Mechanosensitive Channels Activity in a Droplet Interface Bilayer System
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Mechanosensitive Channels Activity in a Droplet Interface Bilayer System Joseph Najem1, Myles Dunlap1, Sergei Sukharev2, and Donald J. Leo3 1 Biomolecular Materials and Systems Laboratory, Virginia Tech, Blacksburg, VA 24061, U.S.A. 2 Department of Biology, University of Maryland, College Park, MD 20742, U.S.A. 3 College of Engineering, University of Georgia, Athens, GA 30609, U.S.A. ABSTRACT This paper presents the first attempts to study the large conductance mechano-sensitive channel (MscL) activity in an artificial droplet interface bilayer (DIB) system. A novel and simple technique is developed to characterize the behavior of an artificial lipid bilayer interface containing mechano-sensitive (MS) channels. The experimental setup is assembled on an inverted microscope and consists of two micropipettes filled with PEG-DMA hydrogel and containing Ag/AgCl wires, a cylindrical oil reservoir glued on top of a thin acrylic sheet, and a piezoelectric oscillator actuator. By using this technique, dynamic tension can be applied by oscillating axial motion of one droplet, producing deformation of both droplets and area changes of the DIB interface. The tension in the artificial membrane will cause the MS channels to gate, resulting in an increase in the conductance levels of the membrane. The results show that the MS channels are able to gate under an applied dynamic tension. Moreover, it can be concluded that the response of channel activity to mechanical stimuli is voltage-dependent and highly related to the frequency and amplitude of oscillations. INTRODUCTION Biomolecular unit cells can be described as small building blocks whose repetition can form the basis of a novel biomolecular material system1. The biomolecular unit cell consists of a lipid bilayer interface formed at the contact of two aqueous droplets encased in lipid monolayers. The droplets are surrounded by a hydrophobic organic solvent (Hexadecane), and are sitting on fixed silver-silver chloride (Ag/AgCl) electrodes2, 3. Many types of biomolecules, such as ion channels can self-assemble in the lipid bilayer interface. Therefore, the Droplet Interface Bilayer (DIB) has been extensively used to systematically study the activity of various biomolecules including alamethicin4, bacteriorhodopsin5, and many more. Other types of biomolecules such as mechanosensitive (MS) channels self-assembled within the DIB should be able to respond to an expansion in the artificial membrane. Therefore, it can be used as a model system to understand how the structure of the protein and its incorporation into the unit cell affects its transduction properties. MS channels residing in the cytoplasmic membrane of Escherichia coli respond to a mechanical tension in the cell membrane6, and fall under three categories according to their conductance level7. MscL, the mechanosensitive channel of large conductance, has been studied both in vivo and in vitro using patch-clamp methods8. In this paper, the incorporation and activation of MscL is investigated. These channels, usually found
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