University Center for Biomedical Research, Universidad de Colima, Colima, México.
J Physiol. 2011 Apr 1;589(Pt 7):1741-53. doi: 10.1113/jphysiol.2010.204107. Epub 2011 Jan 31.
The ability to sense transmembrane voltage is a central feature of many membrane proteins, most notably voltage-gated ion channels. Gating current measurements provide valuable information on protein conformational changes induced by voltage. The recent observation that muscarinic G-protein-coupled receptors (GPCRs) generate gating currents confirms their intrinsic capacity to sense the membrane electrical field. Here, we studied the effect of voltage on agonist activation of M2 muscarinic receptors (M2R) in atrial myocytes and how agonist binding alters M2R gating currents. Membrane depolarization decreased the potency of acetylcholine (ACh), but increased the potency and efficacy of pilocarpine (Pilo), as measured by ACh-activated K+ current, I(KACh). Voltage-induced conformational changes in M2R were modified in a ligand-selective manner: ACh reduced gating charge displacement while Pilo increased the amount of charge displaced. Thus, these ligands manifest opposite voltage-dependent I(KACh) modulation and exert opposite effects on M2R gating charge displacement. Finally, mutations in the putative ligand binding site perturbed the movement of the M2R voltage sensor. Our data suggest that changes in voltage induce conformational changes in the ligand binding site that alter the agonist–receptor interaction in a ligand-dependent manner. Voltage-dependent GPCR modulation has important implications for cellular signalling in excitable tissues. Gating current measurement allows for the tracking of subtle conformational changes in the receptor that accompany agonist binding and changes in membrane voltage.
跨膜电压感应能力是许多膜蛋白的核心特征，尤其是电压门控离子通道。门控电流测量提供了关于电压诱导的蛋白质构象变化的有价值的信息。最近观察到毒蕈碱 G 蛋白偶联受体（GPCR）产生门控电流，证实了它们固有感知细胞膜电场的能力。在这里，我们研究了电压对心房肌细胞中 M2 毒蕈碱受体（M2R）激动剂激活的影响，以及激动剂结合如何改变 M2R 门控电流。膜去极化降低了乙酰胆碱（ACh）的效力，但增加了匹罗卡品（Pilo）的效力和效能，如 ACh 激活的 K+电流，I(KACh) 所测。M2R 的电压诱导构象变化以配体选择性的方式改变：ACh 减少门控电荷位移，而 Pilo 增加位移的电荷量。因此，这些配体表现出相反的电压依赖性 I(KACh) 调制，并对 M2R 门控电荷位移产生相反的影响。最后，假定的配体结合位点的突变扰乱了 M2R 电压传感器的运动。我们的数据表明，电压变化诱导配体结合位点的构象变化，以配体依赖的方式改变激动剂-受体相互作用。电压依赖性 GPCR 调节对兴奋组织中的细胞信号传递具有重要意义。门控电流测量允许跟踪伴随激动剂结合和膜电压变化的受体的细微构象变化。
