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

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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.