Subunit mutations affect ethanol actions on GABA A receptors expressed in Xenopus oocytes

Mutations of specific amino acids were introduced in transmembrane domains (TM) of GABA A receptor α 2 , β 1 and γ 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. Mutant α 2 subunits containing S270I (TM2) or A291W (TM3) made the receptor more sensitive to GABA, as compared to wild‐type α 2 β 1 γ 2L receptor. The mutation S265I (TM2) of β 1 and S280I (TM2) or S301W (TM3) in γ 2L subunits did not alter apparent affinity of the receptor for GABA. M286W (TM3) in the β 1 subunit resulted in a receptor that was tonically open. Using an EC 5 concentration of GABA, the function of the wild‐type receptor with α 2 β 1 γ 2L subunits was potentiated by ethanol (50–200 m M ). The mutations in TM2 or TM3 of the α 2 subunit diminished the potentiation by ethanol. The action of ethanol was also eliminated with a mutation in the TM2 site of the β 1 subunit. Ethanol produced significant inhibition of GABA responses in receptors containing the combination of α 2 and β 1 TM2 mutants with a wild‐type γ 2L subunit. A small but significant reduction in the potentiation by ethanol was observed with γ 2L TM2 and/or TM3 mutants. From these results, we suggest that in heteromeric GABA A receptors composed of the α, β and γ subunits, ethanol may bind in a cavity formed by TM2 and TM3, and that binding to the α or β subunit may be more critical than the γ subunit.British Journal of Pharmacology (1999) 127 , 377–382; doi: 10.1038/sj.bjp.0702563