Radiation inactivation of brain [35S]t-butylbicyclophosphorothionate binding sites reveals complicated molecular arrangements of the GABA/benzodiazepine receptor chloride channel complex.
[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-beta-carboline-3-carboxylate, ZK 93423) and inverse agonists (methyl 6,7-dimethoxy-4-ethyl-beta-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 [3H]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.
Nielsen
M
M
Honore
T
T
Braestrup
C
C
D000818:Animals; D001665:Binding Sites / Q000187:drug effects / Q000528:radiation effects; D001643:Bridged Bicyclo Compounds / Q000378:metabolism*; D019086:Bridged Bicyclo Compounds, Heterocyclic; D001952:Bridged-Ring Compounds / Q000378:metabolism*; D002243:Carbolines / Q000494:pharmacology*; D002531:Cerebellum / Q000187:drug effects / Q000378:metabolism / Q000528:radiation effects; D002540:Cerebral Cortex / Q000187:drug effects / Q000378:metabolism* / Q000528:radiation effects; D002712:Chlorides / Q000378:metabolism; D004974:Etazolate / Q000378:metabolism; D005045:Etomidate / Q000378:metabolism; D066298:In Vitro Techniques; D007473:Ion Channels; D008970:Molecular Weight; D009118:Muscimol / Q000378:metabolism; D009943:Organophosphorus Compounds / Q000378:metabolism; D011869:Radioligand Assay; D051381:Rats; D011963:Receptors, GABA-A / Q000187:drug effects / Q000378:metabolism* / Q000528:radiation effects; D013462:Sulfur Radioisotopes
D001643:Bridged Bicyclo Compounds; D019086:Bridged Bicyclo Compounds, Heterocyclic; D001952:Bridged-Ring Compounds; D002243:Carbolines; D002712:Chlorides; D007473:Ion Channels; D009943:Organophosphorus Compounds; D011963:Receptors, GABA-A; D013462:Sulfur Radioisotopes; D009118:Muscimol; C008207:4-isopropylbicyclophosphate; C037476:tert-butylbicyclophosphorothionate; C042569:ZK 93423; D004974:Etazolate; D005045:Etomidate