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.

项目概要/摘要 线粒体钙单向转运蛋白（the uniporter）是一个多亚基 Ca2+ 离子通道，输入 细胞质 Ca2+ 进入线粒体基质。在哺乳动物细胞中，单向转运蛋白起着至关重要的作用 调节 ATP 生成、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 以近端肌病和学习困难为特征的神经肌肉疾病。这个项目 寻求扩大单向转运蛋白关键分子机制的知识库 在病理生理学中的作用。具体目标包括开发新的电生理学工具，并使用 建立了解决离子传输和门控基本问题的方法。现在， 单向转运蛋白的机制研究受到技术障碍的阻碍： 线粒体使得应用膜片钳电生理学来分析通道变得困难 原生环境。在目标#1中，我们通过将单向转运蛋白定位为替代蛋白来解决这个问题 膜系统，包括重构的磷脂双层和细胞质膜。两个都 系统为高分辨率记录提供了非常简单的电生理学访问 宏观层面和单渠道层面。我们计划全面建立这些工具，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最基本的 单向转运蛋白的机制。在开发新技术的同时，我们还将使用基于 CRISPR 的 我的实验室已使用该策略来解决关键的机械问题。 （一）如何监管 MICU1 亚基在静息细胞条件下使单向转运蛋白失活（目标#2）？ (2) MCU 和 EMRE，单向转运体的膜嵌入亚基，形成开放的 Ca2+ 途径，使 Ca2+ 渗透线粒体膜（目标#3）？几个结果，包括一个突变 出乎意料地废除了 MICU1 的单向转运蛋白失活作用，并发现了独特的 MCU 嵌合体可以在没有 EMRE 存在的情况下传导 Ca2+，使我们能够制定逻辑且可测试的 回答这些重要但也困难的问题的假设。该项目的完成可以 通过调节来提高设计治疗疾病的新疗法所需的科学知识 线粒体 Ca2+ 稳态。此外，此处纯化的人类单向转运蛋白可用于 高通量筛选分析来鉴定单向转运蛋白靶向药理化合物。新的 电生理学方法将允许对药物动力学进行详细分析，这是改善铅所需的 具有潜在治疗用途的化合物。