Uncovering the interplay of calcium and calmodulin in regulation of TRPA1

项目摘要 持续性疼痛影响着美国数百万人，花费了数十亿美元的医疗保健费用， 年生产力。疼痛管理最令人担忧的方面是阿片类药物的广泛使用， 导致了成瘾的流行，每年有成千上万的美国人死亡。这些问题 需要开发治疗慢性疼痛的新疗法， 文献表明，瞬时受体电位锚蛋白1（TRPA 1）是降低糖尿病的真正药物靶点。 慢性疼痛TRPA 1在感觉神经元中表达，在感觉神经元中，TRPA 1响应于刺激而产生急性疼痛信号。 有毒的亲电子化合物然而，TRPA 1作为药物靶点的希望来自于它作为一种积极的药物靶点的作用。 疼痛和炎症循环中的调节因子，其中该反馈循环的失调可能是过渡的基础 从急性疼痛到慢性疼痛。利用基因敲除小鼠的动物研究表明，TRPA 1活性对于 由组织损伤以及哮喘诱导的气道产生的机械和热超敏反应 炎症支持该模型。该提案的总体目标是更好地理解TRPA 1 调节，特别关注钙调节机制。这是一个有争议的 该领域的一个主题，一些研究小组提供了TRPA 1活性受钙离子结合调节的证据 直接与钙调蛋白（CaM）没有贡献，而其他人已经显示CaM结合TRPA 1并调节 其活动。我的初步结果表明，钙调素结合TRPA 1，并调节钙离子结合以及 作为TRPA 1的柔性胞质结构域。我假设有多个TRPA 1结构域参与了 钙调素结合，钙调素差异从事不同的钙浓度。我将检验这个假设 使用在S2-S3环处结合钙的能力受损或具有截短的突变TRPA 1构建体， 在N和C-末端的柔性胞质区域与生物化学、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的总体效果 钙结合环和钙调素两部分。第一组实验将确定 在不同的钙浓度下，CaM对TRPA 1和S2-S3三重突变体的亲和力。第二组 实验将确定在不同钙浓度下CaM与TRPA 1的结合化学计量。目的 2旨在确定参与CaM结合的N和C-末端的柔性胞质区域。结构性 CaM赋予TRPA 1的变化以及这些不同区域如何调节CaM结合将在 目标3。总的来说，我将进行一项全面的研究，调查钙调素结合，S2- S3钙结合位点和柔性胞质区域。TRPA 1监管的更完整的画面将 为合理药物设计提供新的机会，以寻求更有效的慢性疼痛治疗方法， 对抗阿片类药物成瘾

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.