The channel-lining 6' amino acid in the second membrane-spanning region of ionotropic GABA receptors has more profound effects on 4'-ethynyl-4-n-propylbicycloorthobenzoate binding than the 2' amino acid.

The noncompetitive antagonist of ionotropic gamma-aminobutyric acid (GABA) receptors 4'-ethynyl-4-n-propylbicycloorthobenzoate (EBOB) is a useful tool to probe the antagonist-binding site. In the present study, four mutants of the human GABA(A) receptor beta3 subunit were stably expressed in S2 cells and examined for their abilities to bind [(3)H]EBOB to identify the binding site of EBOB. The homo-oligomeric beta3 GABA receptor was used as a housefly GABA receptor model, as the beta3 subunit has a high sequence similarity with the housefly Rdl subunit in the second membrane-spanning (M2) region. The A274S mutation at the -1' position in the M2 region had no effect on [(3)H]EBOB binding. The A277S mutation at the 2' position led to a decrease in the affinity of EBOB for the GABA receptor. The T281V mutant at the 6' position and the A277S/T281V double mutant completely abolished the binding ability. A beta3 GABA receptor homology model predicts these interactions between the receptor and EBOB. These results suggest that EBOB interacts with threonine 281 and alanine 277, and that threonine 281 plays a more critical role in interacting with EBOB than alanine 277.