Functional studies indicate amantadine binds to the pore of the influenza A virus M2 proton-selective ion channel
Functional studies indicate amantadine binds to the pore of the influenza A virus M2 proton-selective ion channel
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DOI：
10.1073/pnas.0804958105
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发表时间：
2008-08-05
影响因子：
11.1
通讯作者：
Lamb, Robert A.
中科院分区：
文献类型：
--
作者：
Jing, Xianghong;Ma, Chunlong;Lamb, Robert A.
甲型和B型流感病毒分别含有质子选择性离子通道A/M2和BM 2，并且A/M2通道活性被药物金刚烷胺及其甲基衍生物金刚乙胺抑制。孔-跨膜结构域的结构已经通过X射线晶体学[Stouffer等人（2008）Nature 451：596-599]和NMR方法[Schnell和Chou（2008）Nature 451：591-595]确定。尽管晶体结构表明通道孔中有一个金刚烷胺分子，但NMR数据显示在朝向膜细胞质侧的螺旋外侧结合有四个金刚乙胺分子。药物结合包括与残基40-45的相互作用，金刚乙胺和天冬氨酸残基44（D44）之间的极性氢键似乎是重要的。这两个不同的药物结合位点导致两种不相容的药物抑制机制。我们将D44和R45诱变为丙氨酸，因为这些突变可能干扰金刚乙胺结合并导致药物不敏感通道。然而，D44 A通道被发现是敏感的金刚烷胺时，测量的电生理记录在非洲爪蟾卵母细胞和哺乳动物细胞，当D44和R45突变被引入流感病毒基因组。此外，将A/M2孔残基24-36移植到BM 2中，产生了被金刚烷胺部分抑制的pH激活的嵌合离子通道。因此，综合我们的功能数据表明，金刚烷胺/金刚乙胺在通道孔外的结合不是与A/M2离子通道的药理学抑制相关的主要部位。
Influenza A and B viruses contain proton-selective ion channels, A/M2 and BM2, respectively, and the A/M2 channel activity is inhibited by the drugs amantadine and its methyl derivative rimantadine. The structure of the pore-transmembrane domain has been determined by both x-ray crystallography [Stouffer et al. (2008) Nature 451:596-599] and by NMR methods [Schnell and Chou (2008) Nature 451:591-595]. Whereas the crystal structure indicates a single amantadine molecule in the pore of the channel, the NMR data show four rimantadine molecules bound on the outside of the helices toward the cytoplasmic side of the membrane. Drug binding includes interactions with residues 40-45 with a polar hydrogen bond between rimantadine and aspartic acid residue 44 (D44) that appears to be important. These two distinct drug-binding sites led to two incompatible drug inhibition mechanisms. We mutagenized D44 and R45 to alanine as these mutations are likely to interfere with rimantadine binding and lead to a drug insensitive channel. However, the D44A channel was found to be sensitive to amantadine when measured by electro-physiological recordings in oocytes of Xenopus laevis and in mammalian cells, and when the D44 and R45 mutations were introduced into the influenza virus genome. Furthermore, transplanting A/M2 pore residues 24-36 into BM2, yielded a pH-activated chimeric ion channel that was partially inhibited by amantadine. Thus, taken together our functional data suggest that amantadine/rimantadine binding outside of the channel pore is not the primary site associated with the pharmacological inhibition of the A/M2 ion channel.