Acid-Sensing Ion Channel gating: Conformations and Consequences

酸敏感离子通道门控：构象和后果

• 批准号: 10654874
• 负责人: David Malcom MacLean
• 金额: $ 38.5万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-01 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10654874
• 关键词: ASIC channel; Ablation; Acids; Address; Amino Acids; Attenuated; Baroreflex; Basic Science; Biophysics; Brain; Cardiac; Cell Death; Chickens; Coupling; Cryoelectron Microscopy; Data; Drug Design; Electrophysiology (science); Event; Exhibits; Extracellular Domain; Fluorescence Resonance Energy Transfer; Fright; Genetic; Impairment; Investigation; Ion Channel Gating; Ion Channel Protein; Ischemic Stroke; Knowledge; Label; Learning; Link; Maps; Mediating; Membrane Proteins; Methods; Molecular; Molecular Conformation; Motion; Movement; Nervous System; Neurons; Optical Methods; Pain; Phenotype; Proteins; Regulation; Reporting; Rest; Role; Signal Transduction; Site; Structure; Testing; Toxin; cancer cell; cell type; desensitization; drug development; epithelial to mesenchymal transition; experimental study; extracellular; fluorescence lifetime imaging; innovation; insight; operation; patch clamp; pharmacologic; protein protein interaction; protonation; response; scaffold; therapeutic development; ASIC channel; Ablation; Acids; Address; Amino Acids; Attenuated; Baroreflex; Basic Science; Biophysics; Brain; Cardiac; Cell Death; Chickens; Coupling; Cryoelectron Microscopy; Data; Drug Design; Electrophysiology (science); Event; Exhibits; Extracellular Domain; Fluorescence Resonance Energy Transfer; Fright; Genetic; Impairment; Investigation; Ion Channel Gating; Ion Channel Protein; Ischemic Stroke; Knowledge; Label; Learning; Link; Maps; Mediating; Membrane Proteins; Methods; Molecular; Molecular Conformation; Motion; Movement; Nervous System; Neurons; Optical Methods; Pain; Phenotype; Proteins; Regulation; Reporting; Rest; Role; Signal Transduction; Site; Structure; Testing; Toxin; cancer cell; cell type; desensitization; drug development; epithelial to mesenchymal transition; experimental study; extracellular; fluorescence lifetime imaging; innovation; insight; operation; patch clamp; pharmacologic; protein protein interaction; protonation; response; scaffold; therapeutic development

PROJECT SUMMARY/ABSTRACT Extracellular pH is a highly dynamic and ubiquitous signal and many cell types exhibit robust electrophysiological responses upon extracellular acidification, particularly in the nervous system. Acid-sensing ion channels (ASICs) are thought to mediate the majority of these responses since various ASIC subunits are expressed at high levels in many neuronal types and genetic ablation of ASIC subunits dramatically reduces acid-evoked responses. Consequently, ASICs are vital players in cell death following ischemic stroke. However, ASICs are more than simply an `extracellular acid alarm'. Genetic deletion, or pharmacological manipulation, of specific ASIC subunits can produce a wide array of phenotypes ranging from attenuated fear learning, deficits in pain and mechanosensation, problems in cardiac autonomic regulation and the baroreflex and impairment of the epithelial to mesenchymal transition in various cancer cells. These myriad roles of ASICs have motivated structural and biophysical investigation, leading to crystal or cryo-EM structures of chicken ASIC1 in the resting, toxin-stabilized open and desensitized states. This structural data, combined with functional experiments, have led to a general outline of how ASICs function. Briefly, protonation of key acidic residues in the extracellular domain, in regions known as the palm domain and the acidic pocket, leads to global rearrangements of the extracellular domain as well as channel gating. Yet we lack a clear picture of the molecular events linking protonation with these observed conformational changes and activation or desensitization. To address this gap in our knowledge, we will combine electrophysiology with the power of photo-responsive non-canonical amino acids to test specific molecular hypotheses of protonation-gating coupling. In addition, there has been no structural data for the ASIC intracellular domains. To address this gap, we will employ a combination of fluorescence lifetime imaging, as well as patch clamp FRET to map the overall topology and motions of the intracellular domain using innovative methods of site specific labelling. Finally, despite some reports of ASIC protein-protein interactions and signal transduction capacity, we lack a clear picture of how ASICs scaffold with other proteins. We will address this final knowledge gap using targeted protein labeling and downstream analysis of interactions. Taken together, these proposed experiments will provide new insights into the operation of these important signaling entities, and may help guide drug development.

项目摘要/摘要 细胞外pH是一种高度动态和无处不在的信号，许多细胞类型表现出可靠的 细胞外酸化后，特别是在神经系统中的电生理反应。酸性感应 由于各种ASIC亚基是 在许多神经元类型和ASIC亚基的遗传消融中以高水平表达 酸诱发的反应。因此，ASIC是缺血性中风后细胞死亡的重要参与者。然而， ASIC不仅仅是“细胞外酸警报”。遗传缺失或药理学操纵 特定的ASIC亚基可以产生各种各样的表型，从衰减的恐惧学习，缺陷等 在疼痛和机械敏化中，心脏自主神经法规的问题以及压力反射和损害 各种癌细胞中上皮向间充质转变。这些无数ASIC的角色都激发了 结构和生物物理研究，导致静止中鸡Asic1的晶体或冷冻EM结构， 毒素稳定的开放和脱敏状态。这些结构数据与功能实验相结合，具有 导致了ASIC如何运作的一般概述。简而言之，细胞外关键酸性残基的质子化 在称为棕榈域和酸性口袋的区域中，域导致全球重排 细胞外结构域以及通道门控。然而，我们缺乏链接的分子事件的清晰图片 这些观察到的构象变化以及激活或脱敏的质子化。解决这个差距 据我们所知，我们将将电生理学与光响应性非官方氨基的力量相结合 酸测试质子化门控偶联的特定分子假设。另外，没有 ASIC细胞内结构域的结构数据。为了解决这一差距，我们将采用 荧光寿命成像以及斑块夹架，以绘制整体拓扑和动作 使用特定地点标记的创新方法的细胞内结构域。最后，尽管有一些ASIC的报道 蛋白质 - 蛋白质相互作用和信号转导能力，我们缺乏明确的图片，即Asics如何脚手架 其他蛋白质。我们将使用目标蛋白质标签和下游分析来解决最终的知识差距 互动。综上所述，这些提议的实验将为这些操作提供新的见解 重要的信号传导实体，并可能有助于指导药物开发。