Molecular Physiology of Mitochondrial Calcium Uniporter

项目摘要 线粒体钙单转运体复合体(MCUcx)是一种高度选择性的四聚体钙离子通道， 在将钙离子从细胞质输送到线粒体基质中的主要作用。虽然人体对钙的摄取 线粒体在刺激ATP生成中起着至关重要的作用，基质钙超载可触发心肌细胞的开放 线粒体通透性转换孔，导致细胞死亡。MCU功能障碍被牵连到 几种病理情况，如心力衰竭、缺血再灌注损伤、神经变性、癌症 和骨骼肌营养不良。最近的生化和结构研究表明，MCUcx是一种 由成孔MCU亚基、必需亚基EmRE和EF-Hand组成的大分子络合物 含有结构域的MICU1-3亚基。MCU复合体的每个亚单位是如何聚集在一起并调节的 线粒体Ca~(2+)内流是近年来许多研究的热点。使用直接线粒体膜片钳 方法学和创新的外源表达系统，我们最近进行了全面的 线粒体内膜中MCUcx的结构与功能分析这个表达系统 由MCUcx每个单独亚基的基因敲除组成(超过6个不同的敲除系被 由CRISPR/CAS9生成)，易于适应不同的膜片钳配置，具有较高的 成功率。利用该系统和膜片钳技术，我们建议回答 领域：1)MCU门在哪里，这个门是如何受钙离子和MCU附属亚基调节的？和2) MCU大分子结构的独特特征(如最近的结构研究所揭示的)是如何 与MCU选通和功能有关？实现这项建议的目标将提供更好的 了解通过MCU调节钙离子内流的机制，并为 以MCU门控为治疗目的的药理学干预的发展。

Project Summary The mitochondrial calcium uniporter complex (MCUcx) is a highly-selective, tetrameric Ca2+ channel that plays a primary role in transporting Ca2+ from the cytoplasm into the mitochondrial matrix. Although Ca2+ uptake by mitochondria plays a crucial role in stimulating ATP production, matrix Ca2+ overload can trigger opening of the mitochondrial permeability transition pore, leading to cell death. MCU dysfunction has been implicated in several pathological conditions such as heart failure, ischemia–reperfusion injury, neurodegeneration, cancer and skeletal muscle dystrophies. Recent biochemical and structural studies have shown that the MCUcx is a macromolecular complex composed of a pore-forming MCU subunit, an essential subunit EMRE, and EF-hand domain-containing MICU1–3 subunits. How each subunit of the MCU complex comes together and regulates mitochondrial Ca2+ entry has been the focus of many recent studies. Using direct mitochondrial patch-clamp methodology and an innovative heterologous expression system, we recently performed a comprehensive structure–function analysis of the MCUcx in the inner mitochondrial membrane. This expression system consists of gene knockout of each individual subunit of the MCUcx (more than 6 different knockout lines were generated by CRISPR/cas9) and is readily amenable to different patch-clamp configurations with a high success rate. Using this system and the patch-clamp technique, we propose to answer central questions in the field: 1) Where is the MCU gate, and how is this gate regulated by Ca2+ and MCU accessory subunits? and 2) How do the unique features in the MCU macromolecular structures (as revealed by recent structural studies) relate to MCU gating and function? Accomplishment of the aims of this proposal will provide better understanding of the mechanisms that regulate Ca2+ entry via the MCU and create an essential framework for the development of pharmacological interventions that target MCU gating for therapeutic purposes.