Fluctuations in Ionic Current Through Membrane Channels

通过膜通道的离子电流的波动

• 批准号: 8096614
• 负责人: FREDERICK J SIGWORTH
• 金额: $ 40.8万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1984
• 资助国家: 美国
• 起止时间: 1984-12-01 至 2013-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8096614
• 关键词: Address; Area; Binding; Brain; Code; Collection; Cryoelectron Microscopy; Data Collection; Data Set; Defect; Diffusion; Disease; Electron Microscope; Electron Microscopy; Epilepsy; Family; Freezing; Gated Ion Channel; Genes; Health; Heart; Human Genome; Hypertension; Image; Inferior; Ion Channel; Kv1.2' channel; Lipids; Liposomes; Macromolecular Complexes; Maps; Mediating; Membrane Potentials; Membrane Proteins; Methods; Migraine; Modality; Molecular; Molecular Conformation; Molecular Structure; Muscle; Organ; Pain; Paralysed; Pharmacotherapy; Potassium; Process; Proteins; Relative (related person); Research; Resolution; Rest; Roentgen Rays; Role; Sensory; Signal Transduction; Solutions; Specific qualifier value; Structural Models; Structure; Techniques; Technology; Testing; Time; Validation; Vesicle; Voltage-Gated Potassium Channel; Work; X-Ray Crystallography; alpha helix; density; heart rhythm; image processing; improved; interest; large-conductance calcium-activated potassium channels; lymphocyte proliferation; method development; novel; particle; protein structure; reconstitution; reconstruction; sensor; simulation; three dimensional structure; voltage; voltage gated channel

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.

描述(申请人提供)：人类基因组中80多个基因编码电压门控离子通道及其6TM离子通道家族中的近亲。在它们的众多作用中，这些通道介导疼痛和其他感官方式，在大脑中执行长途信号，为心跳计时，并控制淋巴细胞增殖。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，也作为对膜电位变化具有独特高度敏感性的蛋白质而引起人们的内在兴趣。单粒子电子冷冻显微镜(Cryo-EM)是一种观察大分子络合物三维结构的方法。尽管分辨率不如X射线结晶学，但低温电磁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不需要形成晶体的巨大优势。我们发展了重建为脂质体的膜蛋白的冷冻-EM单粒子成像方法，最近获得了真核细胞6TM通道的第一个闭态结构-大电导钙激活钾通道(BK)。该结构的分辨率相对较低，受我们能够获取的粒子图像数量较少的限制。在这一应用中，我们首先建议大幅提高数据采集效率，以达到优于1 nm的分辨率。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，这是研究最深入的电压门控钾通道之一。通过在囊泡中产生膜电位，我们将能够捕获该通道在其闭合和开放状态下进行结构确定。与公共健康相关：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。由于有许多(80多种)品种，这些离子通道的缺陷会导致一系列疾病，从癫痫、偏头痛、肌肉瘫痪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不同功能状态下的分子结构。