Differential effects of lipids and lyso-lipids on the mechanosensitivity of the mechanosensitive channels MscL and MscS

Abstract

Mechanosensitive (MS) channels of small (MscS) and large (MscL) conductance are the major players in the protection of bacterial cells against hypoosmotic shock. Although a great deal is known about structure and function of these channels, much less is known about how membrane lipids may influence their mechanosensitivity and function. In this study, we use liposome coreconstitution to examine the effects of different types of lipids on MscS and MscL mechanosensitivity simultaneously using the patch-clamp technique and confocal microscopy. Fluorescence lifetime imaging (FLIM)-FRET microscopy demonstrated that coreconstitution of MscS and MscL led to clustering of these channels causing a significant increase in the MscS activation threshold. Furthermore, the MscL/MscS threshold ratio dramatically decreased in thinner compared with thicker bilayers and upon addition of cholesterol, known to affect the bilayer thickness, stiffness and pressure profile. In contrast, application of micromolar concentrations of lysophosphatidylcholine (LPC) led to an increase of the MscL/MscS threshold ratio. These data suggest that differences in hydrophobic mismatch and bilayer stiffness, change in transbilayer pressure profile, and close proximity of MscL and MscS affect the structural dynamics of both channels to a different extent. Our findings may have far-reaching implications for other types of ion channels and membrane proteins that, like MscL and MscS, may coexist in multiple molecular complexes and, consequently, have their activation characteristics significantly affected by changes in the lipid environment and their proximity to each other.

Fig. 1.

Comparison of the TR and patch fluorescence confocal microscopy of MscS and MscL. Channel activities of MscS and MscL in spheroplasts (AW737) (A) and azolectin (100%) (B) liposomes. Arrowhead and arrow indicate the first opening of MscS and MscL, and dotted and dashed line show the midpoint activation of MscS and MscL, respectively. (C) TR and MR of MscS and MscL (mean ± SEM; n = 6–7). (D, Top) Representative current trace of MscS and MscL coreconstituted into azolectin (99.9%) and rhodamine-PE (0.1%) liposomes recorded at +30-mV pipette potential. The filled squares in a–h between the current and pressure trace indicate resting state (a), first channel opening of MscS (arrowhead) (b), midpoint activation of MscS (dotted line) (c), saturating point of MscS activity (d), first channel opening of MscL (arrow) (e), midpoint activation of MscL (dashed line) (f), saturating point of MscL activity (g), and lysis of the patch membrane (h), respectively. (E) Boltzmann curves for MscS and MscL. The midpoint tension for MscS and MscL is 6.2 ± 0.1 and 12.0 ± 0.3 mN/m, respectively (mean ± SEM; n = 4). These values correspond well to those values obtained previously by other researchers (48, 49). (F) Confocal single frame images of the patch membrane showing the shape of the patch membrane corresponding to the current traces shown in D. Scan rate was 196 ms/scan, with no interval between consecutive scans. Dashed line indicates the resting position of the patch membrane. (Scale bar: 1 µm.)

Fig. 2.

Effects of bilayer thickness on coreconstituted MscS and MscL. (A) Current traces of MscS and MscL coreconstituted into PE18:PC18 (70%:30%) liposomes recorded at +30 mV. Arrowheads point to the first observed MscS opening, whereas the downward pointing arrows indicate the first observed MscL opening used to determine the MscL/MscS TR. (B) Representative current traces of MscS and MscL coreconstituted into PE16:PC16 (70%:30%) liposomes (upper) and spontaneous opening of MscL in the absence of pressure (Lower). Spontaneous openings were noticed in 2/16 patches and are most likely the result of induced membrane tension caused by the formation of the giga-ohm seal (50, 51). (C) Activation threshold of MscS (Top) and MscL (Middle) coreconstituted into azolectin (100%), PE18:PC18 (70%:30%), and PE16:PC16 (70%:30%) liposomes (mean ± SEM; n = 7–8). TR of MscL relative to MscS (mean ± SEM; n = 7–8) (Bottom). Significant differences are indicated by asterisks (**P < 0.01 by t test).

Fig. 3.

Cholesterol effects on MscS and MscL. (A) Superimposed current traces (Upper) of MscS and MscL coreconstituted into azolectin (100%) and azolectin:cholesterol (70%:30%) liposomes in response to negative pressure (Lower) at +30 mV pipette voltage. Black and gray bars indicate the relative change in the activation threshold of MscS and MscL, respectively. First MscS (arrowhead) and MscL (arrow) opening are indicated. (B) Activation threshold of MscS and MscL coreconstituted into azolectin (100%) and azolectin:cholesterol (70%:30%) liposomes (mean ± SEM; n = 9–15). Significant differences are indicated by asterisks in B and C (**P < 0.01 by t test). (C) TR of MscL and MscS reconstituted in azolectin 100% and azolectin:cholesterol 95%:5%, 90%:10%, 80%:20%, and 70%:30% liposomes (mean ± SEM; n = 9–15).

Fig. 4.

LPC effect on MscS and MscL. (A) MscS and MscL activation thresholds were recorded by applying pressure ramps for 10 min after training the patch. After another 10 min, the pressure ramp was reapplied. First MscS (arrowhead) and MscL (arrow) opening are indicated. (B) LPC (5 µM) was applied to the bath after the 10-min interval. Inset shows a single channel trace of MscS at +30-mV pipette voltage. (C) Normalized activation threshold of MscS and MscL coreconstituted into azolectin (100%) liposomes before and after 10-min interval in the absence and presence of 5 µM LPC (mean ± SEM; n = 5). (D) TR before and after addition of 0 and 5 µM LPC (mean ± SEM; n = 5). Significant differences are indicated by asterisks in C and D (**P < 0.01 by t test).

Fig. 5.

Clustering of MscS with MscL. (A) MscS and MscL activation thresholds were measured for individually reconstituted or coreconstituted channels (mean ± SEM; n = 6–9). The difference was significant for MscS (**P < 0.01 by t test). (B) Illustration of the two labeled proteins with the approximate position of each fluorescent label. MscS was labeled with AF568 (acceptor), whereas MscL was labeled with AF488 (donor). (C) FLIM image (70 × 70 μm) of an azolectin (100%) sample reconstituted with a donor only population of MscL M94C labeled with AF488. (D) FLIM image of azolectin (100%) sample containing separate populations of MscL M94C labeled with AF488 and MscS M47C labeled with AF568. Lifetimes are measured for the donor fluorophore (AF488) only. Regions colored in blue show areas where fluorescence lifetimes are shorter as a result of FRET attributable to the close proximity of the MscL and MscS protein populations in the lipid, indicating clustering. FRET is not exhibited uniformly in all regions of the lipid.