Figure 5.

Automatic repeated stepwise tension-clamp and single-channel responses of the TREK-1 channel. (A) The bilayer tension was repeatedly changed between 4 and 8 mN·m^–1 (orange) by simultaneously driving the leaflet tension (blue and green). Typical flicker gating of single-channel TREK-1 was recorded at −100 mV. At 4 mN·m^–1 tension, the TREK-1 channel exhibited infrequent flicker gating. The flicker gating frequency immediately increased after the tension was increased to 8 mN·m^–1. The single-channel currents were Bessel filtered at the cutoff frequency of 5 kHz and were sampled at 25 kHz, which are further digitally filtered (Bessel filter) at 1 kHz cutoff frequency for display. (B) Flicker gating analysis by the BPoF method.⁷¹ The amplitude histograms are shown for the low- and high-tension traces (see the Methods section). The histograms were compiled from the first-order filtered current data (cutoff frequency of 3 Hz for low tension and 10 Hz for high tension). Subsequent fitting with one beta-distribution was unsuccessful, whereas two beta-distributions (blue and red) successfully reproduced the amplitude histogram. Accordingly, two distinct flickerings underly the gating of the TREK-1 channel. Two sets of flickering rates were estimated by assuming two independent flickering relevant to two beta distributions. The estimated rate constants for opening, k_co, and closing, k_oc, were 0.02 and 2.07 ms^–1 for the major component and 0.018 and 5.89 ms^–1 for the minor component for the low tension; k_co and k_oc for the high tension were 0.89 and 1.31 ms^–1 for the major component and 0.21 and 0.64 ms^–1 for the minor component.