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: 10437808
• 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/10437808
• 关键词: ASIC channel; Ablation; Acids; Address; Amino Acids; Attenuated; Baroreflex; Basic Science; Biophysics; Brain; Cardiac; Cell Death; Chickens; Coupling; Cryoelectron Microscopy; Crystallization; 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; Lead; Learning; Link; Maps; Mediating; Membrane Proteins; Methods; Molecular; Molecular Conformation; Motion; Movement; Nervous system structure; Neurons; Optical Methods; Pain; Pharmacology; 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; 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.

项目概要/摘要 细胞外 pH 值是一种高度动态且普遍存在的信号，许多细胞类型都表现出强大的稳定性 细胞外酸化时的电生理反应，特别是在神经系统中。酸感应 离子通道 (ASIC) 被认为介导了大部分反应，因为各种 ASIC 亚基 在许多神经元类型中高水平表达，并且 ASIC 亚基的基因消融极大地减少了 酸诱发反应。因此，ASIC 是缺血性中风后细胞死亡的重要参与者。然而， ASIC 不仅仅是一个“细胞外酸警报器”。基因删除或药理操作 特定的 ASIC 亚基可以产生多种表型，包括恐惧学习减弱、缺陷 疼痛和机械感觉、心脏自主调节问题和压力反射以及神经损伤 各种癌细胞中的上皮细胞到间质细胞的转变。 ASIC 的这些无数角色激励了 结构和生物物理研究，得出静息状态下鸡 ASIC1 的晶体或冷冻电镜结构， 毒素稳定的开放状态和脱敏状态。该结构数据与功能实验相结合， 得出 ASIC 功能的总体概述。简而言之，细胞外关键酸性残基的质子化 域，在称为棕榈域和酸性口袋的区域中，导致全局重新排列 细胞外结构域以及通道门控。然而我们缺乏对分子事件的清晰认识 质子化与这些观察到的构象变化和激活或脱敏。为了解决这个差距 据我们所知，我们将把电生理学与光响应非规范氨基的力量结合起来 酸来测试质子化门控耦合的特定分子假设。此外，还没有出现过 ASIC 细胞内域的结构数据。为了解决这一差距，我们将采用以下组合： 荧光寿命成像以及膜片钳 FRET 来绘制整体拓扑结构和运动 使用位点特异性标记的创新方法来识别细胞内结构域。最后，尽管 ASIC 的一些报告 蛋白质-蛋白质相互作用和信号转导能力，我们缺乏关于 ASIC 如何支架的清晰图景 其他蛋白质。我们将使用靶向蛋白质标记和下游分析来解决这个最终的知识差距 的互动。总而言之，这些拟议的实验将为这些操作提供新的见解 重要的信号实体，可能有助于指导药物开发。