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G-protein coupled receptors (GPCRs) comprise the largest family of transmembrane receptors in the human genome. Thirty-four percent of the drugs approved by the FDA in 2017 have a GPCR as their target, which is why this receptor group is in focus of pharmacological research (Hauser et al., 2017). GPCRs as integral membrane proteins are exposed to a strong electrical field that is subject to natural fluctuations. It is now over 15 years ago, that for the first time the agonist-induced receptor activity modulated by changes in membrane potential (VM) could be shown for a member of this group (Ben-Chaim et al., 2003). Although some time has passed since then and intensive research has been carried out on this topic, many questions arising from this discovery have not yet been answered or only partially. To date, no generally valid explanation of the underlying mechanism of the voltage dependence of GPCRs has been described. In the meantime, different groups of GPCRs have been examined on their voltage dependence. A group with a wide distribution in the human body, with only limited information accessible concerning this aspect before this study was carried out, is the prostanoid receptor group. The starting point for this study was an investigation of megakaryocytes that suggested activation of endogenous thromboxane receptors (TP receptors) in the presence of an agonist upon depolarization (Martinez-Pinna et al., 2005). Due to the lack of linearity in the measurement of calcium levels used for receptor activity, neither a qualitative nor a quantitative analysis of the voltage effect could be carried out. We used in FRET biosensors, which directly reflected the TP receptor activity and thus made it possible to characterize the voltage effect. In the context of this work, a FRET-based TP receptor conformation sensor was cloned. The actual investigation of the voltage dependence was carried out with a combination of FRET measurement as a measure of the receptor activity and simultaneous patch-clamp measurement to control the membrane potential. We observed a robust voltage dependence of the TP receptor both at the receptor level and on the downstream signal level. TP receptor activity doubled upon depolarization from -90 mV to +60 mV in the presence of U46619, a stable analog of prostaglandin H2. The half-maximal voltage effect V0.5 determined for the TP receptor was -46 mV, which was within the physiological range of VM. We were also able to show that the voltage effect mainly modulated the affinity of the TP receptor for U46619. Beside other findings pointing in this direction, we observed that the EC50 for U46619 at -90 mV was left shifted about 4.5 times compared to the EC50 at +60 mV and both curves had the same maximum. We tested the voltage effect on the TP receptor, activated with differently substituted prostanoid derivatives, each of which carried modifications at different positions around the molecule. All ligands tested showed activation upon depolarization, which indicated a more global change in the TP receptor conformation due to depolarization. Our finding fits with the observation that none of the point mutations we performed, in positions important for ligand binding of the TP receptor, led to a change in the voltage dependence of the TP receptor. Consequently, there was no evidence for a specific modulation of the receptor-ligand interaction by changes in membrane potential.
Furthermore, we were able to show that the range and strength of the voltage dependence was restricted by R2957.40, an amino acid that is situated in the agonist pocket. The modulation of ligand-induced receptor activity by VM was not limited to the TP receptor, since the prostaglandin F receptor (FP receptor) activated with U46619 and the prostaglandin E2 receptor - subtype 3 (EP3 receptor ) activated with Iloprost showed a similar reaction to the depolarization as observed for the TP receptor activated with U46619. In contrast, IP receptor activated with Iloprost showed no detectable voltage dependence. The difference in voltage dependence could not be attributed to individual charged amino acids, which is why a more complex difference can be assumed as the reason for the different voltage dependence. Within this study, the PAR1 was also examined with the particularly sensitive Gα13-p115-FRET interaction Assay, here it turned out, that the PAR1 activated with thrombin was not modulated by changes in VM.
While searching for a ligand-specific voltage dependence, Daltroban, a TP receptor antagonist, for which there was evidence of partial agonism, was examined. To our surprise, it turned out that at high concentrations Daltroban converted from an antagonist to partial agonist, which transiently activated the TP receptor. Because of these remarkable findings, the observed Daltroban effect was further investigated. It was shown that the TP receptor had a reduced capability to be activated, after administration of Daltroban, whereby it remained unclear whether Daltroban bound (pseudo-) irreversibly to the receptor. Contacts between Daltroban and the orthosteric binding site played a role in the partial agonistic effect. Different Daltroban derivatives were tested to investigate the structure-activity relationship of Daltroban. However, the underlying mechanism has remained unclear and will be the subject of future research.
Furthermore, a systematic approach to clone FRET-based GPCR receptor sensors was developed in this work, since these sensors can be of great value for future direct studies of the quality and quantity of the voltage effect on the ligand-induced GPCR activity. In this work, FRET-based GPCR receptor sensors for IP receptor, FP receptor and ETB receptor were successfully cloned.