IDENTIFICATION OF CHANNEL-LINING RESIDUES IN THE M2 MEMBRANE-SPANNINGSEGMENT OF THE GABA(A) RECEPTOR ALPHA(1) SUBUNIT

The gamma-aminobutyic acid type A (GABA(A)) receptors are the major in hibitory, postsynaptic, neurotransmitter receptors in the central nerv ous system. The binding of gamma-aminobutyric acid (GABA) to the GABA( A) receptors induces the opening of an anion-selective channel that re mains open for tens of milliseconds before it closes. To understand ho w the structure of the GABA(A) receptor deter-mines the functional pro perties such as ion conduction, ion selectivity and gating we sought t o identify the amino acid residues that line the ion conducting channe l. To accomplish this we mutated 26 consecutive residues (250-275), on e at a time, in and flanking the M2 membrane-spanning segment of tile rat alpha(1) subunit to cysteine. We expressed the mutant alpha(1) sub unit with wild-type beta(1) and gamma(2) subunits in Xenopus oocytes. We probed die accessibility of the engineered cysteine to covalent mod ification by charged, sulfhydryl-specific reagents added extracellular ly. 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 conducti on through the channel, We infer that nine of the residues, alpha(1)Va l257, alpha(1)Thr261, alpha(1)Thr262, alpha(1)Leu264, alpha(1)Thr265, alpha(1)Thr268, alpha(1)Ile271, alpha(1)Ser272 and alpha(1)Asn275 are exposed in the channel, On a helical wheel plot, the exposed residues, except alpha(1)Thr262, lie on one side of the helix in an arc of 120 degrees. We infer that the M2 seg ment forms an a helix that is interr upted in the region of alpha(1)Thr262. The modification of residues as cytoplasmic as alpha(1)Val257 in the closed state of the channel sugg ests that the gate is at least as cytoplasmic as alpha(1)Vak257. The a bility of the positively charged reagent methanethiosulfonate ethylamm onium to reach the level of alpha(1)Thr261 suggests that the charge-se lectivity filter is at least as cytoplasmic as this residue.