Identification of various allosteric interaction sites on M1 muscarinic receptor using 125I-Met35-oxidized muscarinic toxin 7
Identification of various allosteric interaction sites on M1 muscarinic receptor using 125I-Met35-oxidized muscarinic toxin 7
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DOI：
10.1124/mol.105.020883
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发表时间：
2006-05-01
影响因子：
3.6
通讯作者：
Servent, D
中科院分区：
文献类型：
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作者：
Fruchart-Gaillard, C;Mourier, G;Servent, D
合成了单碘化、氧化的MT7ox毒碱毒素7 (MT7ox)，并通过平衡和动力学结合实验直接测定了其对游离或n -甲基东莨菪碱（NMS）占据的hM 1受体的亲和力常数。两种检测方法分别在游离或NMS配体受体状态下得到相同的值，分别为14 pM和0.9 nM，这表明该变构毒素与NMS之间存在很强的负协同作用。用三元配合物模型与[H-3] NMS间接结合实验得到了相同的结果，清楚地证明了这种协同性的互反性质。此外,各种orthosteric的影响和变构剂的离解动能i - 125 - mt7ox测量和显示,除了MT1毒素,所有的配体研究(NMS,阿托品,加拉,番木鳖碱,他克林,staurosporine,和(9,10,12 r) 2、3、9、10日11-hexahydro-10-hydroxy-9-methyl-1-oxo-9, 12 - epoxy-1H-diindolo[1、2、3——fg: 3 ', 2 ' 1 ' kl] pyrrolo[3,我][1,6]benzodiazocine-10-carboxylic酸己酯(KT5720)]互动变构毒蕈碱的毒素7。通过与I-125-MT7ox和[H-3] NMS的平衡结合实验，揭示了这些配体对游离受体的影响，并利用变构三元配合物模型计算了亲和常数pK(x)值。我们的研究结果表明，MT7毒素在一个特定的变构位点与hM(1)受体相互作用，这可能部分重叠了先前确定的“经典”或“非典型”变构剂，并突出了这种新的变构示踪剂在研究毒蕈碱受体变构方面的潜力。
Monoiodinated, Met35-oxidized muscarinic toxin 7 (MT7ox) was synthesized, and its affinity constants for free or N-methyl scopolamine (NMS)-occupied hM 1 receptor were measured directly by equilibrium and kinetic binding experiments. Identical values were obtained with the two types of assay methods, 14 pM and 0.9 nM in free or NMS-liganded receptor states, respectively, highlighting a strong negative cooperativity between this allosteric toxin and NMS. Identical results were obtained with indirect binding experiments with [H-3] NMS using the ternary complex model, clearly demonstrating the reciprocal nature of this cooperativity. Furthermore, the effects of various orthosteric and allosteric agents on the dissociation kinetic of I-125-MT7ox were measured and show that, except for the MT1 toxin, all of the ligands studied [ NMS, atropine, gallamine, brucine, tacrine, staurosporine, and (9S, 10S, 12R)-2,3,9,10,11-hexahydro-10-hydroxy-9-methyl-1-oxo-9,12- epoxy-1H-diindolo[1,2,3- fg: 3', 2', 1'-kl] pyrrolo[ 3,4-i][1,6]benzodiazocine-10-carboxylic acid hexyl ester (KT5720)] interact allosterically with muscarinic toxin 7. Equilibrium binding experiments with I-125-MT7ox and [H-3] NMS were conducted to reveal the effects of these ligands on the free receptor, and affinity constants (pK(x) values) were calculated using the allosteric ternary complex model. Our results suggest that MT7 toxin interacts with hM(1) receptor at a specific allosteric site, which may partially overlap those identified previously for "classic" or "atypical" allosteric agents and highlight the potential of this new allosteric tracer in studying allosterism at muscarinic receptors.