Application of a kinetic model to the apparently complex behavior of negative and positive allosteric modulators of muscarinic acetylcholine receptors
Application of a kinetic model to the apparently complex behavior of negative and positive allosteric modulators of muscarinic acetylcholine receptors
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DOI：
10.1124/jpet.103.059840
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发表时间：
2004-03-01
影响因子：
3.5
通讯作者：
Christopoulos, A
中科院分区：
文献类型：
--
作者：
Avlani, V;May, LT;Christopoulos, A
变构调节剂与G蛋白偶联受体（GPCR）的结合通常由平衡变构三元复合物模型（ATCM）描述。本研究评价了三种调节剂对[H-3] N-甲基东莨菪碱（[H-3] NMS）与人M-2毒蕈碱乙酰胆碱受体（mAChR）结合的影响。每个调制器的结合比预测的ATCM更复杂;抑制剂庚烷-1，7-双-（二甲基-3-邻苯二甲酰亚胺丙基）-溴化铵和gallamine产生的双相曲线，经验上描述的两个网站的结合模型，而增强剂阿库溴铵产生一个钟形曲线。放射性配体解离分析表明，调制器延迟[H-3] NMS动力学，使系统从未达到平衡。随后应用的动力学ATCM容纳和量化所有的实验观察。我们的研究结果证实并扩展了以前的研究使用的动力学ATCM的mAChR变构增强剂，但也强调了如何复杂的曲线显示的变构抑制剂可以被误解的多位点正构结合。其他变构调节剂在GPCR处的类似行为可能反映了非平衡结合假象，而不是与ATCM的偏离。
The binding of allosteric modulators to G protein-coupled receptors (GPCRs) is often described by an equilibrium allosteric ternary complex model (ATCM). This study evaluated the effects of three modulators on the binding of [H-3] N-methylscopolamine ([H-3] NMS) to the human M-2 muscarinic acetylcholine receptor ( mAChR). The binding of each modulator was more complex than predicted by the ATCM; the inhibitors heptane-1,7-bis-(dimethyl-3- phthalimidopropyl)-ammonium bromide and gallamine yielded biphasic curves that were described empirically by a two-site binding model, whereas the enhancer alcuronium yielded a bell-shaped curve. Radioligand dissociation assays revealed that the modulators retarded [H-3] NMS kinetics such that the system never attained equilibrium. Subsequent application of a kinetic ATCM accommodated and quantified all experimental observations. Our findings confirm and extend previous studies on the use of a kinetic ATCM for mAChR allosteric enhancers, but also highlight how complex curves displayed by allosteric inhibitors can be misinterpreted in terms of multisite orthosteric binding. It is possible that similar behavior of other allosteric modulators at GPCRs may reflect nonequilibrium binding artifacts rather than deviation from an ATCM.