Mechanistic studies of the menthol receptor TRPM8: a novel target for analgesic drugs

项目摘要 据估计，慢性疼痛患者占美国人口的三分之一，他们很难找到减少疼痛的方法。 改善他们的日常生活。瞬时受体电位（TRP）通道检测广泛的物理 和化学刺激，并通过它们的整合和对这些刺激的反应， 许多慢性疼痛疾病的病理生理学。值得注意的是，许多来自植物和 有毒动物靶向TRP通道，因此可以用于识别和表征这些通道。 对痛觉的重要影响例如，TRP melastatin 8（TRPM 8），体感受体， 由低温门控，也由天然清凉剂激活，如薄荷醇和桉树脑， 常用的局部镇痛剂。此外，TRPM 8对于冷的发展是必不可少的。 异常性疼痛，由化疗或其他神经损伤引起的对寒冷的衰弱性超敏反应。 尽管TRPM 8和其他TRP通道对从急性疼痛状态向慢性疼痛状态的转变很重要， 配体结合、通道门控和离子渗透的分子基础仍然不完全清楚。 本提案的目的是确定TRPM 8在细胞内传导离子的机制。 细胞膜对各种信号做出反应，包括那些来自植物衍生化合物的信号， 感觉。具体目的是：1）确定TRPM 8在不同构象状态下的原子结构 (K99 2）研究TRPM 8（K99期）的电生理特性，3）探索TRPM 8的机制。 磷脂酰肌醇脂质对TRPM 8的调节（R 00期）。单粒子电子冷冻显微镜（cryo- 将确定TRPM 8单独和与激动剂、拮抗剂或天然毒素复合的EM）结构。 特别是，来自动物毒液的毒素是阐明结构机制的有力工具 潜在的通道门控和调制。旨在确定门控的结构-功能分析 机制和验证配体结合位点，以及纯化的， 重构蛋白质以功能上表征TRPM 8的内在门控。生物活性脂质的调节是一种 TRP通道，包括TRPM 8的统一功能特性，因此磷脂酰肌醇脂质对 将探索TRPM 8，并使用天然质谱法鉴定非共价结合的脂质。 这些目标意义重大，因为它们将增强对疼痛的生物物理和分子理解 感觉，特别是TRPM 8调节如何促进慢性疼痛。最终目的是协助 基于TRPM 8的新型镇痛药物的合理设计。我的导师朱利叶斯博士，也是我的专家 cryo-EM（郑博士），电生理学（Kirichok博士），蛋白质-脂质相互作用（Marty博士），以及 疼痛信号（冯扎斯特罗博士），将提供培训，为我的职业生涯作为一个独立的科学家做准备。

Project Summary Chronic pain sufferers, an estimated one third of the American population, struggle to identify ways to reduce pain and improve their daily lives. Transient receptor potential (TRP) channels detect a wide range of physical and chemical stimuli, and through their integration of and response to these stimuli have an essential role in the pathophysiology of many chronic pain disorders. Notably, many natural products from plants and venomous animals target TRP channels, and can therefore be used to identify and characterize these important contributors to pain sensation. For instance, TRP melastatin 8 (TRPM8), the somatosensory receptor gated by cold temperatures, is also activated by natural cooling agents, such as menthol and eucalyptol, commonly used topical analgesic agents. Furthermore, TRPM8 is essential for the development of cold allodynia, a debilitating hypersensitivity to cold resulting from chemotherapy or other neuropathic insults. Despite the importance of TRPM8 and other TRP channels to the transition from acute to chronic pain states, the molecular basis for ligand binding, channel gating, and ion permeation remain incompletely understood. The objective of this proposal is to determine the mechanisms whereby TRPM8 conducts ions across cellular membranes in response to diverse signals, including those from plant-derived compounds that produce a cold sensation. The specific aims are to: 1) determine atomic structures of TRPM8 in different conformational states (K99 phase), 2) study the electrophysiological properties of TRPM8 (K99 phase), and 3) probe mechanisms of TRPM8 modulation by phosphatidylinositol lipids (R00 phase). Single-particle electron cryo-microscopy (cryo- EM) structures will be determined of TRPM8 alone and in complex with agonists, antagonists, or natural toxins. In particular, toxins from animal venoms are powerful tools for elucidating the structural mechanisms underlying channel gating and modulation. Structure-function analyses aimed at determining gating mechanisms and validating ligand binding sites will be conducted, as will biophysical studies of purified, reconstituted protein to functionally characterize intrinsic gating of TRPM8. Modulation by bioactive lipids is a unifying functional trait of TRP channels, including TRPM8, and thus effects of phosphatidylinositol lipids on TRPM8 will be explored and non-covalently bound lipids will be identified using native mass spectrometry. These goals are significant because they will enhance the biophysical and molecular understanding of pain sensation and, specifically, how TRPM8 modulation contributes to chronic pain. Ultimately, the aim is to assist in the rational design of novel TRPM8-based analgesic drugs. My mentor, Dr. Julius, as well as my expert advisors in cryo-EM (Dr. Cheng), electrophysiology (Dr. Kirichok), protein-lipid interactions (Dr. Marty), and pain signaling (Dr. von Zastrow), will provide training, in preparation for my career as an independent scientist.