Molecular Mechanism of Mitochondrial Membrane Transport

项目摘要 线粒体钙（Ca 2+）摄取是许多基本生理过程的核心。它能刺激ATP 在代谢需要增加的时间期间产生，并提供Ca 2+汇以调节Ca 2+介导的 在细胞内发出信号。线粒体Ca 2+浓度也调节细胞凋亡和失调-- 具体地说，Ca 2+超载是从神经元兴奋性毒性到心力衰竭的病理学的标志， 从癫痫到肌肉萎缩症然而，尽管线粒体Ca 2+摄取在正常人中的重要性， 生理学和疾病，介导这一过程的分子机制是相对最近确定的，许多 一些根本性的问题仍有待回答。 Ca 2+流入线粒体的主要途径是一种称为线粒体钙单向转运体的通道， 包括普遍存在的孔形成亚基MCU和几个调节亚基， （当在复合体中时称为“单重”）。这种新型通道对Ca 2+具有高度选择性，其活性与Ca 2+浓度密切相关。 受胞浆Ca ~（2+）浓度的调节。 我的团队最近确定了一种真菌MCU的高分辨率晶体结构， 通道结构，并揭示了高亲和力的Ca 2+结合位点。此外，我们的人体冷冻电镜结构 uniplex holocomplex揭示了它的结构，并暗示了它的调节机制。与这些 结构和我们开发的方法，我的实验室是独一无二的准备走上机械 了解线粒体钙单向转运体。 在这里，我们建议：1）阐明离子选择性，传导和 抑制; 2）了解通道门控和远程调制的机制; 3）探测 钙离子依赖性调节单链体的分子基础。 这些结果将为我们提供急需的线粒体活性和调节机制的见解， 钙单向转运体，扩大我们对钙离子通道的一般原理的理解。此外，他们还应 提供了一个强有力的框架，以帮助设计MCU抑制剂，这可能是有前途的治疗， 以MCU失调和线粒体Ca 2+过载为标志的疾病和病理。 !

PROJECT SUMMARY Mitochondrial calcium (Ca2+) uptake is central to many fundamental physiological processes. It stimulates ATP production during times of increased metabolic need and provides a Ca2+ sink to modulate Ca2+-mediated signaling locally within a cell. Mitochondrial Ca2+ concentrations also regulate apoptosis and dysregulation–– specifically, Ca2+ overload––is a hallmark of pathologies ranging from neuronal excitotoxicity to heart failure and some epilepsies to muscular dystrophies. Yet despite the importance of mitochondrial Ca2+ uptake in normal physiology and disease, the molecular machinery mediating this process is relatively recently identified and many fundamental questions remain to be answered. The main route of Ca2+ influx to mitochondria is a channel called mitochondrial calcium uniporter, which includes the ubiquitous pore-forming subunit MCU and, depending on the species, several regulatory subunits (termed “uniplex” when in complex). This novel channel is highly selective for Ca2+, and its activity is tightly regulated by cytosolic Ca2+ concentration. My group recently determined a high-resolution crystal structure for a fungal MCU that defined a novel channel architecture and revealed a high-affinity Ca2+-binding site. Moreover, our cryo-EM structure of the human uniplex holocomplex revealed its architecture and hints at the mechanisms by which it is regulated. With these structures and the methods we developed, my lab is uniquely poised to embark on the mechanistic understanding of the mitochondrial calcium uniporter. Here, we propose to: 1) elucidate the structural and biophysical basis of ion selectivity, conduction and inhibition; 2) understand mechanisms of the channel gating and the long-range modulation; and 3) probe the molecular basis of Ca2+-dependent regulation of the uniplex. These results will give us much needed mechanistic insights into the activity and regulation of mitochondrial calcium uniporter, expanding our understanding of general principles of Ca2+ channels. In addition, they should provide a strong framework to aid the design of MCU inhibitors, which may represent promising treatments for diseases and pathologies marked by MCU dysregulation and mitochondrial Ca2+ overload. !