Investigating the molecular mechanisms of Ca2+ selectivity and Ca2+ potentiation of the mitochondrial calcium uniporter

研究线粒体钙单向转运蛋白 Ca2 选择性和 Ca2 增强的分子机制

• 批准号: 10535187
• 负责人: Bryce Delgado
• 金额: $ 4.68万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-26 至 2024-07-25
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10535187
• 关键词: ATP Synthesis Pathway; Affinity; Apoptosis; Architecture; Binding Sites; Biophysical Process; Calcium; Calcium Channel; Cell Death; Cell physiology; Cells; Chemosensitization; Complement; Complex; Cryoelectron Microscopy; Data; Disease; Electrophysiology (science); Environment; Eukaryota; Future; Goals; Human; Individual; Integral Membrane Protein; Investigation; Ions; Kinetics; Knowledge; Lipid Bilayers; Lipids; Measurement; Mediating; Membrane Lipids; Metabolism; Mitochondria; Mole the mammal; Molecular; Mutagenesis; Mutation; Organism; Permeability; Probability; Process; Production; Property; Protein Subunits; Proteins; Regulation; Research Personnel; Resolution; Rest; Role; Shapes; Signal Transduction; Structure; System; Time; Training; Work; calcium uniporter; density; fungus; mutant; particle; prevent; reconstitution; uptake

Project Summary/ Abstract The mitochondrial calcium uniporter mediates Ca2+ uptake into mitochondria and thereby regulates metabolism, cell death, and cytoplasmic Ca2+ signaling. How the Uniporter catalyzes ion permeation and achieves ion selectivity are not well understood. The Uniporter is a supercomplex of protein subunits, containing at least: MCU, EMRE, MICUs 1-3, MCUR1, and MCUb. The mechanism of Ca2+ selectivity and how the subunits control gating of the channel have not been fully resolved, in part, because of the difficulties associated with studying the function of the Uniporter in a purified context. The goal of this proposal is to understand the mechanisms of Ca2+ selectivity and MCUR1 regulation of the Uniporter. I will take a reductionist approach to investigate these processes using purified components. Through a blend of structural (cryo-EM) and functional (electrophysiological) approaches, I will determine critical residues for selectivity of Ca2+ ions in the pore and I will determine if the pore operates in a multi-ion fashion (Aim 1). Additionally, I will determine interactions of MCU-EMRE-MCUR1 (MER) holocomplex that are involved in gating (Aim 2). I hypothesize that high Ca2+ selectivity through the pore is mediated by a multi-ion pore mechanism (Aim 1) and that MCUR1 promotes Ca2+ uptake through inhibition of Ca2+ dependent inactivation (Aim 2). The complementary use of single-particle cryo- EM and planar lipid bilayer electrophysiology will define principles that underline ion selectivity and gating.

项目概要/摘要 线粒体钙单向转运蛋白介导线粒体对 Ca2+ 的摄取，从而调节新陈代谢， 细胞死亡和细胞质 Ca2+ 信号传导。 Uniporter如何催化离子渗透并实现离子 选择性尚不十分清楚。 Uniporter 是蛋白质亚基的超级复合物，至少包含： MCU、EMRE、MICU 1-3、MCUR1 和 MCUb。 Ca2+选择性机制及亚基如何控制 通道门控尚未完全解决，部分原因是与研究相关的困难 Uniporter 在纯化环境中的功能。该提案的目标是了解 Uniporter 的 Ca2+ 选择性和 MCUR1 调节。我将采取还原论的方法来研究这些 使用纯化成分的工艺。通过结构（冷冻电镜）和功能的结合 （电生理学）方法，我将确定孔中 Ca2+ 离子选择性的关键残基，并且我 将确定孔隙是否以多离子方式运行（目标 1）。此外，我将确定以下相互作用： 参与门控的 MCU-EMRE-MCUR1 (MER) 全复合物（目标 2）。我推测高 Ca2+ 通过孔的选择性是由多离子孔机制介导的（目标 1），并且 MCUR1 促进 Ca2+ 通过抑制 Ca2+ 依赖性失活来摄取（目标 2）。单粒子冷冻的补充使用 EM 和平面脂质双层电生理学将定义强调离子选择性和门控的原理。