Molecular mechanisms of the mitochondrial calcium uniporter

项目摘要/摘要 线粒体钙单转运体(单转运体)是一种多亚单位钙离子通道 胞质内的钙离子进入线粒体基质。在哺乳动物细胞中，单转运蛋白起着至关重要的作用。 调节三磷酸腺苷的生成，缓冲细胞内的钙离子，调节细胞死亡的途径。它的 功能障碍与广泛的病理情况有关，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 寻求扩大单一转运体关键的分子机制方面的知识库 在病理生理学中的作用。具体目标包括开发新的电生理工具，并使用 已确定的方法来解决离子传输和门控中的基本问题。目前， 对单一运输商的机械学研究一直受到一个技术障碍的阻碍： 线粒体使应用膜片钳电生理学分析通道变得困难 原生环境。在目标1中，我们通过将单一转运蛋白定位于替代蛋白来解决这个问题。 膜系统，包括重组磷脂双层和细胞质膜。两者都有 系统为高分辨率记录提供了更直接的电生理通道 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来照明最基本的 单行商的机制。在开发新技术的同时，我们还将使用基于CRISPR的 我的实验室已经在使用策略来解决关键的机械问题。(1)监管机构如何 MICU1亚基在静息细胞条件下使单转运蛋白失活(目标2)？(2)MCU和 Emre是单转运蛋白的膜嵌入亚单位，形成了一条开放的钙通道 穿透线粒体膜(目标3)？几个结果，包括一种突变 意外取消MICU1的单转化器停用，并发现唯一的MCU 嵌合体可以在没有Emre在场的情况下进行钙离子交换，允许我们制定逻辑和可测试的 回答这些重要但也很困难的问题的假设。这个项目的完成可以 通过调节提高设计新疗法治疗疾病所需的科学知识 线粒体钙动态平衡。此外，在此纯化的人单转运蛋白可用于 高通量筛选试验用于识别单转运体靶向的药理化合物。新的 电生理学方法将允许对药物动力学进行详细分析，这是改善铅含量所必需的 潜在治疗用途的化合物。

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.