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Activation of bacterial channel MscL in mechanically stimulated droplet interface bilayers.

Najem JS, Dunlap MD, Rowe ID, Freeman EC, Grant JW, Sukharev S, Leo DJ - Sci Rep (2015)

Bottom Line: Geometrical analysis of droplets during compression indicates that both contact angle and total area of the water-oil interfaces contribute to the generation of tension in the bilayer.The measured expansion of the interfaces by 2.5% is predicted to generate a 4-6 mN/m tension in the bilayer, just sufficient for gating.This work clarifies the principles of interconversion between bulk and surface forces in the DIB, facilitates the measurements of fundamental membrane properties, and improves our understanding of MscL response to membrane tension.

View Article: PubMed Central - PubMed

Affiliation: Department of Mechanical Engineering, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061, United States.

ABSTRACT
MscL, a stretch-activated channel, saves bacteria experiencing hypo-osmotic shocks from lysis. Its high conductance and controllable activation makes it a strong candidate to serve as a transducer in stimuli-responsive biomolecular materials. Droplet interface bilayers (DIBs), flexible insulating scaffolds for such materials, can be used as a new platform for incorporation and activation of MscL. Here, we report the first reconstitution and activation of the low-threshold V23T mutant of MscL in a DIB as a response to axial compressions of the droplets. Gating occurs near maximum compression of both droplets where tension in the membrane is maximal. The observed 0.1-3 nS conductance levels correspond to the V23T-MscL sub-conductive and fully open states recorded in native bacterial membranes or liposomes. Geometrical analysis of droplets during compression indicates that both contact angle and total area of the water-oil interfaces contribute to the generation of tension in the bilayer. The measured expansion of the interfaces by 2.5% is predicted to generate a 4-6 mN/m tension in the bilayer, just sufficient for gating. This work clarifies the principles of interconversion between bulk and surface forces in the DIB, facilitates the measurements of fundamental membrane properties, and improves our understanding of MscL response to membrane tension.

No MeSH data available.


Related in: MedlinePlus

V23T-MscL activities recorded using standard patch-clamp technique in E. coli giant MJF465 spheroplasts (a) and in DPhPC liposomes reconstituted with purified protein (b).The mechanical stimuli in both cases are shallow ramps of negative pipette pressure (suction) to 50–170 mm Hg. Both traces recorded in a symmetric 400 mM KCl, 20 mM MgCl2 and 10 mM CaCl2 buffer characterized with the same conductivity as the buffer used for DIB formation. Conductance levels of 3.5–3.8 nS indicated by black arrows correspond to the fully open channels occurring amid various sub-conductive states.
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f3: V23T-MscL activities recorded using standard patch-clamp technique in E. coli giant MJF465 spheroplasts (a) and in DPhPC liposomes reconstituted with purified protein (b).The mechanical stimuli in both cases are shallow ramps of negative pipette pressure (suction) to 50–170 mm Hg. Both traces recorded in a symmetric 400 mM KCl, 20 mM MgCl2 and 10 mM CaCl2 buffer characterized with the same conductivity as the buffer used for DIB formation. Conductance levels of 3.5–3.8 nS indicated by black arrows correspond to the fully open channels occurring amid various sub-conductive states.

Mentions: In order to observe V23T-MscL-6His gating pattern in the native setting, we expressed the channel in MJF 465 E. coli cells4 and recorded traces from giant spheroplasts using standard patch-clamp technique. The gating response of the mutant in spheroplasts to mechanical stimuli (shallow negative pressure ramps ranging between 50 to 170 mm Hg) under an applied potential of  ±40 mV, are shown in Fig. 3a. In a parallel effort, we have purified 6-His tagged V23T-MscL and reconstituted it in DPhPC liposomes. Recordings from liposome ‘blisters’ were performed, with mechanical stimuli applied in a similar fashion as previously mentioned, in order to observe single-channel events (Fig. 3b). To compare conductance levels, both types of recordings were performed in the buffer of the same specific conductivity as used for DIBs. We found that the activities in both cases were essentially identical in terms of unitary conductance (3.5–3.8 nS full openings designated by arrows in Fig. 3), with similar kinetic patterns. When activated at relatively low open probability, V23T-MscL exhibits a variety of short-lived sub-conductive states. According to the previous data633, this mutant has a tension midpoint of 9.5 mN/m (compared to 12–14 mN/m for WT-MscL33), yet the first opening events are reproducibly observed near 6 mN/m. Based on this data, we anticipated that in DIBs V23T-MscL should start flickering at tensions between 6 and 7 mN/m.


