Fluctuations in Ionic Current Through Membrane Channels

描述（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6TM离子通道家族的亲属。在它们的许多作用中，这些通道介导疼痛和其他感觉模式，在大脑中执行长距离信号传导，定时心跳和控制淋巴细胞增殖。它们作为一组丰富的药物和治疗的潜在靶点而引起了人们的极大兴趣，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人们的内在兴趣。单粒子电子低温显微镜（cryo-EM）是一种观察大分子配合物三维结构的方法。虽然分辨率不如x射线晶体学，但冷冻电镜技术在这一领域正在稳步发展；同时，它具有提供蛋白质的“溶液结构”而无需形成晶体的巨大优势。我们已经开发了重组成脂质体的膜蛋白的低温电镜单粒子成像方法，并且最近获得了真核生物6TM通道的第一个封闭状态结构，大电导Ca2+活化钾（BK）通道。该结构的分辨率相对较低，受到我们能够获得的少量粒子图像的限制。在此应用中，我们首先提出大幅提高数据采集效率，以达到优于1nm的分辨率。然后我们将对不同构象状态下的BK通道进行成像。同时，我们将把这些方法应用于研究得最好的电压门控钾通道之一Kv1.2。通过在囊泡中产生膜电位，我们将能够将该通道捕获在其关闭状态和开放状态，以确定其结构。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的电流。由于离子通道种类繁多（超过80种），这些离子通道的缺陷会引起一系列疾病，从癫痫、偏头痛、肌肉麻痹到高血压和心律失常。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来观察其中两个通道在不同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.