Structural Mechanisms of Acid Sensing Ion Channels

项目总结 伤害感是感觉神经元检测痛苦刺激的过程。伤害性感受器激活可以 引发急性疼痛反应，并产生局部炎症，导致组织酸中毒。这一削减 PH值与口腔面部包括冠周炎在内的一系列病理生理反应有关， 牙髓炎和偏头痛。酸敏离子通道(ASIC)是一种分子质子传感器，在 对这种胞外酸化的反应。ASIC在外周和外周血中广泛表达 中枢神经系统(PNS/CNS)。在三叉神经节内，ASIC存在于三叉神经节(TG)神经元中。 神经支配牙髓、面部皮肤和牙周Ruffini末端。有证据表明ASIC3的表达 大鼠实验性牙痛与口面部疼痛的关系可用ASIC缓解 拮抗剂APETx2和阿米洛利。ASICS参与三叉神经节内的伤害性感觉和机械感觉 神经元使其成为一个新的止痛靶点，但关于ASICs的分子仍有许多需要阐明 在强健和有效的药物能够被开发出来之前的机制。虽然ASIC的结构已经被解决， 在原子细节上揭示了这种通道的三聚体性质，它们缺乏细胞内末端。使用小说 荧光方法，如用非天然氨基酸和过渡金属离子FRET进行特异性标记 伴随着电生理学，特殊目标1试图检查分子内的动态重排 通道激活过程中的细胞内域。这套实验将通过回答以下问题来填补空白 ASIC结构没有的问题。我将寻求确定n-的动态重排 通道功能期间的终端。我将把渠道结构中的这些重新安排分配给 该通道采用膜片钳电生理。此外，我将测试一些假设，由 晶体结构。在ASIC的跨膜结构域中存在一种不寻常的结构域交换结构 一些，但不是全部，晶体结构。我可以使用我的新方法来测试这种掉期是否真实存在。 膜。在目标2中，我将试图了解ASIC的异构体组装。文学作品 压倒性的专注于同种异构体，可能是因为一次研究一个亚基很容易。这个 ASIC异构体的生理相关性使其成为研究的关键。然而，异构体的结果 研究很难解释，通常是因为渠道化学计量学未知。《特定目标2》将尝试 勾勒出ASIC异构化的规律。这些实验将开始回答问题，这些问题应该 推动除同分异构体以外的其他异构体的未来研究。是否优先形成异构体，无论是 是2：1还是1：2，或者是非特定的？通道上的哪些位点负责异构化？这些 这些发现将有助于阐明ASIC门控的分子机制，并为我们提供对ASIC门控的新认识。 这些通道的异构体组装。我的目标是最终为针对ASIC的药物设计提供信息 治疗炎症性口腔面部疼痛。

PROJECT SUMMARY Nociception is the process by which sensory neurons detect painful stimuli. Nociceptor activation can initiate both an acute pain response and produce local inflammation leading to tissue acidosis. This reduction in pH is associated with a range of pathophysiological responses in the orofacial region including pericoronitis, pulpitis and migraine. Acid sensing ion channels (ASICs) are molecular proton sensors that are activated in response to this extracellular acidification. ASICs are widely expressed throughout both the peripheral and central nervous systems (PNS/CNS). In the PNS, ASICs are found in the trigeminal ganglion (TG) neurons that innervate tooth pulp, facial skin and periodontal Ruffini endings. There is evidence that ASIC3 expression correlates with orofacial pain following experimental tooth pain in rats, which could be relieved with ASIC antagonists, APETx2 and amiloride. ASICs involvement in nociception and mechanosensation within TG neurons make it a novel analgesic target, but much remains to be elucidated about ASICs molecular mechanisms before robust and effective drugs can be developed. While structures of ASIC have been solved, revealing in atomic detail the trimeric nature of this channel, they lack the intracellular termini. Using novel fluorescence methodologies like specific labeling with an unnatural amino acid and transition metal ion FRET accompanied by electrophysiology, specific aim 1 seeks to examine intramolecular dynamic rearrangements of the intracellular domains during channel activation. This set of experiments will fill a void by answering questions that the ASIC structures do not. I will seek to determine the dynamic rearrangements of the n- terminus during channel function. I will assign these rearrangements in channel structure to functional states of the channel using patch clamp electrophysiology. In addition, I will test some hypotheses, put forth by the crystal structures. There is an unusual domain swapped architecture in the transmembrane domains of ASIC in some, but not all, crystal structures. I can use my novel approach to test for the presence of this swap in real membranes. In aim 2, I will seek to understand the heteromeric assembly of ASICs. The literature overwhelmingly focuses on homomers likely due to the ease of studying one subunit at a time. The physiological relevance of ASIC heteromers makes them critical to study. However, results from heteromeric studies are challenging to interpret, often because channel stoichiometry is unknown. Specific aim 2 will try to delineate the rules of ASIC heteromerization. These experiments will begin to answer questions which should motivate future studies of heteromers in addition to homomers. Do heteromers form preferentially, whether that be 2:1 or 1:2, or is it nonspecific? What sites on the channel are responsible for heteromerization? These findings will help elucidate the molecular mechanisms of ASIC gating and provide a new understanding on the heteromeric assembly of these channels. My goal is to ultimately inform drug design targeting ASIC as a treatment for inflammatory orofacial pain.