Genetic and pharmacological evidence for a role of the ion channel TRPV2 as a regulator of actin-dependent functional traits in rat basophilic leukemia cells.

The transient receptor potential ion channel TRPV2 is broadly expressed in cells originating from hematopoietic stem cells and is vital for an effective innate immunity. TRPV2 is expressed in mast cells, where it was suggested that physical stimuli activate TRPV2 to provoke physical urticaria. Given that only unselective pharmacological tools are available to study TRPV2, functional characterization of TRPV2 in mast cells remains imprecise. We studied mast cell-like rat basophilic leukemia (RBL-2H3) cells following CRISPR-Cas9-genetic deletion of TRPV2. Cells lacking TRPV2 fail to generate membrane currents induced by the nonselective TRPV-agonists 2-aminoethoxydiphenyl borate (2-APB) and probenecid (PBC), and they exhibit reduced proliferation, cell adhesion, migration and phagocytosis. While Transforming Growth Factor β1 (TGF-β1) reduces cortical actin in wildtype RBL2-2H3 cells, the basal level of cortical actin is reduced in TRPV2-knockout cells but not modified by TGF-β1. Deletion of TRPV2 reduces the numbers of membrane ruffles induced by TGF-β1, and it increases phosphorylated ERK (p-ERK) in both unstimulated and TGF-β1-treated cells. The release of β-hexosaminidase induced by IgE antigen-stimulation of FcεRI receptors does not depend on TRPV2. PBC applied alone or in combination with 2-APB, cannabidiol (CBD), or protons induce a TRPV2-dependent degranulation. The intracellular residues His165 and His521 are crucial for this pH-sensitive activation of rat TRPV2. Our data suggest that TRPV2 regulates actin-dependent mast cell-like properties of RBL2-2H3 cells. PBC can be used as a pharmacological tool to induce a TRPV2-dependent but IgE-independent degranulation, and it employs a novel molecular mechanism to activate TRPV2 in a proton-dependent manner.