FIGURE 4.
Region containing the first 40 amino acids in the N-terminal domain modulates TRPM8 sensitivity to cold and menthol. A, representative intracellular calcium imaging traces showing responses to cold and 100 μm menthol in cells expressing TRPM8-myc (mM8-myc, black, left panel), Δ5–39 TRPM8-myc (Δ5–39, red, middle panel), and a chimera 1–40 mTRPV1/41–1104 mTRPM8-myc (1–40 V1/M8, blue right panel). B, summary histogram of the results obtained for the experimental protocol in A. Statistical significance was assessed with a one-way ANOVA test in combination with a Dunnett's post hoc test; ratios are different (***, p < 0.001) with respect to mM8-myc, n = 458; Δ5–39, n = 334; 1–40 V1/M8, n = 223. C, time course of [Ca2+]i response during cooling ramps and applications of menthol at different concentrations in a HEK293 cell co-transfected with mTRPM8-myc and GFP. D, percentage of active population recruited during a cooling ramp in HEK293 cells transfected with mM8-myc, Δ5–39, and 1–40V1/M8 channels. E, histogram of mean ± S.E. temperature thresholds exhibited by HEK293 cells transfected with mM8-myc (n = 548), Δ5–39 (n = 512), and 1–40V1/M8 (n = 336). Temperature thresholds were compared using a one-way ANOVA test in combination with a Dunnett's post hoc test: ***, p < 0.001 with respect to mM8-myc. F, dose-response curves of responses to menthol in transfected HEK293 cells. Upper panel shows the comparison of mM8-myc with Δ5–39. The solid lines represent fits to the Hill equation that yielded an EC50 of 127 ± 7 μm for mM8-myc and 46 ± 17 μm for Δ5–39 (mM8-myc, n = 93; Δ5–39, n = 180). Responses were normalized to the amplitude obtained with maximal stimulation (300 μm menthol plus cold). Lower panel shows the comparison of mM8-myc with 1–40V1/M8 construct in simultaneous experiments (1–40V1/M8, n = 103; EC50 of 60 ± 1 μm). Recordings from wild type and mutant channels were interlaced on the same day.