Molecular physiology of intracellular InsP3R and MCU ion channels

细胞内 InsP3R 和 MCU 离子通道的分子生理学

• 批准号: 10170553
• 负责人: James Kevin FOSKETT
• 金额: $ 44.66万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-05-01 至 2026-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10170553
• 关键词: Address; Agonist; Alzheimer' s Disease; Behavior; Biochemical; Biophysics; Cell physiology; Cells; Cellular Metabolic Process; Cessation of life; Citric Acid Cycle; Complex; Cryoelectron Microscopy; Disease; Electrons; Electrophysiology (science); Endoplasmic Reticulum; Environment; Goals; Health; Heart; Individual; Inner mitochondrial membrane; Inositol; Ion Channel; Ions; Kinetics; Life; Malignant Neoplasms; Mediating; Membrane; Microscopic; Mitochondria; Mitochondrial Proteins; Molecular; Outcome; Oxidative Phosphorylation; Oxidoreductase; Pathway interactions; Physiological; Physiological Processes; Physiology; Play; Positioning Attribute; Production; Property; Proteins; Regulation; Role; Signal Pathway; Signal Transduction; Structure; System; Techniques; biophysical techniques; cancer cell; cell type; familial Alzheimer disease; insight; patch clamp; receptor; tripolyphosphate; uptake

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.

概括 肌醇三磷酸（INSP3）触发的释放的Ca2+（[Ca2+] i）的细胞质浓度的调节 来自内质网（ER）的Ca2+是一个无处不在的信号系统，可调节众多细胞 生理过程。 INSP3介导的[Ca2+] I信号表现为重复的尖峰或振荡，并且 它们可以高度局部或传播以提供信号以离散细胞的离散部分。核心 复杂的信号系统是INSP3R离子通道。我们已经对离子提供了严格的理解 - INSP3R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膜的夹具电生理学，这是我们开创的技术；这些属性是如何 由生理激动剂和蛋白质相互作用调节；以及如何反映这些属性的变化 在生理结果中。 INSP3R介导的Ca2+信号的重要生理靶标是线粒体。 INSP3R通道在细胞代谢的调节中起着基本作用，主要是通过供应释放 Ca2+到线粒体以刺激TCA周期脱氢酶以促进氧化磷酸化（OXPHOS） 和ATP生产。我们发现低级构型INSP3R介导的Ca2+释放到线粒体 对于在大多数细胞类型中维持基底水平的OXPHOS和ATP产生至关重要，并且癌症至关重要 细胞特别依赖这种途径的生存。线粒体Ca2+的主要途径 摄取是线粒体Ca2+ Uniporter（MCU），这是内部线粒体中的Ca2+选择性离子通道 膜（IMM）。至于insp3r，我们采用了生化和强大的生物物理方法 了解MCU的离子通道特性，包括MCU CA2+电流的贴片钳电生理学 在单个线粒体中。我们的总体努力是定量了解分子 INSP3R和MCU通道的生理学，其整合活动控制细胞生理和生命 和死亡决定。最近，INSP3R和MCU的冷冻电子显微镜（Cryo-EM）结构 已解决。因为我们在这些细胞内离子的生物物理学和分子生理中发挥了作用 渠道，我们唯一地定位了这些新信息，以解决有关的重要问题 离子渗透和通道门控的分子机制及其对Ca2+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， 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，并利用从第一次获得的信息 两个目标是提供对ER到线粒体Ca2+转移的定量见解。由于基本 依赖癌细胞对该信号系统及其在家族性阿尔茨海默氏病中的作用，我们预计 这些研究将为信号传导途径提供新的和关键的定量见解 许多细胞生理过程。