Structural Mechanisms of Acid Sensing Ion Channels

酸敏感离子通道的结构机制

• 批准号: 10083726
• 负责人: Megan Cullinan
• 金额: $ 3.6万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-02-01 至 2022-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10083726
• 关键词: ASIC channel; Acidosis; Acute Pain; Afferent Neurons; Amiloride; Analgesics; Architecture; Attenuated; Binding; Biophysics; Cartoons; Cell membrane; Color; Crystallization; Data; Dental Pulp; Development; Drug Design; Electrophysiology (science); Extracellular Domain; Fluorescence; Fluorescence Resonance Energy Transfer; Fright; Future; Genes; Genetic; Goals; Incisor; Inflammation; Inflammatory; Institution; Ion Channel; Ion Channel Gating; Ions; Knock-out; Label; Literature; Location; Maps; Measures; Membrane; Memory; Mentors; Methodology; Migraine; Modeling; Molecular; Molecular Conformation; Mus; Nature; Neuraxis; Neurons; Nociception; Nociceptors; Opioid Receptor; Orofacial Pain; Pain; Pericoronitis; Peripheral; Peripheral Nervous System; Pharmaceutical Preparations; Pharmacology; Photobleaching; Physiological; Process; Protein Isoforms; Protons; Pulpitis; Rattus; Research; Resources; Rest; Ruffinis Corpuscles; Site; Sodium; Stimulus; Structure; Structure of trigeminal ganglion; Subcellular structure; Suggestion; Techniques; Testing; Time; Tissues; Tooth structure; Toothache; Training; Transition Elements; Transmembrane Domain; Vascular Smooth Muscle; Work; base; cell type; desensitization; design; experience; experimental study; extracellular; face skin; flexibility; inflammatory pain; inhibitor/antagonist; interest; knock-down; ligand gated channel; mouse model; new therapeutic target; novel; novel strategies; orofacial; patch clamp; pre-doctoral; programs; response; sensor; stoichiometry; unnatural amino acids

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.

项目摘要 伤害感受是感觉神经元检测到疼痛刺激的过程。伤害感受器激活可以 引发急性疼痛反应并产生局部炎症，导致组织酸中毒。这种减少 在pH中，pH与包括上皮炎，包括上皮炎的一系列病理生理反应有关， 肺炎和偏头痛。酸性传感离子通道（ASIC）是分子质子传感器，在 对这种细胞外酸化的反应。 ASIC在周围和 中枢神经系统（PNS/CNS）。在PN中，在三叉神经节（TG）神经元中发现了ASIC 支配牙齿果肉，面部皮肤和牙周rfuffini结尾。有证据表明ASIC3表达 大鼠实验性牙齿疼痛后，与口面疼痛相关，可以通过ASIC缓解 拮抗剂，Apetx2和Amiloride。 ASIC参与TG内的伤害感受和机械感应 神经元使其成为一个新型的镇痛靶标，但关于Asics分子的尚待阐明。 可以开发出鲁棒和有效药物之前的机制。虽然已经解决了ASIC的结构，但 在原子细节中揭示了该通道的三聚体性质，它们缺乏细胞内末端。使用小说 荧光方法，例如具有非天然氨基酸和过渡金属离子fret的特定标记 伴随电生理学，特定目标1试图检查分子内动态重排 通道激活过程中细胞内结构域。这套实验将通过回答填充空隙 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。我将把这些在通道结构中的重排分配给的功能状态 使用斑块夹电生理学的通道。此外，我将检验一些假设 晶体结构。 ASIC的跨膜域中有一个不寻常的域交换结构 有些但不是全部的晶体结构。我可以使用我的新颖方法来测试这种交换的存在 膜。在AIM 2中，我将寻求了解ASIC的异元组装。文学 绝大多数的重点是可能是由于一次研究一个亚基的易于性。这 ASIC异构体的生理相关性使其对学习至关重要。但是，来自异构体的结果 研究通常是挑战性的，通常是因为渠道化学计量尚不清楚。特定目标2将尝试 描述ASIC异构化规则。这些实验将开始回答问题 除同源物外，还可以激励对异构体的未来研究。是否要优先形成异构体 是2：1或1：2，还是非特异性的？渠道上的哪些站点负责异构化？这些 调查结果将有助于阐明ASIC门控的分子机制，并对 这些通道的杂体组装。我的目标是最终将剂量的药物设计定位为ASIC 治疗炎症性口面疼痛。