Molecular mechanisms of the mitochondrial calcium uniporter

项目总结/摘要 线粒体钙单向转运体（uniporter）是一种多亚基Ca 2+离子通道， 细胞质Ca 2+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， 在调节ATP生成、缓冲细胞内Ca 2+和调节细胞死亡途径中。其 功能障碍涉及广泛的病理状况，包括人类 以近端肌病和学习困难为特征的神经肌肉紊乱。这个项目 寻求扩大知识基础的分子机制的uniporter的关键 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 建立了解决离子传输和门控中基本问题的方法。目前， 单向转运体的机理研究受到技术障碍的阻碍： 线粒体的存在使得难以应用膜片钳电生理学来分析线粒体中的通道。 原生环境。在目标#1中，我们通过将单向转运蛋白靶向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 系统为高分辨率记录提供了更直接的电生理访问， 宏观和单通道水平。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 uniporter的机制。在开发新技术的同时，我们还将使用基于CRISPR的 已经在我的实验室使用的策略来解决关键的机械问题。(1)如何监管 MICU 1亚基在静息细胞条件下是单向转运体（目的#2）？(2)MCU如何与 EMRE是单向转运蛋白的膜包埋亚基，形成了一个开放的Ca 2+通道， 渗透线粒体膜（目标#3）？几个结果，包括一个突变， 出乎意料地消除了MICU 1的单向转运蛋白失活，并发现了一种独特的MCU， 嵌合体可以在没有EMRE存在的情况下传导Ca 2+，使我们能够制定逻辑和可测试的 这些问题的答案，既重要又困难。该项目的完成可以 提高设计新疗法所需的科学知识，通过调节 线粒体Ca 2+稳态。此外，在此纯化的人单向转运蛋白可用于 高通量筛选测定以鉴定靶向单转运子的药理学化合物。新 电生理学方法将允许详细分析药物动力学，需要改善铅 潜在的治疗用途的化合物。

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.