Structural dynamics in cyclic nucleotide-modulated channels

环核苷酸调节通道在心脏和大脑的起搏活动中起重要作用， 神经系统中的嗅觉和视觉信号传导。这些渠道的功能缺陷导致 癫痫、心律不齐和色盲等疾病。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控制（打开/关闭）通道以及如何 脂质调节开闭平衡。我们将结合最先进的技术来实现这一目标 比如单粒子低温电子显微镜（croEM）和高速原子力显微镜（HS-AFM）， 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 为了确定这些通道的单粒子cryoEM高分辨率结构，在不同的 构象（无配体结合、cAMP结合、cGMP结合）和不同脂质（通过改变脂质 纳米盘中的组成）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中的通道， 各种脂质环境，我们将分配功能状态（开放，关闭，等）的结构， 获得的构象。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率结构， 调制的通道，但也有其他构象的结构，这将使我们能够启动建设一个 结构门控模型我们的第二个目标是确定各种通道构象在接近天然的 条件（在脂质双层和生理缓冲液中在环境温度下重构的通道， 使用AFM成像（包括HS-AFM）。我们将确定稳定的构象景观 状态（即在存在或不存在配体的情况下）以及这些通道的构象变化 在配体结合时经历真实的时间，以及不同脂质的平衡如何变化。我们会直接 将这些构象与目标1中通过cryoEM获得的构象进行比较。使用停流荧光 宏观分析的通道在脂质体中，我们将研究如何激活/失活动力学 与HS-AFM测量的真实的时间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节通道的结构门控模型 和2个具有指定的功能状态。

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.