Structure of an acid sensing ion channel in a resting state at high pH.

高 pH 下静息状态的酸传感离子通道的结构。

• 批准号: 9326027
• 负责人: Nathan Yoder
• 金额: $ 4.4万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-01 至 2019-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9326027
• 关键词: ASIC channel; Acidosis; Address; Affinity; Behavior; Binding; Binding Sites; Biological Assay; Calorimetry; Cations; Cells; Complex; Coupled; Crystallization; Data; Development; Divalent Cations; Electrophysiology (science); Exhibits; Family; Glutamates; Goals; Homo; Impairment; Ion Channel; Ions; Ischemia; Knowledge; Learning; Ligands; Location; Maps; Mechanics; Mediating; Membrane Proteins; Memory; Modeling; Molecular; Molecular Conformation; Neuraxis; Neurologic Process; Neuronal Injury; Neuroprotective Agents; Nociception; Pathologic; Pathologic Processes; Pathology; Peripheral Nervous System; Permeability; Physiological Processes; Plant Roots; Prevention; Protons; Regulation; Research; Resolution; Rest; Role; Signal Transduction; Site; Site-Directed Mutagenesis; Stroke; Structural Models; Structure; Structure-Activity Relationship; Synapses; Synaptic plasticity; Testing; Thermodynamics; Thumb structure; Titrations; Traumatic Brain Injury; Validation; Vestibule; Work; Wrist; X-Ray Crystallography; base; desensitization; experimental study; extracellular; improved; insight; member; patch clamp; prevent; stoichiometry; structural biology; therapeutic target

Project Summary/Abstract The Acid Sensing Ion Channel 1a (ASIC1a) is expressed throughout both central and peripheral nervous systems and has been implicated in a variety of neurological processes. This homotrimeric channel responds to extracellular acidosis with fast activation of an inward cationic current followed by rapid desensitization. Most recently, ASIC1a has emerged as a regulator of synaptic plasticity as well as an important therapeutic target for preventing ischemia-induced central nervous system damage common to both stoke and traumatic brain injury. Importantly, a high-resolution structure of the resting (closed) ASIC1a channel and a detailed understanding of the channel's pH-dependent gating mechanism have remained elusive. The overall goal of this proposal is to address these major gaps in our understanding of the structure, function, and modulation of the ASIC1a channel. Previously solved crystal structures of ASIC1a highlight distinct structural conformations associated with both open and desensitized functional states. These results demonstrated that regions of the trimeric channel, primarily thumb, palm, and wrist domains, exhibit structurally dynamic and state-dependent behavior potentially important for channel gating. Additionally, though ASIC1a is primarily Na+-selective, the channel is slightly permeable to and modulated by extracellular Ca2+. Intriguingly, all three above-mentioned gating domains have been implicated in the Ca2+-dependent modulation of ASIC1a activity. It is currently thought that these Ca2+-dependent modulatory effects may have their roots in disruption of channel gating mechanics. A detailed molecular mechanism for this modulation, however, has yet to be determined. The primary goal of this proposal is to utilize x-ray crystallography experiments to determine the high pH, resting state structure of ASIC1a, the location of Ca2+ binding sites, and the channel's pH-dependent gating mechanism. In support of this goal, a combination of whole-cell patch clamp electrophysiology and isothermal titration calorimetry will be used to further characterize the modulatory interaction between ASIC1a and extracellular Ca2+. The inherent difficultly of membrane protein structural biology makes a complete structural representation covering all functional states of an ion channel very rare. At a basic level the work I am proposing will expand our knowledge of ion channel structure/function relationships and improve our understanding of the highly complex regulatory mechanisms present at central nervous system synapses. Additionally, the information gained from this proposal could provide details important for the development of neuroprotective agents used to treat conditions associated with central ischemia including stoke and traumatic brain injury.

项目总结/摘要 酸敏感离子通道1a（ASIC 1a）在中枢和外周神经系统中都有表达。 系统，并已牵连在各种神经过程。这种同源三聚体通道响应 细胞外酸中毒伴随着内向阳离子电流的快速激活和随后的快速脱敏。最 近年来，ASIC 1a已成为突触可塑性的调节因子和重要的治疗靶点 用于预防斯托克和创伤性脑常见的缺血诱导的中枢神经系统损伤 损伤重要的是，静息（闭合）ASIC 1a通道的高分辨率结构和详细的 对通道的pH依赖性门控机制的理解仍然是难以捉摸的。的总目标 这一建议是为了解决我们在理解的结构，功能和调制的这些主要差距， ASIC 1a通道以前解决的ASIC 1a的晶体结构突出了不同的结构构象 与开放和脱敏功能状态相关。这些结果表明， 三聚体通道，主要是拇指，手掌和手腕域，表现出结构动态和状态依赖性 对于通道选通潜在重要的行为。此外，尽管ASIC 1a主要是Na+选择性的， 通道对细胞外Ca 2+具有轻微的渗透性并受其调节。有趣的是，上述三者 门控结构域与ASIC 1a活性的Ca 2+依赖性调节有关。目前 我认为这些依赖于钙离子的调节作用可能源于通道门控的破坏 力学然而，这种调制的详细分子机制尚未确定。的 该提案的主要目标是利用X射线晶体学实验来确定高pH值、静息 ASIC 1a的状态结构、Ca 2+结合位点的位置和通道的pH依赖性门控 机制为了支持这一目标，结合全细胞膜片钳电生理学和等温 滴定量热法将用于进一步表征ASIC 1a和 细胞外Ca ~（2+）。膜蛋白结构生物学的固有困难使得一个完整的结构 覆盖离子通道所有功能状态的表示非常罕见。在一个基本的层面上， 提出将扩大我们对离子通道结构/功能关系的了解，并提高我们的 了解中枢神经系统突触高度复杂的调节机制。 此外，从这一提案中获得的信息可以为制定 用于治疗与中枢缺血有关的病症的神经保护剂，包括斯托克和创伤性 脑损伤