Structural mechanism of polymodal TRP channel activation

项目摘要 辣椒素和热激活的TRPV1离子通道是化学和 热刺激，因此是一个有吸引力的止痛药的目标。尽管有冷冻EM可用 TRPV1的高分辨结构及其激活的分子机制 目前仍不清楚。从低温电磁结构中获取机械信息的一个主要限制 是普遍缺乏分辨率来确定侧链取向和相关的原子 互动。我们最近证明，通过结合Rosetta可以克服这一限制 用热力学突变循环分析等特定部位功能测试进行结构预测 用于约束、验证和改进结构预测。使用这种迭代方法，在 结合包括FRET和斑块荧光测量在内的特定部位的荧光记录，我们的 这项研究旨在确定辣椒素结合域和外孔中的功能相互作用， 更重要的是，为了揭示辣椒素和热诱导过程中这些相互作用的动态变化 激活。我们将特别利用荧光非天然氨基酸(Fuaa)。 将一个小的荧光团引入通道的运动部件的掺入方法。我们的目标是 阐明调节化学和热的关键分子相互作用的结构机制 激活，从而提供一个分子框架来指导药物干预。

Project Summary Capsaicin- and heat-activated TRPV1 ion channel is a primary nociceptor for both chemical and thermal stimuli, hence an attractive target for pain medication. Despite the availability of cryo-EM structures of TRPV1 at up-to-2.9 Å resolutions, molecular mechanisms underlying TRPV1 activation remains unclear. A major limitation for obtaining mechanistic information from the cryo-EM structures is the general lack of resolution to determine side-chain orientation and the associated atomic interaction. We recently demonstrated that the limitation could be overcome by combining Rosetta structural prediction with site-specific functional tests such as thermodynamic mutant cycle analysis that serve to constrain, validate, and improve structure prediction. Using this iterative approach, in combination with site-specific fluorescence recordings including FRET and patch fluorometry, our study aims to identify functional interactions in the capsaicin-binding domain and the outer pore and, more importantly, to reveal dynamic changes of these interactions during capsaicin- and heat-induced activation. We will take particular advantage of the fluorescent unnatural amino acid (FUAA) incorporation method to introduce a small fluorophore to the channel’s moving parts. Our goal is to elucidate structural mechanisms for key molecular interactions that mediate chemical and thermal activation, thus providing a molecular framework to guide pharmaceutical intervention.