Fluctuations in Ionic Current Through Membrane Channels

描述（由申请人提供）：人类基因组中有80多个基因编码电压门控离子通道及其在6 TM离子通道家族中的亲属。在它们的许多作用中，这些通道介导疼痛和其他感觉方式，在大脑中执行长距离信号传递，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引起了极大的兴趣，并且作为对膜电位变化具有独特的高敏感性的蛋白质也引起了固有的兴趣。单粒子电子低温显微镜（cryo-EM）是一种观察大分子复合物三维结构的方法。虽然分辨率不如X射线晶体学，但cryo-EM技术在这一领域正在稳步发展;同时，它具有提供蛋白质“溶液结构”而无需形成晶体的巨大优势。我们已经开发出的方法cryo-EM单粒子成像的膜蛋白重组成脂质体，最近获得了第一个封闭状态的结构的真核6 TM通道，大电导钙激活钾（BK）通道。该结构具有相对较低的分辨率，受到我们能够获得的粒子图像数量较少的限制。在这个应用中，我们首先建议大大提高数据收集效率，以达到优于1 nm的分辨率。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通道之一。通过在囊泡中产生膜电位，我们将能够将该通道捕获在其封闭和开放状态中以用于结构确定。公共卫生相关性：电压门控离子通道充当分子开关，控制大脑、心脏和许多其他器官中的电流。因为有许多（超过80）品种，这些离子通道的缺陷会引起一系列疾病，从癫痫，偏头痛和肌肉麻痹到高血压和心律不齐。为了了解它们是如何工作的，我们建议使用新的电子显微镜技术观察其中两个通道在其各种功能状态下的分子结构。

DESCRIPTION (provided by applicant): 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 structur determination. PUBLIC HEALTH RELEVANCE: Voltage-gated ion channels act as molecular switches, controlling the electrical currents in the brain, heart and many other organs. Because there are many (more than 80) varieties, defects in these ion channels give rise to a spectrum of disorders ranging from epilepsy, migraine and muscular paralysis to hypertension and irregular heart rhythm. To understand how they work, we propose to observe the molecular structure of two of these channels, in their various functional states, using novel electron- microscopy techniques.