Effects of hydrophobic substitutions for N117 and N167 on the resting, open, and inactivated states. Three similar protocols for WT MscS and the mutants N117V and N167V are shown. (left) Upon being subjected to a 1-s pressure ramp, WT MscS displays its characteristic opening and closure, whereas both N117V and N167V refuse to open. (middle) The patches were then subjected to a closure protocol similar to those from Fig. 4 (B and C). N117V and N167V require near-saturating pressure to hold the channels open long enough to monitor closure, and both close much faster than WT MscS. (right) Lastly, the patches were subjected to an inactivation protocol beginning with a saturating pulse followed by a conditioning step and ending with several pulses of saturating pressure to monitor the process of channel recovery from the inactivated state. The N117V and N167V MscS mutants showed more inactivation than WT MscS after the conditioning step, yet both mutants recovered faster, with a characteristic time of recovery (τ) of 0.5 ± 0.02 s (n = 3) for N117V and 0.6 ± 0.2 s (n = 3) for N167V. Both of these recovery rate values were lower than that found for WT MscS (1.4 ± 0.4 s; n = 10).