Structural basis for mitochondrial calcium uniporter function

线粒体钙单向转运蛋白功能的结构基础

• 批准号: 8897438
• 负责人: Dipayan Chaudhuri
• 金额: $ 15.46万
• 依托单位: BOSTON CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-08-01 至 2015-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8897438
• 关键词: ATP Synthesis Pathway; Affinity; Animals; Applications Grants; Arrhythmia; Automobile Driving; Behavior; Binding; Bioinformatics; Biology; Calcium; Cardiovascular Diseases; Cardiovascular system; Cell Death; Cells; Charge; Complement; Cultured Cells; Cysteine; Disease; EF Hand Motifs; Electrophysiology (science); Environment; Event; Failure; Foundations; Functional disorder; Future; Genes; Genetic; Genomic approach; Genomics; Goals; Heart Diseases; Heart failure; Image; Imaging Techniques; Inner mitochondrial membrane; Investigation; Ion Channel; Ion Transport; Ions; Kinetics; Lead; Learning; Location; Membrane; Mentors; Methods; Mitochondria; Mitochondrial Diseases; Mitochondrial Proteins; Modeling; Molecular; Organelles; Patients; Phase; Physiology; Proline; Proteins; Protocols documentation; Qualifying; Regulation; Research Project Grants; Research Training; Rest; Scanning; Series; Side; Signal Transduction; System; Techniques; Testing; Training; Tryptophan; base; calcium uniporter; career; cohort; driving behavior; gene discovery; genetic manipulation; genome editing; imaging modality; innovation; mitochondrial dysfunction; mutant; post-doctoral training; prevent; protein transport; research study; skills; therapeutic target; uptake; voltage clamp

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.

描述(由申请人提供)：这项建议将支持候选人的职业目标，即研究心脏病中异常钙信号(钙信号)引起的线粒体功能障碍。候选人将利用这一项目为这一长期目标奠定基础，首先，完成必要的实验，剖析钙摄取发生的分子机制，其次，培训分离具有这种线粒体功能障碍的患者队列所需的定量基因组和生物信息学方法。在信号转导过程中，线粒体转运钙的主要蛋白质是线粒体钙离子单一转运体，这是一个嵌入内膜的通道。这个频道有两个主要特点。它对钙离子有很高的选择性，不允许其他离子进入静止的细胞质钙离子水平。只有当细胞质水平较高时，例如在信号传递过程中或当钙离子清除不足时，它才会转运钙离子。这种选择性和调节防止了不必要的离子运输，这将导致线粒体解偶联和失败，在心脏病中它们可能会改变。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洞(MCU)和附属亚基(MICU1)的基因进行突变分析。以前的研究一直受到成像方法的阻碍，这些方法无法控制影响钙摄取的次要因素，导致了相互矛盾的模型。这一提议的主要创新是使用线粒体电生理学，它精确地控制这些次要因素。在指导阶段，实验将检验这些假设，即面对膜间隙的高度保守的残基用于结合钙离子并形成一个狭窄的硬孔，阻止其他离子的运输。在独立阶段，实验将检验这一假设，即MICU1亚单位通过驱动通道进入主要关闭状态，在钙升高期间释放这种抑制，而不是更复杂的当前模型，来抑制静息细胞质钙水平的运输。在独立阶段，候选人还将接受基因组方法的培训，以识别提示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，以详细检查线粒体钙信号异常在多大程度上是原因。在此背景下，本申请中提出的实验对于了解线粒体钙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的支持下进行他的培训和研究。