RESPIRATORY VIRUS ION CHANNELS

在平均一年中，10%-20%的美国人口成为 感染了流感病毒，但唯一的抗病毒治疗 可供感染甲型流感病毒的人使用的是金刚乙胺， 其靶标是M2离子通道蛋白。更有效 由于对金刚乙胺具有耐药性，因此需要使用该通道的抑制剂 突变体产生的速度很快。在之前的赠款期间，我们发现 这种通道的抑制发生在细胞质的pH 结构域降低，与M2形成过渡金属络合物 分子。我们建议确定这些抑制的机制 以确定药物作用于M2分子的新靶点。 从功能测量中我们发现，细胞的活动状态 M2离子通道是一种同源四聚体，我们已经确定了 排列沟槽毛孔。我们建议确定 有直接测量的分子，因为这种结构信息 将允许设计更有用的抑制剂。个人 目的是：1.了解M2离子的抑制机制 通道是C-末端结构域pH降低的结果。2.至 用取代基确定M2离子通道的孔结构 半胱氨酸可及性法。3.使用过渡金属络合物来 探索M2离子通道的孔结构。4.应用该方法 M2离子通道跨膜的定点自旋标记 域以确定其静止结构和 在低pH激活时发生。5.了解 跨膜组氨酸37通过M2离子通道进行质子运输。 6.在另一组实验中，我们将测试假设 B型流感病毒的Nb蛋白具有离子通道的功能。

During an average year, 10-20 percent of the US population becomes infected with influenza virus, but the only antiviral treatment available for those infected with influenza A virus is rimantadine, the target for which is the M2 ion channel protein. More effective inhibitors of the channel are needed because rimantadine-resistant mutants are generated rapidly. In the previous grant period we found that inhibition of the channel occurs when the pH of the cytoplasmic domain is lowered and transition metal complexes are formed with the M2 molecule. We propose to determine the mechanism for these inhibitions in order to identify new targets for drug action on the M2 molecule. From functional measurements we have found that the active state of the M2 ion channel is a homotetramer and we have identified residues that line the channel pore. We propose to determine the structure of the molecule with direct measurements because this structural information will allow for the design of more useful inhibitors. The individual aims are: 1. To understand the mechanism for inhibition of M2 ion channel as a result of decreased pH of the C-terminal domain. 2. To determine the M2 ion channel pore structure with the substituted cysteine accessibility method. 3. To use transition metal complexes to probe the pore structure of the M2 ion channel. 4. To apply the approach of site-directed spin labeling of the M2 ion channel transmembrane domain to determine its resting structure and structural changes that occur on activation by low pH. 5. To understand the role of transmembrane histidine 37 in proton transport by the M2 ion channel. 6. In a separate set of experiments we will test the hypothesis that the NB protein of influenza B virus functions as an ion channel.