Functional studies and modeling of pore-lining residue mutants of the influenza a virus M2 ion channel.
Functional studies and modeling of pore-lining residue mutants of the influenza a virus M2 ion channel.
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DOI：
10.1021/bi901799k
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发表时间：
2010-02-02
期刊：
影响因子：
2.9
通讯作者：
Pinto LH
中科院分区：
文献类型：
--
作者：
Balannik V;Carnevale V;Fiorin G;Levine BG;Lamb RA;Klein ML;Degrado WF;Pinto LH
甲型流感病毒的A/M2蛋白形成一个四聚体质子选择性ph门控离子通道。位于通道孔中的H37xxxW41基序负责其门控和质子选择性。通道激活很可能与H37残基的质子化有关，而通道的导电状态以四分体中两个或三个带电荷的His残基为特征。抗病毒药物金刚烷胺可抑制A/M2通道活性。尽管在体外已经观察到大量具有金刚烷胺抗性的a /M2突变体，但在存在或不存在金刚烷胺的高传染性病毒中只观察到少数。因此，我们检查了49个孔衬残基的点突变体，代表了自然和非自然的变体。在爪蟾卵母细胞中测定了它们的离子选择性、金刚烷胺敏感性、比活性和pH依赖性电导。这些测量显示了序列的变化如何导致质子传导的变化。结果与多步骤机制一致，该机制允许蛋白质在广泛的质子浓度范围内微调其ph率谱，假设是由H37四聚体的不同质子化状态产生的。在低pH下产生原生电导以及在pH 7.0下产生最小漏电流的突变非常罕见。此外，结果与金刚烷胺结合位点在通道孔内的位置一致。这些发现有助于确定在考虑设计抑制金刚烷胺耐药甲型流感病毒毒株的新药时应该针对的一组功能合适的突变体。
The A/M2 protein of influenza A virus forms a tetrameric proton selective pH-gated ion channel. The H37xxxW41 motif located in the channel pore is responsible for its gating and proton selectivity. Channel activation most likely involves protonation of the H37 residues, while the conductive state of the channel is characterized by two or three charged His residues in a tetrad. A/M2 channel activity is inhibited by the anti-viral drug amantadine. Although a large number of functional amantadine-resistant mutants of A/M2 have been observed in vitro, only a few are observed in highly transmissible viruses in the presence or absence of amantadine. We therefore examined 49 point mutants of the pore-lining residues, representing both natural and non-natural variants. Their ion selectivity, amantadine sensitivity, specific activity, and pH dependent conductance were measured in Xenopus oocytes. These measurements showed how variations in the sequence lead to variations in the proton conduction. The results are consistent with a multi-step mechanism that allows the protein to fine-tune its pH-rate profile over a wide range of proton concentrations, hypothesized to arise from different protonation states of the H37 tetrad. Mutations that give native-like conductance at low pH as well as minimal leakage current at pH 7.0 were surprisingly rare. Moreover, the results are consistent with a location of the amantadine-binding site inside the channel pore. These findings have helped to define the set of functionally fit mutants that should be targeted when considering the design of novel drugs that inhibit amantadine-resistant strains of influenza A virus.