Figure 5.

TAC activates the menthol- and the icilin-insensitive TRPM8 mutants. A, Averaged ± SEM fura-2 ratio fluorescence of mouse TRPM8-transfected HEK293 cells during cooling, TAC (30 μm) and the combined application of TAC and cooling. Records have been baseline-subtracted and the average trace normalized to the initial response to cold. Top to bottom, Calcium responses of WT TRPM8, the menthol-insensitive TRPM8–Y745H mutant, and a representative recording of the temperature time course in the chamber during the recording. B, Histogram of mean response amplitudes to the different stimuli, normalized to the initial cold response. Note the lack of potentiation of the cold-evoked response by TAC in the menthol-insensitive mutant. Statistical differences were evaluated by an unpaired Student's t test. C, Representative traces of whole-cell recordings exploring the effect of cold and TAC in two different TRPM8 mutants. Top to bottom, Currents, measured at −100 and 100 mV, in WT mouse TRPM8, the menthol-insensitive TRPM8–Y745H mutant, and the icilin-insensitive TRPM8–N799A mutant. Bottom, Representative recording of the temperature change in the chamber during the protocol. D, Histogram of the average current values to the different agonists in TRPM8 WT and the different mutants during the protocol shown in C. For each cell, current responses were normalized to the initial cold-evoked response. Statistical differences for the response to each agonist in the different TRPM8 constructs were evaluated with a one-way ANOVA, followed by Bonferroni's post hoc test. *p < 0.05, **p < 0.01.