Radiation inactivation of brain [ 35S] t-butylbicyclophosphorothionate binding sites reveals complicated molecular arrangements of the GABA/benzodiazepine receptor chloride channel complex

Abstract

[ 35S] t-Butylbicyclophosphorothionate ([ 35S]TBPS), a bicyclic cage convulsant, binds to the anion gating mechanism of the GABA/benzodiazepine receptor chloride channel complex. Using a carefully calibrated radiation inactivation technique, the molecular weight of [ 35S]TBPS binding complexes from frozen rat cerebral cortex was estimated to be 137,000 daltons. The GABA agonist muscimol reduced [ 35S]TBPS binding to 0–10% of the control value, in a way which is independent of the radiation dose. This shows that the GABA receptor ( Mw = 55,000 daltons) is included in the 137,000-dalton [ 35S]-TBPS binding complex; the [ 35S]TBPS binding protein alone accounts for 137,000−55,000 = 82,000 daltons. The pyrazolopyridazine etazolate (SQ 20.009) and etomidate in appropriate concentrations both reduced specific binding of [ 35S]TBPS. The ability of SQ 20.009 and etomidate to reduce [ 35S]TBPS binding was greatly reduced by exposure to low radiation doses, suggesting that SQ 20.009 and etomidate reduce [ 35S]TBPS binding by an allosteric mechanism requiring a molecular structure of 450,000–500,000 daltons. Benzodiazepine agonists (ethyl 4-methoxymethyl-6-benzyloxy-β-carboline-3-carboxylate, ZK 93423) and inverse agonists (methyl 6,7-dimethoxy-4-ethyl-β-carboline-3-carboxylate, DMCM) enhance and reduce [ 35S]TBPS binding, respectively, in repeatedly frozen and washed membrane preparations. The effects of ZK 93423 and DMCM on [ 35S]TBPS binding disappeared upon exposure of membranes to low radiation doses. This suggests that the benzodiazepine receptor site interacts allosterically with the [ 35S]TBPS binding site, requiring a molecular complex of at least c. 400,000 daltons. The [ 35S]TBPS site alone in these latter conditions of membrane preparation (repeatedly frozen/ washed) revealed a molecular weight of 221,000 daltons (TBPS-site + GABA receptor + unknown structures). The number of binding sites for [ 35S]TBPS (145 pmol/g tissue) was only slightly higher than for [ 35H]flunitrazepam (130 pmol/g tissue) in cerebral cortex. These results are all consonant with the conclusion that the GABA/BZ receptor chloride channel complex is composed of highly integrated multimeric subunits, tentatively accounted for by a tetramic complex of molecular weight 548,000 daltons.