Tränkle C, Kostenis E, Mohr K
Department of Pharmacology and Toxicology, Institute of Pharmacy, University of Bonn, Germany.
Naunyn Schmiedebergs Arch Pharmacol. 2001 Aug;364(2):172-8. doi: 10.1007/s002100100441.
Among the five subtypes of muscarinic acetylcholine receptors, the sensitivity towards allosteric modulation is generally higher in M2 and M4 receptors that preferentially couple to inhibitory G-proteins of the Gi/o type than in M1, M3, and M5 that preferentially couple to stimulatory G-proteins such as Gq/11. We aimed to check whether the high allosteric sensitivity of the M2 receptor compared to M3 is related to the differential G-protein coupling preference. As the third intracellular loop (i3) is known to be the major determinant in receptor G-protein coupling specificity, we used wild-type M2 and M3 receptors and the related chimeric constructs with exchanged i3-loops, i.e., M2 containing M3-i3 (M2/M3-i3) and M3 containing M2-i3 (M3/M2-i3). The allosteric effect of the archetypal modulator gallamine on the dissociation and the equilibrium binding of [3H]N-methylscopolamine ([3H]NMS) was measured in membranes of mouse A9L cells stably expressing the wild-type and the chimeric receptors (4 mM Na2HPO4, 1 mM KH2PO4, pH 7.4, 23 degrees C). The dissociation of [3H]NMS was monophasic under all conditions studied. Control values of t 1/2 were (means +/- SEM, n = 4-7): M2: 3.8 +/- 0.2 min, M2/M3-i3: 4.8 +/- 0.3 min, M3:43.3 +/- 4.2 min, M3/M2-i3: 41.1 +/- 3.6 min. At M2 receptors, 0.2 microM gallamine allosterically reduced the apparent rate constant of dissociation k-1 to 51 +/- 5% of the control value (n = 5). At M2/M3-i3 the allosteric potency of gallamine was not significantly changed (0.2 microM gallamine --> k-1 = 61 +/- 4%, n = 7). At M3, a 20-fold higher concentration was required for an equieffective allosteric action (10 microM gallamine --> k-1 = 51 +/- 5%, n = 5). The potency of gallamine at M3/M2-i3 was not increased compared with M3 receptors (10 microM gallamine --> k-1 = 73 +/- 2%, n = 4) but even significantly diminished. [3H]NMS equilibrium binding experiments revealed that neither the binding constants of gallamine at free receptor subtypes (pKA,M2: 7.57 +/- 0.04, n = 4; pKA,M3: 5.56 +/- 0.13, n = 3) nor the factors of negative cooperativity with [3H]NMS (alphaM2 = 31 +/- 1, alphaM3 = 3 +/- 0.4) were affected by the exchanged i3-loops (pKA,M2/M3-i3: 7.65 +/- 0.03, pKA,M3/M2-i2: 5.35 +/- 0.24, alphaM2/M3-i3= 30 +/- 2, alphaM3/M2-i2 = 3 +/- 0.7). In conclusion, the different sensitivities of M2 and M3 receptors towards allosteric modulation by gallamine are not related to the G-protein coupling specificity of the receptors.
在毒蕈碱型乙酰胆碱受体的五种亚型中，与优先偶联刺激性G蛋白（如Gq/11）的M1、M3和M5受体相比，优先偶联Gi/o型抑制性G蛋白的M2和M4受体对变构调节的敏感性通常更高。我们旨在检验M2受体相对于M3受体的高变构敏感性是否与不同的G蛋白偶联偏好有关。由于已知第三细胞内环（i3）是受体G蛋白偶联特异性的主要决定因素，我们使用了野生型M2和M3受体以及交换了i3环的相关嵌合构建体，即含有M3-i3的M2（M2/M3-i3）和含有M2-i3的M3（M3/M2-i3）。在稳定表达野生型和嵌合受体的小鼠A9L细胞膜中（4 mM Na2HPO4，1 mM KH2PO4，pH 7.4，23℃），测量了原型调节剂加拉明对[3H]N-甲基东莨菪碱（[3H]NMS）解离和平衡结合的变构效应。在所有研究条件下，[3H]NMS的解离是单相的。t1/2的对照值为（平均值±标准误，n = 4-7）：M2：3.8±0.2分钟，M2/M3-i3：4.8±0.3分钟，M3：43.3±4.2分钟，M3/M2-i3：41.1±3.6分钟。在M2受体处，0.2μM加拉明变构地将解离的表观速率常数k-1降低至对照值的51±5%（n = 5）。在M2/M3-i3处，加拉明的变构效力没有显著变化（0.2μM加拉明→k-1 = 61±4%，n = 7）。在M3处，需要高20倍的浓度才能产生等效的变构作用（10μM加拉明→k-1 = 51±5%，n = 5）。与M3受体相比，加拉明在M3/M2-i3处的效力没有增加（10μM加拉明→k-1 = 73±2%，n = 4），甚至显著降低。[3H]NMS平衡结合实验表明，加拉明在游离受体亚型处的结合常数（pKA，M2：7.57±0.04，n = 4；pKA，M3：5.56±0.13，n = 3）以及与[3H]NMS的负协同性因子（αM2 = 31±1，αM3 = 3±0.4）均不受交换的i3环影响（pKA，M2/M3-i3：7.65±0.03，pKA，M3/M2-i2：5.35±0.24，αM2/M3-i3 = 30±2，αM3/M2-i2 = 3±0.7）。总之，M2和M3受体对加拉明变构调节的不同敏感性与受体的G蛋白偶联特异性无关。
