Alcohol- and Alcohol Antagonist-Sensitive Human GABAA Receptors: Tracking δ Subunit Incorporation into Functional Receptors

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 delta subunit-containing GABA(A)Rs is uniquely sensitive to ethanol and enhanced at concentrations relevant for human alcohol consumption. Ethanol enhancement of recombinant alpha 4 beta 3 delta receptors is blocked by the behavioral alcohol antagonist 8-azido-5,6-dihydro-5methyl-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 alpha 4 beta 3 delta 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 delta subunits into functional receptors. To track delta subunit incorporation, we used a functional tag, a single amino acid change (H68A) in a benzodiazepine binding residue in which a histidine in the delta subunit is replaced by an alanine residue found at the homologous position in gamma subunits. We demonstrate that the delta H68A substitution confers diazepam sensitivity to otherwise diazepam-insensitive alpha 4 beta 3 delta receptors. The extent of enhancement of alpha 4 beta 3 delta H68A receptors by 1 mu M diazepam, 30 mM EtOH, and 1 mu M beta-carboline-3-carboxy ethyl ester (but not 1 mu M Zn2+ block) is correlated in individual recordings, suggesting that delta 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 delta subunit-incorporation is often incomplete in recombinant systems yet is necessary for high ethanol sensitivity, one of the features of native delta subunit-containing GABA(A)Rs.