Fluctuations in Ionic Current Through Membrane Channels

项目摘要 人类基因组中80多个基因编码电压门控离子通道及其近亲 6TM离子通道系列。在它们的众多作用中，这些通道调节疼痛和其他感官 模式，在大脑中执行远程信号，为心跳计时，并控制淋巴细胞 扩散。它们作为一组丰富的潜在药物和治疗靶点而引起人们的极大兴趣，以及 作为一种对膜电位具有独特高度敏感性的蛋白质，也具有内在的意义 改变。 单粒子电子冷冻显微镜(Cryo-EM)是一种观察三维结构的方法 大分子络合物的结构。虽然分辨率低于X射线结晶学， 低温电磁技术在这一领域正在稳步发展，同时它具有很大的优势 提供蛋白质的“溶液结构”，而不需要形成晶体。 我们已经开发出用于重组膜蛋白的冷冻电磁单颗粒成像方法。 转化为脂质体，最近获得了真核细胞6TM通道的第一个闭态结构， 大电导钙激活钾(BK)通道。结构相对较低 分辨率受到我们能够获取的少量粒子图像的限制。在这 我们首先提出了应用程序，以极大地提高数据收集效率，以达到 分辨率优于1 nm。然后我们将成像处于不同构象状态的BK通道。 同时，我们将把这些方法应用于Kv1.2，一种研究得最好的电压门控钾 频道。通过在囊泡中产生膜电位，我们将能够将该通道捕获在其 用于结构确定的闭合状态和开放状态。

Project Summary More than 80 genes in the human genome code for voltage-gated ion channels and their relatives in the 6TM ion-channel family. Among their many roles these channels mediate pain and other sensory modalities, perform long-distance signaling in the brain, time the heartbeat and control lymphocyte proliferation. They are of great interest as a rich set of potential targets for drugs and therapies, and are also of intrinsic interest as proteins having a uniquely high sensitivity to membrane potential changes. Single-particle electron cryomicroscopy (cryo-EM) is a method for observing the three-dimensional structure of macromolecular complexes. Although the resolution is inferior to X-ray crystallography, cryo-EM technology is making steady progress in this area; meanwhile it has the great advantage of providing "solution structures" of proteins without the necessity of forming crystals. We have developed methods for cryo-EM single-particle imaging of membrane proteins reconstituted into liposomes, and have recently obtained the first closed-state structure of a eukaryotic 6TM channel, the large-conductance Ca2+-activated potassium (BK) channel. The structure has relatively low resolution, limited by the small number of particle images we have been able to acquire. In this application we propose first to greatly increase the data-collection efficiency in order to reach resolutions better than 1 nm. We will then image the BK channel in its various conformational states. In parallel, we will apply the methods to Kv1.2, one of the best-studied voltage-gated potassium channels. By creating membrane potentials in the vesicles, we will be able to trap this channel in its closed as well as open states for structure determination.