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在细胞内流动的精致分子机器 膜响应电压和小分子配体（例如 第二信使和神经递质）。它们是所有电兴奋性的基础 大脑和心脏以及离子通道的缺陷是造成许多人类疾病的原因 失调。尽管进行了数十年的实验和许多高分辨率分子 结构，我们仍然不知道，对于任何通道，电压或配体的机制 依赖门控。缺失的成分似乎是构象能量学。这 不同通道构象的能量控制着时间进程，电压 通道孔开放的依赖性和配体依赖性，最终 细胞的电兴奋性。在本提案中，我们将确定以下机制： 电压依赖性门控和配体依赖性门控，填补了我们的重要空白 了解离子通道生物学。我们将重点关注环核苷酸结合 离子通道结构域（CNBD）家族，它们在结构上相关，但在功能上 各种各样的。虽然一些 CNBD 通道是通过去极化激活的，但其他通道是通过去极化激活的。 被超极化激活，一些成员被 cAMP 激活，还有一些成员被 cAMP 激活 被 cGMP 激活。我们将利用我们开发的突破性 FRET 方法 使用测量分子内距离分布和构象能量学 荧光寿命成像显微镜（FLIM），同时记录 使用膜片钳荧光测定法 (PCF) 进行通道功能。来自多个捐助者的数据- 整个通道的受体位点将被编译成四维图（X， Y、Z 和能量）与配体相关的构象重排 CNBD 通道的依赖性和电压依赖性激活。我们的长期愿景是 了解离子通道变构调节的一般主题，以及 这些实验有望在实现这一目标方面取得快速进展。最终，方法和 我们发现的原理对于阐明所有变构机制具有广泛的用途 蛋白质。