Figure 5.

S26A and S541A+S542A showed minor alterations in their responses to TRPM8 agonists. A, Time course of [Ca²⁺]_i (F₃₄₀/F₃₈₀) of cells submitted to an experimental protocol that consists of a cold pulse, 100 μm menthol, and a combined stimulus of cold and menthol. For each mutant channel displayed, the traces correspond to the Ca²⁺ signal of HEK293 cells transfected with WT mTRPM8-myc channel in control conditions (open black circles) and after a preincubation of 10 min with 200 nm staurosporine (Sta.) (red circles), and transfected with S26A and S541A+S542A mutants in control conditions (open gray circles) and treated with staurosporine (gray circles filled with red). B, C, Plots represent the mean and SEM of cold response, ΔT. Threshold, Menthol-, and Cold+menthol-induced responses of TRPM8 in control conditions or after a 10 min incubation with staurosporine 200 nm, of cells expressing WT channels or unphosphorylated mutants. The values were normalized to the mean response observed in control conditions in parallel experiments. ΔT. Threshold represents the temperature threshold shift displayed according to the mTRPM8-myc values. Positive values indicate shifts to warmer temperatures (B: WT Ctrl., n = 108 WT, Sta., n = 107; S26A Ctrl., n = 249; S26A Sta., n = 85; C: WT Ctrl., n = 89 WT Sta., n = 122; S541A+S542A Ctrl., n = 113; S541A+S542A Sta., n = 101). Statistical analysis was performed using the one-way ANOVA test (Cold S26A: F_(3,545) = 14.53, p < 0.0001; ΔT. threshold S26A: F_(3,545) = 45.40, p < 0.0001; Menthol S26A: F_(3,545) = 57.14, p < 0.0001; Cold+menthol S26A: F_(3,545) = 12.27, p < 0.0001; Cold S541A+S542A: F_(3,421) = 19.22, p <0.0001; ΔT. threshold S541A+S542A: F_(3,421)= 46.42, p < 0.0001; Menthol S541A+S542A: F_(3,421) = 36.05, p < 0.0001; Cold+menthol S541A+S542A: F_(3,421) = 7.16, p = 0.0001), in combination with a Dunnett's post hoc test: *p < 0.05; **p < 0.01; ***p < 0.001; compared with the control.