Department of Pharmaceutical Sciences, University of Toronto, Toronto, Ontario, Canada M5S 3M2.
Biochemistry. 2012 Jun 5;51(22):4518-40. doi: 10.1021/bi3000287. Epub 2012 May 24.
At least four allosteric sites have been found to mediate the dose-dependent effects of gallamine on the binding of [(3)H]quinuclidinylbenzilate (QNB) and N-[(3)H]methylscopolamine (NMS) to M(2) muscarinic receptors in membranes and solubilized preparations from porcine atria, CHO cells, and Sf9 cells. The rate of dissociation of [(3)H]QNB was affected in a bell-shaped manner with at least one Hill coefficient (n(H)) greater than 1, indicating that at least three allosteric sites are involved. The level of binding of [(3)H]QNB was decreased in a biphasic manner, revealing at least two allosteric sites; binding of [(3)H]NMS was affected in a triphasic, serpentine manner, revealing at least three sites, and values of n(H) >1 pointed to at least four sites. Several lines of evidence indicate that all effects of gallamine were allosteric in nature and could be observed at equilibrium. The rates of equilibration and dissociation suggest that the receptor was predominately oligomeric, and the heterogeneity revealed by gallamine can be attributed to differences in its affinity for the constituent protomers of a tetramer. Those differences appear to arise from inter- and intramolecular cooperativity between gallamine and the radioligand.
至少有四个变构位点被发现可以调节加兰他敏对猪心房、CHO 细胞和 Sf9 细胞的膜和溶解制剂中 [(3)H]奎宁定苯甲酸盐 (QNB) 和 N-[(3)H]甲基东莨菪碱 (NMS) 与 M(2)毒蕈碱受体结合的剂量依赖性效应。[(3)H]QNB 的解离速率呈钟形曲线变化，至少有一个 Hill 系数 (n(H)) 大于 1，表明至少有三个变构位点参与其中。[(3)H]QNB 的结合水平呈双相方式降低，揭示至少有两个变构位点；[(3)H]NMS 的结合呈三相反旋曲线方式受到影响，揭示至少有三个位点，n(H) >1 值表明至少有四个位点。有几条证据表明，加兰他敏的所有作用都是变构的，可以在平衡时观察到。平衡和解离的速率表明受体主要是寡聚的，加兰他敏所揭示的异质性可以归因于其对四聚体组成原聚体的亲和力的差异。这些差异似乎源于加兰他敏和放射性配体之间的分子内和分子间协同作用。
