Tryptophan scanning mutagenesis in TM2 of the GABA(A) receptor alpha subunit: effects on channel gating and regulation by ethanol

1 Each residue in the second transmembrane segment (TM2) of the human GABAA receptor alpha(2) subunit was individually mutated to tryptophan. The wild -type or mutant alpha(2) subunits were expressed with the wild-type human G ABA, receptor beta(2) subunit in Xenopus oocytes, and the effects of these mutations were investigated using two-electrode voltage-clamp recording. 2 Four mutations (V257W, T262W, T265W and S270W) produced receptors which w ere active in the absence of agonist, and this spontaneous open channel act ivity was blocked by both picrotoxin and bicuculline, except in the alpha(2 )(V257W)beta(2) mutant receptor, which was not sensitive to picrotoxin. 3 Six mutations (V257W, V260W, T262W, T267W, S270W and A273W) enhanced the agonist sensitivity of the receptor, by 10-100 times compared with the wild -type alpha(2)beta(2) receptor. Other mutations (T261W, V263W, L269W, I271W and S272W) had little or no effect on the apparent affinity of the recepto r to GABA. Eight of the tryptophan mutations (R255, T256, F258, G259, L264, T265, M266 or T268) resulted in undetectable GABA-induced currents. 4 The S270W mutation eliminated potentiation of GABA by ethanol, whereas T2 61W markedly increased the action of ethanol. The T262W mutation produced d irect activation (10% of maximal GABA response) by ethanol in the absence o f GABA, while other mutations did not alter the action of ethanol significa ntly. 5 These results are consistent with a unique role for S270 in the action of ethanol within the TM2 region, and with models of GABA(A) receptor channel function, in which specific residues within TM2 are critical for the regul ation of channel gating (S270, L264), while other residues (L269, 1271 and S272) have little effect on these functions and may be non-critical structu ral residues.