Molecular mechanisms of TRPA1 regulation

项目总结 山葵受体TRPA1是一种非选择性同源四聚体阳离子通道，在原代细胞中表达。 由有毒化学刺激物激活的感觉神经元参与痛觉和 局部发炎。局部炎症信号反过来又会使感觉神经元对痛苦的刺激敏感。在 在疼痛和局部炎症的调节循环中，TRPA1起着积极的调节作用，其 调节失调可能导致慢性疼痛的发展。小鼠TRPA1基因缺失的研究 消除对化学刺激物、机械和热过敏产生的疼痛感觉 组织损伤和哮喘引起的呼吸道炎症支持这一模型。函数增益TRPA1 突变导致人类先天性疼痛障碍，突出了它在疼痛感知中的直接作用。 这使得理解TRPA1的功能和失调具有非常重要的意义。基础科学研究 保尔森实验室的主要目标是确定TRPA1调节和 第二信使的失调，局部炎症信号，通过蛋白质-蛋白质相互作用， 并由新的功能增益突变所赋予。高分辨率TRPA1结构存在于 打开和关闭状态，在正常通道活动期间进行采样。虽然它们代表着一种 尽管取得了重大进展，但这些结构并没有解决TRPA1如何成为 在疾病中被敏化以赋予通道多动症。了解这些机制将开启 为开发新的靶向疗法打开了大门。在未来5年内，我们将使用互补性 生化、生物物理和结构生物学方法，以确定分子基础 一种新的功能获得性TRPA1突变导致的通道过度活跃。我们的初步数据 提示该TRPA1突变蛋白与野生型TRPA1亚基共同组装形成超活性 频道。我们想要了解这个TRPA1突变体引入的结构变化是如何影响 通道功能。此外，我们还将确定钙是如何致敏和/或激活TRPA1的。 和钙调蛋白。许多局部炎症信号间接激活G蛋白下游的TRPA1 促进细胞内钙释放的偶联受体。TRPA1可直接结合钙离子或 通用型钙传感器、钙调素可介导钙敏感。我们的初步数据支持 钙和钙调蛋白在调节TRPA1中的相互作用我们想要了解钙调素是如何 结合与钙结合部位协同作用传递TRPA1钙依赖通道 活动。总的来说，这项工作将加强我们对TRPA1调控和失调的理解 在分子水平上，并将发现针对异常通道的药物开发的新途径 在慢性疼痛和炎症的情况下。

PROJECT SUMMARY The wasabi receptor, TRPA1, is a non-selective homotetrameric cation channel expressed in primary sensory neurons where its activation by noxious chemical irritants contributes to pain perception and local inflammation. Local inflammatory cues, in turn, sensitize sensory neurons to painful stimuli. Within the pain and local inflammation regulatory cycle, TRPA1 serves as a positive regulator and its dysregulation could contribute to the development of chronic pain. Genetic loss of TRPA1 in mice abrogates pain perception to chemical irritants, mechanical and thermal hypersensitivity produced from tissue injury, and asthma-induced airway inflammation supporting this model. Gain-of-function TRPA1 mutations cause congenital painful disorders in humans highlighting its direct role in pain perception. This makes understanding TRPA1 function and dysregulation highly significant. Basic science research in the Paulsen Laboratory broadly aims to determine molecular mechanisms of TRPA1 regulation and dysregulation by second messengers, local inflammatory cues, through protein-protein interactions, and as imparted by novel gain-of-function mutations. High-resolution TRPA1 structures exist in the open and closed states, which are sampled during normal channel activity. While they represent a major advance, these structures do not address the fundamental question of how TRPA1 becomes sensitized to confer channel hyperactivity in disease. Understanding these mechanisms would open the door to develop new targeted therapeutics. During the next 5 years, we will use complementary biochemical, biophysical, and structural biology approaches to determine the molecular basis of channel hyperactivity conferred by a novel gain-of-function TRPA1 mutation. Our preliminary data suggest this TRPA1 mutant protein co-assembles with wild type TRPA1 subunits to form hyperactive channels. We want to understand how the structural alterations introduced by this TRPA1 mutant affect channel function. Additionally, we will determine how TRPA1 is sensitized and/or activated by calcium and calmodulin. Many local inflammatory cues indirectly activate TRPA1 downstream of G-protein coupled receptors that promote intracellular calcium release. TRPA1 could bind calcium directly or the universal calcium sensor, calmodulin could mediate calcium sensing. Our preliminary data support an interplay of calcium and calmodulin in regulating TRPA1 and we want to understand how calmodulin binding works in concert with calcium-binding sites to confer TRPA1 calcium-dependent channel activity. Collectively, this work will enhance our understanding of regulation and dysregulation of TRPA1 at the molecular level and will uncover novel avenues for drug development to target aberrant channels in chronic pain and inflammatory conditions.