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

摘要

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 (mTRPMS), 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 EC_(50) values of the agonists defined their relative potencies: icilin (0.2 +- 0.1 μM) > FrescolatML (3.3 +- 1.5μM) > WS-3 (3.7 +- 1.7 μM) (-)menthol (4.1 +- 1.3 μM) frescolatMAG (4.8 +- 1.1 μM) > cooling agent 10 (6 +- 2.2 μm) (+)menthol (14.4 +- 1.3 μM) > PMD38 (31 +- 1.1 μM) > WS-23 (44 +- 7.3 μM) > Coolact P (66 +- 20 μM) > 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 μM, 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.