Gating Mechanisms of Retinal Rod cGMP Activated Channels

视网膜杆 cGMP 激活通道的门控机制

• 批准号: 8265002
• 负责人: William N Zagotta
• 金额: $ 33.36万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-01-01 至 2014-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8265002
• 关键词: Anosmia; Behavior; Behavior Control; Binding; Blindness; Boxing; Brain; Calmodulin; Cations; Cell Membrane Proteins; Cells; Cellular Membrane; Coupled; Couples; Cyclic AMP; Cyclic GMP; Cyclic Nucleotides; DNA Sequence Rearrangement; Development; Fluorescence; Fluorometry; Funding; Gated Ion Channel; Goals; Grant; Health; Ion Channel; Ions; Label; Ligand Binding; Ligands; Light; Lipids; Measurement; Measures; Membrane; Metals; Methods; Molecular; Molecular Machines; Motion; Mutation; Phosphatidylinositol 4,5-Diphosphate; Phosphatidylinositols; Photoreceptors; Process; Proteins; Relative (related person); Resolution; Role; Second Messenger Systems; Sensory Disorders; Signal Transduction; Smell Perception; Solutions; Subcellular structure; Taste Perception; Testing; Thinking; Time; Transistors; Vertebrate Photoreceptors; Vision; Visual Perception; Work; cyclic-nucleotide gated ion channels; method development; molecular dynamics; molecular rearrangement; nervous system disorder; novel; novel strategies; olfactory receptor; patch clamp; protein function; research study; response; retinal rods; second messenger

DESCRIPTION (provided by applicant): Ion channels are exquisite molecular machines. By opening and closing an ion selective pore across the cell membrane, these proteins ultimately control everything from our senses to our thoughts. Our long term goal is to understand the precise molecular motions that underlie this gating behavior of ion channels. Cyclic nucleotide-gated (CNG) channels produce the primary electrical signal in our photoreceptors in response to light. They are nonselective cation channels that are opened by the direct binding of cyclic nucleotides (cAMP and cGMP) to the channel and modulated by various second messengers. In addition to their role in vision, they are also essential for olfaction and taste, and mutations in these channels cause an assortment of sensory disorders ranging from blindness to anosmia. Their dynamic behavior controls our visual perception, yet the molecular mechanism for their function is largely unknown. This void is due, in part, to a lack of experimental approaches that allow us to "watch" proteins in action in real time at atomic resolution. We aim to fill this void by developing novel fluorescence approaches and applying them to investigate the mechanisms of activation and modulation of CNG channels. We will take advantage of two exciting new developments: 1) our solution of the x-ray crystal structures of the intracellular ligand binding and gating domains of the closely related HCN2 and SpIH channels, and 2) our development of methods for simultaneous current and fluorescence measurements from cell-free membrane patches (termed patch-clamp fluorometry, PCF). Our specific aims are to precisely determine the molecular rearrangement in two important parts of the channel, the cyclic nucleotide- binding domain, and the C-linker, the region that couples binding of cyclic nucleotides to opening of the pore. At the conclusion of these experiments we will know a great deal more about how CNG and related channels work, and will have fully developed new approaches to studying molecular rearrangements applicable to other channels and other proteins. PUBLIC HEALTH RELEVANCE: Ion channels are the transistors of the brain and thereby control everything from our senses to our thoughts. Cyclic nucleotide-gated (CNG) channels produce the primary electrical signal in our rods and cones in response to light, and mutations in these channels cause an assortment of sensory disorders ranging from blindness to anosmia. Our long term goal is to understand how these important proteins work at the molecular level to uncover basic mechanisms of protein function and enable us to develop targeted therapies for neurological diseases.

描述（由申请人提供）：离子通道是精美的分子机。通过打开和关闭整个细胞膜的离子选择性孔，这些蛋白质最终可以控制从我们的感官到思想的一切。我们的长期目标是了解离子通道这种门控行为的确切分子运动。循环核苷酸门控（CNG）通道响应光照响应光，在我们的光感受器中产生主要的电信号。它们是非选择性阳离子通道，通过环状核苷酸（CAMP和CGMP）与通道的直接结合开放，并由各种第二使者调节。除了它们在视觉中的作用外，它们对于嗅觉和口味也是必不可少的，这些渠道中的突变会导致各种感觉障碍，从失明到厌食。他们的动态行为控制着我们的视觉感知，但是其功能的分子机制在很大程度上尚不清楚。该空白部分是由于缺乏实验方法，使我们可以在原子分辨率下实时“观察”蛋白质。我们的目的是通过开发新型的荧光方法来填补这一空隙，并应用它们来研究CNG通道的激活和调节机制。我们将利用两个令人兴奋的新事态发展：1）我们对密切相关的HCN2和SPIH渠道的细胞内配体结合和门控域的X射线晶体结构的解决方案，以及2）我们开发的方法开发了我们的同时电流和荧光测量方法的开发，该方法是无需用培养基贴片贴片贴片贴片（称为贴片斑块荧光纤维纤维计量）。我们的具体目的是精确确定通道两个重要部分的分子重排，环状核苷酸结合结构域和c-linker，c-linker是伴侣结合环核苷酸与孔隙打开的区域。在这些实验的结论中，我们将了解有关CNG和相关渠道如何工作的更多信息，并将为研究适用于其他通道和其他蛋白质的分子重排的新方法。公共卫生相关性：离子渠道是大脑的晶体管，因此控制了从我们的感官到思想的一切。循环核苷酸门控（CNG）通道响应光照响应光，在我们的棒和锥中产生主要的电信号，这些通道中的突变会导致各种感觉障碍，从失明到厌食。我们的长期目标是了解这些重要的蛋白质如何在分子水平上起作用，以发现蛋白质功能的基本机制，并使我们能够开发针对神经系统疾病的靶向疗法。