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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

Gating dependence on the amplitude of droplet displacement and voltage.The droplets are oscillated at three different amplitudes. At each amplitude the applied electric potential is varied between 20 mV and 100 mV. The results in black, corresponding to the lowest amplitude (±65 μm), and show that no MS channel activity occur at all potentials. However, as the amplitude is increased to  ±75 μm, gating events occur at potentials starting from 80 mV up to 100 mV. The results at the highest amplitude (±87.5 μm) are similar to the previous case however the conductance levels are higher which may be a result of opening the MS channels further as the tension in the bilayer is higher at higher oscillation amplitudes.
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f6: Gating dependence on the amplitude of droplet displacement and voltage.The droplets are oscillated at three different amplitudes. At each amplitude the applied electric potential is varied between 20 mV and 100 mV. The results in black, corresponding to the lowest amplitude (±65 μm), and show that no MS channel activity occur at all potentials. However, as the amplitude is increased to  ±75 μm, gating events occur at potentials starting from 80 mV up to 100 mV. The results at the highest amplitude (±87.5 μm) are similar to the previous case however the conductance levels are higher which may be a result of opening the MS channels further as the tension in the bilayer is higher at higher oscillation amplitudes.

Mentions: The gating of the V23T-MscL channels is observed to be dependent on the transmembrane electrical potential, as well as the amplitude of oscillations. These findings are highlighted in Fig. 6 where the current responses of the DIB are recorded for three different oscillation amplitudes (±62.5 μm; ±75 μm; ±87.5 μm), while a frequency of 0.2 Hz is maintained. At each oscillation amplitude, the transmembrane potential is varied between 20 mV and 100 mV. Figure 6 shows the polar plots of different cycles for the three different amplitudes each at a specific transmembrane electrical potential. In the ±62.5 μm displacement case, no gating occurs, where the results resemble those of the channel-free case. This means that the induced bilayer tension is not strong enough to make the channels open. As the amplitude of oscillations is increased to ±75 μm, MscL gating events are observed at transmembrane potentials higher than 80 mV. Similar results are obtained for the ±87.5 μm, however, it is noticed that the conductance levels are higher compared to the lower amplitude case. The results imply that widening of the conductive pore can be achieved through an increase in bilayer tension produced by increased oscillation amplitude. The results presented in this section confirm that both the transmembrane applied potential and the degree of droplet deformation are contributing in increasing the tension in the lipid bilayer membrane.


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)

Gating dependence on the amplitude of droplet displacement and voltage.The droplets are oscillated at three different amplitudes. At each amplitude the applied electric potential is varied between 20 mV and 100 mV. The results in black, corresponding to the lowest amplitude (±65 μm), and show that no MS channel activity occur at all potentials. However, as the amplitude is increased to  ±75 μm, gating events occur at potentials starting from 80 mV up to 100 mV. The results at the highest amplitude (±87.5 μm) are similar to the previous case however the conductance levels are higher which may be a result of opening the MS channels further as the tension in the bilayer is higher at higher oscillation amplitudes.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f6: Gating dependence on the amplitude of droplet displacement and voltage.The droplets are oscillated at three different amplitudes. At each amplitude the applied electric potential is varied between 20 mV and 100 mV. The results in black, corresponding to the lowest amplitude (±65 μm), and show that no MS channel activity occur at all potentials. However, as the amplitude is increased to  ±75 μm, gating events occur at potentials starting from 80 mV up to 100 mV. The results at the highest amplitude (±87.5 μm) are similar to the previous case however the conductance levels are higher which may be a result of opening the MS channels further as the tension in the bilayer is higher at higher oscillation amplitudes.
Mentions: The gating of the V23T-MscL channels is observed to be dependent on the transmembrane electrical potential, as well as the amplitude of oscillations. These findings are highlighted in Fig. 6 where the current responses of the DIB are recorded for three different oscillation amplitudes (±62.5 μm; ±75 μm; ±87.5 μm), while a frequency of 0.2 Hz is maintained. At each oscillation amplitude, the transmembrane potential is varied between 20 mV and 100 mV. Figure 6 shows the polar plots of different cycles for the three different amplitudes each at a specific transmembrane electrical potential. In the ±62.5 μm displacement case, no gating occurs, where the results resemble those of the channel-free case. This means that the induced bilayer tension is not strong enough to make the channels open. As the amplitude of oscillations is increased to ±75 μm, MscL gating events are observed at transmembrane potentials higher than 80 mV. Similar results are obtained for the ±87.5 μm, however, it is noticed that the conductance levels are higher compared to the lower amplitude case. The results imply that widening of the conductive pore can be achieved through an increase in bilayer tension produced by increased oscillation amplitude. The results presented in this section confirm that both the transmembrane applied potential and the degree of droplet deformation are contributing in increasing the tension in the lipid bilayer membrane.

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