Pharmacological characterization and molecular determinants of the activation of transient receptor potential V2 channel orthologs by 2-aminoethoxydiphenyl borate

Despite its expression in different cell types, transient receptor potential V2 (TRPV2) is still the most cryptic members of the TRPV channel family. 2-Aminoethoxydiphenyl borate (2APB) has been shown to be a common activator of TRPV1, TRPV2, and TRPV3, but 2APB-triggered TRPV2 activation remains to be thoroughly characterized. In this study, we have developed an assay based on cell lines stably expressing mouse TRPV2 channels and intracellular calcium measurements to perform a pharmacological profiling of the channel. Phenyl borate derivatives were found to activate mouse TRPV2 with similar potencies and thus were used to screen a panel of channel blockers. Besides the classic TRP inhibitors ruthenium red (RR) and 1-(beta-[3-(4-methoxyphenyl) propoxy]-4-methoxyphenethyl)-1H-imidazole hydrochloride (SKF96365), two potassium channel blockers, tetraethylammonium (TEA) and 4-aminopyridine, and an inhibitor of capacitative calcium entry, 1-(2-(trifluoromethyl) phenyl) imidazole (TRIM), were found to inhibit TRPV2 activation by 100 mu M 2APB. Activation by 300 mu M 2APB, however, could only be inhibited by RR and TRIM. Electrophysiological recordings demonstrated that TEA inhibition was use-dependent, suggesting that high concentrations of 2APB might induce a progressive conformational change of the channel. Comparison of TRPV2 orthologs revealed that the human channel was insensitive to 2APB. Analysis of chimeric constructs of mouse and human TRPV2 channels showed that the molecular determinants of 2APB sensitivity could be localized to the intracellular amino and carboxyl domains. Finally, using lentiviral-driven expression, we demonstrate that hTRPV2 exerts a dominant-negative effect on 2APB activation of native rodent TRPV2 channels and thus may provide an interesting tool to investigate cellular functions of TRPV2 channels.