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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

(a,b) The current response of the bilayer containing V23T-MscL mutant channels at 0.2 Hz (sinusoidal) and an applied transmembrane potential of 90 and 100 mV respectively. The shape of the current response is sinusoidal which corresponds to a sinusoidal change in bilayer capacitance as a consequence of the bilayer area change. The currents spikes at the peak of each cycle (i.e. maximum bilayer area) correspond to sub-conductance gating events of the MS channels. (c) Each of the six cycles is plotted in polar form indicating that the gating events consistently occur between 90 and 120 degrees.
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f4: (a,b) The current response of the bilayer containing V23T-MscL mutant channels at 0.2 Hz (sinusoidal) and an applied transmembrane potential of 90 and 100 mV respectively. The shape of the current response is sinusoidal which corresponds to a sinusoidal change in bilayer capacitance as a consequence of the bilayer area change. The currents spikes at the peak of each cycle (i.e. maximum bilayer area) correspond to sub-conductance gating events of the MS channels. (c) Each of the six cycles is plotted in polar form indicating that the gating events consistently occur between 90 and 120 degrees.

Mentions: The incorporation of V23T-MscL into DIBs is achieved by introducing proteoliposomes into both droplets. A low-threshold V23T mutant of MscL generates reliable activities including sub-conductive states as well as full opening events (Fig. 4a) when mechanically stimulated and a DC potential is applied to the membrane. These events are identical to those recorded using the patch-clamp technique from intact inner E. coli membranes and liposomes reconstituted with the purified V23T-MscL (Fig. 3). The mechanical stimulus is applied through axial sinusoidal displacements of one droplet toward and away from another with a frequency of 0.2 Hz and peak-to-peak amplitude of 150 μm. It is observed that the gating of V23T-MscL mostly occurs at transmembrane potentials above 80 mV and low oscillation frequencies (<1 Hz). Gating occurs exclusively near the point of shortest separation where both the contact area and fractional change in droplet area are maximal, while the lipid bilayer contact angle between the droplets (monolayers) is minimal.


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)

(a,b) The current response of the bilayer containing V23T-MscL mutant channels at 0.2 Hz (sinusoidal) and an applied transmembrane potential of 90 and 100 mV respectively. The shape of the current response is sinusoidal which corresponds to a sinusoidal change in bilayer capacitance as a consequence of the bilayer area change. The currents spikes at the peak of each cycle (i.e. maximum bilayer area) correspond to sub-conductance gating events of the MS channels. (c) Each of the six cycles is plotted in polar form indicating that the gating events consistently occur between 90 and 120 degrees.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: (a,b) The current response of the bilayer containing V23T-MscL mutant channels at 0.2 Hz (sinusoidal) and an applied transmembrane potential of 90 and 100 mV respectively. The shape of the current response is sinusoidal which corresponds to a sinusoidal change in bilayer capacitance as a consequence of the bilayer area change. The currents spikes at the peak of each cycle (i.e. maximum bilayer area) correspond to sub-conductance gating events of the MS channels. (c) Each of the six cycles is plotted in polar form indicating that the gating events consistently occur between 90 and 120 degrees.
Mentions: The incorporation of V23T-MscL into DIBs is achieved by introducing proteoliposomes into both droplets. A low-threshold V23T mutant of MscL generates reliable activities including sub-conductive states as well as full opening events (Fig. 4a) when mechanically stimulated and a DC potential is applied to the membrane. These events are identical to those recorded using the patch-clamp technique from intact inner E. coli membranes and liposomes reconstituted with the purified V23T-MscL (Fig. 3). The mechanical stimulus is applied through axial sinusoidal displacements of one droplet toward and away from another with a frequency of 0.2 Hz and peak-to-peak amplitude of 150 μm. It is observed that the gating of V23T-MscL mostly occurs at transmembrane potentials above 80 mV and low oscillation frequencies (<1 Hz). Gating occurs exclusively near the point of shortest separation where both the contact area and fractional change in droplet area are maximal, while the lipid bilayer contact angle between the droplets (monolayers) is minimal.

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