several positions in the transmembrane domain of GlyRs that are critical for ethanol modulation.; Studies to date in the extracellular domain of alpha1GlyRs found that mutations at position 52 in Loop 2 alter receptor sensitivity to ethanol, abolish receptor sensitivity to an ethanol antagonist and can eliminate subunit-dependent differences in ethanol sensitivity between alpha1 and alpha2GlyRs. These findings suggest that the extracellular domain also represents a target for ethanol in GlyRs.; The present study uses systematic substitution of the amino acid residue at position 52 of alpha1 GlyRs to study possible structure-function relationship between the physical-chemical properties of the residue at position 52 and receptor sensitivity to glycine or ethanol. The current investigation also begins to test for possible relationships between changes in agonist activation produced by these mutations and ethanol sensitivity. To investigate the specificity of the findings for ethanol, we also tested propofol, another general anesthetic, which is thought to act at different sites than ethanol in alpha1GlyR and GABAARs.; To accomplish this, we produced mutant alpha1GlyRs in which the alanine at position 52 in wild-type (WT) alpha1GlyRs was replaced with each of the remaining nineteen naturally occurring amino acids and measured their responses to glycine and modulation by ethanol. Xenopus oocytes expressing WT or mutant alpha1 GlyRs were voltage-clamped and tested for the effects of ethanol using an OpusXpress 8 channel two electrode voltage clamp system (-70mV).; We found that: (1) all mutations produced functional alpha1 GlyRs, with several of the mutations significantly reduced the glycine EC50; (2) most of the mutations significantly reduced receptor sensitivity to ethanol; (3) molecular volume and molecular weight of the amino acid residue at position 52 play roles in determining the responsiveness of alpha1GlyRs to both glycine and ethanol; (4) changes in glycine EC50 were correlated with changes in magnitude of ethanol potentiation; (5) position 52 of alpha1 GlyRs is a target for ethanol action, but not for propofol action.; Together, the present study adds to the evidence that position 52 in the extracellular domain, in addition to the transmembrane domain, plays an important role in ethanol modulation in alpha1 GlyRs. The findings provide insight into structure-function relationships for GlyRs. This work also provides new evidence that ethanol modulates alpha1 GlyR function, at least partly, by affecting agonist activation through position 52