title: bSUM: A bead-supported unilamellar membrane system facilitating unidirectional insertion of membrane proteins into giant vesicles.
abstract: Fused or giant vesicles, planar lipid bilayers, a droplet membrane system, and planar-supported membranes have been developed to incorporate membrane proteins for the electrical and biophysical analysis of such proteins or the bilayer properties. However, it remains difficult to incorporate membrane proteins, including ion channels, into reconstituted membrane systems that allow easy control of operational dimensions, incorporation orientation of the membrane proteins, and lipid composition of membranes. Here, using a newly developed chemical engineering procedure, we report on a bead-supported unilamellar membrane (bSUM) system that allows good control over membrane dimension, protein orientation, and lipid composition. Our new system uses specific ligands to facilitate the unidirectional incorporation of membrane proteins into lipid bilayers. Cryo-electron microscopic imaging demonstrates the unilamellar nature of the bSUMs. Electrical recordings from voltage-gated ion channels in bSUMs of varying diameters demonstrate the versatility of the new system. Using KvAP as a model system, we show that compared with other in vitro membrane systems, the bSUMs have the following advantages: (a) a major fraction of channels are orientated in a controlled way; (b) the channels mediate the formation of the lipid bilayer; (c) there is one and only one bilayer membrane on each bead; (d) the lipid composition can be controlled and the bSUM size is also under experimental control over a range of 0.2-20 µm; (e) the channel activity can be recorded by patch clamp using a planar electrode; and (f) the voltage-clamp speed (0.2-0.5 ms) of the bSUM on a planar electrode is fast, making it suitable to study ion channels with fast gating kinetics. Our observations suggest that the chemically engineered bSUMs afford a novel platform for studying lipid-protein interactions in membranes of varying lipid composition and may be useful for other applications, such as targeted delivery and single-molecule imaging.
authors.affiliation: Department of Cell Biology, Department of Physiology, and Department of Biophysics, The University of Texas Southwestern Medical Center, Dallas, TX 75390 Department of Cell Biology, Department of Physiology, and Department of Biophysics, The University of Texas Southwestern Medical Center, Dallas, TX 75390.
authors.forename: Hui
authors.initials: H
authors.lastname: Zheng
authors.affiliation: Department of Cell Biology, Department of Physiology, and Department of Biophysics, The University of Texas Southwestern Medical Center, Dallas, TX 75390 Department of Cell Biology, Department of Physiology, and Department of Biophysics, The University of Texas Southwestern Medical Center, Dallas, TX 75390.
authors.forename: Sungsoo
authors.initials: S
authors.lastname: Lee
authors.affiliation: Department of Cell Biology, Department of Physiology, and Department of Biophysics, The University of Texas Southwestern Medical Center, Dallas, TX 75390 Department of Cell Biology, Yale University, New Haven, CT 06510.
authors.forename: Marc C
authors.initials: MC
authors.lastname: Llaguno
authors.affiliation: Department of Cell Biology, Department of Physiology, and Department of Biophysics, The University of Texas Southwestern Medical Center, Dallas, TX 75390 Department of Microbiology and Cell Science, University of Florida, Gainesville, FL 32611 qiu-xing.jiang@utsouthwestern.edu qxjiang@ufl.edu.
authors.forename: Qiu-Xing
authors.initials: QX
authors.lastname: Jiang
mesh_terms: D002462:Cell Membrane / Q000737:chemistry*; D007473:Ion Channels / Q000737:chemistry*; D008024:Ligands; D008051:Lipid Bilayers / Q000737:chemistry*; D008055:Lipids / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry*; D036103:Nanotechnology / Q000379:methods; D012822:Silicon Dioxide / Q000737:chemistry
chemical_list: D007473:Ion Channels; D008024:Ligands; D008051:Lipid Bilayers; D008055:Lipids; D008565:Membrane Proteins; D012822:Silicon Dioxide