Figure 1. Cold and menthol induce PtdIns(4,5)P₂ hydrolysis and InsP₃ production in TRPM8-expressing cells.

Fluorescence measurements were performed in HEK293 cells transfected with the CFP- and YFP-tagged PLCδ4 PH domains and TRPM8 (A and B) or pCDNA3 (C) as described in the Methods section. A, upper traces, fluorescence traces detected at 480 and 535 nm; excitation wavelength was 430 nm. Lower trace, ratio of the two individual traces. The consecutive applications of 500 μm menthol are indicated by the horizontal bars. B, similar measurement using cold as a stimulus; upper trace shows temperature, lower trace shows the fluorescence ratio 535/480 nm. C, similar measurement on cells not expressing TRPM8. D, summary of the changes evoked by cold or menthol, in TRPM8-expressing and control cells (n = 7–12). E and F, HEK cells were transfected with the InsP₃ reporter IRIS-1, and the human muscarinic M1 receptor (E) or TRPM8 (F). The ratio of 480/535 nm is shown; applications of 15 μm carbachol (CCh) and 500 μm menthol are indicated by the horizontal bars. IRIS-1 responds to InsP₃ formation with a decrease in FRET; the traces shown are reverted i.e. we show the 480/535 ratio, to visually reflect increased InsP₃ formation. All fluorescence ratio traces are normalized to the ratio level before the application of the stimulus.