Pharmacological profiling of the TRPV3 channel in recombinant and native assays

Abstract

BACKGROUND AND PURPOSETransient receptor potential vanilloid subtype 3 (TRPV3) is implicated in nociception and certain skin conditions. As such, it is an attractive target for pharmaceutical research. Understanding of endogenous TRPV3 function and pharmacology remains elusive as selective compounds and native preparations utilizing higher throughput methodologies are lacking. In this study, we developed medium-throughput recombinant and native cellular assays to assess the detailed pharmacological profile of human, rat and mouse TRPV3 channels. EXPERIMENTAL APPROACHMedium-throughput cellular assays were developed using a Ca 2+-sensitive dye and a fluorescent imaging plate reader. Human and rat TRPV3 pharmacology was examined in recombinant cell lines, while the mouse 308 keratinocyte cell line was used to assess endogenous TRPV3 activity. KEY RESULTSA recombinant rat TRPV3 cellular assay was successfully developed after solving a discrepancy in the published rat TRPV3 protein sequence. A medium-throughput, native, mouse TRPV3 keratinocyte assay was also developed and confirmed using genetic approaches. Whereas the recombinant human and rat TRPV3 assays exhibited similar agonist and antagonist profiles, the native mouse assay showed important differences, namely, TRPV3 activity was detected only in the presence of potentiator or during agonist synergy. Furthermore, the native assay was more sensitive to block by some antagonists. CONCLUSIONS AND IMPLICATIONSOur findings demonstrate similarities but also notable differences in TRPV3 pharmacology between recombinant and native systems. These findings offer insights into TRPV3 function and these assays should aid further research towards developing TRPV3 therapies. LINKED ARTICLESThis article is part of a themed section on the pharmacology of TRP channels. To view the other articles in this section visit http://dx.doi. org/10.1111/bph.2014.171.issue-10 Abbreviations 2-APB, 2-aminoethoxydiphenylborate; 6-TBC, terpenoid 6-tert-butyl-m-cresol; AA, arachidonic acid; DPBA, diphenylborinic anhydride; FLIPR, fluorescent imaging plate reader; FPP, farnesyl pyrophosphate; HEK293, human embryonic kidney cells; IA, incensole acetate; LA, linoleic acid; m308k, mouse 308 keratinocyte; PUFA, poly-unsaturated fatty acid; RR, ruthenium red; RT, reverse transcription; shRNA, small hairpin RNA; TRP, transient receptor potential; TRPV3, transient receptor potential vanilloid subtype 3

Evidence for the functional involvement of members of the TRP channel family in the uptake of Na+ and NH4 + by the ruminal epithelium

“…TRPA1 and TRPV3 both have a considerable conductance for Ca 2+ , with values of p(Ca 2+ )/p(Na + ) in the range of 7 to 10 and 1 to 12 for TRPV3 and TRPA1, respectively [19,38,69], with an influx of Ca 2+ observable after stimulation with appropriate agonists [21]. Accordingly, the effects of menthol and thymol on Ca 2+ fluxes were studied.…”

Targeting nociceptive transient receptor potential channels to treat chronic pain: current state of the field

“…Several companies have pursued selective antagonists of TRPV3 channels including Abbvie (Gomtsyan et al, ), Hydra Biosciences and Glenmark/Sanofi‐Aventis (Grubisha et al, ). The Glenmark/Sanofi‐Aventis compound GRC15300 failed a Phase 2 trial in chronic peripheral neuropathy patients (a 4 week trial), and the collaboration between the two companies was terminated in 2014 (Broad et al, ).…”

“…The Glenmark/Sanofi‐Aventis compound GRC15300 failed a Phase 2 trial in chronic peripheral neuropathy patients (a 4 week trial), and the collaboration between the two companies was terminated in 2014 (Broad et al, ). However, recent reports suggest that these TRPV3 channel antagonists are not full blockers of the channel (Grubisha et al, ), so it is difficult to know how to interpret these results with regards to a potential role of TRPV3 channels in pain.…”

TRP channels in the skin

“…Among the large number of TRP channels expressed in the skin, TRPV3 (and TRPV4) possibly plays the most prominent role in the regulation of skin functions (Nilius and Bíró, ; Nilius et al ., ). Actually, TRPV3 was originally demonstrated to be most abundantly expressed on epidermal keratinocytes both in humans and rodents (Smith et al ., ; Xu et al ., ; Peier et al ., ; Grubisha et al ., ), whereas TRPV4 was found in several tissues (Wissenbach et al ., ; Liedtke et al ., ; Strotmann et al ., ; Delany et al ., ) including keratinocytes (Suzuki et al ., ). No wonder, therefore, that both channels were thought to markedly regulate numerous cutaneous biological processes.…”