2-Aminoethoxydiphenyl Borate Is a Common Activator of TRPV1, TRPV2, and TRPV3

Abstract

The transient receptor potential (TRP) superfamily contains a large number of proteins encoding cation permeable channels that are further divided into TRPC (canonical), TRPM (melastatin), and TRPV (vanilloid) subfamilies. Among the six TRPV members, TRPV1, TRPV2, TRPV3, and TRPV4 form heat-activated cation channels, which serve diverse functions ranging from nociception to osmolality regulation. Although chemical activators for TRPV1 and TRPV4 are well documented, those for TRPV2 and TRPV3 are lacking. Here we show that in the absence of other stimuli, 2-aminoethoxydiphenyl borate (2APB) activates TRPV1, TRPV2, and TRPV3, but not TRPV4, TRPV5, and TRPV6 expressed in HEK293 cells. In contrast, 2APB inhibits the activity of TRPC6 and TRPM8 evoked by 1-oleolyl-2-acetyl-sn-glycerol and menthol, respectively. In addition, low levels of 2APB strongly potentiate the effect of capsaicin, protons, and heat on TRPV1 as well as that of heat on TRPV3 expressed in Xenopus oocytes. In dorsal root ganglia neurons, supra-additive stimulations were evoked by 2APB and capsaicin or 2APB and acid. Our data suggest the existence of a common activation mechanism for TRPV1, TRPV2, and TRPV3 that may serve as a therapeutic target for pain management and treatment for diseases caused by hypersensitivity and temperature misregulation. The transient receptor potential (TRP)1 superfamily of cation channels consists of a large number of recently identified molecules that share sequence homology with the Drosophila protein named after a phototransduction mutant called trp. According to sequence similarities, the TRP channels are further divided into subfamilies, such as TRPC (canonical), TRPM (melastatin), and TRPV (vanilloid) (see reviews in Refs. 1 and 2). These channels are involved in diverse cellular functions including receptor and store-operated Ca 2ϩ entry (3), Ca 2ϩ transport (4, 5), trace metal detection (6), and temperature (7-9) and osmolality (10, 11) sensations. The activation mechanisms for most of the TRP channels remain to be elucidated. Specific ligands have been found for TRPC3, TRPC6, TRPC7, TRPV1, TRPV4, TRPM2, TRPM4, TRPM5, TRPM7, and TRPM8. These include endogenous substances, such as lipids (diacylglycerol (12), anandamide (13, 14), and phosphatidylinositol 4,5-bisphosphate (15)), nucleotides (ADP-ribose (16) (23,24), 2APB was soon found to directly block native store-operated channels (25-27), sarco/ endoplasmic reticulum Ca 2ϩ -ATPase pumps (28), mitochondrial permeability transition pore (29), and a few other ion channels (30). The mechanism of action for 2APB is likely to be complex. In addition to inhibition, low concentrations of 2APB enhanced the activity of store-operated channels (26). At greater than 50 M, 2APB activated a Ca 2ϩ -permeable nonselective cation channel with a 50-picosiemens single channel conductance and very low open probability in rat basophilic leukemia cells (31).2APB has been perceived as a general inhibitor of TRP channels (1). However, except for TRPC3 (24,32), the effects of this drug...

The TRPV3 mutation associated with the hairless phenotype in rodents is constitutively active

“…The full-length cDNA for mouse TRPV3 was obtained and placed in the pAGA3 and pIRES2EGFP vectors as previously described [15]. To express the green fluorescence protein (GFP) fusion protein of TRPV3, the open-reading frame of the TRPV3 cDNA was subcloned into pEGFPC3 vector (BD-Clontech, Palo Alto, CA) at the EcoRI/SalI sites, in which the EcoRI site was filled in with Klenow fragment of DNA polymerase I and fused with the second codon of TRPV3 (AAT for Asn) via blunt-end ligation.…”

“…For T-Rex 293, blasticidin S (10 μg/ml) was also included. Transfections were performed in wells of a 96-well plate using Lipofectamine 2000 (Invitrogen) as previously described [15]. The pIRES2EGFP vector was used for the expression in HEK 293 cells whereas pcDNA4/TO was used for the expression in T-Rex 293 cells.…”

“…However, an endogenous ligand has yet to be found for TRPV3. Another chemical commonly used to activate TRPV3 is 2-aminoethoxydiphenyl borate (2APB) [7,[14][15][16][17][18][19].…”

Selective inhibition of TRPM2 channel by two novel synthesized ADPR analogues

“…Previous reported nonselective inhibitors such as FFA and 2‐APB also show significant effects on other ion channels. FFA can also inhibit TRPM3, TRPM4 and TRPM5,35, 36 while 2‐APB also activates TRPV1, TRPV2 and TRPV3 25. When employing selective inhibitors 7i or 8a on probing TRPM2 function, these unwanted side‐effects will be eliminated, so that the results will be more relevant to TRPM2 channel.…”

“…However, none of them show any specificity against TRPM2 channel 24. For instance, 2‐APB activates TRPV1, TRPV2 and TRPV3 25. In addition, several nucleotide analogues such as adenosine monophosphate (AMP), 8‐bromoadenosine 5′‐diphosphoribose (8‐Br‐ADPR) have also been reported to be TRPM2 inhibitors (Figure 1).…”