3D structure and function of Acid Sensing Ion channels

酸敏离子通道的 3D 结构和功能

• 批准号: 9477168
• 负责人: David Malcom MacLean
• 金额: $ 24.9万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-07-01 至 2020-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9477168
• 关键词: ASIC channel; Acute; Award; Awareness; Binding; Biological Assay; Biology; Biophysics; Brain; Cell Death; Cerebrum; Communities; Complex; Comprehension; Cryoelectron Microscopy; Crystallization; Data; Dependence; Disease; Drug Addiction; Electrophysiology (science); Environment; Event; Family; Fluorescence; Fright; Funding; Goals; Healthcare; Image; Injury; Instruction; Knock-out; Knowledge; Learning; Ligands; Location; Mediating; Memory; Methods; Molecular; Molecular Conformation; Mutate; Mutation; Nervous system structure; Neurobiology; Neurons; Outcome; Pain; Pharmaceutical Preparations; Pharmacology; Phase; Physiological; Population; Positioning Attribute; Process; Protons; Reporting; Research; Resolution; Role; Seizures; Stroke; Structure; Synapses; Testing; Therapeutic; Toxin; Training; Translating; Traumatic injury; Work; base; desensitization; drug development; experimental study; extracellular; insight; luminescence resonance energy transfer; operation; programs; protein purification; public health relevance; reconstruction; response; sensor; stoichiometry; three dimensional structure

 DESCRIPTION (provided by applicant): In the brain, many pathophysiological events are accompanied by acidification of the extracellular environment. Unsurprisingly, neurons often show robust electrophysiological responses during such changes in extracellular pH. Acid sensing ion channels (ASICs) mediate the vast majority of these currents and the last few years have seen a substantial increase in our understanding of ASIC neurobiology. Because of this research, ASICs have been implicated in cell death following stroke, in pain, in fear related memory and in drug addiction amongst other disorders. However, we have not seen a corresponding surge in our understanding of how ASICs operate at a molecular level. Without such knowledge, we cannot translate our growing awareness of neurobiology into useful treatments. The candidate will combine his expertise in electrophysiology and fluorescence with new training in cryo-electron microscopy (cryo-EM) to bridge the gap between ASIC neurobiology and biophysics. This will be done through three Aims. During Specific Aim 1 in the K phase, the candidate will obtain high resolution cryo-EM structures of ASIC1a apo and desensitized states. New training in protein purification will be provided from Dr. Jayaraman and ASIC-specific advice from Dr. Gonzales. Dr. Serysheva will provide expert instruction in cryo-EM imaging and 3D reconstruction to a level where the candidate can perform the experiments independently. These structures will provide the first glimpse of the apo state and determine if steady-state desensitization (provoked by pH's near 7) is the same conformation as desensitization following activation (pH's near 5). The R phase will begin with Aim 2 where the candidate and his trainees will use concatenated subunits to determine the stoichiometry of activation and establish if conformational changes are concerted across subunits. In Aim 3, the candidate and his trainees will obtain high resolution cryo-EM images of heteromeric ASICs, the predominant population in the brain. This combination of functional information and structural data will help fulfill the candidates two near term goals of contributing essential insight into th fundamental operation of ASICs and emerging as a leader in the field of ASIC gating. Moreover, these studies will provide preliminary data to develop a competitive R01 application focusing on ASIC heteromeric structure and function. This K99/R00 award will also better position the candidate to satisfy the long term goals of rendering the effect of ASIC mutations or manipulations predictable and to help drive efforts to develop more selective and useful pharmacological agents.

 描述（申请人提供）：在大脑中，许多病理生理事件都伴随着细胞外环境的酸化。毫不奇怪，在细胞外 pH 值发生变化时，神经元通常会表现出强烈的电生理反应。酸传感离子通道 (ASIC) 介导了绝大多数电流，在过去几年中，我们对 ASIC 神经生物学的了解有了大幅增长。由于这项研究，ASIC 与中风后的细胞死亡、疼痛、恐惧相关记忆以及毒瘾等疾病有关。然而，我们对 ASIC 如何在分子水平上运作的理解并没有相应的提高。如果没有这些知识，我们就无法将日益增长的神经生物学意识转化为有用的治疗方法。该候选人将把他在电生理学和荧光方面的专业知识与冷冻电子显微镜 (cryo-EM) 方面的新培训相结合，以弥合 ASIC 神经生物学和生物物理学之间的差距。 这将通过三个目标来实现。在 K 阶段的特定目标 1 期间，考生将获得 ASIC1a apo 和脱敏状态的高分辨率冷冻电镜结构。 Jayaraman 博士将提供蛋白质纯化方面的新培训，Gonzales 博士将提供针对 ASIC 的建议。 Serysheva 博士将提供冷冻电镜成像和 3D 重建方面的专家指导，使候选人能够独立执行实验。这些结构将提供对 apo 状态的第一眼观察，并确定稳态脱敏（由 pH 接近 7 引起）是否与激活后脱敏（pH 接近 5）相同的构象。 R 阶段将从目标 2 开始，候选人和他的学员将使用串联的亚基来确定激活的化学计量，并确定构象变化是否跨亚基协调一致。在目标 3 中，候选人和他的学员将获得异聚 ASIC（大脑中的主要群体）的高分辨率冷冻电镜图像。功能信息和结构数据的结合将有助于候选人实现两个近期目标：对 ASIC 的基本操作提供重要的见解，并成为 ASIC 门控领域的领导者。此外，这些研究将为开发专注于 ASIC 异聚结构和功能的有竞争力的 R01 应用提供初步数据。该 K99/R00 奖项还将让候选人更好地满足使 ASIC 突变或操作的效果可预测的长期目标，并帮助推动开发更具选择性和有用的药物制剂。