Molecular mechanisms of the mitochondrial calcium uniporter

线粒体钙单向转运蛋白的分子机制

• 批准号: 10192757
• 负责人: Ming-Feng Tsai
• 金额: $ 31.1万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-01 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10192757
• 关键词: Address; Adopted; Animals; Area; Behavior; Binding; Biochemical; Biological Assay; Buffers; Caenorhabditis elegans; Cardiac; Cell Death; Cell Respiration; Cell membrane; Chimera organism; Clustered Regularly Interspaced Short Palindromic Repeats; Co-Immunoprecipitations; Cysteine; Detergents; Disease; Drug Kinetics; Electrophysiology (science); Environment; Functional disorder; Future; Generations; Hand; Homeostasis; Human; Inner mitochondrial membrane; Ion Channel; Ion Transport; Ions; Knowledge; Lead; Learning; Light; Literature; Mammalian Cell; Mediating; Medicine; Membrane; Methods; Mitochondria; Mitochondrial Matrix; Molecular; Morphologic artifacts; Muscle; Mutation; Myopathy; Neuromuscular Diseases; Neurons; Pathologic; Pathology; Pathway interactions; Permeability; Pharmacology; Phospholipids; Physiological; Plasma Cells; Play; Problem Solving; Procedures; Property; Proteins; Regulation; Research; Research Personnel; Resolution; Rest; Role; Series; Signal Transduction; Site; Structure; Support System; System; Techniques; Testing; Therapeutic Uses; Transmembrane Domain; Xenopus oocyte; base; calcium uniporter; design; experimental study; genome editing; high throughput screening; improved; inhibitor/antagonist; knowledge base; method development; mitochondrial membrane; neuromuscular; novel therapeutics; patch clamp; reconstitution; tool

Project Summary/Abstract The mitochondrial calcium uniporter (the uniporter) is a multi-subunit Ca2+ ion channel that imports cytoplasmic Ca2+ into the mitochondrial matrix. In mammalian cells, the uniporter plays a crucial role in regulating ATP generation, buffering intracellular Ca2+, and modulating cell-death pathways. Its dysfunction has been implicated in a wide range of pathological conditions, including a human neuromuscular disorder characterized by proximal myopathy and learning difficulties. This project seeks to expand the knowledge base in the molecular mechanisms underlying the uniporter's key roles in pathophysiology. Specific aims include developing new electrophysiological tools, and using established methods to address fundamental questions in ion transport and gating. Currently, mechanistic studies of the uniporter have been impeded by a technical barrier: The small size of mitochondria makes it difficult to apply patch-clamp electrophysiology to analyze the channel in native environments. In Aim #1, we solved this problem by targeting uniporter proteins to alternative membrane systems, including reconstituted phospholipid bilayers and cell plasma membranes. Both systems offer much straightforward electrophysiological access for high-resolution recordings in macroscopic and single-channel levels. We plan to fully establish these tools so that researchers can begin to adopt classical ion-channel electrophysiology to illuminate most fundamental mechanisms of the uniporter. While developing new techniques, we will also use a CRISPR-based strategy already in use in my lab to attack key mechanistic questions. (1) How does a regulatory MICU1 subunit inactivate the uniporter in resting cellular conditions (Aim #2)? (2) How do MCU and EMRE, the membrane-embedded subunits of the uniporter, form an open Ca2+ pathway for Ca2+ to permeate mitochondrial membranes (Aim #3)? Several results, including a mutation that unexpectedly abolishes uniporter inactivation by MICU1, and the discovery of a unique MCU chimera that can conduct Ca2+ without EMRE present, allow us to formulate logical and testable hypotheses to answer these important but also difficult questions. Completion of this project can improve the scientific knowledge necessary to design new therapies to treat disease by modulating mitochondrial Ca2+ homeostasis. Moreover, human uniporter proteins purified here can be used for high-throughput screening assays to identify uniporter-targeting pharmacological compounds. New electrophysiological methods will allow detailed analysis of drug kinetics, required to improve lead compounds for potential therapeutic use.

项目摘要/摘要 线粒体钙Uniporter（Uniporter）是一种多物品CA2+离子通道，导入 细胞质Ca2+进入线粒体基质。在哺乳动物的细胞中，单位物质起着至关重要的作用 在调节ATP生成时，缓冲细胞内Ca2+和调节细胞死亡途径。它是 功能障碍与多种病理状况有关，包括人类 神经肌肉疾病的特征是近端肌病和学习困难。这个项目 旨在扩大Uniporter键的分子机制中的知识基础 病理生理学中的作用。具体目的包括开发新的电生理工具，并使用 建立的方法来解决离子运输和门控中的基本问题。现在， 统一机的机械研究受到技术障碍的阻碍： 线粒体使得很难应用贴片钳电生理学来分析该通道 本地环境。在AIM＃1中，我们通过将Uniporter蛋白定位到替代方案来解决此问题 膜系统，包括重建的磷脂双层和细胞质膜。两个都 系统为高分辨率录音提供了很多直接的电生理访问 宏观和单通道水平。我们计划完全建立这些工具，以便研究人员 可以开始采用经典的离子通道电生理学来阐明大多数基本 Uniporter的机制。在开发新技术时，我们还将使用基于CRISPR的 我的实验室已经使用的策略来攻击关键机理问题。 （1）如何调节 MICU1亚基在静止的细胞条件下（AIM＃2）使Uniporter失活？ （2）MCU和 EMRE是Uniporter的膜包裹的亚基，形成了Ca2+的开放CA2+途径 渗透线粒体膜（AIM＃3）？几个结果，包括突变 出乎意料地废除了MICU1的Uniporter灭活，并发现了独特的MCU 可以在不存在EMRE的情况下进行CA2+的嵌合体，允许我们制定逻辑和可测试 假设回答这些重要但困难的问题。完成这个项目可以 改善设计新疗法以通过调节来治疗疾病所需的科学知识 线粒体Ca2+稳态。此外，在此纯化的人类单位蛋白可用于 高通量筛选测定法，以识别靶向Uniporter的药理学化合物。新的 电生理方法将允许对药物动力学进行详细分析，以改善铅 潜在治疗用途的化合物。