We next tested the effect of scPPX1 by transiently transfecting scPPX1-pcDNA (0.4 μg) into HEK-293 cells stably expressing TRPM8 (TRPM8-HEK293). Cells were co-transfected with GFP (0.2 μg) to allow detection of transfected cells
(Fig. 1D–F). Control experiments were performed in TRPM8-HEK293 cells expressing GFP alone. In controls, the values of menthol-induced currents obtained at −60 mV were 0.94}0.12 and 0.915}0.122 nA (n = 7) and in scPPX1 expressing cells the values were found to be 0.054}0.001 and 0.051}0.008 nA (n= 8), for the first and the second pulses, respectively.
The current-voltage relationships of TRPM8 channels in the control cells andscPPX1-expressing cells are demonstrated in Figure 1G–I. We found that inwardcurrents of TRPM8 exhibit more profound inhibition by scPPX1 (~83%) than
outward currents (~65%).