Control of Ethanol Sensitivity of the Glycine Receptor α3 Subunit by Transmembrane 2, the Intracellular Splice Cassette and C-Terminal Domain

Previous studies have shown that the effect of ethanol on glycine receptors (GlyRs) containing the alpha 1 subunit is affected by interaction with heterotrimeric G proteins (G beta gamma). GlyRs containing the alpha 3 subunit are involved in inflammatory pain sensitization and rhythmic breathing and have received much recent attention. For example, it is unknown whether ethanol affects the function of this important GlyR subtype. Electrophysiologic experiments showed that GlyR alpha 3 subunits were not potentiated by pharmacologic concentrations of ethanol or by G beta gamma. Thus, we studied GlyR alpha 1-alpha 3 chimeras and mutants to determine the molecular properties that confer ethanol insensitivity. Mutation of corresponding glycine 254 in transmembrane domain 2 (TM2) found in alpha 1 in the alpha 3(A254G)-alpha 1 chimera induced a glycine-evoked current that displayed potentiation during application of ethanol (46 +/- 5%, 100 mM) and G beta gamma activation (80 +/- 17%). Interestingly, insertion of the intracellular alpha 3L splice cassette into GlyR alpha 1 abolished the enhancement of the glycine-activated current by ethanol (5 +/- 6%) and activation by G beta gamma (-1 +/- 7%). Incorporation of the GlyR alpha 1 C terminus into the ethanol-resistant alpha 3S(A254G) mutant produced a construct that displayed potentiation of the glycine-activated current with 100 mM ethanol (40 +/- 6%) together with a current enhancement after G protein activation (68 +/- 25%). Taken together, these data demonstrate that GlyR alpha 3 subunits are not modulated by ethanol. Residue A254 in TM2, the alpha 3L splice cassette, and the C-terminal domain of alpha 3 GlyRs are determinants for low ethanol sensitivity and form the molecular basis of subtype-selective modulation of GlyRs by alcohol.