线粒体钙离子单向转运体MCU的转运和抑制机制研究

Calcium is one of the most versatile and universal signalling agents in the human body. Calcium influx to the mitochondrial matrix is mediated by an inner membrane calcium channel called mitochondrial calcium uniporter (MCU). MCU is a calcium-conducting channel with remarkably high conductance and selectivity, but its primary sequence does not resemble any calcium channel studied to date. MCU activity is membrane potential dependent and sensitive to ruthenium red or its derivative Ru360. Moreover, in metazoans, the calcium-conducting activity of MCU absolutely requires the presence of the single-transmembrane protein EMRE(Essential MCU REgulator). Previously, we reported the structure of the pore region of MCU from Caenorhabditis elegans, determined using nuclear magnetic resonance (NMR) and electron microscopy (EM). MCU is a homo-oligomer with novel channel architecture. The critical DxxE motif forms the pore entrance, which features two carboxylate rings. The new structure prompted many interesting mechanistic questions of MCU activation and regulation. Here we propose a detailed study to determine the calcium selectivity filter of MCU, the mechanism of Ru360 block, and MCU-EMRE interaction. Our research will provide valuable knowledge to decipher the molecular mechanism of calcium uptake by MCU.

钙离子是人体各项生理活动不可缺少的重要信使之一。线粒体内膜上的钙离子通道MCU是将钙离子转运进入线粒体基质的重要途径。MCU具有很高的钙离子转运能力和选择性，其蛋白序列与迄今已知的任何钙离子通道都不相似。钙离子经由MCU进入基质的过程是一个依赖线粒体内膜电势，并对钌红、Ru360非常敏感的过程。此外，在动物线粒体中，MCU需要另外一个跨膜蛋白EMRE才能完成钙离子转运的过程。前期我们利用核磁共振技术结合电镜解析了MCU通道核心区域的三维结构，发现MCU形成了一个同源五聚体，揭示了MCU中DxxE形成了一个双层羧酸环的通道入口。然而MCU精确的钙离子选择机制和转运机制仍然缺乏。本项目中我们将联合应用核磁和生化手段，在分子水平上研究钙离子转运过程中MCU的钙离子选择机制、Ru360抑制机理和EMRE调控机制，使我们对MCU离子通道有更全面的认识和理解。

MCU是线粒体内膜上将钙离子转运进入线粒体基质的钙离子通道。MCU具有很高的钙离子转运能力和选择性，并对钌红、Ru360非常敏感。在动物线粒体中，MCU需要EMRE的辅助才能完成钙离子转运。MCU精确的钙离子选择机制和转运机制仍然缺乏。本项目中我们联合应用结构和生化手段，在分子水平上研究钙离子转运过程中MCU的钙离子选择机制、Ru360抑制MCU转运钙离子的机理和EMRE调控MCU的机制。我们前期通过Mn2+的顺磁滴定，确认了Ca2+的结合位点是通道口的DxxE motif，Ru360也通过结合在DxxE上抑制MCU的活性。我们进一步通过SEC-MALS，ITC，crosslinking等生化实验，测定了人源MCU-EMRE的化学当量为1：1。在把研究对象调整为盘基网柄菌DdMCU后，我们解析了DdMCU-NTD的晶体结构，发现钙离子对DdMCU-NTD的寡聚状态起调控作用，并且获得了DdMCU全长的电镜图。当前的实验结果基本完成了项目的预期目标，完成情况良好。