title: NMR investigation of the isolated second voltage-sensing domain of human Nav1.4 channel.
abstract: Voltage-gated Na+channels are essential for the functioning of cardiovascular, muscular, and nervous systems. The α-subunit of eukaryotic Na+channel consists of ~2000 amino acid residues and encloses 24 transmembrane (TM) helices, which form five membrane domains: four voltage-sensing (VSD) and one pore domain. The structural complexity significantly impedes recombinant production and structural studies of full-sized Na+channels. Modular organization of voltage-gated channels gives an idea for studying of the isolated second VSD of human skeletal muscle Nav1.4 channel (VSD-II). Several variants of VSD-II (~150a.a., four TM helices) with different N- and C-termini were produced by cell-free expression. Screening of membrane mimetics revealed low stability of VSD-II samples in media containing phospholipids (bicelles, nanodiscs) associated with the aggregation of electrically neutral domain molecules. The almost complete resonance assignment of13C,15N-labeled VSD-II was obtained in LPPG micelles. The secondary structure of VSD-II showed similarity with the structures of bacterial Na+channels. The fragment of S4 TM helix between the first and second conserved Arg residues probably adopts 310-helical conformation. Water accessibility of S3 helix, observed by the Mn2+titration, pointed to the formation of water-filled crevices in the micelle embedded VSD-II.15N relaxation data revealed characteristic pattern of μs-ms time scale motions in the VSD-II regions sharing expected interhelical contacts. VSD-II demonstrated enhanced mobility at ps-ns time scale as compared to isolated VSDs of K+channels. These results validate structural studies of isolated VSDs of Na+channels and show possible pitfalls in application of this 'divide and conquer' approach.
authors.affiliation: Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya str., 16/10, Moscow 117997, Russia; Lomonosov Moscow State University, Moscow 119991, Russia.
authors.forename: A S
authors.initials: AS
authors.lastname: Paramonov
authors.affiliation: Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya str., 16/10, Moscow 117997, Russia; Lomonosov Moscow State University, Moscow 119991, Russia.
authors.forename: E N
authors.initials: EN
authors.lastname: Lyukmanova
authors.affiliation: Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya str., 16/10, Moscow 117997, Russia; Moscow Institute of Physics and Technology (State University), Institutskiy Pereulok 9, Dolgoprudny, Moscow Region 141700, Russia.
authors.forename: M Yu
authors.initials: MY
authors.lastname: Myshkin
authors.affiliation: Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya str., 16/10, Moscow 117997, Russia; Lomonosov Moscow State University, Moscow 119991, Russia.
authors.forename: M A
authors.initials: MA
authors.lastname: Shulepko
authors.affiliation: Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya str., 16/10, Moscow 117997, Russia; Lomonosov Moscow State University, Moscow 119991, Russia.
authors.forename: D S
authors.initials: DS
authors.lastname: Kulbatskii
authors.affiliation: Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya str., 16/10, Moscow 117997, Russia; Moscow Institute of Physics and Technology (State University), Institutskiy Pereulok 9, Dolgoprudny, Moscow Region 141700, Russia.
authors.forename: N S
authors.initials: NS
authors.lastname: Petrosian
authors.affiliation: Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya str., 16/10, Moscow 117997, Russia.
authors.forename: A O
authors.initials: AO
authors.lastname: Chugunov
authors.affiliation: Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya str., 16/10, Moscow 117997, Russia; Lomonosov Moscow State University, Moscow 119991, Russia.
authors.forename: D A
authors.initials: DA
authors.lastname: Dolgikh
authors.affiliation: Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya str., 16/10, Moscow 117997, Russia; Lomonosov Moscow State University, Moscow 119991, Russia.
authors.forename: M P
authors.initials: MP
authors.lastname: Kirpichnikov
authors.affiliation: Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya str., 16/10, Moscow 117997, Russia; Moscow Institute of Physics and Technology (State University), Institutskiy Pereulok 9, Dolgoprudny, Moscow Region 141700, Russia.
authors.forename: A S
authors.initials: AS
authors.lastname: Arseniev
authors.affiliation: Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya str., 16/10, Moscow 117997, Russia; Moscow Institute of Physics and Technology (State University), Institutskiy Pereulok 9, Dolgoprudny, Moscow Region 141700, Russia. Electronic address: zakhar-shenkarev@yandex.ru.
authors.forename: Z O
authors.initials: ZO
authors.lastname: Shenkarev
mesh_terms: D000595:Amino Acid Sequence; D002474:Cell-Free System; D006017:Glycolipids / Q000737:chemistry; D006801:Humans; D007295:Inositol Phosphates / Q000737:chemistry; D008345:Manganese / Q000737:chemistry; D008823:Micelles; D018482:Muscle, Skeletal / Q000378:metabolism; D062553:NAV1.4 Voltage-Gated Sodium Channel / Q000737:chemistry* / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular; D017433:Protein Structure, Secondary; D016415:Sequence Alignment
chemical_list: D006017:Glycolipids; D007295:Inositol Phosphates; C111513:LPPG glycoinositolphosphoceramide; D008823:Micelles; D062553:NAV1.4 Voltage-Gated Sodium Channel; C106251:SCN4A protein, human; D008345:Manganese
keywords: Cell-free expression; NMR spectroscopy; Na(+) channels; Voltage-sensing domain