Figure 5. Electrophysiological characterization of TRPM8 and other mutants' sensitivity to menthol and cold stimulus.

Whole-cell current-voltage relationships from voltage ramps (−100 +100mV) were obtained for HEK-293 cells expressing the wild type TRPM8 (n = 7) and the mutant channels: 5S-G (n = 5), S827G (n = 4), S827P (n = 4), L825G (n = 4) Y826G (n = 3), V830G (n = 4); measurements were performed in the presence of 2 mM Ca²⁺. Panel A demonstrates averaged ramp-recordings for menthol-induced currents (500 μM) and panel B shows cold-induced currents (10 °C). Control values for background current for the wild type TRPM8 and mutants are very close, and only background wild-type TRPM8 current was demonstrated. Summary of the menthol-induced whole-cell currents at −100 and +100 mV potentials are shown in panel C, and those for cold-induced currents, in panel D. For each group background current marked in gray color.

Panel E. Representative current traces for −100 +100 mV potentials of the cold-induced and subsequent menthol-induced currents obtained for the wild type TRPM8 and single mutants, S827G and S827P. In comparison to the wild type TRPM8, the menthol-induced activity of the PHB-mutants showed a reduction in current density in the following sequence S827G (p = 0.212 at +100 mV; p=0.0465 at −100 mV) > L825G (p = 0.044 at 100 mV; p = 0.0097 at −100 mV) > 5S-G (p = 0.023 at +100 mV; p = 0.0088 at −100 mV) > S827P (p = 0.012 at +100 mV; p = 0.024 at −100 mV) > V830G (p = 0.0024 at +100 mV; p = 0.0041 at −100 mV) > Y826G (p = 0.0072 at +100 mV; p = 0.0085 at −100 mV) (panels A and C). The cold-induced activity of the mutants was inhibited to a greater degree and exhibited a similar pattern of inhibition: S827G (p = 0.105 at +100 mV; p = 0.141 at −100 mV) > L825G (p = 0.0106 at 100 mV; p = 0.0559 at −100 mV) > 5S-G (p = 0.0054 at +100 mV; p = 0.096 at −100 mV) > S827P (p = 0.0016 at +100 mV; p = 0.186 at −100 mV) > V830G (p = 0.00005 at +100 mV; p = 0.0569 at −100 mV) > Y826G (p = 0.002 at +100 mV; p = 0.092 at −100 mV) (panels B and D). The number of asterisks in the graphs indicates the significance level: one star (*) for p ≤ 0.05, two (**) for ≤ 0.01, and three (***) for ≤ 0.001.

Kinetics of cold activation of TRPM8 and mutants 5S-G, S827G, and L825G and their menthol-induced desensitization: Panel F: Summary plot of experiments showing cold ramp (drop to 10 °C in 15 sec) and menthol-induced (panel G) whole-cell currents for the wild type TRPM8 (n = 10) and mutants 5G (n = 5), S827G (n = 9), L825G (n = 8) at +100 mV potentials. To minimize current desensitization this set of experiments was performed in Ca²⁺-free extracellular solution. Panel G demonstrates slight but non-significant current desensitization for all 3 experimental replications of menthol application.