Structural basis for mitochondrial calcium uniporter function

描述（由申请人提供）：该提案将支持候选人的职业目标，即研究心脏疾病中异常钙（Ca2+）信号引起的线粒体功能障碍。候选人将利用该项目为这一长期目标奠定基础，首先，完成必要的实验以剖析Ca 2+吸收发生的分子机制，其次，培训隔离所需的定量基因组和生物信息学方法具有此类线粒体功能障碍的患者队列。在信号事件期间转运Ca 2+的主要线粒体蛋白是线粒体Ca 2+单向转运体，其是嵌入内膜中的通道。这个渠道有两个主要特点。它对Ca2+具有高度选择性，不允许其他离子进入静息细胞质Ca2+水平。它仅在细胞质水平高时转运Ca2+，例如在信号传导事件期间或Ca2+清除不足时。这种选择性和调节防止不必要的离子转运，这将导致线粒体解偶联和失败，并且它们可能在心脏病中改变。为了确定通道如何执行这两个关键功能，将对最近发现的形成通道的孔（MCU）和辅助亚基（MICU1）的基因进行突变分析。以前的调查已受到阻碍，使用成像方法，不能控制次要因素影响钙的摄取，导致矛盾的模型。这项建议的主要创新是使用线粒体电生理学，它精确地控制了这些次要因素。在指导阶段，实验将测试以下假设：面向膜间空间的高度保守残基用于结合Ca2+并形成狭窄的刚性孔，从而阻止其他离子的运输。在独立阶段，实验将测试的假设，MICU1亚基抑制运输静息细胞质Ca2+水平的驱动通道进入一个主要的关闭状态，释放这种抑制在Ca2+升高，在更复杂的电流模型。在独立阶段，候选人还将接受基因组方法的培训，以确定患有心脏病的患者，这些患者提示线粒体功能障碍。在细胞或动物系统中模拟这种功能障碍将是未来拨款申请的基础，以详细研究异常线粒体Ca2+信号传导的致病程度。在这种情况下，本申请中提出的实验对于理解线粒体Ca2+摄取在基线时是如何调节的是必要的。候选人完全有资格实现上述短期和长期目标。他在离子通道生物学方面有很强的背景，花了相当大的努力学习线粒体电生理学，并计划在线粒体疾病，离子通道生物学和基因组方法专家支持的环境中进行培训和研究。

DESCRIPTION (provided by applicant): This proposal will support the candidate's career goals of studying mitochondrial dysfunction caused by abnormal calcium (Ca2+) signaling in cardiac disease. The candidate will use this project to lay the groundwork for this long-term goal by, first, completing necessary experiments to dissect the molecular mechanisms by which Ca2+ uptake occurs, and, second, training in quantitative genomic and bioinformatic methods necessary for isolating patient cohorts possessing such mitochondrial dysfunction. The major mitochondrial protein transporting Ca2+ during signaling events is the mitochondrial Ca2+ uniporter, a channel embedded in the inner membrane. This channel possesses two key features. It is highly selective for Ca2+, not allowing other ions to enter at resting cytoplasmic Ca2+ levels. And it transports Ca2+ only when cytoplasmic levels are high, such as during signaling events or if Ca2+ clearance is insufficient. This selectivity and regulation prevent unnecessary ion transport, which would lead to mitochondrial uncoupling and failure, and they may be altered in heart disease. To identify how the channel performs these two key functions, a mutational analysis of the recently- discovered genes that form the pore (MCU) and accessory subunits (MICU1) of the channel will be conducted. Prior investigations have been hampered by the use of imaging methods that cannot control for secondary factors influencing Ca2+ uptake, leading to contradictory models. The chief innovation of this proposal is the use of mitochondrial electrophysiology, which controls for precisely these secondary factors. In the mentored phase, experiments will test the hypotheses that highly-conserved residues facing the inter-membrane space serve to bind Ca2+ and form a narrow, rigid pore, preventing the transport of other ions. In the independent phase, experiments will test the hypothesis that the MICU1 subunit inhibits transport at resting cytoplasmic Ca2+ levels by driving the channel into a predominantly closed state, releasing this inhibition during Ca2+ elevations, in contrast to more complicated current models. During the independent phase, the candidate will also receive training in genomic approaches to identify patients with cardiac disease suggesting mitochondrial dysfunction. Modeling this dysfunction in cellular or animal systems will be the basis of future grant applications, to examine in detail to what degree aberrant mitochondrial Ca2+ signaling is causative. In this context, the experiments proposed in this application are necessary to understand how mitochondrial Ca2+ uptake is regulated at baseline. The candidate is well-qualified to carry out the short- and long-term goals described above. He has a strong background in ion-channel biology, has spent considerable effort learning mitochondrial electrophysiology, and plans to conduct his training and research in an environment supported by experts in mitochondrial disease, ion-channel biology, and genomic approaches.