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：
9208793
负责人：
Dipayan Chaudhuri
金额：
$ 24.9万
依托单位：
UNIVERSITY OF UTAH
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 2019-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9208793
关键词：
ATP Synthesis Pathway Affinity Animals Applications Grants Arrhythmia Automobile Driving Behavior Binding Bioinformatics Biology Calcium Cardiovascular Diseases Cardiovascular system Carrier Proteins 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 Mutation Analysis Organelles Patient Isolators Patients Phase Physiology Proline Proteins Protocols documentation Regulation Research Research Project Grants Rest Scanning Series Side Signal Transduction System Techniques Testing Training Tryptophan calcium uniporter career cohort experimental study gene discovery genetic manipulation genome editing imaging modality innovation mitochondrial dysfunction mutant post-doctoral training prevent skills targeted treatment 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研究心脏病中钙 (Ca2+) 信号异常引起的线粒体功能障碍的职业目标。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+ 吸收发生的分子机制，其次进行隔离患者群体所需的定量基因组和生物信息学方法的培训具有这种线粒体功能障碍。在信号事件期间运输 Ca2+ 的主要线粒体蛋白是线粒体 Ca2+ 单向转运蛋白，它是嵌入内膜的通道。该渠道有两个主要特点。它对 Ca2+ 具有高度选择性，不允许其他离子在静息细胞质 Ca2+ 水平下进入。仅当细胞质水平较高时（例如在信号传导事件期间或 Ca2+ 清除不足时），它才会转运 Ca2+。这种选择性和调节可以防止不必要的离子运输，这会导致线粒体解偶联和衰竭，并且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）和辅助亚基（MICU1）的基因进行突变分析。先前的研究因使用无法控制影响 Ca2+ 吸收的次要因素的成像方法而受到阻碍，导致模型相互矛盾。该提案的主要创新是使用线粒体电生理学，它可以精确控制这些次要因素。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、刚性的孔，从而阻止其他离子的传输。在独立阶段，实验将测试以下假设：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+ 水平下的转运，并在 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培训，以识别患有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成为未来资助申请的基础，以详细检查异常的线粒体 Ca2+ 信号传导在多大程度上是起因的。在这种情况下，本申请中提出的实验对于了解线粒体 Ca2+ 摄取在基线时如何受到调节是必要的。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拥有深厚的背景，花费了大量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支持的环境中进行培训和研究。