Differences in kinetics of xanomeline binding and selectivity of activation of G proteins at M1 and M2 muscarinic acetylcholine receptors
Differences in kinetics of xanomeline binding and selectivity of activation of G proteins at M1 and M2 muscarinic acetylcholine receptors
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DOI：
10.1124/mol.106.023762
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发表时间：
2006-08-01
影响因子：
3.6
通讯作者：
Dolezal, Vladimir
中科院分区：
文献类型：
--
作者：
Jakubik, Jan;El-Fakahany, Esam E.;Dolezal, Vladimir
Xanomeline 是一种功能选择性 M-1/M-4 毒蕈碱乙酰胆碱受体激动剂，但仍以高亲和力与所有五种毒蕈碱受体亚型结合。该配体与毒蕈碱 M-1 受体相互作用的一种新模式，其特征是在广泛冲洗后持续结合和受体激活。在本研究中，使用中国仓鼠卵巢细胞中表达的人M-1和M-2受体以及[H-3]N-甲基东莨菪碱作为示踪剂，我们发现xanomeline也发生在M-2受体上，其亲和力与M-1受体相似（K-I分别为294和296 nM）。然而，Xanomeline 与 M-2 受体的耐洗涤结合的形成动力学明显慢于与 M-1 受体的结合。 Xanomeline 是一种有效的快速作用完全激动剂，可刺激 M-1 受体上的鸟苷 5'-O-(3-[S-35]thio) 三磷酸结合，而在 M-2 受体上，仅在预孵育 1 小时后，它才表现为有效的部分激动剂（卡巴胆碱最大反应的 40%）。 Xanomeline 对 M-2 受体的激动作用的发展慢于其减弱卡巴胆碱反应的能力。我们还证明 xanomeline 在 M-1 受体上比在 M-2 受体上更好地区分 G 蛋白亚型。我们的数据支持这样的观点，即 xanomeline 与毒蕈碱受体上的多个位点相互作用，导致不同的构象，对配体结合和受体激活表现出不同的影响。这些构象均具有时间和浓度依赖性，并且在 M-1 和 M-2 受体之间有所不同。
Xanomeline is a functionally selective M-1/M-4 muscarinic acetylcholine receptor agonist that nevertheless binds with high affinity to all five subtypes of muscarinic receptors. A novel mode of interaction of this ligand with the muscarinic M-1 receptors characterized by persistent binding and receptor activation after extensive washout has been shown previously. In the present study, using human M-1 and M-2 receptors expressed in Chinese hamster ovary cells and [H-3]N-methylscopolamine as a tracer, we show that persistent binding of xanomeline also occurs at the M-2 receptor with similar affinity as at the M-1 receptor (K-I = 294 and 296 nM, respectively). However, kinetics of formation of xanomeline wash-resistant binding to M-2 receptors was markedly slower than to M-1 receptors. Xanomeline was a potent fast-acting full agonist in stimulating guanosine 5'-O-(3-[S-35]thio) triphosphate binding at M-1 receptors, whereas at M-2 receptors it behaved as a potent partial agonist (40% of carbachol maximal response) only upon preincubation for 1 h. Development of xanomeline agonistic effects at the M-2 receptor was slower than its ability to attenuate carbachol responses. We also demonstrate that xanomeline discriminates better between G protein subtypes at M-1 than at M-2 receptors. Our data support the notion that xanomeline interacts with multiple sites on the muscarinic receptor, resulting in divergent conformations that exhibit differential effects on ligand binding and receptor activation. These conformations are both time- and concentration-dependent and vary between the M-1 and the M-2 receptor.