Substitutions of the highly conserved M2 leucine create spontaneously opening ρ γ-aminobutyric acid receptors

All members of the receptor-operated ion channel family that includes γ-aminobutyric acid (GABA), glycine, nicotinic acetylcholine, and serotonin type 3 receptors have a conserved leucine near the center of the presumed second membrane-spanning domain. This leucine has been postulated to play a role in the gating of the pore. In this study, we examined the effects of mutating this leucine (L301) on the function of human homomeric ρ1 GABA receptors. Oocytes expressing γ GABA receptors in which this leucine was substituted with alanine (A), glycine (G), serine (S), threonine (T), valine, or tyrosine, but not isoleucine or phenylalanine, demonstrated larger-than- normal resting conductances in the absence of GABA. This resting conductance had a reversal potential (and shifted reversal potential with chloride substitution) indistinguishable from that of the wild-type γ1 GABA-activated current. This resting conductance was antagonized by picrotoxin and, in the case of the A, G, S, and T substitutions, by GABA itself. Although the 1/2 competitive antagonist 3-aminopropyl(methyl)-phosphinic acid did not block the resting conductance, this compound did competitively inhibit the GABA- mediated antagonism of the resting conductance. At higher concentrations, both 3-aminopropyl(methyl)-phosphinic acid and GABA directly activated the A, G, S, and T mutant receptors. Taken together, these data suggest that substitution of this highly conserved leucine with either small or polar residues produced γ1 GABA receptors that can open in the absence of GABA and support the hypothesis that this leucine may play a key role in the gating of the pore.