COMPARISON OF INTERACTIONS OF [H-3] MUSCIMOL, T-BUTYLBICYCLOPHOSPHORO[S-35]THIONATE, AND [H-3] FLUNITRAZEPAM WITH CLONED GAMMA-AMINOBUTYRIC ACID(A) RECEPTORS OF THE ALPHA(1)BETA(2) AND ALPHA(1)BETA(2)GAMMA(2) SUBTYPES

The alpha1beta2 and alpha1beta2gamma2 subtypes, common subtypes of gamma-aminobutyric acid (GABA)A receptors in the brain, are known to share many ligands, but only the latter interacts with benzodiazepines. In this study, we attempted to examine whether the presence of the gamma2 subunit in the cloned receptors alters the binding properties of GABA and t-butylbicyclophosphorothionate (TBPS) (a highly sensitive probe for conformational changes in the chloride ionophore of GABA(A) receptors) and their interactions. Using a high-level expression system of SF-9 cells infected with baculovirus, we produced a group of cloned GABA(A) receptors with variations in the ratio (0 to 3) of the virion carrying the cDNA for the gamma2 subunit to those carrying the cDNAs for the alpha1 and beta2 subunits. The number of benzodiazepine binding sites increased as the level of the gamma2 virion was raised and reached that of GABA high affinity sites at a gamma2 to alpha1beta2 ratio of 0.5 or more. It appears that the conversion of the alpha1beta2 to the alpha1beta2gamma2 subtype is favorable and complete in the presence of a sufficient level of the gamma2 subunit, assuming the number of the GABA sites to be equal to the total number of the cloned GABA(A) receptors. In all preparations, the dissociation constants for flunitrazepam, muscimol, and TBPS were fairly constant, and the maximal number of binding sites for TBPS appeared to be equal to that for muscimol, with no dependence on the gamma2 virion levels. The effect of GABA on TBPS binding, however, were markedly altered by the gamma2 subunit. With the alpha1beta2 subtype GABA at concentrations occupying its high affinity sites markedly stimulated but at higher concentrations (micromolar ranges) inhibited TBPS binding, whereas with the alpha1alpha2gamma2 subtype GABA inhibited TBPS binding without the early stimulatory phase. We also confirmed the selective interaction of Zn2+ (50 mum) with the alpha1beta2 subtype, as probed with TBPS binding, and observed a progressive disappearance of Zn2+ sensitivity as the gamma2 virion level increased. These results show that addition of the gamma2 subunit to the alpha1beta2 subtype does not disturb the primary binding sites for GABA and TBPS but markedly modifies GABA-induced conformational changes in the chloride channel, as detected with TBPS binding, and alters allosteric sites (i.e., creating benzodiazepine sites and obliterating Zn2+ sites), in agreement with earlier studies [Nature (Lond.) 338:582-585 (1989); Neuron 5:781-788 (1990)].