Molecular physiology of intracellular InsP3R and MCU ion channels

总结 三磷酸肌醇（InsP 3）触发释放对胞浆Ca ~（2+）浓度（[Ca ~（2+）]i）的调节 内质网（ER）中Ca 2+的释放是一个普遍存在的信号系统，它调节许多细胞 生理过程。InsP 3介导的[Ca 2 +]i信号表现为重复的尖峰或振荡， 它们可以高度局部化或传播以向细胞的离散部分提供信号。这件事的核心 复杂的信号系统是InsP 3R离子通道。我们已经提供了对离子的严格理解- InsP 3R的通道特性，通过使用强大的定量单通道贴片研究通道， 天然ER膜的钳位电生理学，这是我们开创的技术;这些特性是如何 受生理激动剂和蛋白质相互作用的调节;以及这些特性的变化如何反映 在生理结果上。InsP 3R介导的Ca 2+信号的重要生理靶点是线粒体。 InsP 3R通道在细胞代谢的调节中起着重要作用，主要是通过提供释放的 Ca 2+进入线粒体，刺激TCA循环酶，促进氧化磷酸化（OXPHOS） ATP的生产。我们发现，低水平的组成型InsP 3R介导的Ca 2+释放到线粒体， 对于维持大多数细胞类型中OXPHOS和ATP产生的基础水平是必需的，并且癌症 细胞的生存特别依赖于这种途径。线粒体Ca 2+的主要途径 摄取是线粒体Ca 2+单向转运体（MCU），一种线粒体内的Ca 2+选择性离子通道 膜（IMM）。至于InsP 3R，我们采用了生物化学和强大的生物物理方法， 了解MCU的离子通道特性，包括MCU钙电流的膜片钳电生理学 在单个的有丝分裂体中。我们最大的努力是定量地了解 InsP 3R和MCU通道的生理学，其综合活动控制细胞生理学和生命 和死亡决定。最近，InsP 3R和MCU的低温电子显微镜（cryo-EM）结构 已经解决了由于我们在这些细胞内离子的生物物理学和分子生理学方面的努力， 渠道，我们处于独特的地位，可以利用这些新信息来解决以下重要问题 离子渗透和通道门控的分子机制及其对Ca ~（2+）和Ca ~（2+）的调节 渠道我们的目标是了解InsP 3R通道门控调控的分子机制， 获得对MCU通道离子渗透和门控的分子机制的基本新见解 调节，包括通过相互作用的线粒体蛋白，并利用从第一个获得的信息， 两个目标，提供定量的见解ER到线粒体Ca 2+转移。由于基本的 癌细胞对这种信号系统的依赖及其在家族性阿尔茨海默病中的作用，我们预计， 这些研究将为信号通路提供新的和关键的定量见解， 许多细胞生理过程。

SUMMARY Modulation of the cytoplasmic concentration of Ca2+ ([Ca2+]i) by inositol trisphosphate (InsP3)-triggered release of Ca2+ from the endoplasmic reticulum (ER) is a ubiquitous signaling system that regulates numerous cell physiological processes. InsP3-mediated [Ca2+]i signals are manifested as repetitive spikes or oscillations, and they can be highly localized or propagate to provide signals to discrete parts of the cell. At the heart of this complex signaling system is the InsP3R ion channel. We have provided rigorous understanding of the ion- channel properties of the InsP3R, by studying the channel using powerful quantitative single-channel patch- clamp electrophysiology of native ER membranes, a technique that we pioneered; how those properties are regulated by physiological agonists and protein interactions; and how changes in these properties are reflected in physiological outcomes. An important physiological target of InsP3R-mediated Ca2+ signals are mitochondria. InsP3R channels play a fundamental role in the regulation of cell metabolism, primarily by supplying released Ca2+ to mitochondria to stimulate TCA-cycle dehydrogenases to promote oxidative phosphorylation (OXPHOS) and ATP production. We discovered that low-level constitutive InsP3R-mediated Ca2+ release to mitochondria is essential for maintaining basal levels of OXPHOS and ATP production in most cell types, and that cancer cells have a particular reliance on this pathway for their survival. The primary pathway for mitochondrial Ca2+ uptake is the mitochondrial Ca2+ uniporter (MCU), a Ca2+-selective ion channel in the inner mitochondrial membrane (IMM). As for the InsP3R, we have employed biochemical and powerful biophysical approaches to understand the ion-channel properties of MCU, including patch-clamp electrophysiology of MCU Ca2+ currents in individual mitoplasts. Our overarching effort has been to quantitatively understand the molecular physiologies of the InsP3R and MCU channels whose integrated activities control cellular physiology and life and death decisions. Recently, cryo-electron microscopic (cryo-EM) structures of both the InsP3R and MCU have been solved. Because of our exertise in the biophysics and molecular physiology of these intracellular ion channels, we are uniquely positioned to exploit this new information to address important questions regarding the molecular mechanisms of ion permeation and channel gating and their regulation of both Ca2+ ion channels. Our goals are to understanding the molecular mechanisms of InsP3R channel gating regulation, to gain fundamental new insights into the molecular mechanisms of MCU channel ion permeation and gating regulation, including by interacting mitochondrial proteins, and to exploit the information gained from the first two goals to provide quantitative insights into ER-to-mitochondrial Ca2+ transfer. Because of the fundamental reliance of cancer cells on this signaling system and its role in familial Alzheimer's disease, we expect that these studies will provide new and critical quantitative insights into a signaling pathway that is important in many cell physiological processes.