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

1 TRPM8 (CMR1) is a Ca2+-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(R) assay. In all, 10 substances (linalool, geraniol, hydroxycitronellal, WS-3, WS-23, FrescolatMGA, FrescolatML, PMD38, Cool-actP and Cooling Agent 10) were found to be agonists. 3 The EC50 values of the agonists defined their relative potencies: icilin (0.2 +/- 0.1 muM)>FrescolatML (3.3 +/- 1.5 muM) > WS-3 (3.7 +/- 1.7 muM) (-)menthol (4.1 +/- 1.3 muM) frescolatMAG (4.8 +/- 1.1 muM) > cooling agent 10 (6 +/- 2.2 muM) (+)menthol (14.4 +/- 1.3 muM) > PMD38 (31 +/- 1.1 muM) > WS-23 (44 +/- 7.3 muM) > Coolact P (66 +/- 20 muM) > 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 (IC50: 0.8 +/- 1.0, 3.5 +/- 1.1 and 18 + 1.1 muM, respectively). 5 The Ca2+ 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.