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

项目摘要 慢性疼痛患者估计占美国人口的三分之一，他们正在努力寻找减少疼痛的方法 疼痛和改善他们的日常生活。瞬时受体电位(Trp)通道检测到广泛的物理 和化学刺激，并通过它们的整合和对这些刺激的反应，在 许多慢性疼痛障碍的病理生理学。值得注意的是，许多来自植物和 有毒动物以Trp通道为靶标，因此可以用来识别和表征这些通道 对痛感有重要贡献。例如，躯体感觉受体Trp Melastatin 8(TRPM8) 受低温控制，也被自然降温剂激活，如薄荷醇和桉油醇， 常用的局部止痛药。此外，TRPM8对于冷病毒的发展也是必不可少的。 痛觉过敏，一种由于化疗或其他神经性侮辱而导致的对感冒的虚弱过敏。 尽管TRPM8和其他Trp通道在从急性疼痛状态向慢性疼痛状态的转变中非常重要， 配体结合、通道门控和离子渗透的分子基础仍不完全清楚。 这项建议的目标是确定TRPM8跨细胞传导离子的机制 膜对各种信号的反应，包括那些来自产生感冒的植物衍生化合物的信号 轰动一时。具体目的是：1)测定TRPM8在不同构象状态下的原子结构 (K99相)；2)研究TRPM8(K99相)的电生理特性； 磷脂酰肌醇脂类(R00相)对TRPM8的调节作用。单粒子电子冷冻显微镜(低温电子显微镜) EM)结构将被确定为TRPM8单独或与激动剂、拮抗剂或天然毒素形成复合体。 特别是，来自动物毒液的毒素是阐明结构机制的有力工具。 基础通道选通和调制。旨在确定浇注方式的结构-功能分析 将进行机制和验证配基结合部位，以及纯化的生物物理研究， 重组蛋白用于表征TRPM8的内在门控功能。生物活性脂类的调节是一种 统一Trp通道的功能特性，包括TRPM8，从而磷脂酰肌醇脂类对 将探索TRPM8，并将使用自然质谱学鉴定非共价结合的脂类。 这些目标意义重大，因为它们将加强对疼痛的生物物理和分子理解 感觉，具体地说，TRPM8调制如何有助于慢性疼痛。归根结底，目标是协助 合理设计以TRPM8为基础的新型止痛药。我的导师朱利叶斯博士和我的专家 冷冻-EM(郑博士)、电生理学(Kirichok博士)、蛋白质-脂肪相互作用(Marty博士)和 疼痛信号(冯·萨斯特罗博士)将提供培训，为我作为一名独立科学家的职业生涯做准备。

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.