Identification of the functional core of the influenza A virus A/M2 proton-selective ion channel
Identification of the functional core of the influenza A virus A/M2 proton-selective ion channel
复制标题
DOI：
10.1073/pnas.0905726106
复制
发表时间：
2009-07-28
影响因子：
11.1
通讯作者：
Pinto, Lawrence H.
中科院分区：
文献类型：
--
作者：
Ma, Chunlong;Polishchuk, Alexei L.;Pinto, Lawrence H.
甲型流感病毒 M2 蛋白 (A/M2) 是一种同源四聚体 pH 激活的质子转运蛋白/通道，介导内体包裹病毒内部的酸化。这种 97 个残基的蛋白质具有单个跨膜 (TM) 螺旋，可结合形成同源四聚体，与抗流感药物金刚烷胺结合。然而，细胞膜中组装和质子运输所需的最小片段尚未确定。因此，检查了爪蟾卵母细胞中表达的截短突变体的电导特性。跨越残基 21-61 的短片段 M2(21-61) 被插入细胞质膜，具有与全长 A/M2 没有区别的特异金刚烷胺敏感质子转运活性；更短片段 M2(21-51)-FLAG 的表位标记版本的比活性在全长蛋白质的 2 倍范围内。此外，发现包括跨越残基22-46的肽的合成片段能够以金刚烷胺敏感的方式将质子转运到脂质体中。此外，功能上重要的 His-37 残基 pK(a) 值在蛋白质四聚体形式中受到高度干扰，这是胶束和双层中 TM 肽和全长 A/M2 中保守的特性。这些数据表明，折叠、药物结合和质子易位的决定因素被包装在一个非常小的肽中，现在可以放心地研究它。
The influenza A virus M2 protein (A/M2) is a homotetrameric pH-activated proton transporter/channel that mediates acidification of the interior of endosomally encapsulated virus. This 97-residue protein has a single transmembrane (TM) helix, which associates to form homotetramers that bind the anti-influenza drug amantadine. However, the minimal fragment required for assembly and proton transport in cellular membranes has not been defined. Therefore, the conductance properties of truncation mutants expressed in Xenopus oocytes were examined. A short fragment spanning residues 21-61, M2(21-61), was inserted into the cytoplasmic membrane and had specific, amantadine-sensitive proton transport activity indistinguishable from that of full-length A/M2; an epitope-tagged version of an even shorter fragment, M2(21-51)-FLAG, had specific activity within a factor of 2 of the full-length protein. Furthermore, synthetic fragments including a peptide spanning residues 22-46 were found to transport protons into liposomes in an amantadine-sensitive manner. In addition, the functionally important His-37 residue pK(a) values are highly perturbed in the tetrameric form of the protein, a property conserved in the TM peptide and full-length A/M2 in both micelles and bilayers. These data demonstrate that the determinants for folding, drug binding, and proton translocation are packaged in a remarkably small peptide that can now be studied with confidence.