Structure-function studies of allosteric agonism at M2 muscarinic acetylcholine receptors
Structure-function studies of allosteric agonism at M2 muscarinic acetylcholine receptors
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DOI：
10.1124/mol.107.037630
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发表时间：
2007-08-01
影响因子：
3.6
通讯作者：
Christopoulos, Arthur
中科院分区：
文献类型：
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作者：
May, Lauren T.;Avlani, Vimesh A.;Christopoulos, Arthur
M-2毒蕈碱乙酰胆碱受体（mAChR）具有至少一个依赖于残基（172）EDGE（175）、Tyr（177）和Thr（423）的变构调节剂的结合位点。然而，这些残基对变构激动剂而不是调节剂的作用的贡献是未知的。我们创建了突变体M2 mAChR，其中172 EDGE 175序列的电荷已被中和，并且每个Tyr 177和Thr 423被丙氨酸取代。放射性配体结合实验表明，这些突变对原型调节剂没食子胺、阿库溴铵和庚烷-1，7-双-[二甲基-3 '-邻苯二甲酰亚胺丙基]-溴化铵（C（7）7/3-phth）具有显著的抑制作用，但对正构拮抗剂[H-3] N-甲基东莨菪碱的影响极小。相反，变构激动剂4-[3-氯苯基]氨基甲酰氧基）-2-丁炔基三甲基氯化铵（McN-A-343），4-正丁基-1-[4-（2-甲基苯基）4-氧代-1-丁基]哌啶盐酸盐（AC-42），和新的AC-42衍生物1-[3-（2-甲基苯基）-4-氧代-1-丁基]哌啶盐酸盐（AC-42），（4-丁基-1-哌啶基）丙基] 3，4-二氢-2（1H）-喹啉酮（77-LH-28-1）在组合的EDGE-YT突变处表现出亲和力或高亲和力位点比例增加，表明与原型调节剂的结合模式不同。随后的细胞外信号调节激酶（ERK）1/2磷酸化和鸟苷5 '-（γ-[S-35]硫代）三磷酸（[35 S] GTP γ S）结合的功能测定显示，突变对正构激动剂乙酰胆碱（ACh）和毛果芸香碱的影响最小，但McN-A-343的功效和77-LH-28-1的效力显著增加。另外的诱变实验发现，这些效应主要由Tyr（177）和Thr（423）介导，而不是由（172）EDGE（175）序列介导。每个变构激动剂和乙酰胆碱之间的功能相互作用的特点是高负协同性，但在合并的EDGE-YT突变M-2 mAChR的变构激动剂的亲和力增加是一致的。因此，本研究揭示了关键变构位点残基对M-2 mAChR的变构激动剂与变构调节剂的结合和功能的不同作用。
The M-2 muscarinic acetylcholine receptor ( mAChR) possesses at least one binding site for allosteric modulators that is dependent on the residues (172)EDGE(175), Tyr(177), and Thr(423). However, the contribution of these residues to actions of allosteric agonists, as opposed to modulators, is unknown. We created mutant M2 mAChRs in which the charge of the 172EDGE175 sequence had been neutralized and each Tyr177 and Thr423 was substituted with alanine. Radioligand binding experiments revealed that these mutations had a profound inhibitory effect on the prototypical modulators gallamine, alcuronium, and heptane-1,7- bis-[dimethyl- 3'- phthalimidopropyl]- ammonium bromide (C(7)7/3-phth) but minimal effects on the orthosteric antagonist [H-3] N-methyl scopolamine. In contrast, the allosteric agonists 4-I-[3-chlorophenyl] carbamoyloxy)-2-butynyltrimethylammnonium chloride (McN-A-343), 4-n-butyl-1-[4-(2-methylphenyl)4- oxo-1-butyl] piperidine hydrogen chloride (AC-42), and the novel AC-42 derivative 1-[3-(4-butyl-1-piperidinyl) propyl]3,4- dihydro-2(1H)-quinolinone (77-LH-28-1) demonstrated an increased affinity or proportion of high-affinity sites at the combined EDGE-YT mutation, indicating a different mode of binding to the prototypical modulators. Subsequent functional assays of extracellular signal-regulated kinase (ERK) 1/2 phosphorylation and guanosine 5'-(gamma-[S-35] thio) triphosphate ([ 35S] GTP gamma S) binding revealed minimal effects of the mutations on the orthosteric agonists acetylcholine (ACh) and pilocarpine but a significant increase in the efficacy of McN-A-343 and potency of 77-LH-28-1. Additional mutagenesis experiments found that these effects were predominantly mediated by Tyr(177) and Thr(423), rather than the (172)EDGE(175) sequence. The functional interaction between each of the allosteric agonists and ACh was characterized by high negative cooperativity but was consistent with an increased allosteric agonist affinity at the combined EDGE-YT mutant M-2 mAChR. This study has thus revealed a differential role of critical allosteric site residues on the binding and function of allosteric agonists versus allosteric modulators of M-2 mAChRs.