Activation of bacterial channel MscL in mechanically stimulated droplet interface bilayers.

Najem JS, Dunlap MD, Rowe ID, Freeman EC, Grant JW, Sukharev S, Leo DJ - Sci Rep (2015)

V23T-MscL activities recorded using standard patch-clamp technique in E. coli giant MJF465 spheroplasts (a) and in DPhPC liposomes reconstituted with purified protein (b).The mechanical stimuli in both cases are shallow ramps of negative pipette pressure (suction) to 50–170 mm Hg. Both traces recorded in a symmetric 400 mM KCl, 20 mM MgCl2 and 10 mM CaCl2 buffer characterized with the same conductivity as the buffer used for DIB formation. Conductance levels of 3.5–3.8 nS indicated by black arrows correspond to the fully open channels occurring amid various sub-conductive states.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4562232&req=5

f3: V23T-MscL activities recorded using standard patch-clamp technique in E. coli giant MJF465 spheroplasts (a) and in DPhPC liposomes reconstituted with purified protein (b).The mechanical stimuli in both cases are shallow ramps of negative pipette pressure (suction) to 50–170 mm Hg. Both traces recorded in a symmetric 400 mM KCl, 20 mM MgCl2 and 10 mM CaCl2 buffer characterized with the same conductivity as the buffer used for DIB formation. Conductance levels of 3.5–3.8 nS indicated by black arrows correspond to the fully open channels occurring amid various sub-conductive states.
Mentions: In order to observe V23T-MscL-6His gating pattern in the native setting, we expressed the channel in MJF 465 E. coli cells4 and recorded traces from giant spheroplasts using standard patch-clamp technique. The gating response of the mutant in spheroplasts to mechanical stimuli (shallow negative pressure ramps ranging between 50 to 170 mm Hg) under an applied potential of  ±40 mV, are shown in Fig. 3a. In a parallel effort, we have purified 6-His tagged V23T-MscL and reconstituted it in DPhPC liposomes. Recordings from liposome ‘blisters’ were performed, with mechanical stimuli applied in a similar fashion as previously mentioned, in order to observe single-channel events (Fig. 3b). To compare conductance levels, both types of recordings were performed in the buffer of the same specific conductivity as used for DIBs. We found that the activities in both cases were essentially identical in terms of unitary conductance (3.5–3.8 nS full openings designated by arrows in Fig. 3), with similar kinetic patterns. When activated at relatively low open probability, V23T-MscL exhibits a variety of short-lived sub-conductive states. According to the previous data633, this mutant has a tension midpoint of 9.5 mN/m (compared to 12–14 mN/m for WT-MscL33), yet the first opening events are reproducibly observed near 6 mN/m. Based on this data, we anticipated that in DIBs V23T-MscL should start flickering at tensions between 6 and 7 mN/m.

Bottom Line: Geometrical analysis of droplets during compression indicates that both contact angle and total area of the water-oil interfaces contribute to the generation of tension in the bilayer.The measured expansion of the interfaces by 2.5% is predicted to generate a 4-6 mN/m tension in the bilayer, just sufficient for gating.This work clarifies the principles of interconversion between bulk and surface forces in the DIB, facilitates the measurements of fundamental membrane properties, and improves our understanding of MscL response to membrane tension.

View Article: PubMed Central - PubMed

Affiliation: Department of Mechanical Engineering, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061, United States.

ABSTRACT
MscL, a stretch-activated channel, saves bacteria experiencing hypo-osmotic shocks from lysis. Its high conductance and controllable activation makes it a strong candidate to serve as a transducer in stimuli-responsive biomolecular materials. Droplet interface bilayers (DIBs), flexible insulating scaffolds for such materials, can be used as a new platform for incorporation and activation of MscL. Here, we report the first reconstitution and activation of the low-threshold V23T mutant of MscL in a DIB as a response to axial compressions of the droplets. Gating occurs near maximum compression of both droplets where tension in the membrane is maximal. The observed 0.1-3 nS conductance levels correspond to the V23T-MscL sub-conductive and fully open states recorded in native bacterial membranes or liposomes. Geometrical analysis of droplets during compression indicates that both contact angle and total area of the water-oil interfaces contribute to the generation of tension in the bilayer. The measured expansion of the interfaces by 2.5% is predicted to generate a 4-6 mN/m tension in the bilayer, just sufficient for gating. This work clarifies the principles of interconversion between bulk and surface forces in the DIB, facilitates the measurements of fundamental membrane properties, and improves our understanding of MscL response to membrane tension.

No MeSH data available.


Related in: MedlinePlus