Molecular link between MCU and Mrs2p channels for mitochondrial ion homeostasis and energy metabolism

MCU 和 Mrs2p 通道之间用于线粒体离子稳态和能量代谢的分子联系

• 批准号: 10171884
• 负责人: Shanmughapriya Santhanam
• 金额: $ 24.9万
• 依托单位: PENNSYLVANIA STATE UNIV HERSHEY MED CTR
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-08-01 至 2023-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10171884
• 关键词: Affinity; Attenuated; Binding; Binding Sites; Biochemical Process; Bioenergetics; Biological; Biological Models; Buffers; CRISPR/Cas technology; Calcium; Cardiac; Cardiac Myocytes; Cations; Cell Death; Cell membrane; Cell model; Cells; Cessation of life; Complex; Coupling; Crystallization; Data; Disease; Divalent Cations; Energy Metabolism; Energy Supply; Ensure; Equilibrium; Event; Exhibits; F0 ATPase; Failure; Feedback; Gene Targeting; Generations; Genome Stability; Heart; Heart Diseases; Homeostasis; ITPR1 gene; Individual; Injury; Inositol; Ions; Kinetics; Knock-in; Knock-out; Knowledge; Link; Magnesium; Mediating; Membrane; Metabolic; Metabolism; Mitochondria; Mitochondrial Matrix; Molecular; Muscle Cells; Myocardial Infarction; Myocardium; N-terminal; Necrosis; Oxidative Phosphorylation; Oxidoreductase; Pathologic; Pathology; Patients; Physiological; Physiological Processes; Physiology; Play; Positioning Attribute; Potassium Channel; Protein Family; Proteins; Publishing; Recurrence; Regulation; Reproducibility; Role; Ryanodine Receptors; Site; Structure; Survivors; System; Therapeutic; Workload; base; calcium uniporter; cell type; gain of function; grasp; insight; mitochondrial metabolism; monomer; mutant; mutant mouse model; preservation; receptor; tripolyphosphate; uptake

Summary In cardiomyocytes the high metabolic demand of contractility emphasizes the need for an efficient and tightly controlled energy producing system. Oxidative Phosphorylation (OxPhos) serves the need of myocardium and as such the site of OxPhos is the mitochondria that represents a central control dogma to ensure that energy demands are met. In mitochondria, Ca2+ is proposed to be the link between EC coupling (ECC) and OxPhos and has been shown to modulate mitochondrial metabolism through the activation of Ca2+-dependent dehydrogenases. It is a long standing mystery on how mitochondrial calcium ([Ca ]m) uptake is tightly regulated during physiology and 2+ pathology. The [Ca ]m uptake is facilitated by the large electrochemical gradient across the inner mitochondrial 2+ membrane and mediated by the Mitochondrial Calcium Uniporter (MCU). MCU is a hetero-oligomeric complex and known to be regulated by several of its interacting partners MICUs, MCUR1, EMRE and MCUb. But there is a lack of knowledge on the exact molecular mechanism of MCU regulation. Our recent structural insight of the MCU channel revealed an acidic patch where cations can bind and regulate MCU activity. Consistent with this, other Ca2+ channels including L-type, ryanodine receptors (RyRs), Inositol 1,4,5 triphosphate receptors (IP3Rs), and Ca release activated 2+ Ca2+ channels are known to be regulated by a negative feedback mechanism. Our discovery of this long-sought regulatory mechanism, uniquely positions us to study the divalent cation based regulation of MCU during pathophysiological condition. Thus I hypothesize that under physiological conditions mitochondrial matrix Mg2+- binding-induced inactivation of MCU may be a protective buffering mechanism for [Ca ]m overload mediated cell 2+ death that is pertinent to cardiomyocyte energy metabolism. Mg2+ being the most abundant divalent cation is known to play important roles in regulating Ca2+ and K+ channels of plasma membrane. In mitochondria, matrix Mg2+ homeostasis is maintained by a selective CorA transport family protein, Mrs2p. Thus, the current proposal aims to delineate the mechanism by which mitochondrial matrix magnesium ([Mg ]m) contributes to the regulation of MCU 2+ activity, mitochondrial Ca2+ homeostasis and bioenergetics. To uncover the molecular link between and MCU and Mrs2p channels, I will generate knockout, and functional domain (loss/gain of function) knock-in mutant model systems using CRISPR/Cas9 mediated gene targeting to study the regulation of MCU-mediated [Ca ]m 2+ uptake by matrix Mg2+. We hypothesize that mitochondrial matrix will be overloaded with Ca2+ in cells that lack Mrs2p and its functional domain (GMN). Conversely, we also hypothesize that the knock-in mutant corresponding to the acidic patch of Mrs2p will serve as a gain-of- function mutant and will alleviate MCU- mediated Ca2+ overload during pathological conditions including I/R injury and protect cardiomyocytes from necrotic cell death. The proposed study will reveal how MCU regulation by divalent cations provide a therapeutic strategy for I/R injury.

