Mechanisms overcoming the resistance of human TRPV2 to chemical activation

Transient receptor potential vanilloid 2 (TRPV2) is a thermosensitive cation channel widely expressed across diverse cell types. There, it contributes to numerous physiological processes and plays a role in the pathophysiology of inflammation, immunity, various cancers and neuronal development. The inherent resistance of human TRPV2 (hTRPV2) to stimuli that readily activate its rodent orthologs currently limits progress in translational and clinical research on this channel. Here, we present engineered variants of hTRPV2 that were designed to enhance channel activation induced by 2-aminoethoxydiphenylborate (2-APB), cannabidiol, and the TRPV2 agonist AV2-1, thereby identifying sites that allow for the most effective channel regulation. Through a series of rational mutations and chimeric constructs, combined with electrophysiology and molecular modeling, we identified two key allosteric sites in hTRPV2 that determine the resistance of the channel to activating stimuli. We discovered that two intracellular membrane-proximal salt bridges prevent effective activation of hTRPV2: (i) one formed between residues of the putative 2-APB-binding site and the bottom of S6 helix (R533-E647), and (ii) another formed between residues from the TRP helix and the proximal N-terminus (D655-R369). We also propose possible mechanisms by which the non-conserved pore turret contributes to channel activation. Our results may facilitate the rational drug design towards potent and selective hTRPV2 ligands and help to elucidate the functions of this channel in organs or cell types of interest.