Department of Cardiovascular Physiology, Ruhr-University Bochum, Universitätsstr. 150, 44780 Bochum, Germany.
Department of Pharmaceutical Chemistry, Philipps-University Marburg, Marbacher Weg 6-10, 35032 Marburg, Germany.
Cell Signal. 2018 Jan;42:114-126. doi: 10.1016/j.cellsig.2017.10.011. Epub 2017 Oct 19.
Muscarinic receptors (M-Rs) for acetylcholine (ACh) belong to the class A of G protein-coupled receptors. M-Rs are activated by orthosteric agonists that bind to a specific site buried in the M-R transmembrane helix bundle. In the active conformation, receptor function can be modulated either by allosteric modulators, which bind to the extracellular receptor surface or by the membrane potential via an unknown mechanism. Here, we compared the modulation of M-Rs and M-Rs induced by changes in voltage to their allosteric modulation by chemical compounds. We quantified changes in receptor signaling in single HEK 293 cells with a FRET biosensor for the G protein cycle. In the presence of ACh, M-R signaling was potentiated by voltage, similarly to positive allosteric modulation by benzyl quinolone carboxylic acid. Conversely, signaling of M-R was attenuated by voltage or the negative allosteric modulator gallamine. Because the orthosteric site is highly conserved among M-Rs, but allosteric sites vary, we constructed "allosteric site" M/M-R chimeras and analyzed their voltage dependencies. Exchanging the entire allosteric sites eliminated the voltage sensitivity of ACh responses for both receptors, but did not affect their modulation by allosteric compounds. Furthermore, a point mutation in M-Rs caused functional uncoupling of the allosteric and orthosteric sites and abolished voltage dependence. Molecular dynamics simulations of the receptor variants indicated a subtype-specific crosstalk between both sites, involving the conserved tyrosine lid structure of the orthosteric site. This molecular crosstalk leads to receptor subtype-specific voltage effects.
毒蕈碱型乙酰胆碱受体（M-R）属于 G 蛋白偶联受体 A 类。M-R 被乙酰胆碱（ACh）的变构激动剂激活，变构激动剂与 M-R 跨膜螺旋束中的特定埋藏位点结合。在活性构象中，受体功能可以通过变构调节剂进行调节，变构调节剂与细胞外受体表面结合，或者通过未知机制与膜电位结合。在这里，我们比较了 M-R 和 M-R 对电压变化的调节与其变构调节的异同。我们使用用于 G 蛋白循环的 FRET 生物传感器在单个 HEK 293 细胞中量化了受体信号转导的变化。在 ACh 存在的情况下，M-R 信号通过电压增强，类似于苯并喹诺酮羧酸的正变构调节。相反，M-R 的信号通过电压或负变构调节剂胍胺衰减。由于 M-R 之间的变构位点高度保守，但变构位点不同，我们构建了“变构位点”M/M-R 嵌合体，并分析了它们的电压依赖性。交换整个变构位点消除了两种受体对 ACh 反应的电压敏感性，但不影响它们对变构化合物的调节。此外，M-R 中的一个点突变导致变构和变构位点的功能分离，并消除了电压依赖性。受体变体的分子动力学模拟表明，两种位点之间存在亚型特异性的串扰，涉及变构位点的保守酪氨酸盖结构。这种分子串扰导致受体亚型特异性的电压效应。
