Sunstrom N A, Premkumar L S, Premkumar A, Ewart G, Cox G B, Gage P W
John Curtin School of Medical Research, Australian National University, Canberra, Australia.
J Membr Biol. 1996 Mar;150(2):127-32. doi: 10.1007/s002329900037.
The influenza B virus protein, NB, was expressed in Escherichia coli, either with a C-terminal polyhistidine tag or with NB fused to the C-terminus of glutathione S-transferase (GST), and purified by affinity chromatography. NB produced ion channel activity when added to artificial lipid bilayers separating NaCl solutions with unequal concentrations (150-500 mM cis, 50 mM trans). An antibody to a peptide mimicking the 25 residues at the C-terminal end of NB, and amantadine at high concentration (2-3 mM), both depressed ion channel activity. Ion channels had a variable conductance, the lowest conductance observed being approximately 10 picosiemens. At a pH of 5.5 to 6.5, currents reversed at positive potentials indicating that the channel was more permeable to sodium than to chloride ions (PNa/PCl approximately 9). In asymmetrical NaCl solutions at a pH of 2.5, currents reversed closer to the chloride than to the sodium equilibrium potential indicating that the channel had become more permeable to chloride than to sodium ions (PCl/PNa approximately 4). It was concluded that, at normal pHs, NB forms cation-selective channels.
乙型流感病毒蛋白NB在大肠杆菌中表达，带有C端多组氨酸标签，或者与谷胱甘肽S-转移酶（GST）的C端融合，然后通过亲和层析进行纯化。当将NB添加到分隔不同浓度NaCl溶液（顺式150 - 500 mM，反式50 mM）的人工脂质双层中时，会产生离子通道活性。针对模拟NB C端25个残基的肽的抗体以及高浓度（2 - 3 mM）的金刚烷胺，均会抑制离子通道活性。离子通道具有可变的电导率，观察到的最低电导率约为10皮西门子。在pH为5.5至6.5时，电流在正电位处反转，表明该通道对钠离子的通透性比对氯离子更高（PNa/PCl约为9）。在pH为2.5的不对称NaCl溶液中，电流反转更接近氯离子而非钠离子的平衡电位，表明该通道对氯离子的通透性比对钠离子更高（PCl/PNa约为4）。得出的结论是，在正常pH值下，NB形成阳离子选择性通道。
