Institute for Pharmacology and Clinical Pharmacy, Faculty of Pharmacy, Philipps-University Marburg, Marburg, Germany.
Institute for Pharmacology and Clinical Pharmacy, Faculty of Pharmacy, Philipps-University Marburg, Marburg, Germany
Mol Pharmacol. 2020 Apr;97(4):267-277. doi: 10.1124/mol.119.118372. Epub 2020 Jan 31.
G protein-coupled receptors (GPCRs) are the largest class of transmembrane receptors and serve as signal mediators to transduce information from extracellular signals such as neurotransmitters, hormones, or drugs to cellular responses. They are exposed to the strong electrical field of the plasma membrane. In the last decade voltage modulation of ligand-induced GPCR activity has been reported for several GPCRs. Using Foerster resonance energy transfer-based biosensors in patch clamp experiments, we discovered a robust voltage dependence of the thromboxane receptor (TP receptor) on the receptor level as well as on downstream signaling. TP receptor activity doubled upon depolarization from -90 to +60 mV in the presence of U46619, a stable analog of prostaglandin H Half-maximal effective potential (V) determined for TP receptor was -46 mV, which is within the physiologic range. We identified that depolarization affected the agonist affinity for the TP receptor. Depolarization enhanced responses of several structural analogs of U46619 with modifications to a similar extent all around the molecule, indicating that voltage modulates the general conformation of TP receptor. By means of site direct mutagenesis, we identified TP receptor R295, which showed alteration of voltage sensitivity of TP receptor upon mutation. Voltage sensitivity was not limited to TP receptor because prostaglandin F receptor activated with U46619 and prostaglandin E receptor subtype 3 activated with iloprost showed a similar reaction to depolarization as TP receptor. However, prostacyclin receptor activated with iloprost showed no detectable voltage dependence. SIGNIFICANCE STATEMENT: Prostanoids mediate many of their physiological effects via transmembrane receptors expressed in the plasma membrane of excitable cells. We found that agonist-mediated activation of prostaglandin F receptors and prostaglandin E receptors as well as thromboxane receptors are activated upon depolarization, whereas prostacyclin receptors are not. The voltage-induced modulation of thromboxane receptor activity was observed on the level of receptor conformation and downstream signaling. The range of voltage dependence was restricted by R295 in the agonist-binding pocket.
G 蛋白偶联受体（GPCRs）是最大的跨膜受体家族，作为信号介质，将细胞外信号（如神经递质、激素或药物）传递到细胞反应。它们暴露于质膜的强电场中。在过去的十年中，已经报道了几种 GPCR 对配体诱导的 GPCR 活性的电压调节。我们使用基于荧光共振能量转移的生物传感器在膜片钳实验中，发现血栓素受体（TP 受体）在受体水平以及下游信号转导中存在强大的电压依赖性。在 U46619 存在下，从 -90 到 +60 mV 的去极化使 TP 受体的活性增加了一倍，U46619 是前列腺素 H 的稳定类似物。TP 受体的半最大有效电位（V）确定为 -46 mV，这在生理范围内。我们发现去极化会影响激动剂与 TP 受体的亲和力。去极化增强了 U46619 的几种结构类似物的反应，这些类似物在分子周围以相似的程度进行了修饰，这表明电压调节了 TP 受体的一般构象。通过定点突变，我们确定了 TP 受体 R295，该突变改变了 TP 受体的电压敏感性。电压敏感性不仅限于 TP 受体，因为用 U46619 激活的前列腺素 F 受体和用 iloprost 激活的前列腺素 E 受体亚型 3 对去极化的反应与 TP 受体相似。然而，用 iloprost 激活的前列环素受体没有检测到可察觉的电压依赖性。意义：前列腺素介导其许多生理作用通过表达在可兴奋细胞质膜中的跨膜受体。我们发现，激动剂介导的前列腺素 F 受体和前列腺素 E 受体以及血栓素受体的激活在去极化时被激活，而前列环素受体则没有。在受体构象和下游信号转导水平上观察到了血栓素受体活性的电压诱导调节。电压依赖性的范围受激动剂结合口袋中 R295 的限制。
