Structural energetics of voltage- and ligand-dependent gating in ion channels

离子通道中电压和配体依赖性门控的结构能量学

• 批准号: 10549486
• 负责人: William N Zagotta
• 金额: $ 53.54万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-01-01 至 2027-11-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10549486
• 关键词: Allosteric Regulation; Binding; Biology; Brain; Cell membrane; Cells; Cyclic AMP; Cyclic GMP; Cyclic Nucleotides; Data; Defect; Dependence; Disease; Family; Fluorescence Resonance Energy Transfer; Fluorometry; Four-dimensional; Goals; Heart; Human; Ion Channel; Ions; Ligands; Maps; Measures; Methods; Molecular; Molecular Conformation; Molecular Machines; Molecular Structure; Neurotransmitters; Physiology; Proteins; Resolution; Role; Second Messenger Systems; Site; Stimulus; Time; Work; experimental study; fluorescence lifetime imaging; member; patch clamp; response; small molecule; voltage

Abstract Ion channels are exquisite molecular machines that regulate the flow of ions across cell membranes in response to stimuli such as voltage and small molecule ligands (e.g. second messengers, and neurotransmitters). They underlie all electrical excitability in the brain and heart, and defects in ion channels are responsible for many human disorders. Despite decades of experiments and many high-resolution molecular structures, we still do not know, for any channel, the mechanisms for voltage- or ligand- dependent gating. The missing ingredient seems to be conformational energetics. The energetics of the different channel conformations governs the time course, voltage- dependence, and ligand-dependence of opening of the channel pore, and ultimately electrical excitability of the cell. In this proposal we will determine the mechanisms of voltage-dependent gating and ligand-dependent gating and fill important gaps in our understanding of ion channel biology. We will focus on the cyclic nucleotide-binding domain (CNBD) family of ion channels, which are structurally related, but functionally diverse. Whereas some CNBD channels are activated by depolarization, others are activated by hyperpolarization, and some members are activated by cAMP yet others are activated by cGMP. We will leverage breakthrough FRET methods we developed for measuring intramolecular distance distributions and conformational energetics using fluorescence lifetime imaging microscopy (FLIM), simultaneous with recordings of channel function using patch-clamp fluorometry (PCF). The data from multiple donor- acceptor sites throughout the channels will be compiled into a four-dimensional map (X, Y, Z, and energy) of the conformational rearrangements associated with ligand- dependent and voltage-dependent activation of CNBD channels. Our long-term vision is to understand the general themes that underlie allosteric regulation of ion channels, and these experiments promise rapid progress toward this goal. Ultimately, the methods and principles we discover will be of broad utility for elucidating mechanisms for all allosteric proteins.

抽象的 离子通道是调节离子跨细胞流动的精美分子机器 膜响应刺激（例如电压和小分子配体）（例如， 第二使者和神经递质）。他们是所有电兴奋性的基础 大脑和心脏以及离子渠道中的缺陷是许多人的原因 疾病。尽管实验数十年和许多高分辨率分子 对于任何通道，我们仍然不知道结构的电压或配体机理 依赖的门。缺失的成分似乎是构象的能量学。这 不同渠道构象的能量学控制了时间过程，电压 - 依赖性和通道孔口的配体依赖性，最终 细胞的电兴奋性。在此提案中，我们将确定 依赖电压的门控和配体依赖的门控，并填补我们的重要空白 了解离子通道生物学。我们将专注于环状核苷酸结合 离子通道的域（CNBD）家族在结构上相关，但在功能上 各种各样的。尽管某些CNBD通道被去极化激活，而其他CNBD通道则是 被超极化激活，有些成员被CAMP激活 由CGMP激活。我们将利用我们为 测量分子内距离分布和使用构象的能量学 荧光寿命成像显微镜（FLIM），同时记录 使用Patch-Clamp荧光测定法（PCF）的通道函数。来自多个捐助者的数据 整个通道的受体站点将被编译到四维地图中（x， 与配体相关的构象重排的y，z和能量 CNBD通道的依赖和电压依赖性激活。我们的长期愿景是 了解基于离子渠道的变构调节的一般主题，以及 这些实验有望朝着这一目标迅速进步。最终，方法和 我们发现的原理将是所有变构的阐明机制的广泛实用性 蛋白质。