Constitutive activation of the m5 muscarinic receptor by a series of mutations at the extracellular end of transmembrane 6.
Constitutive activation of the m5 muscarinic receptor by a series of mutations at the extracellular end of transmembrane 6.
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DOI：
10.1021/bi970565g
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发表时间：
1997-08
期刊：
影响因子：
2.9
通讯作者：
T. Spalding;E. Burstein;James W. Wells;M. Brann
中科院分区：
文献类型：
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作者：
T. Spalding;E. Burstein;James W. Wells;M. Brann
M5毒蕈碱乙酰胆碱受体被广泛的氨基酸取代的残基（丝氨酸465），位于第六跨膜结构域和细胞外环的交界处组成型激活。在测试的13个取代中，11个产生了组成型活性的显著增加。用大的（苯丙氨酸和缬氨酸）或碱性残基（精氨酸和赖氨酸）替换丝氨酸465增加了受体的组成活性至野生型受体对激动剂卡巴胆碱的最大响应的55和110%之间。其他替换（例如，半胱氨酸和亮氨酸）使组成型活性增加到中等水平（30%），而小的酸性残基（甘氨酸、天冬氨酸和谷氨酸）引起小的或不显著的增加。每个突变受体的组成活性的增加与卡巴胆碱的结合和功能测定中的效力的增加相关，最组成性激活的受体显示卡巴胆碱的EC50降低40倍。负性拮抗剂阿托品以相同的效力结合并逆转所有突变体受体的组成性活性。这些数据被拟合到受体功能的双态模型。这些数据与丝氨酸465取代的主要作用一致，即选择性地使受体的非活性状态不稳定，从而有利于在不存在激动剂的情况下形成活性状态。我们的数据强烈支持这种受体功能的双态模型，并确定了该域在毒蕈碱受体激活中的关键作用。
The m5 muscarinic acetylcholine receptor was constitutively activated by a wide range of amino acid substitutions at a residue (serine 465) that is positioned at the junction of the sixth transmembrane domain and the extracellular loop. Of 13 substitutions tested, 11 produced significant increases in constitutive activity. Replacement of serine 465 with large (phenylalanine and valine) or basic residues (arginine and lysine) increased the constitutive activity of the receptor to between 55 and 110% of the maximum response of the wild-type receptor to the agonist carbachol. Other substitutions (e.g., cysteine and leucine) increased the constitutive activity to an intermediate level (30%), while small and acidic residues (glycine, aspartate, and glutamate) caused small or insignificant increases. The increase in the constitutive activity of each mutant receptor correlated with an increase in the potency of carbachol in both binding and functional assays, with the most constitutively activated receptors showing a 40-fold decrease in the EC50 of carbachol. The negative antagonist atropine bound to and reversed the constitutive activity of all mutant receptors with equal potency. These data were fitted to a two-state model of receptor function. The data are consistent with the primary effect of substitutions to serine 465 being to selectively destabilize the inactive state of the receptor, thus favoring formation of the active state in the absence of agonists. Our data strongly support this two-state model of receptor function and identify a critical role of this domain in the activation of muscarinic receptors.