Uncovering the interplay of calcium and calmodulin in regulation of TRPA1

揭示钙和钙调蛋白在 TRPA1 调节中的相互作用

基本信息

批准号：
10387088
负责人：
Justin Sanders
金额：
$ 4.68万
依托单位：
YALE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-01-01 至 2023-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10387088
关键词：
ANK1 gene Acute Pain Affect Afferent Neurons Affinity American Americas Animals Area Asthma Binding Binding Sites Biochemical Biophysics Calcium Calcium Binding Calcium ion Calmodulin Calmodulin 1 Cell membrane Cessation of life Chemosensitization Complex Cytoplasmic Tail Data Development Disease Drug Design Drug Targeting Elements Epidemic Event Feedback Goals Golgi Apparatus Healthcare Hypersensitivity Impairment In Vitro Inflammation Ion Channel Knock-out Knockout Mice Literature Mechanics Modeling Mus N-terminal Opiate Addiction Opioid Pain Pain management Persistent pain Persons Phosphorylation Physiological Play Polysaccharides Post-Translational Protein Processing Process Productivity Protein Dephosphorylation Regulation Role Sepharose Site Structure TRPA channel Techniques Testing Therapeutic Work addiction airway inflammation chronic pain chronic pain management cost desensitization drug development effective therapy experimental study fight against flexibility glycosylation insight mutant novel therapeutics pain chronification pain signal prevent response sensor stoichiometry structural biology tissue injury

项目摘要

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.

项目概要持续的疼痛影响着数百万人的美国医疗保健和损失数十亿美元疼痛管理最令人担忧的方面是阿片类药物的广泛使用。导致毒瘾流行，每年导致数以万计的美国人死亡。需要开发治疗慢性疼痛的治疗小说，并且越来越多的人文献表明瞬时受体电位锚蛋白 1 (TRPA1) 是一个真正的药物靶点，可减少 TRPA1 在感觉神经元中表达，在感觉神经元中产生急性疼痛信号以响应。然而，TRPA1 作为药物靶点的前景来自于它的积极作用。疼痛和炎症循环中的调节器，该反馈循环的失调可能是转变的基础使用基因敲除小鼠进行的动物研究表明，TRPA1 活性对于从急性疼痛到慢性疼痛都是必需的。组织损伤以及哮喘引起的气道产生的机械和热过敏该提案的首要目标是更好地了解 TRPA1。特别关注钙调节机制的调节一直是一个有争议的问题。该领域的主题，一些小组提供了 TRPA1 活性通过结合钙离子调节的证据直接作用，不受钙调蛋白 (CaM) 的影响，而其他研究表明 CaM 与 TRPA1 结合并调节我的初步结果表明 CaM 与 TRPA1 结合，并且也受到钙离子结合的调节。作为 TRPA1 的柔性细胞质结构域，我注意到有多个 TRPA1 结构域参与其中。 CaM 结合在不同的钙浓度下有差异，我将检验这一假设。使用突变 TRPA1 构建体，该构建体在 S2-S3 环上结合钙的能力受损或有截断 N 端和 C 端灵活的细胞质区域，结合生物化学、生物物理和目标 1 中提出的实验将确定 S2-S3 的总体效果。 TRPA1 上的钙结合环和 CaM 分两部分进行第一组实验将确定结合。不同钙浓度下 CaM 对 TRPA1 和 S2-S3 突变体三重的亲和力第二组。实验将确定不同钙浓度下 CaM 与 TRPA1 的结合化学计量。图 2 旨在鉴定参与 CaM 结合的 N 和 C 末端的柔性细胞质区域。 CaM 赋予 TRPA1 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 CaM 结合、S2- S3 钙结合位点和灵活的细胞质区域将更完整地描述 TRPA1 调节。为合理药物设计寻找更有效的慢性疼痛治疗方法提供了新的机会与阿片类药物成瘾作斗争。