内质网离子通道TRIC的运行机制及其调控研究

Ca2+ release from sarcoplasmic reticulum and endoplasmic reticulum (SR/ER) is involved in various physiological processes such as muscle contraction and neurotransmission. Since Ca2+ release via SR/ER Ca2+ channels would generate a negative potential on the SR/ER luminal side, the molecular mechanism to balance the transient negative potential had been predicted for the efficient Ca2+ release from SR/ER. The TRIC family proteins are trimeric intracellular cation channels, localized at SR/ER. They are recently identified as counter-cation channels to facilitate Ca2+ release from ER and SR. In higher eukaryotes, including human and mouse, two different subtypes of TRICs, TRIC-A and TRIC-B were identified and functionally characterized. Both of TRIC-A and TRIC-B function as monovalent cation channels with a weak preference for K+, and are regulated by membrane-voltage. TRIC channels are associated with malignant hypertension and osteogenesis imperfecta, and accordingly they are recognized as a potential drug target. Despite their physiological importance, the lack of a high resolution structure of TRIC proteins hinders the understanding of the molecular mechanism of cation conduction of TRIC proteins such as ion selectivity and ion channel gating mechanisms. .In our proposal, we will conduct the structural analyses of TRIC proteins as well as the structure-based electrophysiological analyses to understand the molecular mechanism of cation conduction of TRIC proteins. In addition, by utilizing the technical platform for the structural and functional analyses of TRIC proteins, we will identify chemical compounds targeting TRIC proteins, and will determine their complexes with TRIC proteins to provide the structural basis for the development of new compounds for TRIC channels.

由细胞内质网释放的钙离子与肌肉收缩、神经传递等多种生命活动息息相关。伴随钙离子的释放，内质网内部的负电荷逐渐累计。直到近年来，TRIC离子通道的发现才解释了内质网消除这些负电荷以保证钙离子高效释放的机理。TRIC包括TRIC-A和TRIC-B两个亚型，都属于电压门控离子通道，能够选择性的传递一价阳离子、尤其是钾离子进入内质网内。TRIC被认为与高血压和成骨不全症等疾病有关，作为药物研发的重要靶点受到广泛的关注。但是目前为止尚未取得TRIC的高分辨率立体结构，其离子通道的运行机制、选择性传递一价阳离子特别是钾离子的机理以及离子通道被激活的详细机制均不明确。本课题通过X射线结晶学和电生理学研究TRIC的运行机制。以TRIC的立体结构为基础，筛选对TRIC具有调控作用的化合物，并解析这些化合物与TRIC的复合体结构，研究这些化合物与TRIC具体的结合方式，为新药物的研发提供基础。

TRIC家族蛋白是三聚体胞内阳离子通道，定位于内质网上。直到近年来，TRIC离子通道的发现才解释了内质网消除这些负电荷以保证钙离子高效释放的机理。在高等真核生物中，包括人和鼠，TRIC包括TRIC-A和TRIC-B两个亚型，都属于电压门控离子通道，能够选择性的传递一价阳离子、尤其是钾离子进入内质网内。TRIC被认为与高血压和成骨不全症等疾病有关，作为药物研发的重要靶点受到广泛的关注。尽管在生理上很重要，但是目前为止尚未取得TRIC的高分辨率结构，致使其离子通道的门控机制、选择性传递一价阳离子的详细机理均不明确。.在本项目中，我们获得了原核TRIC通道在关闭状态下的晶体结构，并对原核和真核TRIC通道进行了基于结构的功能分析。电生理、生化和生物物理学分析表明，TRIC通道在每个亚基内都有一个离子传导孔。与对称相关的TM2和TM5螺旋在保守的甘氨酸残基处发生扭结，这些扭结对通道活性具有重要意义。此外TM2和TM5螺旋的扭结在每个亚基界面处产生横向孔道。出乎意料的是，横向孔道被脂质分子占据，因为其在真核TRIC蛋白中是保守的从而可能适合靶向药物。总体来说，本项目提供了此离子通道超家族分子机制的结构和功能框架，以及对未来药物开发的潜在解析。.在本项目的实施过程中，我们进一步建立了一个成熟的膜蛋白结构解析系统。我们解析得到了膜蛋白的晶体结构和冷冻电镜结构。在项目实施过程中，共发表SCI论文6篇（含共同通讯作者），其中一区论文4篇。2名博士后已顺利出站，1名博士生及2名硕士生将于今年毕业。

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