Subunit mutations affect ethanol actions on GABAA receptors expressed in Xenopus oocytes

1 Mutations of specific amino acids were introduced in transmembrane domains (TM) of GABA(A) receptor alpha(2), beta(1) and gamma(2L) subunits. The effects of these mutations on the action of ethanol were studied using the Xenopus oocyte expression system and two-electrode voltage-clamp recording techniques. 2 Mutant alpha(2) subunits containing S270I (TM2) or A291W (TM3) made the receptor more sensitive to GABA, as compared to wild-type alpha(2)beta(1)gamma(2L) receptor. The mutation S265I (TM2) of beta(1) and S280I (TM2) or S301W (TM3) in gamma(2L) subunits did not alter apparent affinity of the receptor for GABA. M286W (TM3) in the beta(1) subunit resulted in a receptor that was tonically open. 3 Using an ECS concentration of GABA, the function of the wild-type receptor with alpha(2)beta(1)gamma(2L) subunits was potentiated by ethanol (50 - 200 mM). The mutations in TM2 or TM3 of the at subunit diminished the potentiation by ethanol. The action of ethanol was also eliminated with a mutation in the TM2 site of the beta(1) subunit. Ethanol produced significant inhibition of GABA responses in receptors containing the combination of alpha(2) and beta(1) TM2 mutants with a wild-type gamma(2L) subunit. A small but significant reduction in the potentiation by ethanol was observed with gamma(2L) TM2 and/or TM3 mutants. 4 From these results, we suggest that in heteromeric GABA(A) receptors composed of the alpha, beta and gamma subunits, ethanol may bind in a cavity formed by TM2 and TM3, and that binding to the alpha or beta subunit may be more critical than the gamma subunit.