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 M₁-Rs and M₃-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_q protein cycle. In the presence of ACh, M₁-R signaling was potentiated by voltage, similarly to positive allosteric modulation by benzyl quinolone carboxylic acid. Conversely, signaling of M₃-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” M₃/M₁-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 M₃-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.

乙酰胆碱（ACh）的毒蕈碱受体（M-Rs）属于G蛋白偶联受体的A类。M-Rs通过正构激动剂激活，该激动剂与埋在MR跨膜螺旋束中的特定位点结合。在活性构象中，受体功能可以通过与细胞外受体表面结合的变构调节剂或通过未知机制的膜电位来调节。在这里，我们将电压变化引起的M ₁ -Rs和M ₃ -Rs的调制与化合物的变构调制进行了比较。我们量化在受体信号传导的改变在单一HEK 293个细胞与生物传感器FRET对G _q蛋白周期。在存在乙酰胆碱的情况下，M ₁-R信号传导通过电压增强，类似于苄基喹诺酮羧酸的正变构调节。相反，M ₃ -R的信号传导被电压或负变构调节剂没食子胺削弱。由于正构位点在M-Rs中高度保守，而别构位点却有所不同，因此我们构建了“别构位点” M ₃ / M ₁ -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ACh反应对两个受体的电压敏感性，但不影响变构化合物对它们的调节。此外，M _3中的点突变-Rs引起变构位和正构位的功能解偶联并消除了电压依赖性。受体变体的分子动力学模拟表明两个位点之间存在亚型特异性串扰，涉及正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电压效应。