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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

The amplitude histogram of V23T-MscL opening events obtained from multiple traces including 110 oscillation cycles.The droplets are subjected to periodic oscillation (0.2 Hz and 87.5 μm amplitude) and a transmembrane potential of 100 mV for several minutes and a total of 52 events were analysed. Under these conditions the probability of seeing an opening event per cycle was 0.47. The histogram shows that low-conductance sub-states are more likely to occur under the given testing conditions.
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f5: The amplitude histogram of V23T-MscL opening events obtained from multiple traces including 110 oscillation cycles.The droplets are subjected to periodic oscillation (0.2 Hz and 87.5 μm amplitude) and a transmembrane potential of 100 mV for several minutes and a total of 52 events were analysed. Under these conditions the probability of seeing an opening event per cycle was 0.47. The histogram shows that low-conductance sub-states are more likely to occur under the given testing conditions.

Mentions: We have also analysed the amplitude distribution of opening events recorded over 110 cycles under ‘near-optimal’ stimulation with 0.2 Hz/87.5 μm sinusoidal droplet oscillations and a transmembrane potential of 100 mV (Fig. 5). Most of the events represent low sub-conductive states, consistent with low-open probability patch-clamp traces presented in Fig. 3 and previous analysis6. Full-openings of 3.1 nS amplitude constitute only 3% of all events.


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)

The amplitude histogram of V23T-MscL opening events obtained from multiple traces including 110 oscillation cycles.The droplets are subjected to periodic oscillation (0.2 Hz and 87.5 μm amplitude) and a transmembrane potential of 100 mV for several minutes and a total of 52 events were analysed. Under these conditions the probability of seeing an opening event per cycle was 0.47. The histogram shows that low-conductance sub-states are more likely to occur under the given testing conditions.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f5: The amplitude histogram of V23T-MscL opening events obtained from multiple traces including 110 oscillation cycles.The droplets are subjected to periodic oscillation (0.2 Hz and 87.5 μm amplitude) and a transmembrane potential of 100 mV for several minutes and a total of 52 events were analysed. Under these conditions the probability of seeing an opening event per cycle was 0.47. The histogram shows that low-conductance sub-states are more likely to occur under the given testing conditions.
Mentions: We have also analysed the amplitude distribution of opening events recorded over 110 cycles under ‘near-optimal’ stimulation with 0.2 Hz/87.5 μm sinusoidal droplet oscillations and a transmembrane potential of 100 mV (Fig. 5). Most of the events represent low sub-conductive states, consistent with low-open probability patch-clamp traces presented in Fig. 3 and previous analysis6. Full-openings of 3.1 nS amplitude constitute only 3% of all events.

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