Figure 5.

The actin cytoskeleton tonically represses TREK-1 channel mechano-gating. (A) Channel activity of WT TREK-1 (wild type) was monitored in the cell-attached patch configuration at a holding potential of 0 mV. The activity of WT TREK-1 (black, n=12), S333D (blue, n=15), S333A (green, n=20) and E306A (red, n=15) was compared at increasing negative pressure values. Pressure–effect curves were fitted with Boltzmann relationships with P_0.5 of −62.4, −60.9, −39.5 and −31.5 and slope factors k of 15.9, 11.7, 14.0 and 8.8 for WT, S333D, S333A and E306A, respectively. (B) The activity of TREK-1 elicited by a membrane stretch of −60 mmHg at 0 mV was monitored in the cell-attached configuration in the absence (ctrl in the presence of dimethyl sulphoxide (DMSO)) and in the presence of 3 μM latrunculin A (ltrc) for 1 h (white bars). (C) TREK-1 channel activity was monitored in the cell-attached configuration (top traces) and 2 min after excision in the inside-out patch configuration (middle traces) at increasing negative pressures from 0 to −60 mmHg (bottom traces) at a holding potential of 0 mV. (D) TREK-1 channel activity was monitored in the cell-attached patch configuration in the control condition containing DMSO (black, n=12), after treatment for 1 h with 3 μM latrunculin A (green, n=15), after 4 h of treatment with 10 μM nocodazole (blue, n=10) and after patch excision in the inside-out patch configuration (red, n=10) at 0 mV. Pressure–effect curves were fitted with Boltzmann relationships with P_0.5 values of −62.4, −70.7, −65.1 and −46.1 mmHg and a slope factor k of 15.9, 16.6, 12.2 and 13.9 for WT TREK-1 cell-attached patch configuration, with latrunculin A, with nocodazole and after excision in the inside-out configuration, respectively. (E) Effect of membrane excision in the inside-out patch configuration before and after 1 h of treatment with 3 μM latrunculin A (white bars) on WT TREK-1, S333D, S333A and E306A mutants at 0 mV.