Kinetic regulation of convulsant (TBPS) binding by GABAergic agents.

The kinetics of specific [35S]-t-butylbicyclophosphorothionate (TBPS) binding was studied in rat brain synaptosomal membrane preparations. The effects of a representative depressant barbiturate, R-(-)-N-(1)-Me-5-Phe-5-Pr-barbituric acid [R(-)MPPB, and gamma-aminobutyric acid (GABA) were biphasic on TBPS binding, but the enhancements of binding by low concentrations of R(-)MPPB or GABA disappeared when binding equilibrium was reached. The slope factors of TBPS displacement and the IC50 values of R(-)MPPB and GABA decreased when TBPS binding approached equilibrium. Up to 70 min, binding in the presence of 300 microM R(-)MPPB exceeded, then remained below, the level of the control. R(-)MPPB decreased the apparent association half-life of TBPS binding from 41.5 min to 11.9 min. The GABA agonist, muscimol, in the concentration range of 20 and 200 nM, progressively accelerated the rate of TBPS dissociation. This effect was completely reversed by 20 microM bicuculline methochloride. GABA antagonists, bicuculline methochloride (20 microM) and R 5135 (20 nM), alone decelerated the rate of TBPS dissociation. Dissociation of TBPS was also initiated by dilution in the presence of various salts. Replacement of Cl- by Br- ions (0.5 molar) accelerated the rate of dissociation, whereas replacement of K+ by Na+ ions had no effect. This indicates the role of Eccles anions, not of cations, in TBPS binding and the possible involvement of the chloride inophore. A kinetic model is discussed for the allosteric modulation of TBPS binding by various GABAergic agents. Interconvertible populations of TBPS sites are proposed with rapid and slow kinetics. Model calculations involving modulation in the same direction of the on and off rates of binding can reproduce observed phenomena. The model predicts that GABA agonists, barbiturate and pyrazolopyridine depressants, as well as 1,4-benzodiazepine agonists, would allosterically increase the proportion of a rapid kinetic population of TBPS sites. In contrast, the model predicts that a decrease in the contribution of the rapid phase might be brought about by GABA antagonists and beta-carboline inverse agonists. The slow and rapid kinetic populations of TBPS sites might represent the closed and open states of the chloride ionophores, respectively.