Abstract
The effects of site-directed mutations in NB, a protein encoded by the influenza B virus that has been shown to form cation-selective ion channels at pH 6.0, were studied on ion channel characteristics in artificial lipid bilayers. It was thought that the residues in the hydrophobic region of NB we selected for mutation might be involved in the transport of cations across the channel and that changes in these residues might affect channel properties such as gating and ion-selectivity. Serine residues at positions 20 and 28, threonine at position 24 and cysteine at position 26 were replaced by alanine. We found that the mutation S20A gave channels that did not gate and that remained open most of the time. Proton permeability of NB channels, as detected by fluorescence quenching, was also altered by the mutation S20A: channels were no longer proton-permeable. The other mutations, S28A, T24A and C26A, did not have any detectable effect on the activity or proton permeability of channels formed by NB. The results indicate that serine 20 may have an important role in normal function of NB channels.
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T. Betakova M.V. Nermut A.J. Hay (1996) ArticleTitleThe NB protein is an integral component of the membrane of influenza B virus. J. Gen. Virol. 77 2689–2694 Occurrence Handle1:CAS:528:DyaK28Xmsl2htr4%3D Occurrence Handle8922461
W.P. Burmeister F. Baudin S. Cusack R.W. Ruigrok (1993) ArticleTitleComparison of structure and sequence of influenza B/Yamagata and B/Beijing neuraminidases shows a conserved “head” but much greater variability in the “stalk” and NB protein. Virology 192 683–686 Occurrence Handle10.1006/viro.1993.1088 Occurrence Handle1:CAS:528:DyaK3sXkvV2qtLk%3D Occurrence Handle8421908
L. Byerly Y. Suen (1989) ArticleTitleCharacterization of proton currents in neurones of the snail, Lymnaea stagnalis. J. Physiol. 413 75–89 Occurrence Handle1:STN:280:By%2BD1Mrps1A%3D Occurrence Handle2557442
T.E. Decoursey V.V. Cherny (1995) ArticleTitleVoltage-activated proton currents in membrane patches of rat alveolar epithelial cells. J. Physiol. 489 299–307 Occurrence Handle1:CAS:528:DyaK28XitFOntQ%3D%3D Occurrence Handle8847626
G.D. Ewart T. Sutherland P.W. Gage G.B. Cox (1996) ArticleTitleThe vpu protein of human immunodeficiency virus type 1 forms cation-selective ion channels. J. Virol. 70 7108–7115 Occurrence Handle1:CAS:528:DyaK28XlslWjsLo%3D Occurrence Handle8794357
WB. Fischer M. Pitkeathly B.A. Wallace L.R. Forrest G.R. Smith M.S.P. Sansom (2000) ArticleTitleTransmembrane peptide NB of influenza B: A simulatiom, structure and conductance study. Biochemistry 39 12708–12716 Occurrence Handle1:CAS:528:DC%2BD3cXmsFGisLc%3D Occurrence Handle11027151
W.B. Fischer M. Pitkeathly M.S.P. Sansom (2001) ArticleTitleAmantadin blocks channel activity of the transmembrane segment of the NB protein from influenza B. Eur. Biophys. J. 30 416–420 Occurrence Handle10.1007/s002490100157 Occurrence Handle1:CAS:528:DC%2BD3MXntVKkt78%3D Occurrence Handle11718294
J. Galzi A. Devillers-Thiery N Hussy S Bertrand J Changeux D. Bertrand (1992) ArticleTitleMutations in the channel domain of a neuronal nicotinic receptor convert ion selectivity from cationic to anionic. Nature 359 500–504 Occurrence Handle1:CAS:528:DyaK38XmsVGks7o%3D Occurrence Handle1383829
H.R. Guy F. Conti (1990) ArticleTitlePursuing the structure and function of voltage-gated channels. Trends Neurosci. 6 201–206
B.A. Haddock J.A. Downie (1974) ArticleTitleThe reconstitution of functional respiratory chains in membranes from electron-transport-deficient mutant of Escherichia coli as demonstrated by quenching of atebrin fluorescence. Biochem. J. 142 703–706 Occurrence Handle1:CAS:528:DyaE2MXhtVOltL0%3D Occurrence Handle4377217
