Structure and function of Transient Receptor Potential Channels

项目总结 瞬时受体电位(Trp)通道代表多模式细胞传感器，它整合了化学， 温度、机械应力和膜电压刺激，并将其转换为离子电流进行调节 我们的视觉、听觉、味觉、嗅觉和触觉有助于感知温度和疼痛。 Trp通道与许多人类疾病的发病机制有关，包括癌症和 代表着最受追捧的毒品目标之一。尽管最近在Trp通道结构方面取得了成功 确定、了解它们的遗传多样性、功能和调控仍远未完成。 这些有限的知识是设计基于Trp通道的治疗策略的关键障碍 规范和促进合理用药设计的进展。我们计划研究Trp通道的结构和功能 使用不同的生物物理和生化方法的组合。我们的具体目标是：1)建立 TRPV6基因多态和疾病变异的分子基础，2)决定TRPV6的结构机制 抑制，以及3)确定在门控和调节中潜在的相似和不同的结构元素 TRPV6和其他Trp通道。Trp通道是结构-功能研究的具有挑战性的目标，因为 它们代表的是大尺寸的多聚体整膜蛋白，通常表达水平较低。至 为了实现我们的目标，我们将使用结构和功能相结合的方法，包括现代低温- 电子显微镜(低温电子显微镜)、X射线结晶学、蛋白质工程、基于荧光的尺寸排除 层析(FSEC)、钙成像、荧光光谱和电生理学。我们会表示 真核细胞系中的Trp通道、它们的突变体和遗传变体使用不同的膜来纯化它们 模拟系统，并在不同刺激下确定低温电子显微镜和晶体结构。到时候我们会的 结合新生的结构信息和功能数据识别Trp通道的分子机制 门控，抑制和调节钙离子，温度和脂质。实现我们的目标将显著提高 了解TRP通道的结构和功能，从而为理论研究提供新的动态模板 预测，在硅胶拟合和新药的化学合成中。

PROJECT SUMMARY Transient Receptor Potential (TRP) channels represent polymodal cellular sensors, which integrate chemical, temperature, mechanical stress and membrane voltage stimuli and convert them into ionic currents to regulate our senses of vision, hearing, taste, smell and touch and contribute to the perception of temperature and pain. TRP channels are implicated in the pathogenesis of numerous human diseases, including cancers, and represent one of the most ardently pursued drug targets. Despite recent successes in TRP channel structure determination, understanding of their genetic diversity, function and regulation is still far from being complete. Such limited knowledge represents a critical barrier to devising therapeutic strategies based on TRP channel regulation and to the progress in the rational drug design. We plan to study TRP channel structure and function using a combination of different biophysical and biochemical methods. Our specific aims are: 1) establish molecular bases of TRPV6 polymorphisms and disease variants, 2) determine structural mechanisms of TRPV6 inhibition, and 3) identify structural elements underlying similarities and difference in gating and regulation of TRPV6 and other TRP channels. TRP channels are challenging targets for structure-functional studies because they represent multimeric integral membrane proteins of a large size with typically low expression levels. To achieve our goals, we will use a combination of structural and functional approaches, including modern cryo- electron microscopy (cryo-EM), X-ray crystallography, protein engineering, Fluorescence-based Size Exclusion Chromatography (FSEC), calcium imaging, fluorescent spectroscopy and electrophysiology. We will express TRP channels, their mutants and genetic variants in eukaryotic cell lines, purify them using different membrane mimetic systems, and determine cryo-EM and crystal structures in the presence of different stimuli. We will then combine the nascent structural information with functional data to discern molecular mechanisms of TRP channel gating, inhibition and regulation by Ca2+, temperature and lipids. Achieving our aims will significantly improve understanding of TRP channel structure and function, resulting in a new dynamic template for theoretical prediction, in silico fitting and chemical synthesis of new drugs.