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.

项目摘要/摘要 酸感应离子通道1a（ASIC1A）在整个中心神经和周围神经均表达 系统并已与各种神经过程有关。此同型频道响应 通过快速激活向内阳离子电流，然后快速脱敏。最多 最近，ASIC1A已成为突触可塑性的调节剂以及重要的治疗靶标 用于防止缺血引起的中枢神经系统损害，大脑和创伤性大脑 受伤。重要的是，静止（封闭）ASIC1A通道的高分辨率结构和详细的结构 了解通道依赖pH的门控机制的理解仍然难以捉摸。总体目标 该建议是在我们对结构，功能和调制的理解中解决这些主要差距 ASIC1A频道。以前解决的ASIC1A的晶体结构突出显示了不同的结构构象 与开放和脱敏的功能状态相关。这些结果表明， 三聚体通道，主要是拇指，棕榈和腕带，在结构上依赖于状态和状态依赖 行为对于通道门控可能很重要。另外，尽管ASIC1A主要是Na+选择性，但 通道略微渗透到细胞外Ca2+的调节。有趣的是，这三个上述 门控域已与ASIC1A活性的Ca2+依赖性调制有关。目前是 认为这些依赖于CA2+的调节作用可能具有破坏通道门控的根源 力学。然而，该调制的详细分子机制尚未确定。这 该建议的主要目标是利用X射线晶体学实验来确定高pH值 ASIC1A的状态结构，Ca2+结合位点的位置和通道的pH依赖性门 机制。为了支持这个目标，全细胞贴片夹电生理学和等温组合 滴定量热法将用于进一步表征ASIC1A和 细胞外Ca2+。膜蛋白结构生物学的固有困难使得完整的结构 涵盖离子通道的所有功能状态的表示非常罕见。在基本层面上我是工作 提议将扩大我们对离子渠道结构/功能关系的了解并改善我们的 了解中枢神经系统突触中存在的高度复杂的调节机制。 此外，从该提案中获得的信息可能会为开发的详细信息提供重要的详细信息 神经保护剂用于治疗与中央缺血有关的疾病，包括斯托克和创伤 脑损伤。