Molecular physiology of intracellular InsP3R and MCU ion channels

摘要 三磷酸肌醇(InsP3)触发释放对细胞内钙离子浓度([Ca~(2+)]i)的调节 内质网钙离子是一个无处不在的信号系统，调节着众多细胞 生理过程。InsP3介导的[钙]i信号表现为重复的尖峰或振荡，以及 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。在这件事的核心 复杂的信号系统是InsP3R离子通道。我们提供了对离子的严格理解- InsP3R的通道特性，通过使用强大的定量单通道贴片来研究通道- 我们首创的一项技术--天然ER膜的钳制电生理学；这些特性如何 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是如何反映的 在生理结果上。InsP3R介导的钙信号的一个重要生理靶点是线粒体。 InsP3R通道在调节细胞新陈代谢中起着基础性作用，主要是通过提供释放的 Ca2激活TCA循环脱氢酶促进氧化磷酸化(OXPHOS) 和三磷酸腺苷生产。我们发现低水平的结构性InsP3R介导的钙释放到线粒体 在大多数细胞类型中维持OXPHOS和ATP产生的基础水平是必不可少的，而癌症 细胞的生存特别依赖于这一途径。线粒体钙的主要途径 摄取是线粒体钙单转运体(MCU)，是线粒体内部的一种钙选择性离子通道 膜(IMM)。至于InsP3R，我们使用了生化和强大的生物物理方法来 了解MCU的离子通道特性，包括MCU钙电流的膜片钳电生理 在单个有丝分裂体中。我们最主要的努力是定量地了解分子 InsP3R和MCU通道的生理学，其综合活动控制细胞的生理和生命 以及死亡的决定。最近，InsP3R和MCU的冷冻电子显微镜(Cryo-EM)结构 都已经解决了。因为我们在这些细胞内离子的生物物理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钙离子的调节 频道。我们的目标是了解InsP3R通道门控调节的分子机制，以 对MCU通道离子渗透和门控的分子机制有了新的认识 调节，包括通过相互作用线粒体蛋白质，并利用从第一个获得的信息 提供对内质网到线粒体钙转移的定量洞察的两个目标。因为基本的 癌细胞对这一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 这些研究将提供对信号通路的新的和关键的定量见解，该信号通路在 许多细胞的生理过程。

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.