title: Multifunctional, Micropipette-based Method for Incorporation And Stimulation of Bacterial Mechanosensitive Ion Channels in Droplet Interface Bilayers.
abstract: MscL, a large conductance mechanosensitive channel (MSC), is a ubiquitous osmolyte release valve that helps bacteria survive abrupt hypo-osmotic shocks. It has been discovered and rigorously studied using the patch-clamp technique for almost three decades. Its basic role of translating tension applied to the cell membrane into permeability response makes it a strong candidate to function as a mechanoelectrical transducer in artificial membrane-based biomolecular devices. Serving as building blocks to such devices, droplet interface bilayers (DIBs) can be used as a new platform for the incorporation and stimulation of MscL channels. Here, we describe a micropipette-based method to form DIBs and measure the activity of the incorporated MscL channels. This method consists of lipid-encased aqueous droplets anchored to the tips of two opposing (coaxially positioned) borosilicate glass micropipettes. When droplets are brought into contact, a lipid bilayer interface is formed. This technique offers control over the chemical composition and the size of each droplet, as well as the dimensions of the bilayer interface. Having one of the micropipettes attached to a harmonic piezoelectric actuator provides the ability to deliver a desired oscillatory stimulus. Through analysis of the shapes of the droplets during deformation, the tension created at the interface can be estimated. Using this technique, the first activity of MscL channels in a DIB system is reported. Besides MS channels, activities of other types of channels can be studied using this method, proving the multi-functionality of this platform. The method presented here enables the measurement of fundamental membrane properties, provides a greater control over the formation of symmetric and asymmetric membranes, and is an alternative way to stimulate and study mechanosensitive channels.
authors.affiliation: Department of Mechanical Engineering, Virginia Polytechnic Institute and State University.
authors.forename: Joseph S
authors.initials: JS
authors.lastname: Najem
authors.affiliation: School of Biomedical Engineering and Sciences, Virginia Polytechnic Institute and State University.
authors.forename: Myles D
authors.initials: MD
authors.lastname: Dunlap
authors.affiliation: Department of Biology, University of Maryland.
authors.forename: Anthony
authors.initials: A
authors.lastname: Yasmann
authors.affiliation: College of Engineering, University of Georgia.
authors.forename: Eric C
authors.initials: EC
authors.lastname: Freeman
authors.affiliation: Department of Engineering Sciences and Mechanics, Virginia Polytechnic Institute and State University.
authors.forename: John W
authors.initials: JW
authors.lastname: Grant
authors.affiliation: Department of Biology, University of Maryland.
authors.forename: Sergei
authors.initials: S
authors.lastname: Sukharev
authors.affiliation: College of Engineering, University of Georgia; donleo@uga.edu.
authors.forename: Donald J
authors.initials: DJ
authors.lastname: Leo