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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) The current response of the lipid bilayer (free of MscL channels) as droplets are oscillated at 0.2 Hz and 150 μm peak-to-peak amplitude. The current response is sinusoidal which corresponds to the change in the bilayer capacitance correlated with a change in the lipid bilayer area resulting from the sinusoidal oscillations of the droplets. (b) The current response of the lipid bilayer containing V23T-MscL channels when high transmembrane potentials are applied without mechanically stimulating the droplets.
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f2: (a) The current response of the lipid bilayer (free of MscL channels) as droplets are oscillated at 0.2 Hz and 150 μm peak-to-peak amplitude. The current response is sinusoidal which corresponds to the change in the bilayer capacitance correlated with a change in the lipid bilayer area resulting from the sinusoidal oscillations of the droplets. (b) The current response of the lipid bilayer containing V23T-MscL channels when high transmembrane potentials are applied without mechanically stimulating the droplets.

Mentions: A specialized test setup was developed to enable controllable actuation of the bilayer as well as accurate mechanical and electrical measurements of the interface. The test setup consisted of one droplet anchored to the tip of a mobile capillary mounted on a piezoelectric actuator and a second droplet anchored to a fixed substrate. The tension in the artificial lipid bilayer membrane is modulated by horizontally oscillating the droplet anchored to the piezoelectric actuator (Fig. 1), thereby distorting the shapes of the droplets (i.e. increasing the surface area of each droplet) and changing the contact angle between the water-oil interfaces. In this work, branched diphytanoyl phosphatidylcholine (DPhPC) is used to form bilayers. DPhPC bilayers neither oxidize nor exhibit phase transitions with temperature and thus are stable; they also have the advantage of high interfacial tension32. It is well established that the electrical properties of a lipid bilayer are modelled accurately by a high membrane resistance (typically in the giga-ohm range) in parallel with the membrane capacitance27. Therefore, low-frequency sinusoidal oscillations applied to the DIB result in a harmonic variation in bilayer capacitance that correlates with a change in the bilayer area (Fig. 2a). The electrical response of the DIB, free of MscL channels, was recorded simultaneously with video imaging of the droplets, while mechanically oscillated at frequencies ranging from 0.1 Hz up to 75 Hz, and peak-to-peak amplitudes ranging between 125 μm and 175 μm. These observations serve to form a basic understanding of the components of the mechanoelectrical response of the DIB without MscL, and serve to provide a ‘baseline’ control for the subsequent recordings with MscL channels reconstituted within the membrane. No measurable conductive component or gating-like spikes are observed in the control (Fig. 2a). This insures that the sub-conductive state events observed (when V23T-MscL is incorporated) are not simply random artefacts resulting from the bilayer oscillations and electrical recordings. The electrical responses of DIBs with V23T-MscL incorporated are also recorded in a broad range of transmembrane potentials (0–150 mV) with no mechanical stimulus applied (Fig. 2b). As previously observed, no gating-like spikes were recorded, indicating that creating tension in the membrane is essential for the gating of V23T-MscL. Note that all low amplitude (~5 pA) channel gating-like events, especially the ones in the 110 mV trace, likely represent transient conductive defects in the dynamic membrane structure stabilized by the electric field. With the droplets of approximately 0.5 mm in diameter, the initial bilayer area was approximately 0.0024 mm2. The generated currents in channel-free controls were small, reflecting a highly resistive lipid bilayer (~10 GΩ).


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) The current response of the lipid bilayer (free of MscL channels) as droplets are oscillated at 0.2 Hz and 150 μm peak-to-peak amplitude. The current response is sinusoidal which corresponds to the change in the bilayer capacitance correlated with a change in the lipid bilayer area resulting from the sinusoidal oscillations of the droplets. (b) The current response of the lipid bilayer containing V23T-MscL channels when high transmembrane potentials are applied without mechanically stimulating the droplets.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: (a) The current response of the lipid bilayer (free of MscL channels) as droplets are oscillated at 0.2 Hz and 150 μm peak-to-peak amplitude. The current response is sinusoidal which corresponds to the change in the bilayer capacitance correlated with a change in the lipid bilayer area resulting from the sinusoidal oscillations of the droplets. (b) The current response of the lipid bilayer containing V23T-MscL channels when high transmembrane potentials are applied without mechanically stimulating the droplets.
Mentions: A specialized test setup was developed to enable controllable actuation of the bilayer as well as accurate mechanical and electrical measurements of the interface. The test setup consisted of one droplet anchored to the tip of a mobile capillary mounted on a piezoelectric actuator and a second droplet anchored to a fixed substrate. The tension in the artificial lipid bilayer membrane is modulated by horizontally oscillating the droplet anchored to the piezoelectric actuator (Fig. 1), thereby distorting the shapes of the droplets (i.e. increasing the surface area of each droplet) and changing the contact angle between the water-oil interfaces. In this work, branched diphytanoyl phosphatidylcholine (DPhPC) is used to form bilayers. DPhPC bilayers neither oxidize nor exhibit phase transitions with temperature and thus are stable; they also have the advantage of high interfacial tension32. It is well established that the electrical properties of a lipid bilayer are modelled accurately by a high membrane resistance (typically in the giga-ohm range) in parallel with the membrane capacitance27. Therefore, low-frequency sinusoidal oscillations applied to the DIB result in a harmonic variation in bilayer capacitance that correlates with a change in the bilayer area (Fig. 2a). The electrical response of the DIB, free of MscL channels, was recorded simultaneously with video imaging of the droplets, while mechanically oscillated at frequencies ranging from 0.1 Hz up to 75 Hz, and peak-to-peak amplitudes ranging between 125 μm and 175 μm. These observations serve to form a basic understanding of the components of the mechanoelectrical response of the DIB without MscL, and serve to provide a ‘baseline’ control for the subsequent recordings with MscL channels reconstituted within the membrane. No measurable conductive component or gating-like spikes are observed in the control (Fig. 2a). This insures that the sub-conductive state events observed (when V23T-MscL is incorporated) are not simply random artefacts resulting from the bilayer oscillations and electrical recordings. The electrical responses of DIBs with V23T-MscL incorporated are also recorded in a broad range of transmembrane potentials (0–150 mV) with no mechanical stimulus applied (Fig. 2b). As previously observed, no gating-like spikes were recorded, indicating that creating tension in the membrane is essential for the gating of V23T-MscL. Note that all low amplitude (~5 pA) channel gating-like events, especially the ones in the 110 mV trace, likely represent transient conductive defects in the dynamic membrane structure stabilized by the electric field. With the droplets of approximately 0.5 mm in diameter, the initial bilayer area was approximately 0.0024 mm2. The generated currents in channel-free controls were small, reflecting a highly resistive lipid bilayer (~10 GΩ).

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