title: Disulfide mapping the voltage-sensing mechanism of a voltage-dependent potassium channel.
abstract: Voltage-dependent potassium (Kv) channels allow for the selective permeability of potassium ions in a membrane potential dependent manner, playing crucial roles in neurotransmission and muscle contraction. Kv channel is a tetramer, in which each subunit possesses a voltage-sensing domain (VSD) and a pore domain (PD). Although several lines of evidence indicated that membrane depolarization is sensed as the movement of helix S4 of the VSD, the detailed voltage-sensing mechanism remained elusive, due to the difficulty of structural analyses at resting potential. In this study, we conducted a comprehensive disulfide locking analysis of the VSD using 36 double Cys mutants, in order to identify the proximal residue pairs of the VSD in the presence or absence of a membrane potential. An intramolecular SS-bond was formed between 6 Cys pairs under both polarized and depolarized environment, and one pair only under depolarized environment. The multiple conformations captured by the SS-bond can be divided by two states, up and down, where S4 lies on the extracellular and intracellular sides of the membrane, respectively, with axial rotation of 180°. The transition between these two states is caused by the S4 translocation of 12 Å, enabling allosteric regulation of the gating at the PD.
authors.affiliation: Graduate School of Pharmaceutical Sciences, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
authors.forename: Tomohiro
authors.initials: T
authors.lastname: Nozaki
authors.affiliation: Graduate School of Pharmaceutical Sciences, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
authors.forename: Shin-Ichiro
authors.initials: SI
authors.lastname: Ozawa
authors.affiliation: Graduate School of Pharmaceutical Sciences, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
authors.forename: Hitomi
authors.initials: H
authors.lastname: Harada
authors.affiliation: Graduate School of Pharmaceutical Sciences, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
authors.forename: Tomomi
authors.initials: T
authors.lastname: Kimura
authors.affiliation: Graduate School of Pharmaceutical Sciences, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.|Faculty of Pharmacy, Keio University, Shibakoen, Minato-ku, Tokyo 105-8512, Japan.
authors.forename: Masanori
authors.initials: M
authors.lastname: Osawa
authors.affiliation: Graduate School of Pharmaceutical Sciences, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
authors.forename: Ichio
authors.initials: I
authors.lastname: Shimada
mesh_terms: D041522:Aeropyrum; D019943:Amino Acid Substitution; D019843:Archaeal Proteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D001643:Bridged Bicyclo Compounds / Q000737:chemistry; D003553:Cystine / Q000737:chemistry / Q000235:genetics; D005456:Fluorescent Dyes / Q000737:chemistry; D015640:Ion Channel Gating; D008081:Liposomes / Q000737:chemistry; D008564:Membrane Potentials; D024642:Potassium Channels, Voltage-Gated / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D000072756:Protein Conformation, alpha-Helical; D013050:Spectrometry, Fluorescence
chemical_list: D019843:Archaeal Proteins; D001643:Bridged Bicyclo Compounds; D005456:Fluorescent Dyes; D008081:Liposomes; D024642:Potassium Channels, Voltage-Gated; D003553:Cystine; C028381:monobromobimane