Figure 3.
Figure 3. The dependencies of the rates of MscS inactivation and recovery on tension in excised patches. (A) The current traces and the stimulus protocols that involve 30-s steps to varied amplitude (from 25 to 150 mmHg in 25-mmHg increments) with interspersed short saturating (180-mmHg) pulses testing for the availability of channels. The numbers 1, 2, and 3 correspond to the pressure steps of 100, 125, and 150 mmHg, respectively. The tips of current responses to test pulses are marked with different arrows. The activation midpoint for this patch determined using 1-s ramp was 140 mmHg. (B) Fitting of MscS transition kinetics recorded at a 100-mmHg pressure step to the three-state model: O→CA→I. Normalization dictates that O + CA + I = 1. The circles designate the position of the continuous trace (A) representing open population (O), the diamonds represent noninactivated population (O + CA), and the intermediate closed-adapted (CA) fraction (dashed line) is calculated by fitting the two other populations to the model (see Materials and methods). (C) A similar fit of the trace obtained at higher background tension (125 mmHg) showing slower adaptation but faster inactivation rate. (D) Membrane tension slows down MscS recovery from inactivation. MscS population was inactivated by a 60-s conditioning step (the last second is shown), and the degree of inactivation was tested by the short saturating test pulse at the end of the step. The pressure was then dropped to different levels, and four saturating test pulses were applied at different time points. The current responses, reflecting the recovered fraction of the population, were then fitted with monoexponential functions. (E) The rates of inactivation and recovery plotted against membrane tension. The slopes dlnk/dγ = ΔA/kT gave estimations of ΔAC→B = 4.6 nm2 and ΔAI→B = 2.6 nm2; the protein area changes from the bottoms of the closed (C) or inactivated (I) well to the transition barrier (B). The values averaged over three independent experiments and their sum representing total protein expansion associated with the CA→I transition are given in Results.

The dependencies of the rates of MscS inactivation and recovery on tension in excised patches. (A) The current traces and the stimulus protocols that involve 30-s steps to varied amplitude (from 25 to 150 mmHg in 25-mmHg increments) with interspersed short saturating (180-mmHg) pulses testing for the availability of channels. The numbers 1, 2, and 3 correspond to the pressure steps of 100, 125, and 150 mmHg, respectively. The tips of current responses to test pulses are marked with different arrows. The activation midpoint for this patch determined using 1-s ramp was 140 mmHg. (B) Fitting of MscS transition kinetics recorded at a 100-mmHg pressure step to the three-state model: O→CA→I. Normalization dictates that O + CA + I = 1. The circles designate the position of the continuous trace (A) representing open population (O), the diamonds represent noninactivated population (O + CA), and the intermediate closed-adapted (CA) fraction (dashed line) is calculated by fitting the two other populations to the model (see Materials and methods). (C) A similar fit of the trace obtained at higher background tension (125 mmHg) showing slower adaptation but faster inactivation rate. (D) Membrane tension slows down MscS recovery from inactivation. MscS population was inactivated by a 60-s conditioning step (the last second is shown), and the degree of inactivation was tested by the short saturating test pulse at the end of the step. The pressure was then dropped to different levels, and four saturating test pulses were applied at different time points. The current responses, reflecting the recovered fraction of the population, were then fitted with monoexponential functions. (E) The rates of inactivation and recovery plotted against membrane tension. The slopes dlnk/dγ = ΔA/kT gave estimations of ΔAC→B = 4.6 nm2 and ΔAI→B = 2.6 nm2; the protein area changes from the bottoms of the closed (C) or inactivated (I) well to the transition barrier (B). The values averaged over three independent experiments and their sum representing total protein expansion associated with the CA→I transition are given in Results.

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