Fig. 6

MOR enhances TRPM8 sensitivity to cold and menthol through PKCβ-dependent phosphorylation. a Representative co-immunoprecipitation of GFP-tagged PKCβ with the full-length HA-tagged TRPM8 in HEK cells for different conditions of transfection (n = 3 independent experiments). Lower panel, input fraction immunoblotted with HA antibody (note the unspecific lower band that appears in the untransfected condition. The upper band is only found in condition of TRPM8-HA transfection). Middle panel, the imunoprecipitated GFP- PKCβ was detected with a GFP antibody. Upper panel shows the western blot of the TRPM8-HA western blot of the immunoprecipitated fraction. b Representative calcium imaging traces of TRPM8 WT and TRPM8SS1040-1041AA mutant (orange) co-transfected with MOR and stimulated by two repeated applications of cold (blue) or menthol (green) after MS application. Note the absence of desensitization with the WT but not the mutant channel. c Whisker plots showing the mean ratio (R2/R1) of the amplitude response to cold or menthol, represented in B, in HEK cells transfected with MOR and TRPM8 WT or TRPM8 mut (SS1040-1041AA mutant) (orange) after morphine treatment (wt: 95.46 ± 1.39% (blue) vs. mutant: 54.16 ± 2.03% (orange), n = 61 and 50 respectively, in response to cold; wt: 97.5 ± 1.79% (green) vs. mutant: 54.24 ± 1.51% (orange), n = 62 and 60, respectively, in response to menthol). Statistical analysis was performed using One-Way ANOVA followed by Bonferroni post hoc test (**p < 0.01). Error bars indicate ±SEM