Structural dynamics in cyclic nucleotide-modulated channels

环核苷酸调节通道的结构动力学

• 批准号: 9368089
• 负责人: Crina M Nimigean
• 金额: $ 40.26万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-01 至 2021-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9368089
• 关键词: Arrhythmia; Atomic Force Microscopy; Bacteria; Binding; Biological Assay; Brain; Buffers; Classification; Color blindness; Cryoelectron Microscopy; Cyclic AMP; Cyclic GMP; Cyclic Nucleotides; Cytoplasmic Tail; Defect; Disease; Electrophysiology (science); Environment; Epilepsy; Equilibrium; Goals; Grant; Heart; Homologous Gene; Image; Imagery; Ion Channel; Kinetics; Lasers; Lead; Length; Ligand Binding; Ligands; Lipid Bilayers; Lipids; Liposomes; Maintenance; Measures; Methods; Modality; Modeling; Molecular; Molecular Conformation; Monitor; Nervous system structure; Perfusion; Physiological; Physiological Processes; Physiology; Play; Probability; Research; Resolution; Role; Signal Transduction; Speed; Structure; Surface; System; Techniques; Temperature; Time; Visual; Visual Signal Transduction Pathway; cyclic-nucleotide gated ion channels; design; experimental study; insight; member; microscopic imaging; nanodisk; novel; novel therapeutics; particle; pressure; reconstitution; response; stopped-flow fluorescence

Cyclic nucleotide-modulated channels play major roles in pacemaking activity in heart and brain as well as in olfactory and visual signal transduction in the nervous system. Defects in the functioning of these channels lead to diseases such as epilepsy, cardiac arrhythmia, and color blindness. The overall objective of this grant is to understand how binding of cyclic nucleotides gates (opens/closes) the channels at the molecular level and how lipids modulate the open-closed equilibrium. We will accomplish this by combining state-of-the-art techniques like single-particle cryo electron microscopy (croEM) with high-speed atomic force microscopy (HS-AFM) and functional assays like single-channel electrophysiology and stopped flow fluorescence assays. Our first aim is to determine using single-particle cryoEM high-resolution structures of these channels, in different conformations (no ligand bound, cAMP-bound, cGMP-bound) and with different lipids (by varying the lipid composition in nanodiscs) and assigning these structures to specific ion channel functional states determined using single-channel electrophysiology. Using lipid bilayer single-channel recordings of the channels in various lipid environments, we will assign functional states (open, closed, etc) to the structures and conformations obtained. This aim will not only yield the first ever atomic-resolution structure of a cyclic nucleotide- modulated channel but also structures of other conformations that will allow us to initiate the building of a structural gating model. Our second aim is to determine the various channel conformations in close-to-native conditions (channels reconstituted in lipid bilayers and in physiological buffer at ambient temperature and pressure) using AFM imaging (including HS-AFM). We will determine the conformational landscape at steady states (i.e. in the presence or absence of ligand) as well as the conformational changes that these channels undergo in real time upon ligand binding and how the equilibrium changes with different lipids. We will directly compare these conformations with those obtained by cryoEM in Aim 1. Using stopped-flow fluorescence macroscopic assays of the channels in liposomes, we will investigate how the activation/inactivation kinetics compares with the real time conformational dynamics measured by HS-AFM. The final goal is to formulate structural gating models for cyclic nucleotide-modulated channels using the conformations determined in aims 1 and 2 with assigned functional states.

环核苷酸调节通道在心脏和大脑的起搏活动以及 神经系统中的嗅觉和视觉信号转导。这些渠道的功能缺陷会导致 癫痫、心律失常和色盲等疾病。这笔赠款的总体目标是 了解环核苷酸的结合如何在分子水平上门（打开/关闭）通道以及如何 脂质调节开闭平衡。我们将通过结合最先进的技术来实现这一目标 例如单粒子冷冻电子显微镜 (croEM) 和高速原子力显微镜 (HS-AFM) 以及 功能测定，如单通道电生理学和停流荧光测定。我们的首要目标是 使用单粒子冷冻电镜确定这些通道的高分辨率结构，在不同的 构象（无配体结合、cAMP 结合、cGMP 结合）和不同的脂质（通过改变脂质 纳米圆盘中的成分）并将这些结构分配给确定的特定离子通道功能状态 使用单通道电生理学。使用脂质双层单通道记录 各种脂质环境，我们将为结构分配功能状态（开放、封闭等） 获得的构象。这一目标不仅将产生第一个原子分辨率的环状核苷酸结构 调制通道以及其他构象的结构，这将使我们能够开始构建 结构门控模型。我们的第二个目标是确定接近原生的各种通道构象 条件（在环境温度和生理缓冲液中在脂质双层和生理缓冲液中重建的通道） 压力）使用 AFM 成像（包括 HS-AFM）。我们将确定稳定时的构象景观 状态（即存在或不存在配体）以及这些通道的构象变化 配体结合时实时发生以及平衡如何随不同脂质而变化。我们将直接 将这些构象与目标 1 中通过冷冻电镜获得的构象进行比较。使用停流荧光 脂质体通道的宏观测定，我们将研究激活/失活动力学如何 与 HS-AFM 测量的实时构象动力学进行比较。最终目标是制定 使用目标 1 中确定的构象的环核苷酸调节通道的结构门控模型 2 个具有指定的功能状态。