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，研究异常钙（Ca2+）信号在心脏病中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，完成必要的实验来剖析Ca2+摄取发生的分子机制，其次，在分离具有线粒体功能障碍的患者群体所需的定量基因组和生物信息学方法方面进行培训。在信号事件中运输Ca2+的主要线粒体蛋白是线粒体Ca2+单转运蛋白，这是一种嵌入内膜的通道。这个频道有两个主要特点。它对Ca2+具有高度选择性，不允许其他离子在静止的细胞质Ca2+水平下进入。它只在细胞质水平高时运输Ca2+，例如在信号事件期间或Ca2+清除不足时。这种选择性和调节防止不必要的离子运输，这将导致线粒体解偶联和失败，它们可能在心脏病中发生改变。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孔（MCU）和附属亚基（MICU1）的基因进行突变分析。先前的研究受到使用成像方法的阻碍，这些方法无法控制影响Ca2+摄取的次要因素，导致相互矛盾的模型。这项提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，实验将测试面向膜间空间的高度保守残基用于结合Ca2+并形成狭窄，刚性孔，阻止其他离子运输的假设。在独立阶段，实验将测试MICU1亚基通过驱动通道进入主要关闭状态来抑制静息细胞质Ca2+水平的运输的假设，在Ca2+升高期间释放这种抑制，与更复杂的当前模型相反。在独立阶段，候选人还将接受基因组方法的培训，以识别可能存在线粒体功能障碍的心脏病患者。在细胞或动物系统中对这种功能障碍进行建模将是未来拨款申请的基础，以详细检查线粒体Ca2+信号异常在多大程度上是致病的。在这种情况下，在本应用程序中提出的实验是必要的，以了解线粒体Ca2+摄取是如何在基线调节。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，花了相当多的精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的支持下进行培训和研究。

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.