Identification of channel-lining residues in the M2 membrane-spanning segment of the GABA_A receptor α₁ subunit

The γ-aminobutyric acid type A (GABA_A) receptors are the major inhibitory, postsynaptic, neurotransmitter receptors in the central nervous system. The binding of γ-aminobutyric acid (GABA) to the GABA_A receptors induces the opening of an anion-selective channel that remains open for tens of milliseconds before it closes. To understand how the structure of the GABAA receptor determines the functional properties such as ion conduction, ion selectivity and gating we sought to identify the amino acid residues that line the ion conducting channel. To accomplish this we mutated 26 consecutive residues (250-275), one at a time, in and flanking the M2 membrane-spanning segment of the rat α₁ subunit to cysteine. We expressed the mutant α₁ subunit with wild-type β₁ and γ₂ subunits in Xenopus oocytes. We probed the accessibility of the engineered cysteine to covalent modification by charged, sulfhydryl-specific reagents added extracellularly. We assume that among residues in membrane-spanning segments, only those lining the channel would be susceptible to modification by polar reagents and that such modification would irreversibly alter conduction through the channel. We infer that nine of the residues, α₁Val257, α₁Thr261, α₁Thr262, α₁Leu264, α₁Thr265, α₁Thr268, α₁Ile271, α₁Ser272 and α₁Asn275 are exposed in the channel. On a helical wheel plot, the exposed residues, except α₁Thr262, lie on one side of the helix in an arc of 120°. We infer that the M2 segment forms an α helix that is interrupted in the region of α₁Thr262. The modification of residues as cytoplasmic as α₁Val257 in the closed state of the channel suggests that the gate is at least as cytoplasmic as α₁Val257. The ability of the positively charged reagent methanethiosulfonate ethylammonium to reach the level of α₁Thr261 suggests that the charge-selectivity filter is at least as cytoplasmic as this residue.