Effects of GABA and various allosteric ligands on TBPS binding to cloned rat GABA A receptor subtypes

1 [ 35 S] t ‐butylbicyclophosphorothionate (TBPS) is a high affinity ligand for the picrotoxin site of GABA A receptors. Here we examined TBPS binding to the cloned receptors made of α 1 , α 3 or α 6 in combination with β2 or β2 and γ2 subunits, in the presence of GABA and several allosteric ligands (diazepam, methyl 6,7‐dimethoxy‐4‐methyl‐β‐carboline‐3‐carboxylate (DMCM), 3α,21‐dihydroxy‐5α‐pregnan‐20‐one (5α‐THDOC), pentobarbitone and Zn). The cloned receptors were transiently expressed in SF‐9 insect cells by infecting with recombinant baculoviruses. 2 In αβ subtypes, GABA at nanomolar concentrations enhanced TBPS binding but inhibited binding at micromolar concentrations. Half maximal GABA concentrations for enhancement or inhibition of TBPS binding were correlated with high and low affinity GABA binding sites, respectively, in individual subtypes. The maximal enhancement of binding also varied according to the α isoform (α3β2>>α1β2>α6β2). In αβγ subtypes, TBPS binding was unaffected by GABA at nanomolar concentrations, but was inhibited by GABA at micromolar concentrations. Addition of γ2 thus appeared to abolish conformational coupling between high affinity GABA sites and TBPS sites, and also altered low affinity GABA sites; in particular, the half maximal GABA concentration for inhibition of TBPS binding changed from > 100 (α6β2) to 1 μ m (α6β2γ2). 3 Allosteric ligands also altered TBPS binding to sensitive GABA A receptor subtypes. For instance, diazepam only in the α1β2γ2 and α3β2γ2 subtypes, and 5α‐THDOC in all the subtypes enhanced TBPS binding in the absence of GABA, and intensified the inhibitory effect of GABA. Pentobarbitone exhibited only the latter effect in all the subtypes we examined. 4 DMCM and Zn, inhibitors of GABA‐induced Cl currents in αβγ and αβ subtypes, respectively, produced opposite effects to agonists, decreasing TBPS binding in the absence of GABA and attenuating (or eliminating in the case of Zn) the inhibitory effect of GABA on TBPS binding. 5 These results show that GABA binding sites and their conformational coupling with TBPS sites are differentially affected by the α isoform (particularly α6 as compared to α1 or α3) and by quaternary interactions involving the γ2 subunit. Moreover, changes in TBPS binding by allosteric ligands include not only direct (allosteric) effects on TBPS sites but also indirect effects via GABA sites, and are consistent with their known subtype selectivity and functionality from previous studies.