RESPIRATORY VIRUS ION CHANNELS

流感仍然是一种常规的流行病，偶尔也会大流行， 疾病是导致发病率和死亡率的主要原因之一 在世界各地，特别是在老年人和有 心脏病和肺病。流感病毒编码两个小整数 结构相似和不寻常的膜蛋白，M2蛋白 甲型流感病毒和乙型流感病毒的NB糖蛋白。这个 拟议的研究是基于我们的论证，即M2蛋白 形成一个离子通道，我们发现离子通道的活性是 与Nb蛋白相关。(1)我们建议研究以下结构- 通过离子测定研究M2蛋白的功能关系 定点突变体的通道活性(包括比活性) 经过改造，使它们对抗病毒药物金刚烷胺或 缺乏某些翻译后的修饰。我们还建议 创建最小尺寸的活性离子通道并重建离子 脂质双分子层中的通道活性。(2)离子通道的作用 甲型流感病毒生命周期中M2蛋白的活性 将通过测量离子通道的pH激活来表征 在表达该蛋白的哺乳动物细胞中，比较pH激活 甲型流感病毒不同毒株的M2蛋白 发生构象变化的血凝素(HA)分子 通过比较离子通道在不同pH值下的酸态 流感“高产”毒株M2蛋白的性质 一种病毒。(3)Nb-缔合物的结构-功能关系 将研究离子通道的活性。我们将比较它们的特性 野生型和各种突变体的离子通道活性 Nb蛋白的形式以确定Nb蛋白本身是否是离子 频道。碳水化合物加成、钙离子调节和细胞周期调控的作用 NB功能中跨膜结构域中的极性残基 蛋白质将通过分析特定部位的突变进行研究。 Nb相关离子通道活性的比活性将是 测量，通道活动所需的最小蛋白质将是 测定，并在脂质双分子层中重建通道活性。 这些研究将提供有关以下方面的重要信息： M2和Nb蛋白，可能有助于靶向药物作用，并将 提供有关未来结构(如核磁共振和核磁共振)的基本信息 结晶学)对这些最小通道的研究可以作为基础。

Influenza continues to be a regular epidemic, and occasionally pandemic, disease which ranks among the leading causes of morbidity and mortality throughout the world, particularly in the elderly and in individuals with heart and lung disease. Influenza viruses encode two small integral membrane proteins of similar and unusual structure, the M2 protein of influenza A virus and the NB glycoprotein of influenza B virus. The proposed research is based upon our demonstration that the M2 protein forms an ion channel and our finding that ion channel activity is associated with the NB protein. (1) We propose to study the structure- function relationship of the M2 protein by making measurements of ion channel activity (including specific activity) of site-directed mutants modified to render them insensitive to the antiviral drug amantadine or lacking in certain post-translational modifications. We also propose to create an active ion channel of the minimal size and to reconstitute ion channel activity in lipid bilayers. (2) The role of the ion channel activity of the M2 protein in the life-cycle of the influenza A virus will be characterized by measuring the pH-activation of the ion channel in mammalian cells that express the protein, comparing the pH-activation of the M2 proteins of various strains of influenza A virus having hemagglutinin (HA) molecules which undergo conformational changes to the acid state at different values of pH and by comparing the ion channel properties of M2 proteins derived from "high yield" strains of influenza A virus. (3) The structure-function relationship of the NB-associated ion channel activity will be studied. We will compare the properties of the ion channel activity associated with the wild-type and various mutant forms of the NB protein to determine if the NB protein is itself an ion channel. The roles of carbohydrate addition, Ca2+ regulation and the polar residues in the transmembrane domain in the function of the NB protein will be investigated using analysis of site-specific mutants. The specific activity of the NB-associated ion channel activity will be measured, the minimal protein needed for channel activity will be determined, and the channel activity reconstituted in lipid bilayers. These studies will provide essential information about the role of the M2 and NB proteins that may help target them for drug action and will provide basic information upon which future structural (e.g., NMR and crystallographic) studies of these minimal channels can be based.