Characterization of the mouse cold-menthol receptor TRPM8 and vanilloid receptor type-1 VR1 using a fluorometric imaging plate reader (FLIPR) assay

Abstract

1. TRPM8 (CMR1) is a Ca(2+)-permeable channel, which can be activated by low temperatures, menthol, eucalyptol and icilin. It belongs to the transient receptor potential (TRP) family, and therefore is related to vanilloid receptor type-1 (VR1, TRPV1). We tested whether substances which are structurally related to menthol, or which produce a cooling sensation, could activate TRPM8, and compared the responses of TRPM8 and VR1 to these ligands. 2. The effects of 70 odorants and menthol-related substances on recombinant mouse TRPM8 (mTRPM8), expressed in HEK293 cells, were examined using a FLIPR assay. In all, 10 substances (linalool, geraniol, hydroxycitronellal, WS-3, WS-23, FrescolatMGA, FrescolatML, PMD38, CoolactP and Cooling Agent 10) were found to be agonists. 3. The EC(50) values of the agonists defined their relative potencies: icilin (0.2+/-0.1 microM)>FrescolatML (3.3+/-1.5 microM) > WS-3 (3.7+/-1.7 microM) >(-)menthol (4.1+/-1.3 microM) >frescolatMAG (4.8+/-1.1 microM) > cooling agent 10 (6+/-2.2 microM) >(+)menthol (14.4+/-1.3 microM) > PMD38 (31+/-1.1 microM) > WS-23 (44+/-7.3 microM) > Coolact P (66+/-20 microM) > geraniol (5.9+/-1.6 mM) > linalool (6.7+/-2.0 mM) > eucalyptol (7.7+/-2.0 mM) > hydroxycitronellal (19.6+/-2.2 mM). 4. Known VR1 antagonists (BCTC, thio-BCTC and capsazepine) were also able to block the response of TRPM8 to menthol (IC(50): 0.8+/-1.0, 3.5+/-1.1 and 18+/-1.1 microM, respectively). 5. The Ca(2+) response of hVR1-transfected HEK293 cells to the endogenous VR1 agonist N-arachidonoyl-dopamine was potentiated by low pH. In contrast, menthol- and icilin-activated TRPM8 currents were suppressed by low pH. 6. In conclusion, in the present study, we identified 10 new agonists and three antagonists of TRPM8. We found that, in contrast to VR1, TRPM8 is inhibited rather than potentiated by protons.

Figure 1

[Ca²⁺]_i responses to TRPM8 agonists. [Ca²⁺]_i fluxes induced by icilin (5 μM), WS-3 (30 μM), (−)menthol (30 μM) and eucalyptol (5 mM) were monitored using the FLIPR® assay in mTRPM8-transfected HEK293 cells. [Ca²⁺]_i responses were measured as changes in fluorescence intensity (FI) before and after the addition of agonists. The data shown are representative plots of the fluorescence signals against time during assays.

Figure 2

Efficacy of TRPM8 agonists. [Ca²⁺]_i responses were measured as maximal increases in fluorescence, expressed as percentages of the maximum icilin response. They are given as means±s.e.m. (n=4–8).

Figure 3

Antagonists of TRPM8. The VR1 antagonists (capsazepine, thio-BCTC and BCTC) inhibited the [Ca²⁺]_i increases induced by 20 μM (−)menthol via mTRPM8 channels in a concentration-dependent manner. [Ca²⁺]_i was monitored as described above. Responses were measured as peak increases in fluorescence, and expressed as percentages of the uninhibited response (mean±s.e.m., n=4).

Figure 4

pH sensitivity of hVR1 and mTRPM8. (a) [Ca²⁺]_i was monitored in hVR1-transfected HEK293 cells before and after the addition of the agonists ANA (2 μM), NADA (2 μM), or capsaicin (0.1 μM). Responses were measured as increases in peak fluorescence intensity. Agonists were applied in buffers at either pH 7.5 or pH 6.3 (n=3). ^*P<0.01 and P<0.005, unpaired t-test. (b) [Ca²⁺]_i was monitored in mTRPM8-transfected HEK293 cells before and after the addition of (−)menthol (20 μM) or icilin (0.5 μM). Agonist responses were measured as increases in peak fluorescence intensity. Agonists were applied at pH 8.0, pH 7.5, pH 6.8, or pH 6.3 (n=3). ^*P<0.05 and P<0.005, unpaired t-test.