Acid-Sensing Ion Channel gating: Conformations and Consequences

项目摘要/摘要 细胞外pH是一个高度动态和普遍存在的信号，许多细胞类型都表现出很强的生命力 细胞外酸化时的电生理反应，特别是在神经系统中。酸敏 离子通道(ASIC)被认为介导了这些反应的大部分，因为各种ASIC亚单位是 在许多神经元类型中高水平表达，ASIC亚单位的遗传消融显著减少 酸引起的反应。因此，ASICs是缺血性卒中后细胞死亡的重要参与者。然而， ASIC不仅仅是一个“胞外酸警报”。基因缺失，或药物操纵， 特定的ASIC亚基可以产生一系列的表型，从减弱的恐惧学习，缺陷 在疼痛和机械感觉中，心脏自主神经调节和压力反射和损伤的问题 多种癌细胞上皮向间充质的转变。ASIC的这些无数角色促使 结构和生物物理研究，导致鸡ASIC1在休眠状态下的晶体或冷冻-EM结构， 毒素稳定的开放和不敏感状态。这些结构数据与功能实验相结合，具有 导致了ASIC如何工作的大体轮廓。简而言之，胞外关键酸性残基的质子化 结构域在被称为棕榈域和酸性口袋的区域中，导致全球范围内 胞外域以及通道门控。然而，我们对分子事件之间的联系缺乏清晰的认识 具有这些观察到的构象变化的质子化和活化或脱敏。为了弥补这一差距 在我们的知识中，我们将结合电生理学和光响应性非正则氨基的力量。 酸来测试质子化-门控耦合的特定分子假说。此外，目前还没有 ASIC细胞内域的结构数据。为了解决这一差距，我们将结合使用 荧光寿命成像，以及膜片钳FRET，以映射整体拓扑和运动 使用创新的位点特异性标记方法进行细胞内结构域标记。最后，尽管有一些ASIC的报道 蛋白质之间的相互作用和信号转导能力，我们缺乏一个清晰的图像ASIC是如何支架 其他蛋白质。我们将通过有针对性的蛋白质标记和下游分析来解决这一最终的知识差距 互动的方式。综上所述，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，并可能有助于指导药物开发。

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.