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

总结 在心肌细胞中，收缩性的高代谢需求强调了对有效和紧密的收缩的需要。 控制能源生产系统。氧化磷酸化（OxPhos）服务于心肌的需要， 这种OxPhos的位置是线粒体，代表着一种中央控制法则，以确保能量需求 得到满足。在线粒体中，Ca 2+被认为是EC偶联（ECC）和OxPhos之间的联系，并且已经显示 通过激活Ca 2+依赖性磷酸酶调节线粒体代谢。它是一项长期 线粒体钙（[Ca ]m）摄取在生理过程中是如何严格调节的， 2+个 病理[Ca ]m的摄取是由线粒体内部的大的电化学梯度促进的 2个以上 线粒体钙单向转运体（MCU）介导的。MCU是异源寡聚复合物， 已知由其几个相互作用伙伴MICU、MCUR 1、EMRE和MCUb调节。但缺乏 了解MCU调节的确切分子机制。我们最近对MCU渠道的结构性见解 揭示了一个酸性补丁，阳离子可以结合和调节MCU的活动。与此一致，其他Ca 2+通道 包括L型、兰尼碱受体（RyRs）、肌醇1，4，5三磷酸受体（IP 3Rs）和钙释放激活的 2+个 钙离子通道是一种负反馈机制。我们发现了这个长期以来 调控机制，独特的定位，我们研究二价阳离子为基础的调控MCU期间， 病理生理状态因此，我假设在生理条件下，线粒体基质Mg 2 +- 结合诱导的MCU失活可能是[Ca ]m超载介导的细胞保护性缓冲机制 2个以上 与心肌细胞能量代谢有关的死亡。已知Mg 2+是最丰富的二价阳离子 在调节质膜Ca ~（2+）和K ~+通道中起重要作用。在线粒体中，基质Mg 2 + 体内平衡由选择性CorA转运家族蛋白Mrs 2 p维持。因此，目前的建议旨在 描述线粒体基质镁（[Mg ]m）参与MCU调节的机制 2+个 活性、线粒体Ca 2+稳态和生物能量学。为了揭示与MCU之间的分子联系， Mrs 2 p通道，我将产生敲除和功能结构域（功能丧失/获得）敲入突变体 使用CRISPR/Cas9介导的基因靶向研究MCU介导的[Ca ]m调节的模型系统 2个以上 基质Mg 2+的吸收。我们假设，线粒体基质将超载与Ca 2+的细胞， 缺少Mrs 2 p及其功能域（GMN）。相反，我们也假设敲入突变体 对应于Mrs 2 p的酸性斑块将作为功能获得性突变体，并将减轻MCU。 介导的Ca 2+超负荷在病理条件下，包括I/R损伤和保护心肌细胞， 坏死性细胞死亡拟议的研究将揭示二价阳离子如何调节MCU， I/R损伤策略。

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.