Pharmacological profiling of the TRPV3 channel in recombinant and native assays

Abstract

Background and purpose: Transient 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 approach: Medium-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 results: A 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 implications: Our 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.

Keywords: FLIPR; TRP channel; TRPV3; mouse 308 keratinocytes.

Figure 1

Ca²⁺ responses () to increasing concentrations of DPBA in human recombinant TRPV3 (A) and untransfected (B) HEK293 cells. In HEK293 cells, responses were also recorded in the absence of extracellular Ca²⁺ () and in the absence of extracellular Ca²⁺ where internal Ca²⁺ stores had been depleted by pre-incubation with 1 μM thapsigargin (). Data are displayed as raw fluorescence units of the maximum minus minimum response from each experiment. Each data point represents the mean response ± SEM from four separate experiments. **P < 0.001 versus responses in the presence of Ca²⁺. RFU, relative fluorescence units.

Figure 2

(A) Ca²⁺ responses to DPBA in untransfected () and recombinant HEK293 cells stably expressing human TRPV3 (), monoclonal rat TRPV3 clones 47 or clone 66 ( and ) or polyclonal rat TRPV3 (). Data are displayed as raw fluorescence units of the maximum minus minimum response from each experiment. Each data point represents the mean response ± SEM from three replicate wells. (B) Characterization of TRPV3 responses in transiently transfected HEK293 cells by fluorescence-based, intracellular Ca²⁺ imaging. Shown are averages from traces obtained from single-cell responses to DPBA for the various transfection conditions. ATP serves as an internal control for activation of P2 receptors leading to increases in intracellular calcium.

Figure 3

In m308ks, DPBA alone does not activate TRPV3. Mouse keratinocytes were exposed to increasing concentrations of DPBA and changes in fluorescence monitored. Responses were recorded in the presence () and absence () of extracellular Ca²⁺ and also after pre-incubation with 10 μM FTP-THQ () or 10 μM RR (). Data are displayed as raw fluorescence units of the maximum minus minimum response from each experiment. Each data point represents the mean response ± SEM from three separate experiments. RFU, relative fluorescence units.

Figure 4

In m308ks, certain TRPV3 agonists synergize with DPBA to activate TRPV3. Ca²⁺ responses obtained in m308k cells in response to (A) carvacrol, (B) camphor, (C) thymol, (D) eugenol, (E) 6-TBC, (F) 2-APB alone () or together with 50 μM DPBA (). Responses to co-agonist application were also assessed after pre-incubation with FTP-THQ () or 10 μM RR (). Data are displayed as raw fluorescence units of the maximum minus minimum response from each experiment. Each data point represents the mean response ± SEM from three separate experiments. RFU, relative fluorescence units.

Figure 5

Fatty acids LA and AA synergize with DPBA and 2-APB to activate TRPV3 in m308ks. (A) Effects of the fatty acids LA (40, 160 μM) and AA (10, 30 μM) alone or in combination with DPBA in m308ks. (B) Concentration response curves to DPBA, 2-APB or camphor in the absence () or presence of 160 μM LA or 30 μM AA (). Responses to agonist in the presence of LA or AA were also assessed after pre-incubation with 10 μM FTP-THQ () or 10 μM RR (). Data are displayed as raw fluorescence units of the maximum minus minimum response from each experiment. Each data point represents the mean response ± SEM from a minimum of three separate experiments. *P < 0.05, ***P < 0.001 one-way anova with Tukey's post hoc test. RFU, relative fluorescence units.

Figure 6

TRPV3 KD in m308k cells. (A) TRPV3 mRNA levels were measured by RT-qPCR in WT, non-target shRNA and TRPV3 KD m308k cell lines. mRNA levels are shown as a percentage of WT, set at 100%. (B) Ca²⁺ responses obtained in WT (), non-target shRNA () and TRPV3 KD () m308 keratinocytes. Cells were exposed to increasing concentrations of DPBA applied in combination with AA, LA or carvacrol and changes in intracellular Ca²⁺ fluorescence were monitored. Responses in WT () and KD () cells were also assessed after pre-incubation with 10 μM FTP-THQ. Data are displayed as raw fluorescence units of the maximum minus minimum response from each experiment. Each data point represents the mean response ± SEM from five separate experiments. RFU, relative fluorescence units.

Figure 7

Inhibition of Ca²⁺ signalling in response to 40 μM DPBA in (A) human TRPV3, (B) rat TRPV3 and 50 μM DPBA + 160 μM LA in (C) m308ks in the presence of increasing concentrations of RR (), FTP-THQ () or CPC-MPP (). Data are displayed as percentage responses to DPBA alone. Each data point represents the mean response ± SEM from 4–6 separate experiments.

Figure 8

Analysis of the mechanism of inhibition of Ca²⁺ responses to 50 μM DPBA and camphor in m308k cells. Cells were treated with increasing concentrations of camphor in combination with 50 μM DPBA either alone () or after pre-incubation with fixed concentrations of RR (A) or FTP-THQ (B). Data are displayed as percentage responses to DPBA and 5 mM camphor alone. Each data point represents the mean response ± SEM from 5–6 separate experiments.