Molecaular mechanisms underlying the activation of muscarinic M1-, M3- and M5-receptors by the membrane potential

G蛋白偶联受体（gpcr）是定位于质膜并被细胞外配体激活的药理学相关信号蛋白。最近的研究表明，膜电位改变了激动剂的结合和受体的激活。GPCR如何感知膜电位的潜在分子机制尚不清楚。本研究通过单细胞荧光显微镜在电压箝位条件下监测毒蕈碱M1-、M3-和m5受体的激活，探讨膜电位如何调节毒蕈碱M1-、M3-和m5受体的激活和信号转导。具体目的有：(1)电压敏感受体结构的鉴定。膜的去极化激活M1受体，但使M3受体失活。使用M1/M3受体嵌合体用于识别作为电压传感器的受体分子的结构。用光学生物传感器监测配体介导的嵌合体激活，该传感器直接报告Gq蛋白在确定的膜电位（全细胞电压钳）下的活性。嵌合体的产生是由分子动力学计算支持的，该计算预测假定的电压传感器是受体的柔性结构，可以在电场中跨膜移动。(2)膜电位作为分析配体-受体相互作用的工具。膜的去极化导致碳甾醇激活的m5受体失活，但诱导匹罗卡品结合的m5受体活化。去极化引起的这些功效差异表明，两种激动剂与受体的分子相互作用不同。为了量化配体特异性效果，M3-和m5受体的生物传感器暴露于不同的激动剂并进行膜去极化。在分子对接计算的帮助下，进一步分析了这些电压敏感性的差异，从而预测了配体与受体结合袋之间精确的分子相互作用。(3)受体信号的电压依赖性。Gq蛋白的激活控制着许多转录途径。由于膜的去极化激活了m1受体，本研究旨在探讨Gq蛋白下游的细胞信号是否表现出电压依赖性。表达M1受体的细胞经受长时间的去极化，转录活性通过特定的基于荧光素酶的报告基因测定来评估。综上所述，本课题获得的实验结果将有助于阐明GPCR中电压依赖性的分子机制，并为分子受体药理学研究提供新的思路。

G protein-coupled receptors (GPCRs) represent pharmacologically relevant signal proteins that are localized to the plasma membrane and activated by extracellular ligands. Recent studies demonstrate that the membrane potential modifies agonist binding and receptor activation. The underlying molecular mechanism how a GPCR senses the membrane potential is unknown. This proposal investigates how the membrane potential regulates activation and signaling of muscarinic M1-, M3- and M5-receptors by monitoring receptor activation with single cell fluorescence microscopy under voltage-clamp conditions. The specific aims are: (1) Identification of voltage-sensitive receptor structures. A depolarization of the membrane activates M1 receptors but deactivates M3 receptors. The use of M1/M3 receptor chimeras is used to identify structures of the receptor molecules that serve as voltage sensors. Ligand-mediated activation of the chimeras is monitored with an optical biosensor that directly reports Gq protein activity at defined membrane potentials (whole cell voltage-clamp). The generation of the chimeras is supported by molecular dynamic calculations, which predict putative voltage sensors as flexible structures of the receptors that can move within the electrical field across the membrane. (2) The membrane potential as a tool to analyze ligand-receptor interactions. Depolarization of the membrane leads to deactivation of Carbachol-activated M5-receptors, but induces activation of Pilocarpine-bound M5-receptors. These differences in efficacy caused by depolarization suggest that both agonists bind to the receptor with different molecular interactions. To quantify ligand-specific efficacies, biosensors of the M3- and M5-receptors are exposed to different agonists and subjected to membrane depolarization. Those differences in voltage sensitivity are further analyzed with the help of molecular docking calculations, which predict the precise molecular interaction between ligands and the binding pocket of the receptors. (3) Voltage dependence of receptor signaling. The activation of Gq proteins controls many transcriptional pathways. Because a depolarization of the membrane activates the M1-receptor, this aim addresses whether cellular signals downstream of Gq proteins exhibit voltage-dependence. Cells that express the M1 receptor are subjected to long lasting depolarization and transcriptional activity is assessed with specific luciferase-based reporter assays. In conclusion, the experimental results obtained in the proposed project will help to elucidate the molecular mechansism of voltage dependence in GPCR and will shed new light on molecular receptor pharmacology.