Figure 6

Electrophysiology confirms the differential effects of the Y745H mutation on BCTC and SKF96365 antagonism. Whole-cell I-V curves from voltage ramps (-100/+150 mV) of TRPM8-wt and TRPM8-Y745H expressing HEK293 cells during cooling in the presence and absence of A, 0.6 μM BCTC; and B, 3 μM SKF96365. Wash traces recorded 3 minutes after removal of the antagonist from the bath are included to show the reversible nature of the inhibition. C, Summary histogram of experiments depicted in A and B, showing the block of cold-evoked currents by 0.6 μM BCTC and 3 μM SKF96365 at a membrane potential of +80 mV. D-E, Parameters obtained from fits of I-V data to equation (i). D, Antagonist-induced change in maximum conductance during cooling in cells expressing TRPM8-wt and TRPM8-Y745H. The data are normalized to the value during cooling in control solution of the same cells, (g_cold+blocker/g_cold). E, Antagonist-induced shifts of the midpoint of voltage activation (V_1/2) of TRPM8-wt vs. TRPM8-Y745H during cooling. The data are represented with respect to the value of V_1/2 in the absence of blocker (ΔV_1/2 = V_{1/2, cold+blocker} - V_{1/2, cold}). In panels C-E, statistical significance was assessed with Student's unpaired t-test, n = 2-6.