title: Mapping of sites facing aqueous environment of voltage-gated proton channel at resting state: a study with PEGylation protection.
abstract: Hv1 (also named, voltage-sensor only protein, VSOP) lacks an authentic pore domain, and its voltage sensor domain plays both roles in voltage sensing and proton permeation. The activities of a proton channel are intrinsic to protomers of Hv1, while Hv1 is dimeric in biological membranes; cooperative gating is exerted by interaction between two protomers. As the signature pattern conserved among voltage-gated channels and voltage-sensing phosphatase, Hv1 has multiple arginines intervened by two hydrophobic residues on the fourth transmembrane segment, S4. S4 moves upward relative to other helices upon depolarization, causing conformational change possibly leading to the formation of a proton-selective conduction pathway. However, detailed mechanisms of proton-selectivity and gating of Hv1 are unknown. Here we took an approach of PEGylation protection assay to define residues facing the aqueous environment of mouse Hv1 (mHv1). Accessibilities of two maleimide molecules, N-ethylmaleimide (NEM) and 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid (AMS), were examined on cysteine introduced into individual sites. Only the first arginine on S4 (R1: R201) was inaccessible by NEM and AMS in mHv1. This is consistent with previous results of electrophysiology on the resting state channel, suggesting that the accessibility profile represents the resting state of mHv1. D108, critical for proton selectivity, was accessible by AMS and NEM, suggesting that D108 faces the vestibule. F146, a site critical for blocking by a guanidinium-reagent, was accessible by NEM, suggesting that F146 also faces the inner vestibule. These findings suggest an inner vestibule lined by several residues on S2 including F146, D108 on S1, and the C-terminal half of S4.
authors.affiliation: Laboratory of Integrative Physiology, Department of Physiology, Graduate School of Medicine, Osaka University, Yamada-oka 2-2, Suita, Osaka 565-0871, Japan.
authors.forename: Tatsuki
authors.initials: T
authors.lastname: Kurokawa
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Okamura
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D005033:Ethylmaleimide / Q000737:chemistry*; D057809:HEK293 Cells; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D015640:Ion Channel Gating; D007473:Ion Channels / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D017354:Point Mutation; D011092:Polyethylene Glycols / Q000737:chemistry*; D054730:Protein Interaction Domains and Motifs; D017433:Protein Structure, Secondary; D011522:Protons; D013045:Species Specificity; D013267:Stilbenes / Q000737:chemistry*; D013329:Structure-Activity Relationship; D013451:Sulfonic Acids / Q000737:chemistry*; D014867:Water / Q000737:chemistry
chemical_list: C510235:HVCN1 protein, human; C540876:Hv1 proton channel, mouse; D007473:Ion Channels; D011522:Protons; D013267:Stilbenes; D013451:Sulfonic Acids; D014867:Water; C079532:4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid; D011092:Polyethylene Glycols; D005033:Ethylmaleimide
keywords: 4-acetamido-4′-maleimidylstilbene-2,2′-disulfonic acid; AMS; Gating; Ion channel; Membrane potential; N-ethylmaleimide; NEM; Proton; Voltage; pH