title: Alcohol- and alcohol antagonist-sensitive human GABAA receptors: tracking δ subunit incorporation into functional receptors.
abstract: GABA(A) receptors (GABA(A)Rs) have long been a focus as targets for alcohol actions. Recent work suggests that tonic GABAergic inhibition mediated by extrasynaptic δ subunit-containing GABA(A)Rs is uniquely sensitive to ethanol and enhanced at concentrations relevant for human alcohol consumption. Ethanol enhancement of recombinant α4β3δ receptors is blocked by the behavioral alcohol antagonist 8-azido-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5-a][1,4]benzodiazepine-3-carboxylic acid ethyl ester (Ro15-4513), suggesting that EtOH/Ro15-4513-sensitive receptors mediate important behavioral alcohol actions. Here we confirm alcohol/alcohol antagonist sensitivity of α4β3δ receptors using human clones expressed in a human cell line and test the hypothesis that discrepant findings concerning the high alcohol sensitivity of these receptors are due to difficulties incorporating δ subunits into functional receptors. To track δ subunit incorporation, we used a functional tag, a single amino acid change (H68A) in a benzodiazepine binding residue in which a histidine in the δ subunit is replaced by an alanine residue found at the homologous position in γ subunits. We demonstrate that the δH68A substitution confers diazepam sensitivity to otherwise diazepam-insensitive α4β3δ receptors. The extent of enhancement of α4β3δH68A receptors by 1 μM diazepam, 30 mM EtOH, and 1 μM β-carboline-3-carboxy ethyl ester (but not 1 μM Zn(2+) block) is correlated in individual recordings, suggesting that δ subunit incorporation into recombinant GABA(A)Rs varies from cell to cell and that this variation accounts for the variable pharmacological profile. These data are consistent with the notion that δ subunit-incorporation is often incomplete in recombinant systems yet is necessary for high ethanol sensitivity, one of the features of native δ subunit-containing GABA(A)Rs.
authors.affiliation: Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, CA 90095-1735, USA.
authors.forename: Pratap
authors.initials: P
authors.lastname: Meera
authors.forename: Richard W
authors.initials: RW
authors.lastname: Olsen
authors.forename: Thomas S
authors.initials: TS
authors.lastname: Otis
authors.forename: Martin
authors.initials: M
authors.lastname: Wallner
mesh_terms: D000494:Allosteric Regulation; D000818:Animals; D001386:Azides / Q000494:pharmacology*; D001569:Benzodiazepines / Q000494:pharmacology*; D002243:Carbolines / Q000494:pharmacology; D002413:Cations, Divalent; D002460:Cell Line; D003975:Diazepam / Q000494:pharmacology; D000431:Ethanol / Q000037:antagonists & inhibitors* / Q000494:pharmacology*; D006801:Humans; D009154:Mutation; D018408:Patch-Clamp Techniques; D021122:Protein Subunits / Q000096:biosynthesis / Q000235:genetics; D051381:Rats; D011963:Receptors, GABA-A / Q000096:biosynthesis* / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D014162:Transfection; D015032:Zinc / Q000494:pharmacology
chemical_list: D001386:Azides; D002243:Carbolines; D002413:Cations, Divalent; D021122:Protein Subunits; D011963:Receptors, GABA-A; D011994:Recombinant Proteins; D001569:Benzodiazepines; D000431:Ethanol; C024915:beta-carboline-3-carboxylic acid ethyl ester; C042957:Ro 15-4513; D015032:Zinc; D003975:Diazepam