I.D. Kerr R. Sankararamakrishnan O.S. Smart M.S.P. Sansom (1994) ArticleTitleParallel helix bundles and ion channels: molecular modeling via simulated annealing and restrained molecular dynamics. Biophys. J. 67 1501–1515 Occurrence Handle1:CAS:528:DyaK2MXhtVGnsr8%3D Occurrence Handle7529585
R.A. Lamb L.H. Pinto (1997) ArticleTitleDo vpu and vpr of human immunodeficiency virus type1 and NB of influenza B virus have ion channel activities in the viral life cycles? Virology 229 1–11 Occurrence Handle1:CAS:528:DyaK2sXhs1Kjsr0%3D Occurrence Handle9123850
J.D. Lear Z.R. Wasserman W.F. Degrado (1988) ArticleTitleSynthetic amphiphilic peptide models for protein ion channels [published erratum appears in Science 1989 Sep 29; 245(4925):1437]. Science 240 1177–1181 Occurrence Handle1:CAS:528:DyaL1cXkvVygtr0%3D Occurrence Handle2453923
R.J. Leonard C.G. Labarca P. Charnet N. Davidson H.A. Lester (1988) ArticleTitleEvidence that the M2 membrane-spanning region lines the ion channel pore of the nicotinic receptor. Science 242 1578–1581 Occurrence Handle1:CAS:528:DyaL1MXntFagsQ%3D%3D Occurrence Handle2462281
Y.A. Lu P Clavijo M. Galantino Z.Y. Shen W. Liu J.P. Tam (1991) ArticleTitleChemically unambiguous peptide immunogen: preparation, orientation and antigenicity of purified peptide conjugated to the multiple antigen peptide system. Mol. Immunol. 28 623–630 Occurrence Handle1:CAS:528:DyaK38Xkslai Occurrence Handle1861679
C. Miller (1986) Ion channel reconstitution. Plenum Press New York
M.S.P. Sansom (1992) ArticleTitleAn investigation of the role of serine and threonine sidechains in ion channel proteins. Eur. Biophys. J. 21 281–298 Occurrence Handle1:CAS:528:DyaK38XmsVOnsb0%3D Occurrence Handle1385107
R.J. Sugrue A.J. Hay (1991) ArticleTitleStructural characteristics of the M2 protein of influenza A viruses: evidence that it forms a tetrameric channel. Virology 180 617–624 Occurrence Handle1:CAS:528:DyaK3MXnvVeluw%3D%3D Occurrence Handle1989386
N.A. Sunstrom L.S. Premkumar A. Premkumar G. Ewart G.B. Cox P.W. Gage (1996) ArticleTitleIon channels formed by NB, an influenza B virus protein. J. Membrane Biol 150 127–132 Occurrence Handle10.1007/s002329900037 Occurrence Handle1:CAS:528:DyaK28XitVKlt74%3D
M.A. Williams R.A. Lamb (1986) ArticleTitleDetermination of the orientation of an integral membrane protein and sites of glycosylation by oligonucleotide-directed mutagenesis: influenza B virus NB glycoprotein lacks a cleavable signal sequence and has an extracellular NH2-terminal region. Mol. Cell Biol. 6 4317–4328 Occurrence Handle1:CAS:528:DyaL2sXksF2ltg%3D%3D Occurrence Handle3025652
S.L. Zebedee R.A. Lamb (1988) ArticleTitleInfluenza A virus M2 protein: monoclonal antibody restriction of virus growth and detection of M2 in virions. J. Virol. 62 2762–2772 Occurrence Handle1:CAS:528:DyaL1cXltVOmt7s%3D Occurrence Handle2455818
O.P. Zhirnov (1990) ArticleTitleSolubilization of matrix protein M1/M from virions occurs at different pH for orthomyxo- and paramyxoviruses. Virology 176 274–279 Occurrence Handle1:CAS:528:DyaK3cXktVaksLo%3D Occurrence Handle2158693
O.P. Zhirnov (1992) ArticleTitleIsolation of matrix protein M1 from influenza viruses by acid-dependent extraction with nonionic detergent. Virology 186 324–330 Occurrence Handle1:CAS:528:DyaK38Xjslemuw%3D%3D Occurrence Handle1727609
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Premkumar, A., Ewart, G., Cox, G. et al. An Amino-Acid Substitution in the Influenza-B NB Protein Affects Ion-Channel Gating . J. Membrane Biol. 197, 135–143 (2004). https://doi.org/10.1007/s00232-004-0648-0
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DOI: https://doi.org/10.1007/s00232-004-0648-0