Figure 6. Menthol-induced activity of wild type TRPM8, and 5S-G, S827P, and Y826G mutant channels in the Planar Lipid Bilayers.

Representative single-channel current recordings of TRPM8 and the mutant channels incorporated in planar lipid bilayers formed from POPC/POPE (3:1) in n-decane, between symmetric bathing solutions of 150 mM KCl, 0.2 mM MgCl₂ in 20mM HEPES buffer, pH 7.4 at 22 °C. Between 0.2 and 0.5 μl of 0.2 μg/ml the protein was incorporated into POPC/POPE micelles, and added to the cis compartment (ground). Clamping potential was +100 mV. The closed state is delineated by a horizontal line under the traces on the left.

Panel A: representative currents traces in the presence of 4 μM of DiC₈ PtdIns(4,5)P₂ and 500 μM of menthol. Panel B: Open probabilities of the wt TRPM8 and mutants obtained at +100 mV.

Cold activation of 5S-G mutant channels in Planar Lipid Bilayer and P_o change for the wild type TRPM8 and mutants:

Panel C: Representative single-channel current recordings of 5S-G mutant channels activated by cold in the presence of 4 μM diC₈PIP₂. Clamping potential was +100 mV. Panel D: Open probability for the wild-type TRPM8 and 5S-G mutant obtained during cooling the planar lipid bilayers. Panel E: Two-phase TRPM8 temperature dependence and single-phase of 5S-G mutant in log(P_o) versus T plot. Panel F: Van't Hoff plot of the equilibrium constant K_eq demonstrates two-phase channel transitions for wild type TRPM8 and a single transition obtained for 5S-G mutant. Changes in enthalpy and entropy of TRPM8 and 5S-G activation are indicated on the graph.