Structures and gating mechanisms of TRP ion channels

 描述(申请人提供)：Trp通道代表一组对广泛的化学和物理刺激做出反应的非选择性阳离子通道。它是一个庞大而多样的超家族，仅次于钾通道。色氨酸香草酸(Trp vanilloid，TRPV)是一类温度敏感的Trp通道，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/或其他类型的细胞中表达，在感受环境有害刺激如高温、酸性pH和天然或合成刺激物方面发挥重要作用。然而，尽管色氨酸离子通道在许多生理和病理生理过程中扮演着有趣的角色，但其结构研究一直是非常具有挑战性的。色氨酸通道超家族的一个主要瓶颈是难以获得有序的三维(3D)晶体，这是X射线结晶学确定结构的先决条件，X射线结晶学是最成功和成熟的蛋白质结构确定方法。最近在单粒子电子冷冻显微镜(CryoEM)方面的技术突破使得对各种大小和对称性的大分子的近原子分辨结构的确定不再局限于高度对称的大对象。它还可以在不需要形成3D晶体的情况下确定完整的膜蛋白的原子结构。我们测定了TRPV1离子通道的原子结构，这是整个Trp通道家族中的第一个原子结构，也是用单粒子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低温电子显微镜作为我们的主要结构分析工具，结合分子生物学、生物化学和生物物理学的其他方法，来阐明Trp通道亚家族TRPV的功能机制。我们将解决以下具体问题：(1)辣椒素受体TRPV1离子通道的多峰激活机制；(2)TRPV通道的热敏感性机制；以及(3)提高单粒子低温电子显微镜的分辨率，进一步可视化配体和离子结合。这些目标的实质性完成将促进我们对Trp通道功能的了解，并促进单粒子低温EM技术的发展。

 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.