Rinne Andreas, Mobarec Juan Carlos, Mahaut-Smith Martyn, Kolb Peter, Bünemann Moritz
Department of Cardiovascular Physiology, Ruhr University Bochum, Universitätsstr. 150, D-44780 Bochum, Germany.
Department of Pharmaceutical Chemistry, Philipps University Marburg, Marbacher Weg 6, D-35032 Marburg, Germany.
Sci Signal. 2015 Nov 3;8(401):ra110. doi: 10.1126/scisignal.aac7419.
Signaling by many heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs) is either enhanced or attenuated by changes in plasma membrane potential. To identify structural correlates of the voltage sensitivity of GPCR signaling, we chose muscarinic acetylcholine receptors (the M1, M3, and M5 isoforms) as a model system. We combined molecular docking analysis with Förster resonance energy transfer (FRET)-based assays that monitored receptor activity under voltage clamp conditions. When human embryonic kidney (HEK) 293 cells expressing the individual receptors were stimulated with the agonist carbachol, membrane depolarization enhanced signaling by the M1 receptor but attenuated signaling by the M3 and M5 receptors. Furthermore, whether membrane depolarization enhanced or inhibited receptor signaling depended on the type of agonist. Membrane depolarization attenuated M3 receptor signaling when the receptor was bound to carbachol or acetylcholine, whereas depolarization enhanced signaling when the receptor was bound to either choline or pilocarpine. Docking calculations predicted that there were two distinct binding modes for these ligands, which were associated with the effect of depolarization on receptor function. From these calculations, we identified a residue in the M3 receptor that, when mutated, would alter the binding mode of carbachol to resemble that of pilocarpine in silico. Introduction of this mutated M3 receptor into cells confirmed that the membrane depolarization enhanced, rather than attenuated, signaling by the carbachol-bound receptor. Together, these data suggest that the directionality of the voltage sensitivity of GPCR signaling is defined by the specific binding mode of each ligand to the receptor.
许多异源三聚体鸟嘌呤核苷酸结合蛋白（G蛋白）偶联受体（GPCR）的信号传导会因质膜电位的变化而增强或减弱。为了确定GPCR信号电压敏感性的结构相关性，我们选择毒蕈碱型乙酰胆碱受体（M1、M3和M5亚型）作为模型系统。我们将分子对接分析与基于Förster共振能量转移（FRET）的检测方法相结合，该方法在电压钳制条件下监测受体活性。当用激动剂卡巴胆碱刺激表达单个受体的人胚肾（HEK）293细胞时，膜去极化增强了M1受体的信号传导，但减弱了M3和M5受体的信号传导。此外，膜去极化是增强还是抑制受体信号传导取决于激动剂的类型。当受体与卡巴胆碱或乙酰胆碱结合时，膜去极化减弱M3受体信号传导，而当受体与胆碱或毛果芸香碱结合时，去极化增强信号传导。对接计算预测这些配体存在两种不同的结合模式，这与去极化对受体功能的影响有关。通过这些计算，我们在M3受体中鉴定出一个残基，当该残基发生突变时，会在计算机模拟中改变卡巴胆碱的结合模式，使其类似于毛果芸香碱的结合模式。将这种突变的M3受体导入细胞证实，膜去极化增强了与卡巴胆碱结合的受体的信号传导，而不是减弱了信号传导。总之，这些数据表明，GPCR信号电压敏感性的方向性是由每种配体与受体的特定结合模式决定的。