摘要 在心肌细胞中，对收缩能力的高代谢要求强调了对高效和紧密的 受控能源生产系统。氧化磷酸化(OxPhos)满足心肌和AS的需要 OxPhos的这样一个部位就是线粒体，它代表了确保能量需求的中央控制教条 都相遇了。在线粒体中，Ca~(2+)被认为是EC偶联(ECC)和OxPhos之间的联系，并已被证明 通过激活依赖于钙的脱氢酶来调节线粒体代谢。这是一个很长的 关于线粒体钙([Ca]m)摄取是如何在生理和生理过程中受到严格调控的谜团 2+ 病理学。线粒体内部的大的电化学梯度促进了[Ca]m的摄取 2+ 线粒体钙统一转运体(MCU)是由线粒体钙转运体(MCU)介导的。MCU是一种杂低聚复合体， 已知受其相互作用的几个伙伴MICUS、MCUR1、EmRE和MCUb的调控。但目前还缺乏 了解MCU调节的确切分子机制。我们最近对MCU渠道的结构性洞察 揭示了一个酸性斑块，其中阳离子可以结合和调节MCU的活性。与此一致的是，其他钙离子通道 包括L受体、兰尼定受体(RyRs)、肌醇1，4，5三磷酸受体(IP3Rs)和激活的钙释放 2+ 已知钙离子通道受负反馈机制的调节。我们发现了这一长期寻找的 调控机制，为我们研究基于二价阳离子的MCU调控奠定了独特的基础 病理生理状况。因此，我假设在生理条件下，线粒体基质镁离子- 结合诱导的MCU失活可能是[Ca]m超载介导的细胞保护缓冲机制 2+ 与心肌细胞能量代谢有关的死亡。镁离子是已知的最丰富的二价阳离子 在质膜钙、钾通道的调节中发挥重要作用。线粒体中的基质镁离子 动态平衡是由选择性的CORA转运蛋白家族蛋白MRS 2P维持的。因此，目前的提案旨在 阐明线粒体基质镁([mg]m)参与MCU调节的机制 2+ 活性、线粒体钙动态平衡和生物能量学。为了揭示和MCU之间的分子联系 Mrs2p通道，我会产生基因敲除，和功能域(功能丧失/功能获得)敲入突变体 CRISPR/Cas9介导的基因打靶模型系统研究MCU介导的[Ca]m调节 2+ 被基质镁离子摄取。我们假设线粒体基质将因细胞内的钙超载而超载 缺乏mrs2p及其功能域(GMN)。相反，我们还假设敲入突变体 与mrs2p的酸性斑块相对应，将作为功能增益突变体，缓解MCU- I/R损伤等病理条件下介导的钙超载及其对心肌细胞的保护作用 坏死性细胞死亡。这项拟议的研究将揭示二价阳离子调节MCU如何提供治疗作用 I/R损伤的策略。