THERMODYNAMICS AND KINETICS OF T-BUTYLBICYCLOPHOSPHOROTHIONATE BINDING DIFFERENTIATE CONVULSANT AND DEPRESSANT BARBITURATE STEREOISOMERS ACTING VIA GABA(A) IONOPHORES

The temperature dependence of [S-35]-t- butylbicyclophosphorothionate (TBPS) binding to the convulsant sites of the GABA, receptor complex w as studied in membrane preparations of rat forebrain. Although specifi c [S-35]TBPS binding was maximal around 20 degrees C, the rate constan ts of dissociation decreased monotonously between 37 degrees C and 2 d egrees C. The displacing potencies of the convulsant S(+) enantiomer o f 1-methyl-5-phenyl-5-propyl-barbituric acid (MPPB) (IC50 = 1250 +/- 3 0 mu M) and the depressant R(-) MPPB (IC50 = 310 +/- 5 mu M) did not s how significant changes between 19 degrees C and 37 degrees C. Therefo re barbiturate binding seems to be driven by entropic, rather than ent halpic changes. An excess of MPPB enantiomers elicited accelerated and polyphasic dissociations of [S-35]TBPS as compared to the monophasic dissociation by TBPS. Arrhenius analysis was applied to the measurable initial rate constants of dissociation. Arrhenius plots were linear b etween 2 degrees C and 37 degrees C. Activation parameters were simila r when [S-35] TBPS dissociation was triggered by the convulsants TBPS and S(+) MPPB. It can be attributed to similar conformations of the cl osed ionophore complex. In contrast, the depressant R(-) MPPB strongly decreased the activation energy of TBPS dissociation from the open io nophore ternary complex. In whole-cell patch-clamp experiments R(-) MP PB, but not S(+) MPPB, elicited chloride currents in rat primary corti cal cultures with an EC(50) value of 560 +/- 30 mu M and a Hill coeffi cient of 2.9 +/- 0.2. These currents were similar to those elicited by GABA and blocked by TBPS. A kinetic scheme is proposed for the dissoc iation of TBPS and to explain the different effects of MPPB enantiomer s. Submillimolar R(-) MPPB is supposed to bind to (about three) barbit urate sites on GABA(A)-ionophores and to open them in a cooperative ma nner to result in a decreased activation energy for accelerated displa cement of convulsant binding.