Manipulating the permeation of charged compounds through the MscL nanovalve

Abstract

MscL is a bacterial mechanosensor that serves as a biological emergency release valve, releasing cytoplasmic solutes to the environment on osmotic downshock. Previous studies have recognized that this channel has properties that make it ideal for use as a triggered nanovalve for vesicular-based targeted drug-release devices. One can even change the modality of the sensor. Briefly, the introduction of charges into the MscL pore lumen gates the channel in the absence of membrane tension; thus, by inserting compounds that acquire a charge on exposure to an alternative stimulus, such as light or pH, into the pore of the channel, controllable nanoswitches that detect these alternative modalities have been engineered. However, a charge in the pore lumen could not only encourage actuation of the nanopore but also have a significant influence on the permeation of large charged compounds, which would thus have important implications for the efficiency of drug-release devices. In this study, we used in vivo and electrophysiological approaches to demonstrate that the introduction of a charge into pore lumen of MscL does indeed influence the permeation of charged molecules. These effects were more drastic for larger compounds and, surprisingly, were related to the orientation of the MscL channel in the membrane.

Figure 1.

Ionic preferences of wild-type and mutant MscL nanopores. A) Current-voltage plots of MscL under asymmetric ionic conditions (40 mM MgCl₂, 10 mM CaCl₂, and 5 mM HEPES/KOH, pH 6.0, plus 200 mM KCl in pipette, 600 mM KCl in bath). Reversal potentials for Cl⁻ and K⁺ are +17.5 and −27.8 mV, respectively. Graphs show current-voltage relationships of wild-type (WT) MscL and mutant MscL K31E, D39C, and G26C treated with either MTSES⁻ or MTSET⁺ (n=6). B) Reversal potentials determined by current-voltage plots in A; n = 6. **P < 0.01, ***P < 0.001 vs. WT MscL; 2-tailed t test.

Figure 2.

In vivo efflux of osmolytes from E. coli expressing G26C MscL. Osmolytes in E. coli efflux out of cell after G26C MscL is gated by either MTSES⁻ or MTSET⁺ treatment. Efflux efficiency is expressed as percentage of cell osmolytes before treatment. A) Efflux of K⁺ and glutamate⁻ (Glu⁻). B) Efflux of Glu⁻ and trehalose (Tre); n = 7. **P < 0.01, ***P < 0.001; 2-tailed t test.

Figure 3.

Permeation of Na₂-succinate through MscL. A) Representative current traces of wild-type (WT) MscL (left) and MTSES⁻-modulated (middle) and MTSET⁺-modulated G26C MscL (right) recorded in configuration in which pipette was filled with low-KCl buffer (10 mM KCl, 40 mM MgCl₂, and 5 mM HEPES/KOH, pH 6) and bath solution contained low-KCL buffer supplemented with 300 mM Na₂-succinate with pH adjusted to 6, at which succinate has 2 negative charges, expressed as succinate²⁻. Positive current was mostly due to permeation of Na⁺, because positive membrane potential only allows Na⁺ to pass through MscL from bath. Full single-channel openings and baselines of recordings are indicated as follows: o, open; c, closed; s, substate of channel. Scale bar in D applies to all traces. B) Current-voltage relationships of single-channel currents for permeation of Na⁺ through wild type (WT) and G26C MscL treated with MTSES⁻ or MTSET⁺. C) Conductance of Na⁺ as determined from slopes of current-voltage relationships in B. D–F) Panels are comparable to A–C, respectively; voltage was reversed so that permeation of succinate²⁻ from the bath could be measured. Same pressures were applied for A and D (indicated in D). Pressure needed to open G26C MscL for access of MTSET⁺ was 213 mmHg. n = 6. *P < 0.05, **P < 0.01, ***P < 0.001 vs. WT MscL; 2-tailed t test.

Figure 4.

Permeation of spermine 4HCl through MscL. A) Representative current traces of wild-type (WT) MscL (left) and MTSES⁻-modulated (middle) and MTSET⁺-modulated G26C MscL (right) recorded in configuration in which pipette was filled with low-KCl buffer (10 mM KCl, 40 mM MgCl₂, and 5 mM HEPES/KOH, pH 6) and bath solution contained low-KCL buffer supplemented with 100 mM spermine 4HCl with pH adjusted to 6, at which spermine has 4 positive charges, expressed as spermine⁴⁺. Positive current is mostly due to permeation of spermine⁴⁺, because positive membrane potential only allows spermine⁴⁺ to pass through MscL from bath. Full single-channel openings and baselines of recordings are indicated as follows: o, open; c, closed; s, substate of channel. Pressures needed to open the channels are shown at bottom. Inset below G26C MTSET⁺ trace shows expansion of indicated section, with scale bar at left. Scale bar in D applies to all other traces. B) Current-voltage relationships of single-channel currents for permeation of spermine⁴⁺ through wild-type (WT) and G26C MscL treated with MTSES⁻ or MTSET⁺. C) Conductance of spermine⁴⁺ as determined by slopes of current-voltage relationships in B. D–F) Panels are comparable to A–C, respectively; voltage was reversed so that permeation of Cl⁻ from bath could be measured. Same pressures were applied for A and D (indicated in D). Pressure needed to open G26C MscL for access of MTSET⁺ was 64 mmHg. n = 6. *P < 0.05, **P < 0.01, ***P < 0.001 vs. WT MscL; 2-tailed t test.

Figure 5.

Comparison of spermine 4HCl permeation from periplasmic vs. cytoplasmic side of the MscL channel. Permeation of spermine⁴⁺ and Cl⁻ is expressed as conductance of MscL under negative and positive membrane potential, respectively. Values are means ± se; n = 6. *P < 0.05, **P < 0.01 vs. corresponding wild-type (WT) MscL group; 2-tailed t test.