title: Reconstitution of Human Ion Channels into Solvent-free Lipid Bilayers Enhanced by Centrifugal Forces.
abstract: Artificially formed bilayer lipid membranes (BLMs) provide well-defined systems for functional analyses of various membrane proteins, including ion channels. However, difficulties associated with the integration of membrane proteins into BLMs limit the experimental efficiency and usefulness of such BLM reconstitution systems. Here, we report on the use of centrifugation to more efficiently reconstitute human ion channels in solvent-free BLMs. The method improves the probability of membrane fusion. Membrane vesicles containing the human ether-a-go-go-related gene (hERG) channel, the human cardiac sodium channel (Nav1.5), and the human GABAA receptor (GABAAR) channel were formed, and the functional reconstitution of the channels into BLMs via vesicle fusion was investigated. Ion channel currents were recorded in 67% of the BLMs that were centrifuged with membrane vesicles under appropriate centrifugal conditions (14-55 × g). The characteristic channel properties were retained for hERG, Nav1.5, and GABAAR channels after centrifugal incorporation into the BLMs. A comparison of the centrifugal force with reported values for the fusion force revealed that a centrifugal enhancement in vesicle fusion was attained, not by accelerating the fusion process but by accelerating the delivery of membrane vesicles to the surface of the BLMs, which led to an increase in the number of membrane vesicles that were available for fusion. Our method for enhancing the probability of vesicle fusion promises to dramatically increase the experimental efficiency of BLM reconstitution systems, leading to the realization of a BLM-based, high-throughput platform for functional assays of various membrane proteins.
authors.affiliation: Graduate School of Biomedical Engineering, Tohoku University, Aoba, Sendai, Japan. Electronic address: ahirano@bme.tohoku.ac.jp.
authors.forename: Ayumi
authors.initials: A
authors.lastname: Hirano-Iwata
authors.affiliation: Graduate School of Biomedical Engineering, Tohoku University, Aoba, Sendai, Japan.
authors.forename: Yutaka
authors.initials: Y
authors.lastname: Ishinari
authors.affiliation: Graduate School of Biomedical Engineering, Tohoku University, Aoba, Sendai, Japan.
authors.forename: Miyu
authors.initials: M
authors.lastname: Yoshida
authors.affiliation: Graduate School of Biomedical Engineering, Tohoku University, Aoba, Sendai, Japan.
authors.forename: Shun
authors.initials: S
authors.lastname: Araki
authors.affiliation: Graduate School of Biomedical Engineering, Tohoku University, Aoba, Sendai, Japan.
authors.forename: Daisuke
authors.initials: D
authors.lastname: Tadaki
authors.affiliation: Graduate School of Biomedical Engineering, Tohoku University, Aoba, Sendai, Japan.
authors.forename: Ryusuke
authors.initials: R
authors.lastname: Miyata
authors.affiliation: Hang-Ichi Corporation, Naka, Yokohama, Japan.
authors.forename: Kenichi
authors.initials: K
authors.lastname: Ishibashi
authors.affiliation: Frontier Research Institute for Interdisciplinary Sciences, Tohoku University, Aoba, Sendai, Japan.
authors.forename: Hideaki
authors.initials: H
authors.lastname: Yamamoto
authors.affiliation: Department of Electric and Electronic Engineering, School of Engineering, Tokyo University of Technology, Hachioji, Tokyo, Japan.
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Kimura
authors.affiliation: Graduate School of Biomedical Engineering, Tohoku University, Aoba, Sendai, Japan; Laboratory for Nanoelectronics and Spintronics, Research Institute of Electrical Communication, Tohoku University, Aoba, Sendai, Japan.
authors.forename: Michio
authors.initials: M
authors.lastname: Niwano
mesh_terms: D000818:Animals; D016466:CHO Cells; D002498:Centrifugation; D003412:Cricetulus; D000072237:ERG1 Potassium Channel / Q000378:metabolism*; D057809:HEK293 Cells; D057166:High-Throughput Screening Assays; D006801:Humans; D066298:In Vitro Techniques; D008051:Lipid Bilayers / Q000378:metabolism*; D008561:Membrane Fusion; D008564:Membrane Potentials; D018625:Microscopy, Atomic Force; D062554:NAV1.5 Voltage-Gated Sodium Channel / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D011963:Receptors, GABA-A / Q000378:metabolism*
chemical_list: D000072237:ERG1 Potassium Channel; C000606913:KCNH2 protein, human; D008051:Lipid Bilayers; D062554:NAV1.5 Voltage-Gated Sodium Channel; D011963:Receptors, GABA-A; C568320:SCN5A protein, human