Structures and gating mechanisms of TRP ion channels

 描述（由申请人提供）：TRP通道代表一组非选择性阳离子通道，对广泛的化学和物理刺激作出反应。它是一个庞大而多样的超家族，仅次于钾通道。TRP香草酸（TRPV）是热敏TRP通道的一个亚家族，使体感神经元能够检测环境温度的变化。它们在感觉神经和/或其他细胞类型中表达，在感受环境有害刺激如高温、酸性pH和天然或合成刺激物中发挥重要作用。然而，尽管它们在许多生理和病理生理过程中的有趣的作用，TRP离子通道的结构研究一直非常具有挑战性。一个主要的瓶颈是难以获得TRP通道超家族的任何成员的有序三维（3D）晶体，这是通过X射线晶体学进行结构测定的先决条件，X射线晶体学是蛋白质结构测定的最成功和完善的方法。单粒子电子低温显微镜（cryoEM）的最新技术突破使各种尺寸和对称性的大分子的近原子分辨率结构测定成为可能，不再局限于高度对称的大型物体。它还能够在不需要形成3D晶体的情况下确定完整膜蛋白的原子结构。我们确定了TRPV 1离子通道的原子结构，这是整个TRP通道家族的第一个原子结构，也是通过单粒子cryoEM确定的完整膜蛋白的第一个原子结构。我们将使用近原子分辨率的单粒子cryoEM作为我们的主要结构分析工具，与其他方法在分子生物学，生物化学和生物物理学，阐明机制，管理的一个亚家族的TRP通道，TRPV的功能。我们将解决以下具体问题：（1）TRPV 1离子通道（辣椒素受体）的多模态激活机制是什么？（2）TRPV通道的热敏性机制是什么？（3）提高单粒子cryoEM的分辨率，进一步可视化配体和离子结合。这些目标的实质性完成将推进我们对TRP通道功能的了解，并推进单粒子cryoEM技术。

 DESCRIPTION (provided by applicant): TRP channels represent a group of non-selective cation channels that respond to a wide range of chemical and physical stimuli. It is a large and diverse superfamily, second only to potassium channels. TRP Vanilloid (TRPV) is a subfamily of thermosensitive TRP channels that enable somatosensory neurons to detect changes in ambient temperature. They are expressed in sensory nerves and/or other cell types, where they play important roles in sensing environmental noxious stimuli such as high temperature, acidic pH and natural or synthetic irritants. However, despite their intriguing roles in a number of physiological and pathophysiological processes, structural studies of TRP ion channels have been very challenging. A major bottleneck has been the difficulties in obtaining well-ordered three-dimensional (3D) crystals for any member of the TRP channel superfamily, which is a prerequisite for structure determination by X-ray crystallography as the most successful and well-established method for protein structure determination. Recent technological breakthroughs in single particle electron cryomicroscopy (cryoEM) have enabled near atomic resolution structure determination of macromolecules of various sizes and symmetry, no longer limited to highly symmetrical large object. It also enabled atomic structure determination of integral membrane proteins without requiring formation of 3D crystals. We determined the atomic structure of TRPV1 ion channel, which is the first atomic structure of the entire TRP channel family and the first atomic structure of an integral membrane protein determined by single particle cryoEM. We will use near atomic resolution single particle cryoEM as our main structural analysis tool, together with other methods in molecular biology, biochemistry and biophysics, to elucidate the mechanism that governs the functions of a subfamily of TRP channels, TRPV. We will address following specific questions: (1) what is the mechanism of polymodal activation of TRPV1 ion channel, a receptor for capsaicin; (2) what is the mechanism of thermosensitivity of TRPV channels; and (3) to improve the resolution of single particle cryoEM further to visualize ligands and ions binding. Substantial completion of these aims will advance our knowledge about the TRP channel function as well as advancing the technology of single particle cryoEM.