Experimental and predicted kinetic structure of TRPM8 channels at 30°C. (A) Representative single-channel currents recorded at 20 and 30°C (100 mV). Upward steps indicate channel opening. (B) 2-D dwell-time histogram (top left) and dependency difference plot (top right) at 30°C for the same channel analyzed for A. The predicted 2-D dwell-time distribution (bottom left) and dependency difference plot (bottom right) were obtained by simulating and analyzing 100,000 open and closed intervals using scheme I with the parameters in C. (C, top) Open and closed dwell-time histograms at 20°C (gray lines) and 30°C (black lines; 100 mV; 25 bins per decade). The plots have been normalized to a fixed number of events (70,000) to facilitate comparison, and a moving bin mean has been used to reduce stochastic variation in the histogram. (bottom) Scheme I (see below for estimated parameters) describes (continuous lines) the open (left) and closed (right) 1-D dwell-time histograms. The rate constants used to obtain these predicted 1-D and 2-D plots were O1-C3 = 2,467 s⁻¹, C3-O1 = 2,899 s⁻¹, O2-C4 = 6,069 s⁻¹, C4-O2 = 690 s⁻¹, C3-C4 = 1,443 s⁻¹, C4-C3 = 115 s⁻¹, C4-C5 = 663 s⁻¹, C5-C4 = 97 s⁻¹, C5-C6 = 4.16 s⁻¹, C6-C5 = 18.8 s⁻¹, C6-C7 = 0.33 s⁻¹, and C7-C6 = 2.91 s⁻¹. (D) Schematic representation of the change in the rate constants of scheme I with decreasing temperature. The arrows and circles can be interpreted as in Fig. 7 B. The values for these changes are shown in Table S2.