title: Identification of channel-lining residues in the M2 membrane-spanning segment of the GABA(A) receptor alpha1 subunit.
abstract: The gamma-aminobutyric acid type A (GABA(A)) receptors are the major inhibitory, postsynaptic, neurotransmitter receptors in the central nervous system. The binding of gamma-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 GABA(A) 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 alpha1 subunit to cysteine. We expressed the mutant alpha1 subunit with wild-type beta1 and gamma2 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, alpha1 Val257, alpha1 Thr26l, alpha1 Thr262, alpha1 Leu264, alpha1 Thr265, alpha1 Thr268, alpha1 Ile27l, alpha1 Ser272 and alpha1 Asn275 are exposed in the channel. On a helical wheel plot, the exposed residues, except alpha1 Thr262, lie on one side of the helix in an arc of 120 degrees. We infer that the M2 segment forms an alpha helix that is interrupted in the region of alpha1 Thr262. The modification of residues as cytoplasmic as alpha1 Val257 in the closed state of the channel suggests that the gate is at least as cytoplasmic as alpha1 Val257. The ability of the positively charged reagent methanethiosulfonate ethylammonium to reach the level of alpha1 Thr261 suggests that the charge-selectivity filter is at least as cytoplasmic as this residue.
authors.affiliation: Center for Molecular Recognition, College of Physicians and Surgeons, Columbia University, New York, New York 10032, USA.
authors.forename: M
authors.initials: M
authors.lastname: Xu
authors.forename: M H
authors.initials: MH
authors.lastname: Akabas
mesh_terms: D002731:4-Chloromercuribenzenesulfonate / Q000494:pharmacology; D000818:Animals; D002729:Chloromercuribenzoates / Q000494:pharmacology; D005020:Ethyl Methanesulfonate / Q000031:analogs & derivatives / Q000494:pharmacology; D005260:Female; D007202:Indicators and Reagents / Q000494:pharmacology; D007461:Iodoacetates / Q000494:pharmacology; D019807:Iodoacetic Acid; D015640:Ion Channel Gating / Q000502:physiology*; D007473:Ion Channels / Q000737:chemistry* / Q000187:drug effects / Q000235:genetics; D016297:Mutagenesis, Site-Directed / Q000502:physiology; D009153:Mutagens / Q000494:pharmacology; D009865:Oocytes / Q000502:physiology; D018408:Patch-Clamp Techniques; D051381:Rats; D011963:Receptors, GABA-A / Q000737:chemistry* / Q000187:drug effects / Q000235:genetics; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D014981:Xenopus; D020245:p-Chloromercuribenzoic Acid
chemical_list: D002729:Chloromercuribenzoates; D007202:Indicators and Reagents; D007461:Iodoacetates; D007473:Ion Channels; D009153:Mutagens; D011963:Receptors, GABA-A; D013439:Sulfhydryl Reagents; C093171:methanethiosulfonate ethylammonium; D020245:p-Chloromercuribenzoic Acid; D002731:4-Chloromercuribenzenesulfonate; D005020:Ethyl Methanesulfonate; D019807:Iodoacetic Acid