Fluctuations in Ionic Current Through Membrane Channels

项目摘要 人类基因组中有80多个基因编码电压门控离子通道及其亲属， 6 TM离子通道系列。在它们的许多角色中，这些通道介导疼痛和其他感觉 模式，在大脑中执行长距离信号，计时心跳和控制淋巴细胞 增殖它们作为药物和治疗的一组丰富的潜在靶点， 作为对膜电位具有独特高敏感性的蛋白质， 变化 单粒子电子低温显微镜（cryo-EM）是一种观察三维结构的方法。 大分子复合物的结构。虽然分辨率不如X射线晶体学， 冷冻电镜技术在这一领域取得了稳步的进展，同时它具有以下优点： 提供蛋白质的“溶液结构”而不需要形成晶体。 我们已经开发了用于重组膜蛋白的冷冻EM单粒子成像的方法， 进入脂质体，最近获得了真核6 TM通道的第一个封闭状态结构， 大电导钙激活钾（BK）通道。结构相对较低 分辨率，受到我们能够获得的粒子图像数量较少的限制。在这 应用程序，我们建议首先大大提高数据收集效率，以达到 分辨率优于1 nm。然后，我们将图像的BK通道在其各种构象状态。 同时，我们将把这些方法应用于Kv1.2，这是研究得最好的电压门控钾离子通道之一。 渠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 用于结构测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.