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

1. [35S]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 alpha 1, alpha 3 or alpha 6 in combination with beta 2 or beta 2 and gamma 2 subunits, in the presence of GABA and several allosteric ligands (diazepam, methyl 6,7-dimethoxy-4-methyl-beta-carboline-3-carboxylate (DMCM), 3 alpha,21-dihydroxy-5 alpha-pregnan-20-one (5 alpha-THDOC), pentobarbitone and Zn). The cloned receptors were transiently expressed in SF-9 insect cells by infecting with recombinant baculoviruses. 2. In alpha beta 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 alpha isoform (alpha 3 beta 2 >> alpha 1 beta 2). In alpha beta gamma subtypes, TBPS binding was unaffected by GABA at nanomolar concentrations, but was inhibited by GABA at micromolar concentrations. Addition of gamma 2 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 (alpha 6 beta 2) to 1 microM (alpha 6 beta 2 gamma 2). 3. Allosteric ligands also altered TBPS binding to sensitive GABA(A) receptor subtypes. For instance,diazepam only in the alpha 1 beta2 gamma 2 and alpha 3 beta 2 gamma 2 subtypes, and 5 alpha-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 alpha beta gamma and alpha beta 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 alpha isoform (particularly alpha 6 as compared to alpha l or alpha 3) and by quaternary interactions involving the gamma 2 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.