Use of acetylcholine mustard to study allosteric interactions at the M(2) muscarinic receptor.
Use of acetylcholine mustard to study allosteric interactions at the M(2) muscarinic receptor.
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DOI：
10.1124/jpet.108.141234
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发表时间：
2008-11
期刊：
影响因子：
--
通讯作者：
Ehlert FJ
中科院分区：
文献类型：
--
作者：
Suga H;Figueroa KW;Ehlert FJ
我们使用毒蕈碱放射性配体[3H]N-甲基东莨菪碱探索了乙酰胆碱的氮芥衍生物与CHO细胞中表达的人M2毒蕈碱受体的相互作用。乙酰胆碱芥子气在 37°C 时会导致 [3H]N-甲基东莨菪碱结合发生浓度依赖性一级损失，半最大速率常数发生在 24 µM 处，最大速率常数为 0.16 min−1。我们研究了各种配体对乙酰胆碱芥子气对 M2 受体烷基化速率的影响。 N-甲基东莨菪碱和 McN-A-343（4-（三甲氨基）-2-丁炔基-（3-氯苯基）氨基甲酸酯）竞争性地减慢烷基化速率，而没食子胺的抑制作用在高浓度下达到平台，表明变构抑制。相反，WIN 51708（17-β-羟基-17-α-乙炔基-5-α-雄甾烷[3,2-b]嘧啶并[1,2-a]苯并咪唑）没有效果。我们还在 0°C 下测量了乙酰胆碱芥子气对 [3H]NMS 结合的抑制作用，在这种条件下，几乎没有或没有可检测到的共价结合。在这些实验中，乙酰胆碱芥子气中氮丙啶鎓离子的解离常数估计为 12.3 µM。相比之下，芥子母体和乙酰胆碱芥子的醇解产物则没有效果。我们的结果表明，测量配体对小定点亲电子试剂对正构位点失活速率的影响是区分竞争性抑制与变构作用的有效方法。
We explored the interaction of a nitrogen mustard derivative of acetylcholine with the human M2 muscarinic receptor expressed in CHO cells using the muscarinic radioligand, [3H]N-methylscopolamine. Acetylcholine mustard caused a concentration-dependent, first order loss of [3H]N-methylscopolamine binding at 37°C, with the half maximal rate constant occurring at 24 µM and a maximal rate constant of 0.16 min−1. We examined the effects of various ligands on the rate of alkylation of M2 receptors by acetylcholine mustard. N-methylscopolamine and McN-A-343 (4-(trimethylamino)-2-butynyl-(3-chlorophenyl)carbamate) competitively slowed the rate of alkylation, whereas the inhibition by gallamine reached a plateau at high concentrations, indicating allosteric inhibition. In contrast, WIN 51708 (17-β-hydroxy-17-α-ethynyl-5-α-androstano[3,2-b]pyrimido[1,2-a]benzimidazole) had no effect. We also measured the inhibition of [3H]NMS binding by acetylcholine mustard at 0°C, conditions under which there is little or no detectable covalent binding. In these experiments, the dissociation constant of the aziridinium ion of acetylcholine mustard was estimated to be 12.3 µM. In contrast, the parent mustard and alcoholic hydrolysis product of acetylcholine mustard were without effect. Our results show that measurement of the effects of ligands on the rate of inactivation of the orthosteric site by a small site-directed electrophile is a powerful method for discriminating competitive inhibition from allosterism.
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影响因子：
3.6
作者：
Lazareno, S;Popham, A;Birdsall, NJM
通讯作者：
Birdsall, NJM
影响因子：
3.6
作者：
May, Lauren T.;Avlani, Vimesh A.;Christopoulos, Arthur
通讯作者：
Christopoulos, Arthur
影响因子：
2.1
作者：
HIRST, M;JACKSON, CH
通讯作者：
JACKSON, CH
DOI：
10.1124/jpet.108.136960
发表时间：
2008-06-01
影响因子：
3.5
作者：
Ehlert, Frederick J.;Griffin, Michael T.
通讯作者：
Griffin, Michael T.
影响因子：
6.1
作者：
LAZARENO, S;FARRIES, T;BIRDSALL, NJM
通讯作者：
BIRDSALL, NJM