Acid-Sensing Ion Channel gating: Conformations and Consequences

项目概要/摘要 细胞外pH是一种高度动态和普遍存在的信号，许多细胞类型表现出鲁棒性。 细胞外酸化后的电生理反应，特别是在神经系统中。酸敏感 离子通道（ASIC）被认为介导大多数这些反应，因为各种ASIC亚基被 在许多神经元类型中以高水平表达，并且ASIC亚基的遗传消融显著降低了 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，在这方面， ASIC不仅仅是一种“细胞外酸警报”。基因缺失或药理学操作， 特定的ASIC亚基可以产生广泛的表型，从减弱的恐惧学习，缺陷， 在疼痛和机械感觉，心脏自主调节和压力反射的问题和损害， 各种癌细胞中上皮细胞向间质细胞的转化。ASIC的这些无数角色促使了 结构和生物物理研究，导致鸡ASIC 1在休息时的晶体或冷冻-EM结构， 毒素稳定的开放和脱敏状态。这些结构数据，结合功能实验， 从而得出了ASIC功能的大致轮廓。简而言之，细胞外蛋白中关键酸性残基的质子化 域，在被称为棕榈域和酸性口袋的区域，导致了全球的重排， 细胞外结构域以及通道门控。然而，我们缺乏一个清晰的图片的分子事件连接 质子化与这些观察到的构象变化和激活或脱敏。为了弥补这一差距 据我们所知，我们将把联合收割机电生理学与光响应非规范氨基的能力结合起来， 酸来测试质子化门控耦合的特定分子假设。此外，没有 ASIC胞内结构域的结构数据。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映射的整体拓扑结构和运动的 使用位点特异性标记的创新方法，对细胞内结构域进行了研究。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，我们缺乏一个清晰的画面，如何ASIC支架与 其他蛋白质。我们将使用靶向蛋白标记和下游分析来解决这一最后的知识差距 的互动。总之，这些拟议的实验将提供新的见解，这些操作 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.