Molecular Mechanisms of Mitochondrial Ca2+ Transport

线粒体Ca~(2+)处理对细胞内钙稳态、能量代谢和细胞存活至关重要 死亡机制。线粒体钙转运受损也被认为是导致 各种疾病的发展。在几十年来令人沮丧的关于分子调节的模棱两可之后 2010-2011年线粒体钙摄取，EF-Hand结构域蛋白MICU1和MCU被鉴定为 分别是钙离子转运蛋白不可或缺的调节剂和成孔成分。现在，它正在成为 有可能解决关于复杂的 钙转运系统及MICU1、MCU等单转运蛋白的生理意义。我们 已经开始调查MICU1的作用方式，与第一份报告相反，我们发现MICU1 不是允许钙离子通过单一转运体吸收所必需的，而是对保持单一转运体关闭在 低细胞质[Ca~(2+)]([Ca~(2+)]c)浓度，并支持单转运体作为 [Ca~(2+)]c升高。这些研究最近在《细胞》杂志上发表了一篇广为人知的论文 新陈代谢。为了进一步研究MICU1的生理意义，我们产生了一个条件 MICU1基因敲除小鼠具有显著的表型，将为拟议的研究提供几个模型。 我们的假设是：(1)MICU1利用其C-末端的正电荷氨基酸残基簇来 与MCU膜间空间环的Dime结构域相互作用，突出一个可能是 新的药物靶点，(2)MICU1和MICU2与其EF-手不同地结合钙和/或镁，提供 它们对MCU协同激活的不同影响的可能机制，以及(3)MICU1是 在肝细胞氧化代谢方面，体内有效解码钙信号所必需的 进食和禁食条件。为了探索MICU1在钙离子转运中的作用，我们将应用高级 荧光成像方法，包括结合细胞评估的一些新方法 生物能量学和细胞存活调节通路的活性在同一模型中。该提案依赖于 在细胞系和小鼠组织中使用强大的MICU1靶向系统。拟议的工作将 揭示单转运蛋白复杂的钙依赖和时间依赖性控制的机制，并将揭示 MICU1的S在保持线粒体健康和对钙的反应中的作用。

Mitochondrial Ca2+ handling is central to cellular calcium homeostasis, energy metabolism and cell survival/cell death mechanisms. Impaired mitochondrial Ca2+ transport is also thought to be an important contributor in the development of a variety of diseases. After decades of frustrating ambiguity about the molecules mediating mitochondrial Ca2+ uptake, in 2010-2011, MICU1, an EF-hand domain protein, and MCU were identified as an indispensible regulator and pore forming component of the Ca2+ uniporter, respectively. Now, it is becoming possible to tackle the fundamental questions about the molecular mechanisms that underlie a sophisticated Ca2+ transport system and the physiological significance of MICU1, MCU and other uniporter components. We have started to investigate the mode of action of MICU1, and contrary to the first report, we found that MICU1 is not required to allow Ca2+ uptake through the uniporter but, instead, is critical to keep the uniporter closed at low cytoplasmic [Ca2+] ([Ca2+]c) concentrations and to support cooperative activation of the uniporter as the [Ca2+]c increases. These studies have been recently published in a widely recognized paper in Cell Metabolism. To further the study of the physiological significance of MICU1 we have generated a conditional MICU1 knockout mouse that has a striking phenotype and will provide several models for the proposed study. Our hypotheses are that (1) MICU1 employs its C-terminal cluster of positively charged amino acid residues to interact with the DIME domain of the intermembrane space loop of MCU, highlighting a site that might be a novel drug target, (2) MICU1 and MICU2 differently bind Ca2+ and/or Mg2+ with their EF-hands, providing a possible mechanisms for their distinct effects on the cooperative activation of the MCU, and (3) MICU1 is required in vivo for effective decoding of calcium signals in terms of oxidative metabolism in hepatocytes under fed and fasting conditions. To explore the role of MICU1 in Ca2+ handling we will apply advanced fluorescence imaging approaches, including some novel methods combined with assessment of cell bioenergetics and the activity of cell survival regulating pathways in the same models. The proposal relies on the use of a powerful targeting system of MICU1 in both cell lines and mouse tissues. The proposed work will uncover the mechanism of the sophisticated Ca2+- and time-dependent control of the uniporter and will expose MICU1's role in keeping mitochondria healthy and responsive to Ca2+.