Abstact

BACKGROUND AND PURPOSE: Transient receptor potential vanilloid 2 (TRPV2) is a non-selective cation channel implicated in immune cell functions. However, progress in understanding TRPV2 has been limited by a lack of potent and selective pharmacological tools, particularly those targeting the human variant. We aimed to identify and characterise a novel small-molecule activator of TRPV2. EXPERIMENTAL APPROACH: We screened a compound library using Ca(2+) imaging in HEK293 cells stably expressing mouse TRPV2. The lead compound AV2-1 was validated by concentration-response analyses, microfluorometric Ca(2+) assays, and electrophysiological recordings. Structural insights were obtained from cryoEM of TRPV2 in complex with AV2-1, and mutagenesis was performed to confirm binding site residues. The efficacy of AV2-1 was assessed in human peripheral blood-derived macrophages by Ca(2+) imaging, whole-cell electrophysiology and TIRF microscopy to detect Ca(2+) microdomains. KEY RESULTS: AV2-1 is a novel TRPV2 activator showing robust efficacy across mouse, rat and human orthologues. Structural analysis reveals that AV2-1 stabilises the channel in its active conformation by binding to an established intracellular pocket via TRPV2-specific residues His165 and Cys157, as confirmed by mutagenesis experiments. AV2-1 induces TRPV2-dependent global [Ca(2+)](i) signals, ionic currents and localised subplasmalemmal Ca(2+) microdomains in human peripheral blood-derived macrophages. CONCLUSION AND IMPLICATIONS: AV2-1 represents a novel pharmacological tool for probing TRPV2 function in immune cells, combining improved selectivity and potency with low toxicity. Its ability to activate human TRPV2 and elicit physiologically relevant Ca(2+) signals highlights its potential for advancing TRPV2 research and for therapeutic exploration in immune modulation and disease contexts.