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

G蛋白偶联受体(GPCRs)是一类药理上相关的信号蛋白，定位于细胞膜并被细胞外配体激活。最近的研究表明，膜电位调节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。本研究通过在电压钳条件下用单细胞荧光显微镜监测受体的激活来研究膜电位如何调节M_1、M_3和M_5受体的激活和信号传递。具体目标是：(1)鉴定电压敏感受体结构。膜的去极化激活M1受体，但使M3受体失活。使用M1/M3受体嵌合体来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的激活，该传感器直接报告GQ蛋白在定义的膜电位(全细胞电压钳)下的活性。嵌合体的产生得到了分子动力学计算的支持，分子动力学计算预测，假设的电压传感器是受体的灵活结构，可以在电场中穿过膜移动。(2)膜电位作为分析配体-受体相互作用的工具。膜的去极化导致卡巴胆碱激活的M5受体失活，但诱导匹罗卡品结合的M5受体激活。去极化引起的这些疗效差异表明，这两种激动剂都以不同的分子相互作用与受体结合。为了量化配体的特异性效应，将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，这些电压灵敏度的差异被进一步分析，这些计算预测了配体和受体结合口袋之间的精确分子相互作用。(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。因为膜的去极化激活了M1受体，所以这个目的是研究GQ蛋白下游的细胞信号是否表现出电压依赖性。表达M1受体的细胞受到长期去极化的影响，转录活性通过基于特定荧光素酶的报告分析进行评估。综上所述，本项目的实验结果将有助于阐明GPCR中电压依赖的分子机制，并将为分子受体药理学提供新的线索。

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.