Pharmacological Characterization and Molecular Determinants of the Activation of Transient Receptor Potential V2 Channel Orthologs by 2-Aminoethoxydiphenyl Borate

Abstract

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-(␤-[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 M 2APB. Activation by 300 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 lentiviraldriven 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.Transient receptor potential (TRP) proteins are cationic channels that were first discovered in Drosophila melanogaster, in which they play a crucial role in the initial steps of visual transduction (Hardie, 2001;Montell, 2005a). Since then, more than 30 mammalian TRP channels have been identified and are classified into seven families, which are TRPC, TRPV, TRPM, TRPN, TRPA and the two distantly related TRPP and TRPML (Montell, 2005b). All members of the TRP superfamily share the same membrane topology as voltage-gated potassium channels; they contain six hydrophobic domains spanning the plasma membrane and an additional P-loop structure between the fifth and the sixth transmembrane segments that participates in the pore-forming domain of the channel. TRP proteins possess long cytoThis work was supported in part by Centre National de la Recherche Scientifique and Institut National de la Santé et de la Recherche Médicale. J.V. and P.A. were supported by student fellowships from Association de la Recherche sur le Cancer.Article, publication date, and citation information can be found at

Store-dependent and -independent Modes Regulating Ca2+ Release-activated Ca2+ Channel Activity of Human Orai1 and Orai3

“…Store-independent Activation of Orai3 by 2-APB-It has been reported that 2-APB can activate several TRP channels, including TRPV1, TRPV2, and TRPV3 (27)(28)(29)(30). Here we show that 2-APB can also activate Orai3 channels, confirming and extending a previous report (31).…”

“…2-Aminoethyl diphenylborinate (2-APB) is a membranepermeable modulator of many ion channels, including Ins(1,4,5)P 3 receptors (26) and TRPV1, TRPV2, and TRPV3 channels (27)(28)(29)(30). In native and overexpression systems, it can potentiate SOCE or CRAC current at low concentrations (Ͻ5 M) and inhibit both at higher concentrations (14,15,(31)(32)(33).…”

Structure of the Full-Length TRPV2 Channel by Cryo-EM

“…The observed differences in the Ca 2 þ measurements between both truncated TRPV2 orthologues could be attributed to many different experimental variables. TRPV2 activation by 2-APB is known to be species dependent 37,38 ; therefore the response of rat TRPV2 to 2-APB may be inherently different from that of rabbit TRPV2. A parallel characterization of how 2-APB interacts with rat versus rabbit TRPV2 may provide insights into how 2-APB activates TRPV2 orthologues.…”

Transient Receptor Potential Canonical Channels Regulate the Induction of Cerebellar Long-Term Depression

“…While SKF96365 has been used extensively as a tool to define the functional roles of TRPC channels in various cell and tissue types (Bengtson et al, 2004;Fowler et al, 2007), previous studies reported notable overlapping effects on numerous channels in various organs (Merritt et al, 1990;Hong and Chang, 1994;Schwarz et al, 1994;Juvin et al, 2007;Liu et al, 2007). A high concentration of SKF96365 (30 M) partly affected low voltageactivated T-type calcium channels in Purkinje cells (Singh et al, 2010).…”