Structural dynamics in cyclic nucleotide-modulated channels

环核苷酸调节通道的结构动力学

• 批准号: 9894550
• 负责人: Crina M Nimigean
• 金额: $ 10.05万
• 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/9894550
• 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

ABSTRACT 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.

摘要 环核苷酸调节的通道在心脏和大脑的起搏活动中起主要作用 神经系统中的嗅觉和视觉信号转导。这些渠道的功能缺陷导致 到癫痫、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打开/关闭通道以及如何 脂类调节开合平衡。我们将通过结合最先进的技术来实现这一点 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 功能分析，如单通道电生理和停流荧光分析。我们的首要目标是 为了确定使用单粒子低温电磁高分辨率结构的这些通道，在不同的 构象(无配体结合、cAMP结合、cGMP结合)和具有不同脂类(通过改变脂类 纳米盘中的组合物)，并将这些结构分配给所确定的特定离子通道功能状态 使用单通道电生理学。使用脂类双层单通道记录在 不同的脂质环境，我们将分配功能状态(打开，关闭等)的结构和 获得了构象。这一目标不仅将产生有史以来第一个环核苷酸的原子分辨结构- 调制的通道，以及其他构象的结构，使我们能够启动 结构浇注模型。我们的第二个目标是确定接近天然的各种通道构象 条件(在常温下，在脂质双层和生理缓冲液中重建的通道和 压力)使用AFM成像(包括HS-AFM)。我们将稳定地确定构象地貌 状态(即存在或不存在配体)以及这些通道的构象变化 实时进行配基结合，以及平衡如何随不同的脂类发生变化。我们会直接 将这些构象与目标1中的CryoEM获得的构象进行比较。使用停流荧光 脂质体中通道的宏观分析，我们将研究激活/失活动力学 与HS-AFM实时测量的构象动力学进行了比较。最终目标是制定 使用AIMS 1中确定的构象的环核苷酸调制通道的结构门控模型 2个具有指定的功能状态。