title: GABA(A) receptor transmembrane amino acids are critical for alcohol action: disulfide cross-linking and alkyl methanethiosulfonate labeling reveal relative location of binding sites.
abstract: Alcohols and inhaled anesthetics modulate GABA(A) receptor (GABA(A)R) function via putative binding sites within the transmembrane regions. The relative position of the amino acids lining these sites could be either inter- or intra-subunit. We introduced cysteines in relevant TM locations and tested the proximity of cysteine pairs using oxidizing and reducing agents to induce or break disulfide bridges between cysteines, and thus change GABA-mediated currents in wild-type and mutant α1β2γ2 GABA(A)Rs expressed in Xenopus laevis oocytes. We tested for: (i) inter-subunit cross-linking: a cysteine located in α1TM1 [either α1(Q229C) or α1(L232C)] was paired with a cysteine in different positions of β2TM2 and TM3; (ii) intra-subunit cross-linking: a cysteine located either in β2TM1 [β2(T225C)] or in TM2 [β2(N265C)] was paired with a cysteine in different locations along β2TM3. Three inter-subunit cysteine pairs and four intra-subunits cross-linked. In three intra-subunit cysteine combinations, the alcohol effect was reduced by oxidizing agents, suggesting intra-subunit alcohol binding. We conclude that the structure of the alcohol binding site changes during activation and that potentiation or inhibition by binding at inter- or intra-subunit sites is determined by the specific receptor and ligand.
authors.affiliation: Cellular and Molecular Biology, Waggoner Center for Alcohol and Addiction Research, The University of Texas at Austin, Austin, Texas, USA.
authors.forename: Cecilia M
authors.initials: CM
authors.lastname: Borghese
authors.forename: Jessica A
authors.initials: JA
authors.lastname: Hicks
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Lapid
authors.forename: James R
authors.initials: JR
authors.lastname: Trudell
authors.forename: R Adron
authors.initials: RA
authors.lastname: Harris
mesh_terms: D000596:Amino Acids / Q000502:physiology*; D000818:Animals; D001665:Binding Sites / Q000187:drug effects; D002492:Central Nervous System Depressants / Q000378:metabolism / Q000494:pharmacology*; D003001:Cloning, Molecular; D003432:Cross-Linking Reagents; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D055724:Electrophysiological Phenomena; D000431:Ethanol / Q000378:metabolism / Q000494:pharmacology*; D018682:GABA Agents / Q000494:pharmacology; D006801:Humans; D008698:Mesylates / Q000378:metabolism*; D008958:Models, Molecular; D009865:Oocytes / Q000378:metabolism; D010084:Oxidation-Reduction; D011963:Receptors, GABA-A / Q000187:drug effects* / Q000235:genetics; D014982:Xenopus laevis; D005680:gamma-Aminobutyric Acid / Q000494:pharmacology
chemical_list: D000596:Amino Acids; D002492:Central Nervous System Depressants; D003432:Cross-Linking Reagents; D004220:Disulfides; D018682:GABA Agents; D008698:Mesylates; D011963:Receptors, GABA-A; D000431:Ethanol; C074502:methanethiosulfonate; D005680:gamma-Aminobutyric Acid; D003545:Cysteine
keywords: GABAA receptor; binding site; cross-link; ethanol; homology model; methanethiosulfonate