title: Oriented Reconstitution of the Full-Length KcsA Potassium Channel in a Lipid Bilayer for AFM Imaging.
abstract: Here, we have developed a method of oriented reconstitution of the KcsA potassium channel amenable to high-resolution AFM imaging. The solubilized full-length KcsA channels with histidine-tagged (His-tag) C-terminal ends were attached to a Ni2+-coated mica surface, and then detergent-destabilized liposomes were added to fill the interchannel space. AFM revealed that the membrane-embedded KcsA channels were oriented with their extracellular faces upward, seen as a tetrameric square shape. This orientation was corroborated by the visible binding of a peptide scorpion toxin, agitoxin-2. To observe the cytoplasmic side of the channel, a His-tag was inserted into the extracellular loop, and the oppositely oriented channels provided wholly different images. In either orientation, the channels were individually dispersed at acidic pH, whereas they were self-assembled at neutral pH, indicating that the oriented channels are allowed to diffuse in the membrane. This method is readily applicable to membrane proteins in general for AFM imaging.
authors.affiliation: PRESTO, Japan Science and Technology Agency (JST) , 4-1-8 Honcho, Kawaguchi-shi, Saitama 332-0012, Japan.|Department of Molecular Physiology and Biophysics, Faculty of Medical Sciences, University of Fukui , 23-3 Matsuokashimoaizuki, Yoshida-gun, Fukui 910-1193, Japan.
authors.forename: Ayumi
authors.initials: A
authors.lastname: Sumino
authors.affiliation: Department of Physics, Kanazawa University , Kakuma-machi, Kanazawa 920-1192, Japan.|Bio-AFM Frontier Research Center , Kanazawa 920-1192, Japan.
authors.forename: Takayuki
authors.initials: T
authors.lastname: Uchihashi
authors.affiliation: Department of Molecular Physiology and Biophysics, Faculty of Medical Sciences, University of Fukui , 23-3 Matsuokashimoaizuki, Yoshida-gun, Fukui 910-1193, Japan.
authors.forename: Shigetoshi
authors.identifier: http://orcid.org/0000-0002-8438-6750
authors.initials: S
authors.lastname: Oiki
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000378:metabolism; D003902:Detergents / Q000737:chemistry; D006657:Histones / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D008051:Lipid Bilayers / Q000737:chemistry*; D008081:Liposomes / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry; D018625:Microscopy, Atomic Force / Q000379:methods*; D008958:Models, Molecular; D011188:Potassium / Q000737:chemistry; D015221:Potassium Channels / Q000737:chemistry*; D012604:Scorpion Venoms / Q000737:chemistry*
chemical_list: D001426:Bacterial Proteins; D003902:Detergents; D006657:Histones; D008051:Lipid Bilayers; D008081:Liposomes; D008565:Membrane Proteins; D015221:Potassium Channels; D012604:Scorpion Venoms; C087603:agitoxin 2; D011188:Potassium