Radiation inactivation of brain [35S]t-butylbicyclophosphorothionate binding sites reveals complicated molecular arrangements of the GABA/benzodiazepine receptor chloride channel complex.
Radiation inactivation of brain [35S]t-butylbicyclophosphorothionate binding sites reveals complicated molecular arrangements of the GABA/benzodiazepine receptor chloride channel complex.
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脑[35S]叔丁基二环硫代磷酸酯结合位点的辐射失活揭示了 GABA/苯二氮卓受体氯离子通道复合物的复杂分子排列。
DOI：
10.1016/0006-2952(85)90223-0
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发表时间：
1985
影响因子：
5.8
通讯作者：
C. Braestrup
中科院分区：
文献类型：
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作者：
M. Nielsen;T. Honoré;C. Braestrup
[35S]双环硫代磷酸叔丁酯([35S]TBP)是一种双环笼状惊厥药，与GABA/苯二氮卓类受体氯通道复合体的阴离子门控机制结合。用一种精心校准的辐射灭活技术，从冷冻的大鼠大脑皮层中估计出[35S]Tbps结合络合物的分子量为137,000道尔顿。GABA激动剂Muscimol以一种与辐射剂量无关的方式将[35S]Tbps结合减少到控制值的0-10%。这表明GABA受体(MW=55,000道尔顿)包含在137,000-道尔顿[35S]-Tbps结合复合体中；仅[35S]Tbps结合蛋白就占137,000−55,000=82,000道尔顿。适当浓度的吡唑并氮卓酮(SQ 20.009)和依托咪酯均可降低[35S]Tbps的特异性结合。SQ 20.009和依托咪酯降低[35S]Tbps结合的能力在低辐射剂量下大大降低，这表明SQ 20.009和依托咪酯通过一种需要450,000-500,000道尔顿分子结构的变构机制来降低[35S]Tbps结合。苯二氮卓类激动剂(乙基4-methoxymethyl-6-benzyloxy-β-carboline-3-carboxylate，ZK 93423)和反向激动剂(甲基6，7-dimethoxy-4-ethyl-β-carboline-3-carboxylate，DMCM)在反复冷冻和洗涤的膜制剂中分别增强和减少[35S]Tbps的结合。ZK 93423和DMCM对膜[35S]Tbps结合的影响在低剂量辐射下消失。这表明苯二氮卓类受体部位与[35S]Tbps结合部位变构相互作用，至少需要一个分子复合体。40万道尔顿。仅在后一种膜制备条件下(反复冷冻/洗涤)的[35S]Tbps位点就显示了221,000道尔顿的分子量(Tbps位点+GABA受体+未知结构)。[35S]Tbps在大脑皮层的结合位点数(145pmol/g组织)仅略高于[35H]氟硝西潘(130pmol/g组织)。这些结果都与GABA/BZ受体氯通道复合体是由高度整合的多聚体亚基组成的结论一致，初步认为是一个分子量为548,000道尔顿的四聚体复合体。
[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 leastc. 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.