Adaptive behavior of bacterial mechanosensitive channels is coupled to membrane mechanics

Abstract

Mechanosensitive channel of small conductance (MscS), a tension-driven osmolyte release valve residing in the inner membrane of Escherichia coli, exhibits a complex adaptive behavior, whereas its functional counterpart, mechanosensitive channel of large conductance (MscL), was generally considered nonadaptive. In this study, we show that both channels exhibit similar adaptation in excised patches, a process that is completely separable from inactivation prominent only in MscS. When a membrane patch is held under constant pressure, adaptation of both channels is manifested as a reversible current decline. Their dose-response curves recorded with 1-10-s ramps of pressure are shifted toward higher tension relative to the curves measured with series of pulses, indicating decreased tension sensitivity. Prolonged exposure of excised patches to subthreshold tensions further shifts activation curves for both MscS and MscL toward higher tension with similar magnitude and time course. Whole spheroplast MscS recordings performed with simultaneous imaging reveal activation curves with a midpoint tension of 7.8 mN/m and the slope corresponding to approximately 15-nm(2) in-plane expansion. Inactivation was retained in whole spheroplast mode, but no adaptation was observed. Similarly, whole spheroplast recordings of MscL (V23T mutant) indicated no adaptation, which was present in excised patches. MscS activities tried in spheroplast-attached mode showed no adaptation when the spheroplasts were intact, but permeabilized spheroplasts showed delayed adaptation, suggesting that the presence of membrane breaks or edges causes adaptation. We interpret this in the framework of the mechanics of the bilayer couple linking adaptation of channels in excised patches to the relaxation of the inner leaflet that is not in contact with the glass pipette. Relaxation of one leaflet results in asymmetric redistribution of tension in the bilayer that is less favorable for channel opening.

Figure 1.

MscS and MscL adapt when expressed in E. coli spheroplasts separately or together. (A and B) A comparison of dose–response curves taken with a series of pressure steps and a 1-s linear ramp from 0 to saturating pressure. Pairs of dose–response curves are shown for MscS (A) and MscL (B) individually expressed in MJF465 spheroplasts. Insets show current responses of the excised patch to the series of pressure steps, with a machine-limited 10-ms raise time. The ramp-generated curves are always right-shifted by 10–20% on the pressure scale. For MscS (A), maximal current was 1.4 nA and ramp p_0.5 = 95 mmHg. For MscL (B), saturating current was 3.14 nA and p_0.5 = 255 mmHg with the ramp stimulus. (C–E) MscS and MscL show similar tension- and time-dependent decline of currents when coexpressed in PB113 spheroplasts carrying a native copy of the mscL gene. Both channels were probed with 3-s pressure steps of varying amplitude. MscS activates in the lower range of pressures (C), whereas MscL (D) had to be probed with pressures twice as high, as seen from the pressure protocol (E). At pressures beyond 150 mmHg, MscS current completely saturates and MscL currents were recorded on top of a flat MscS response. As seen from the coinciding responses to saturating pulse (D), the inactivation of MscS and MscL after 3-s stimulation is negligibly small.

Figure 2.

MscS and MscL both adapt, displaying similar right shifts of activation curves with similar time courses. (A) A pressure ramp response of an excised patch containing both MscS and MscL, followed by a prolonged (59-s) step of subthreshold pressure with a repeated ramp in the end. The midpoints scored with the second ramp are noticeably higher. (B) Superimposed traces in response to a 500-ms ramp show that the midpoint shift for both channels increases gradually with the duration of the subthreshold step varied between 0.2 and 59 s. (C) Plot of activation midpoints as a function of intervening step duration. The midpoints for MscS (bottom) and MscL (top) change concomitantly with characteristic times of ∼2 s.

Figure 3.

MscS adaptation is absent in whole spheroplast recording configuration. A typical dataset recorded in an excised patch illustrating adaptation: (A) current decay as a result of adaptation and (C) a substantial (∼16%) midpoint shift in an experiment with two ramps separated by an 80-s step of subthreshold pressure. In the first case, a short (0.5-s) saturating test pulse was applied to the patch at the end of the step to reveal the non-inactivated fraction of the channel population. (B and D) A similar dataset obtained in whole spheroplast recording mode under steps or ramps of positive pressure. The responses to sustained pressure steps recorded on the same entire spheroplast show no current decay, yet the saturating pressure pulse at the end reveals that part of the population enters a tension-insensitive inactivated state (B). The two saturating ramps separated by a 40-s step of subthreshold pressure display identical responses with the same midpoint (D). In A and B, channels were stimulated with a 9-s step of intermediate pressure, followed by a 0.5-s saturating test pulse.

Figure 4.

The MscL (V23T) channel quickly adapts in excised patches but shows no adaptation in whole cell mode. (A) Fast adaptation in response to a step of pressure in excised patch. (B) Responses to two ramps separated by a 45-s near-threshold conditioning pulse (ramp-step-ramp). The response to the second ramp is right-shifted by 30 mmHg. (C) Whole spheroplast response to a step shows no adaptation. (D) The ramp-step-ramp response of whole spheroplast indicates no midpoint shift.

Figure 5.

Responses of MscS in spheroplast-attached patches to a step stimulus. (A) Response of an intact “shiny” spheroplast shows no adaptive current decline. The phase-contrast appearance is different due to different gradients of refractive index across the membrane. Inset shows that unitary MscS currents (10 pA at −30 mV) appear three times smaller than in excised patches (30 pA) due to the series resistance of the spheroplast membrane. (B) Adaptive response of a “gray” partially lysed spheroplast. The character of adaptation is delayed compared with excised patches (Fig. 3 A). The figure represents typical responses out of five gray and three shiny spheroplasts tested. (C) A cartoon illustrating the spheroplast-attached configuration.

Figure 6.

Whole spheroplast activation curves recorded with spheroplast imaging. (Top) Typical images of three spheroplasts of different sizes and their respective activation curves presented in the pressure scale. The fitting of the image was done using a custom-written MATLAB program. (Bottom) Curves from three spheroplasts presented in the tension scale. Tensions at each pressure were calculated according to Laplace’s law for the sphere (γ = p*r/2). The fit of the averaged curve to the Boltzmann-type equation (see Materials and methods) produces the values of ΔE = 1.17 × 10⁻¹⁹ J (28 kT) and ΔA = 14.7 nm² (solid black line). The combined fit indicated average midpoint γ_0.5 = 7.8 mN/m.

Figure 7.

A cartoon illustrating relaxation of the inner leaflet of the membrane. The top right corner shows a configuration with an excised patch inside the pipette. Only the outer leaflet is in contact with the pipette, whereas the inner leaflet is free to relax. Anisotropic pressure gradient tends to promote “rolling” of the lipids across the membrane edge. The bottom panel shows whole spheroplast configuration with more isotropically distributed pressure. The molecular models illustrate cytoplasmic position of the gate in both channels relative to the membrane midplane (dashed line).