Structure and function of Transient Receptor Potential Channels

项目摘要 瞬时受体电位（TRP）通道代表多模态细胞传感器，其整合化学， 温度、机械应力和膜电压刺激，并将它们转换成离子电流来调节 我们的视觉，听觉，味觉，嗅觉和触觉，并有助于感知温度和疼痛。 TRP通道涉及许多人类疾病的发病机制，包括癌症， 是最热门的药物靶点之一尽管TRP渠道结构最近取得了成功， 然而，目前对它们的遗传多样性、功能和调节的确定和了解还远未完成。 这种有限的知识是设计基于TRP通道的治疗策略的关键障碍 法规和合理药物设计的进展。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物化学方法的组合。我们的具体目标是：1）建立 TRPV 6多态性和疾病变体的分子基础，2）确定TRPV 6的结构机制 抑制，以及3）识别门控和调节中的相似性和差异的基础结构元件， TRPV 6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， 它们代表具有典型低表达水平的大尺寸多聚体整合膜蛋白。到 实现我们的目标，我们将使用结构和功能的方法相结合，包括现代低温， 电子显微镜（cryo-EM），X射线晶体学，蛋白质工程，基于双折射的尺寸排阻 色谱法（FSEC）、钙成像、荧光光谱和电生理学。我们将表达 TRP通道，其突变体和真核细胞系中的遗传变体，使用不同的膜纯化它们， 模拟系统，并确定在不同的刺激存在下的冷冻EM和晶体结构。然后我们将 联合收割机将TRP通道的结构信息与功能数据相结合，以揭示TRP通道的分子机制 门控、抑制和调节由Ca 2+、温度和脂质。实现我们的目标将大大改善 了解TRP通道的结构和功能，从而产生一个新的动态模板， 新药的预测、计算机拟合和化学合成。

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.