Determining the subcellular localization and function of cardiac TRPV2 Ca²⁺ channels through fluorescent microscopy (545.4)

Abstract

Transient receptor potential vanilloid 2 (TRPV2) is a ligand‐gated ion channel that is expressed in cadiomyocytes and increases contractility following stimulation with probenecid, an agonist for TRPV2. However, the subcellular location and mechanism of activation of this receptor are unkown. We used two independent approaches in cardiac‐derived HL‐1 cells to elucidate the localization and mechanism of TRPV2. To study TRPV2‐mediated Ca2+ transport into the cytosol, HL‐1 cells were first treated with a fluorescent Ca2+ dye and multiple fluorescently tagged moieties of probenecid, some permeable and others impermeable to the cytoplasmic membrane, to determine whether probenecid‐activated TRPV2 channels are located on the cytoplasmic or internal cell membranes. In addition, fluorescently labeled TRPV2 was overexpressed to directly assess its subcellular localization. Results show immediate and maximal cytosolic Ca2+ influx after TRPV2 stimulation, consistent with TRPV2‐mediated Ca2+‐induced Ca2+ release. This effect was blocked by tranilast, a TRPV2 antagonist. In conclusion, we have built upon our previous in vivo findings that TRPV2 stimulation increases cytosolic Ca2+ concentrations consistent with Ca2+‐induced Ca2+ release that may underpin the observed TRPV2‐dependent increase in cardiac contractility.