Uncovering the interplay of calcium and calmodulin in regulation of TRPA1

项目总结 持续的疼痛影响着美国数百万人，并造成数十亿美元的医疗费用和损失 每年的生产力。疼痛管理最令人担忧的方面是阿片类药物的广泛使用， 已经导致了成瘾的流行，每年有数万美国人死亡。这些问题 有必要开发新的治疗方法来治疗慢性疼痛，而且越来越多的 文献表明，瞬时受体潜在锚蛋白1(TRPA1)是一种真正的药物靶点，可以减少 慢性疼痛。TRPA1在感觉神经元中表达，在那里它产生急性疼痛信号以响应 有毒的亲电化合物。然而，TRPA1作为药物靶点的S承诺来自于它作为一个积极的 疼痛和炎症环路中的调节因子，这种反馈环路的失调可能是过渡的基础 从急性疼痛到慢性疼痛。利用基因敲除小鼠进行的动物研究表明，TRPA1活性是 组织损伤和哮喘所致的机械和热过敏反应 支持这一模型的炎症。这项提案的首要目标是更好地理解TRPA1 调节，特别关注钙调节的机制。这是一场有争议的 该领域的主题，一些小组提供了TRPA1活性受结合钙离子调节的证据 在没有钙调蛋白(CaM)贡献的情况下，直接与TRPA1结合并调节 它的活动。我的初步结果表明，CaM与TRPA1结合，并受钙离子结合的调节 作为TRPA1的柔韧胞质结构域。我假设有多个TRPA1域参与 CaM结合：CaM在不同的钙浓度下不同地结合。我将检验这一假设 使用突变的TRPA1结构，该结构损害了在S2-S3环结合钙的能力或具有截断 在N和C末端的灵活细胞质区域与生化、生物物理和 结构生物学技术。目标1中提出的实验将确定S2-S3的整体效果 钙结合环和CaM对TRPA1的影响分为两部分。第一组实验将确定绑定 钙调素在不同钙浓度下对TRPA1和S2-S3三重突变体的亲和力。第二套 实验将确定不同钙浓度下CaM与TRPA1的结合化学计量比。目标 2旨在鉴定参与CaM结合的N和C末端的灵活细胞质区域。结构性的 中将探讨CaM对TRPA1的影响以及这些不同区域如何调节CaM结合 目的3.总体而言，我将进行一项全面的研究，调查CaM结合、S2- S3钙结合部位和灵活的细胞质区域。对TRPA1监管的更全面的了解将 为合理设计药物提供新的机会，以寻求更有效的慢性疼痛和 与阿片成瘾作斗争。

PROJECT SUMMARY Persistent pain affects millions of people in America and costs billions of dollars in healthcare and lost productivity annually. The most concerning aspect of pain management is the widespread use of opioids which has led to an addiction epidemic and the deaths of tens of thousands of Americans each year. These problems necessitate the development of novel therapeutics in the treatment of chronic pain, and a growing body of literature suggests that transient receptor potential ankyrin 1 (TRPA1) is a bona fide drug target for reducing chronic pain. TRPA1 is expressed in sensory neurons where it generates acute pain signals in response to noxious, electrophilic compounds. However, TRPA1’s promise as a drug target comes from its role as a positive regulator in a pain and inflammation loop where dysregulation of this feedback loop could underlie the transition from acute to chronic pain. Animal studies utilizing knockout mice show that TRPA1 activity is necessary for mechanical and thermal hypersensitivity produced from tissue injury as well as asthma-induced airway inflammation supporting this model. The overarching goal of this proposal is to better understand TRPA1 regulation with special focus being given to the mechanisms of calcium regulation. This has been a contentious topic in the field with some groups providing evidence that TRPA1 activity is regulated by binding calcium ions directly with no contribution from calmodulin (CaM) while others have shown CaM binds to TRPA1 and regulates its activity. My preliminary results suggest CaM binds to TRPA1 and is modulated by calcium ion binding as well as flexible cytoplasmic domains of TRPA1. I hypothesize there are multiple TRPA1 domains involved in CaM binding that CaM differentially engages at distinct calcium concentrations. I will test this hypothesis using mutant TRPA1 constructs that have impaired ability to bind calcium at the S2-S3 loop or have truncations of the flexible cytoplasmic regions at the N and C-termini with a combination biochemical, biophysical, and structural biology techniques. Experiments proposed in Aim 1 will determine the overall effect of the S2-S3 calcium binding loop and CaM on TRPA1 in two parts. The first set of experiments will determine the binding affinity of CaM to TRPA1 and the S2-S3 triple mutant at varying calcium concentrations. The second set of experiments will determine the binding stoichiometry of CaM to TRPA1 at varying calcium concentrations. Aim 2 seeks to identify the flexible cytoplasmic regions of the N and C-termini involved in CaM binding. The structural changes that CaM imparts on TRPA1 and how these different regions modulate CaM binding will be explored in Aim 3. Overall, I will perform a comprehensive study investigating the relationship between CaM binding, the S2- S3 calcium binding site, and the flexible cytoplasmic regions. A more complete picture of TRPA1 regulation will provide new opportunities for rational drug design in the search for more effective treatments of chronic pain and the fight against opioid addiction.