Pathophysiological Roles of the TRPV4 Channel in the Heart

The transient receptor potential vanilloid 4 (TRPV4) channel is a non-selective cation channel that is mostly permeable to calcium (Ca2+), which participates in intracellular Ca2+ handling in cardiac cells. It is widely expressed through the body and is activated by a large spectrum of physicochemical stimuli, conferring it a role in a variety of sensorial and physiological functions. Within the cardiovascular system, TRPV4 expression is reported in cardiomyocytes, endothelial cells (ECs) and smooth muscle cells (SMCs), where it modulates mitochondrial activity, Ca2+ homeostasis, cardiomyocytes electrical activity and contractility, cardiac embryonic development and fibroblast proliferation, as well as vascular permeability, dilatation and constriction. On the other hand, TRPV4 channels participate in several cardiac pathological processes such as the development of cardiac fibrosis, hypertrophy, ischemia–reperfusion injuries, heart failure, myocardial infarction and arrhythmia. In this manuscript, we provide an overview of TRPV4 channel implications in cardiac physiology and discuss the potential of the TRPV4 channel as a therapeutic target against cardiovascular diseases

Mechanosensitivity in Pulmonary Circulation: Pathophysiological Relevance of Stretch-Activated Channels in Pulmonary Hypertension

A variety of cell types in pulmonary arteries (endothelial cells, fibroblasts, and smooth muscle cells) are continuously exposed to mechanical stimulations such as shear stress and pulsatile blood pressure, which are altered under conditions of pulmonary hypertension (PH). Most functions of such vascular cells (e.g., contraction, migration, proliferation, production of extracellular matrix proteins, etc.) depend on a key event, i.e., the increase in intracellular calcium concentration ([Ca2+]i) which results from an influx of extracellular Ca2+ and/or a release of intracellular stored Ca2+. Calcium entry from the extracellular space is a major step in the elevation of [Ca2+]i, involving a variety of plasmalemmal Ca2+ channels including the superfamily of stretch-activated channels (SAC). A common characteristic of SAC is that their gating depends on membrane stretch. In general, SAC are non-selective Ca2+-permeable cation channels, including proteins of the TRP (Transient Receptor Potential) and Piezo channel superfamily. As membrane mechano-transducers, SAC convert physical forces into biological signals and hence into a cell response. Consequently, SAC play a major role in pulmonary arterial calcium homeostasis and, thus, appear as potential novel drug targets for a better management of PH

Cell Confluence Modulates TRPV4 Channel Activity in Response to Hypoxia

Transient receptor potential vanilloid 4 (TRPV4) is a polymodal Ca2+-permeable channel involved in various hypoxia-sensitive pathophysiological phenomena. Different tools are available to study channel activity, requiring cells to be cultured at specific optimal densities. In the present study, we examined if cell density may influence the effect of hypoxia on TRPV4 activity. Transiently TRPV4-transfected HEK293T cells were seeded at low or high densities corresponding to non-confluent or confluent cells, respectively, on the day of experiments, and cultured under in vitro normoxia or hypoxia. TRPV4-mediated cytosolic Ca2+ responses, single-channel currents, and Ca2+ influx through the channel were measured using Ca2+ imaging/microspectrofluorimetric assay, patch-clamp, and Bioluminescence Resonance Energy Transfer (BRET), respectively. TRPV4 plasma membrane translocation was studied using confocal microscopy, biotinylation of cell surface proteins, and BRET. Our results show that hypoxia exposure has a differential effect on TRPV4 activation depending on cell confluence. At low confluence levels, TRPV4 response is increased in hypoxia, whereas at high confluence levels, TRPV4 response is strongly inhibited, due to channel internalization. Thus, cell density appears to be a crucial parameter for TRPV4 channel activity

Genetically-encoded BRET probes shed light on ligand bias–induced variable ion selectivity in TRPV1 and P2X5/7

International audienceWhether ion channels experience ligand-dependent dynamic ion selectivity remains of critical importance since this could support ion channel functional bias. Tracking selective ion permeability through ion channels, however, remains challenging even with patch-clamp electrophysiology. In this study, we have developed highly sensitive bioluminescence resonance energy transfer (BRET) probes providing dynamic measurements of Ca2+ and K+ concentrations and ionic strength in the nanoenvironment of Transient Receptor Potential Vanilloid-1 Channel (TRPV1) and P2X channel pores in real time and in live cells during drug challenges. Our results indicate that AMG517, BCTC, and AMG21629, three well-known TRPV1 inhibitors, more potently inhibit the capsaicin (CAPS)-induced Ca2+ influx than the CAPS-induced K+ efflux through TRPV1. Even more strikingly, we found that AMG517, when injected alone, is a partial agonist of the K+ efflux through TRPV1 and triggers TRPV1-dependent cell membrane hyperpolarization. In a further effort to exemplify ligand bias in other families of cationic channels, using the same BRET-based strategy, we also detected concentration- and time-dependent ligand biases in P2X7 and P2X5 cationic selectivity when activated by benzoyl-adenosine triphosphate (Bz-ATP). These custom-engineered BRET-based probes now open up avenues for adding value to ion-channel drug discovery platforms by taking ligand bias into account

High-Throughput Screening of TRPV1 Ligands in the Light of the Bioluminescence Resonance Energy Transfer Technique

International audienceIon channels are attractive drug targets for many therapeutic applications. However, high-throughput screening (HTS) of drug candidates is difficult and remains very expensive. We thus assessed the suitability of the bioluminescence resonance energy transfer (BRET) technique as a new HTS method for ion-channel studies by taking advantage of our recently characterized intra- and intermolecular BRET probes targeting the transient receptor potential vanilloid type 1 (TRPV1) ion channel. These BRET probes monitor conformational changes during TRPV1 gating and subsequent coupling with calmodulin, two molecular events that are intractable using reference techniques such as automated calcium assay (ACA) and automated patch-clamp (APC). We screened the small-sized Prestwick chemical library, encompassing 1200 compounds with high structural diversity, using either intra- and intermolecular BRET probes or ACA. Secondary screening of the detected hits was done using APC. Multiparametric analysis of our results shed light on the capability of calmodulin inhibitors included in the Prestwick library to inhibit TRPV1 activation by capsaicin. BRET was the lead technique for this identification process. Finally, we present data exemplifying the use of intramolecular BRET probes to study other transient receptor potential (TRP) channels and non-TRPs ion channels. Knowing the ease of use of BRET biosensors and the low cost of the BRET technique, these assays may advantageously be included for extending ion-channel drug screening. SIGNIFICANCE STATEMENT: This study screened a chemical library against TRPV1 ion channel using bioluminescence resonance energy transfer (BRET) molecular probes and compared the results with the ones obtained using reference techniques such as automated calcium assay and automated patch-clamp. Multiparametric analysis of our results shed light on the capability of calmodulin antagonists to inhibit chemical activation of TRPV1 and indicates that BRET probes may advantageously be included in ion channel drug screening campaigns

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