Sykes David A, Dowling Mark R, Charlton Steven J
Novartis Institutes for Biomedical Sciences, Wimblehurst Road, Horsham, West Sussex, UK. .
Mol Pharmacol. 2009 Sep;76(3):543-51. doi: 10.1124/mol.108.054452. Epub 2009 Jun 4.
Although there are several empirical approaches that enable the comparison of relative agonist efficacy, the molecular basis that underlies differences in the ability of G protein-coupled receptor agonists to elicit a response is still largely unexplained. Several models have been described that incorporate the kinetics of receptor-mediated initiation of the G protein cycle, but these have not directly addressed the influence of agonist-binding kinetics. To test this, we investigated the relationship between the efficacy of seven M(3) muscarinic receptor agonists and their rate of dissociation (k(off)) from the M(3) receptor. The association and dissociation rate constants of the agonists were determined using a l-[N-methyl]-[(3)H]scopolamine methyl chloride competition binding assay in the presence of GTP. The agonists displayed a range of association and dissociation rates. Relative agonist efficacy was measured at two points after M(3) receptor activation: the stimulation of guanosine 5'-O-(3-[(35)S]thio)triphosphate binding to G alpha subunits, and the subsequent increase in intracellular calcium levels. These experiments revealed a range of intrinsic efficacy, from the low-efficacy pilocarpine and oxotremorine to high-efficacy acetylcholine. There was no relationship between agonist efficacy and the equilibrium binding affinity of each agonist (K(d)). When efficacy was compared with the dissociation rate constant, however, the two were highly correlated, suggesting a relationship between the duration of agonist binding at the receptor and the intrinsic efficacy. These data suggest that kinetic models incorporating the mean lifetime of specific complexes will be required to fully explain the nature of agonist efficacy.
尽管有几种实证方法可用于比较相对激动剂效力，但G蛋白偶联受体激动剂引发反应能力差异背后的分子基础仍 largely unexplained。已经描述了几种纳入受体介导的G蛋白循环起始动力学的模型，但这些模型尚未直接解决激动剂结合动力学的影响。为了测试这一点，我们研究了七种M(3)毒蕈碱受体激动剂的效力与其从M(3)受体解离速率（k(off)）之间的关系。在存在GTP的情况下，使用l-[N-甲基]-[(3)H]东莨菪碱甲基氯竞争结合试验测定激动剂的结合和解离速率常数。这些激动剂表现出一系列的结合和解离速率。在M(3)受体激活后的两个时间点测量相对激动剂效力：刺激鸟苷5'-O-(3-[(35)S]硫代)三磷酸与Gα亚基的结合，以及随后细胞内钙水平的升高。这些实验揭示了一系列内在效力，从低效的毛果芸香碱和氧化震颤素到高效的乙酰胆碱。每种激动剂的效力与其平衡结合亲和力（K(d)）之间没有关系。然而，当将效力与解离速率常数进行比较时，两者高度相关，这表明激动剂在受体上的结合持续时间与内在效力之间存在关系。这些数据表明，需要纳入特定复合物平均寿命的动力学模型来充分解释激动剂效力的本质。
