title: The TM2 6' position of GABA(A) receptors mediates alcohol inhibition.
abstract: Ionotropic GABA(A) receptors (GABA(A)Rs), which mediate inhibitory neurotransmission in the central nervous system, are implicated in the behavioral effects of alcohol and alcoholism. Site-directed mutagenesis studies support the presence of discrete molecular sites involved in alcohol enhancement and, more recently, inhibition of GABA(A)Rs. We used Xenopus laevis oocytes to investigate the 6' position in the second transmembrane region of GABA(A)Rs as a site influencing alcohol inhibition. We asked whether modification of the 6' position by substitution with larger residues or methanethiol labeling [using methyl methanethiosulfonate (MMTS)] of a substituted cysteine, reduced GABA action and/or blocked further inhibition by alcohols. Labeling of the 6' position in either α2 or β2 subunits reduced responses to GABA. In addition, methanol and ethanol potentiation increased after MMTS labeling or substitution with tryptophan or methionine, consistent with elimination of an inhibitory site for these alcohols. Specific alcohols, but not the anesthetic etomidate, competed with MMTS labeling at the 6' position. We verified a role for the 6' position in previously tested α2β2 as well as more physiologically relevant α2β2γ2s GABA(A)Rs. Finally, we built a novel molecular model based on the invertebrate glutamate-gated chloride channel receptor, a GABA(A)R homolog, revealing that the 6' position residue faces the channel pore, and modification of this residue alters volume and polarity of the pore-facing cavity in this region. These results indicate that the 6' positions in both α2 and β2 GABA(A)R subunits mediate inhibition by short-chain alcohols, which is consistent with the presence of multiple counteracting sites of action for alcohols on ligand-gated ion channels.
authors.affiliation: Waggoner Center for Alcohol and Addiction Research, University of Texas, 1 University Station A4800, Austin, TX 78712-05159, USA. wdavidjohnsonii@mail.utexas.edu
authors.forename: W David
authors.initials: WD
authors.lastname: Johnson
authors.forename: Rebecca J
authors.initials: RJ
authors.lastname: Howard
authors.forename: James R
authors.initials: JR
authors.lastname: Trudell
authors.forename: R Adron
authors.initials: RA
authors.lastname: Harris
mesh_terms: D000438:Alcohols / Q000378:metabolism / Q000494:pharmacology; D019943:Amino Acid Substitution / Q000502:physiology; D000818:Animals; D001665:Binding Sites / Q000502:physiology; D001667:Binding, Competitive; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D003553:Cystine / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics; D004357:Drug Synergism; D055724:Electrophysiological Phenomena / Q000187:drug effects / Q000502:physiology; D000431:Ethanol / Q000378:metabolism / Q000494:pharmacology*; D005045:Etomidate / Q000494:pharmacology; D058787:GABA-A Receptor Antagonists / Q000378:metabolism / Q000494:pharmacology*; D006801:Humans; D008715:Methionine / Q000235:genetics; D008741:Methyl Methanesulfonate / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D056004:Molecular Dynamics Simulation; D009865:Oocytes / Q000187:drug effects / Q000378:metabolism; D018408:Patch-Clamp Techniques; D011963:Receptors, GABA-A / Q000737:chemistry / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D014364:Tryptophan / Q000235:genetics; D014982:Xenopus laevis; D005680:gamma-Aminobutyric Acid / Q000494:pharmacology
chemical_list: D000438:Alcohols; D018076:DNA, Complementary; D058787:GABA-A Receptor Antagonists; C484841:GABRA2 protein, human; C495710:GABRB2 protein, human; C546279:GABRG2 protein, human; D011963:Receptors, GABA-A; C014674:methyl methanethiosulfonate; D000431:Ethanol; D003553:Cystine; D005680:gamma-Aminobutyric Acid; D014364:Tryptophan; D008715:Methionine; D008741:Methyl Methanesulfonate; D003545:Cysteine; D005045:Etomidate
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