Structural basis for mitochondrial calcium uniporter function

线粒体钙单向转运蛋白功能的结构基础

基本信息

批准号：
8959727
负责人：
Dipayan Chaudhuri
金额：
$ 13.72万
依托单位：
BOSTON CHILDREN'S HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-08-01 至 2016-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8959727
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 Genome 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 imaging modality innovation mitochondrial dysfunction mutant post-doctoral training prevent protein transport research study skills therapeutic target uptake voltage clamp

项目摘要

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）：该提案将支持候选人在心脏病中由异常钙（CA2+）信号引起的线粒体功能障碍的职业目标。候选人将使用该项目为这个长期目标奠定基础，首先完成必要的实验，以剖析发生CA2+摄取的分子机制，其次，第二种培训定量基因组和生物信息学方法的培训，用于隔离具有这种线粒体功能障碍的患者同类群。信号事件期间的主要线粒体蛋白转运Ca2+是线粒体Ca2+ Uniporter，这是一种嵌入内膜中的通道。该频道具有两个关键功能。对于Ca2+，它具有很高的选择性，不允许其他离子在静止的细胞质Ca2+水平下输入。它仅在胞质水平高时才传输Ca2+，例如在信号事件期间或CA2+清除率不足时。这种选择性和调节可以防止不必要的离子运输，这会导致线粒体的解偶联和失败，并且可能会在心脏病中改变。为了确定通道如何执行这两个关键函数，将对新近发现的基因（MCU）和辅助亚基（MICU1）进行突变分析。使用成像方法无法控制影响Ca2+摄取的次要因素，从而阻碍了先前的研究，从而导致了矛盾的模型。该提案的主要创新是使用线粒体电生理学的使用，它可以控制这些次要因素。在指导阶段，实验将检验假设，即面向膜间空间的高度保存的残基有助于结合Ca2+并形成狭窄，刚性的孔，从而阻止了其他离子的运输。在独立阶段，实验将检验以下假设：MICU1亚基通过将通道带入主要封闭状态，在Ca2+升高期间释放这种抑制，与更复杂的电流模型相反，抑制了静止的细胞质Ca2+水平的转运。在独立阶段，候选人还将接受基因组方法的培训，以鉴定患有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赠款应用的基础，以详细检查多种程度的线粒体Ca2+信号传导是原因。在这种情况下，本应用程序中提出的实验对于了解基线时如何调节线粒体Ca2+摄取是必要的。候选人合格地执行上述短期和长期目标。他在离子通道生物学方面具有强大的背景，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的专家支持的环境中进行培训和研究。