TRPM8 channels are present in blood vessels where they are likely to be involved in the regulation of vascular tone (Yang et al., 2006; Inoue et al., 2006). Their activity depends on temperature, voltage and chemical signaling. However, single channel mechanisms of TRPM8 gating remain unknown. We examined the influence of voltage on TRPM8 gating using patch-clamp recording techniques (all values were expressed in means ± S.E.M.). In HEK293 cells stably expressing TRPM8, whole-cell currents during voltage steps (-100 to 150 mV, 10 mV increments) and ramps (-100 to 100 mV) showed a strong outward rectification. Rectification of several TRP channels has been suggested to arise from the voltage-dependence of gating (Nilius et al., 2005). Thus, single channel currents (cell-attached configuration) were measured at different voltages (room temperature, filtered at 2 kHz and sampled at 10 kHz) giving single channel conductance of 60.2±4.3 pS (n=6), and an increase in open probability (Po) from 0.03±0.01 at 40 mV (n=7) to 0.41±0.06 at 160 mV (n=7). To further study the mechanism underlying this voltage-dependent increase in Po, shut and open dwell times at 80 and 140 mV were compared. There was only a modest increase in the mean open time from 0.70±0.08 to 1.10±0.28 ms (n=4 patches; not significant, P=0.229, two-tailed t-test) whereas the mean shut time decreased from 15.55±2.15 to 4.04±1.08 ms (n=4; P=0.003) between 80 and 140 mV, respectively. Dwell-time histograms (transitions > 0.16 ms) were fitted with sums of exponentials (2 open and 4 shut in each case). They showed that membrane depolarization caused a reduction in the relative contributions of the long shut components. Thus, the two longest components (τ > 10 ms) accounted for 40% of all closures at 80 mV, but for only 14% at 140 mV. In contrast, the open time values were little affected by voltage. The 2-D dwell-time distributions (Rothberg and Magleby, 1998) and dependency plots (Magleby and Song, 1992) were consistent with voltage-dependent restructuring of channel gating. These results show that the main effect of membrane depolarization on TRPM8 gating is an increase in the frequency of channel opening. Furthermore, voltage-dependence of TRPM8 gating arises from the effects of membrane potential on the closed dwell times, while open times are essentially voltage-independent.