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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 α1 subunit is affected by interaction with heterotrimeric G proteins (Gβγ). GlyRs containing the α3 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 α3 subunits were not potentiated by pharmacologic concentrations of ethanol or by Gβγ. Thus, we studied GlyR α1–α3 chimeras and mutants to determine the molecular properties that confer ethanol insensitivity. Mutation of corresponding glycine 254 in transmembrane domain 2 (TM2) found in α1 in the α3A254G –α1 chimera induced a glycine-evoked current that displayed potentiation during application of ethanol (46 ± 5%, 100 mM) and Gβγ activation (80 ± 17%). Interestingly, insertion of the intracellular α3L splice cassette into GlyR α1 abolished the enhancement of the glycine-activated current by ethanol (5 ± 6%) and activation by Gβγ (−1 ± 7%). Incorporation of the GlyR α1 C terminus into the ethanol-resistant α3SA254G 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 α3 subunits are not modulated by ethanol. Residue A254 in TM2, the α3L splice cassette, and the C-terminal domain of α3 GlyRs are determinants for low ethanol sensitivity and form the molecular basis of subtype-selective modulation of GlyRs by alcohol.