FIGURE 2.

The effect of negative charges neutralization of C-terminal RKKEE cluster on gating properties of MscL. A, B, C, and D show representative current traces and the corresponding pressure traces of the liposome reconstituted E. coli MscL activated by suction applied to the patch pipette at a pipette potential of +30 mV. (A) Wild-type MscL at physiological pH; (B) MscL harboring the charge reversal mutations; (C) MscL with protonated glutamine residues; (D) MscL with glutamine substituted negative charges. C marks the closed state of channels, whereas O_n marks open levels of n number of channels. (E) Current-voltage relationship for MscL at physiological, acidic, and alkaline pH (n = 4). (F) Boltzmann distribution curves for MscL channels showing rightward shift in the pressure axis in channels where negative charges were neutralized. Boltzmann distribution function of the form: P_o = P_max/[1 + exp α (p_1/2 − p)] was fitted to the data shown above. P_o and P_max are the open and maximal open probabilities; p is the applied negative pressure (in mm Hg), p_1/2 is the pressure at which the channels are open half the time, and α is the slope factor describing the sensitivity to negative pressure of the channels.