Premkumar A, Ewart G D, Cox G B, Gage P W
Division of Molecular Bioscience, John Curtin School of Medical Research, Australian National University, P.O. Box 334, Canberra City, ACT 2601, Australia.
J Membr Biol. 2004 Feb 1;197(3):135-43. doi: 10.1007/s00232-004-0648-0.
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.
乙型流感病毒编码的一种名为NB的蛋白质已被证明在pH 6.0时可形成阳离子选择性离子通道，本研究针对该蛋白质中定点突变对人工脂质双层中离子通道特性的影响展开。我们认为，选定用于突变的NB疏水区域中的残基可能参与阳离子跨通道运输，这些残基的变化可能会影响通道特性，如门控和离子选择性。将第20位和第28位的丝氨酸残基、第24位的苏氨酸残基以及第26位的半胱氨酸残基替换为丙氨酸。我们发现，S20A突变产生的通道无法门控，且大部分时间保持开放状态。通过荧光猝灭检测到，S20A突变也改变了NB通道的质子通透性：通道不再具有质子通透性。其他突变，即S28A、T24A和C26A，对NB形成的通道的活性或质子通透性没有任何可检测到的影响。结果表明，丝氨酸20可能在NB通道的正常功能中发挥重要作用。
