The γ-aminobutyric acid type A (GABAA) receptors are the major inhibitory, postsynaptic, neurotransmitter receptors in the central nervous system. The binding of γ-aminobutyric acid (GABA) to the GABAA 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.
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