title: Thermodynamics and kinetics of t-butylbicyclophosphorothionate binding differentiate convulsant and depressant barbiturate stereoisomers acting via GABAA ionophores.
abstract: The temperature dependence of [35S]-t-butylbicyclophosphorothionate (TBPS) binding to the convulsant sites of the GABAA receptor complex was studied in membrane preparations of rat forebrain. Although specific [35S]TBPS binding was maximal around 20 degrees C, the rate constants of dissociation decreased monotonously between 37 degrees C and 2 degrees C. The displacing potencies of the convulsant S(+) enantiomer of 1-methyl-5-phenyl-5-propyl-barbituric acid (MPPB) (IC50 = 1250 +/- 30 microM) and the depressant R(-) MPPB (IC50 = 310 +/- 5 microM) did not show significant changes between 19 degrees C and 37 degrees C. Therefore barbiturate binding seems to be driven by entropic, rather than enthalpic changes. An excess of MPPB enantiomers elicited accelerated and polyphasic dissociations of [35S]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 between 2 degrees C and 37 degrees C. Activation parameters were similar when [35S] TBPS dissociation was triggered by the convulsants TBPS and S(+) MPPB. It can be attributed to similar conformations of the closed ionophore complex. In contrast, the depressant R(-) MPPB strongly decreased the activation energy of TBPS dissociation from the open ionophore ternary complex. In whole-cell patch-clamp experiments R(-) MPPB, but not S(+) MPPB, elicited chloride currents in rat primary cortical cultures with an EC50 value of 560 +/- 30 microM and a Hill coefficient 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 dissociation of TBPS and to explain the different effects of MPPB enantiomers. Submillimolar R(-) MPPB is supposed to bind to (about three) barbiturate sites on GABAA-ionophores and to open them in a cooperative manner to result in a decreased activation energy for accelerated displacement of convulsant binding.
authors.affiliation: Department of Molecular Pharmacology, Central Research Institute for Chemistry, Hungarian Academy of Sciences, Budapest, Hungary.
authors.forename: G
authors.initials: G
authors.lastname: Maksay
authors.forename: P
authors.initials: P
authors.lastname: Molnár
authors.forename: M
authors.initials: M
authors.lastname: Simonyi
mesh_terms: D000818:Animals; D001667:Binding, Competitive / Q000187:drug effects; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000378:metabolism* / Q000633:toxicity; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D003292:Convulsants / Q000378:metabolism* / Q000633:toxicity; D004594:Electrophysiology; D007476:Ionophores / Q000378:metabolism / Q000633:toxicity; D007700:Kinetics; D008297:Male; D018408:Patch-Clamp Techniques; D010634:Phenobarbital / Q000031:analogs & derivatives / Q000378:metabolism / Q000633:toxicity; D016548:Prosencephalon / Q000187:drug effects / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D011963:Receptors, GABA-A / Q000187:drug effects / Q000378:metabolism*; D013237:Stereoisomerism; D013574:Synaptosomes / Q000187:drug effects / Q000378:metabolism; D013816:Thermodynamics
chemical_list: D019086:Bridged Bicyclo Compounds, Heterocyclic; D003292:Convulsants; D007476:Ionophores; D011963:Receptors, GABA-A; C008555:1-methyl-5-phenyl-5-propylbarbituric acid; C037476:tert-butylbicyclophosphorothionate; D010634:Phenobarbital