Cell Confluence Modulates TRPV4 Channel Activity in Response to Hypoxia

Abstract

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.

Piezo1 Channel Activation Reverses Pulmonary Artery Vasoconstriction in an Early Rat Model of Pulmonary Hypertension: The Role of Ca2+ Influx and Akt-eNOS Pathway

“…A possible compensation mechanism by the calcium-induced calcium release by the reticulum may explain this discrepancy. Upregulation of Piezo1 channels may appear later, such as for TRPV4 calcium channels [ 29 ]. In agreement, evidence was provided that the Piezo1 protein level was upregulated in the PH pulmonary artery [ 14 ].…”

Pathophysiological Roles of the TRPV4 Channel in the Heart

“…TRPV4 is expressed in ECs, where its potentiation results in vasodilatation [149], [150]. TRPV4 is sensitive to shear stress [151] and is able to be activated by blood flow and induce Ca 2+ entry when expressed in the endothelium. In ECs, Ca 2+ entry activates the endothelial nitric oxide synthase (eNOS) pathway and the production of nitric oxide (NO), which can, in turn, diffuse to smooth muscle cells (SMCs).…”

“…Accordingly, the transfection of TRPV4 channels in HEK293 cells confers them sensitivity to flow and induces shear-stress-dependent Ca 2+ entry [100], confirming the important role of TRPV4 in sensing shear stress. TRPV4-transfected HEK-293T cells are also sensitive to cell confluence, modulating TRPV4's response to hypoxia at high cell densities, suggesting another important feature of TRPV4 activation, i.e., hypoxia [151]. Indeed, in vessels, hypoxia induces vasodilatation in the systemic circulation, whereas it induces vasoconstriction in the pulmonary circulation.…”