Molecular Physiology of Mitochondrial Calcium Uniporter

线粒体钙单向转运蛋白的分子生理学

• 批准号: 10586576
• 负责人: Vivek Garg
• 金额: $ 33.99万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-06-15 至 2027-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10586576
• 关键词: Binding; Biochemical; Bioenergetics; Biological Assay; Buffers; CRISPR/Cas technology; Cell Death; Cell Line; Cells; Complex; Cysteine; Cytoplasm; Data; Development; Disease; Dominant-Negative Mutation; EF-Hand Domain; Electrophysiology (science); Enzymes; Exercise; Fibroblasts; Functional disorder; Goals; Heart Diseases; Heart failure; Homeostasis; Immune System Diseases; Individual; Inner mitochondrial membrane; Intervention; Kinetics; Knock-out; Knockout Mice; Learning; Location; Luminal region; Macromolecular Complexes; Malignant Neoplasms; Measures; Mediating; Membrane; Metabolic Diseases; Methodology; Methods; Mitochondria; Mitochondrial Matrix; Molecular; Molecular Structure; Movement Disorders; Mus; Muscular Dystrophies; Mutagenesis; Mutation; Myopathy; Nerve Degeneration; Neuromuscular Diseases; Oxidation-Reduction; Patch-Clamp Techniques; Pathologic; Pathway interactions; Permeability; Physiological; Physiology; Play; Probability; Production; Proteins; Publishing; Regulation; Reperfusion Injury; Resolution; Rest; Role; Side; Skeletal Muscle; Stroke; Structure; System; Techniques; Testing; Therapeutic; Tissues; Work; biophysical properties; calcium uniporter; density; direct application; drug development; human disease; innovation; insight; knockout gene; loss of function mutation; mitochondrial dysfunction; mitochondrial membrane; mitochondrial permeability transition pore; mutant; nervous system disorder; novel; overexpression; paralogous gene; patch clamp; pharmacologic; physiologic model; prevent; response; stoichiometry; success; targeted treatment; therapeutic target; uptake

Project Summary The mitochondrial calcium uniporter complex (MCUcx) is a highly-selective, tetrameric Ca2+ channel that plays a primary role in transporting Ca2+ from the cytoplasm into the mitochondrial matrix. Although Ca2+ uptake by mitochondria plays a crucial role in stimulating ATP production, matrix Ca2+ overload can trigger opening of the mitochondrial permeability transition pore, leading to cell death. MCU dysfunction has been implicated in several pathological conditions such as heart failure, ischemia–reperfusion injury, neurodegeneration, cancer and skeletal muscle dystrophies. Recent biochemical and structural studies have shown that the MCUcx is a macromolecular complex composed of a pore-forming MCU subunit, an essential subunit EMRE, and EF-hand domain-containing MICU1–3 subunits. How each subunit of the MCU complex comes together and regulates mitochondrial Ca2+ entry has been the focus of many recent studies. Using direct mitochondrial patch-clamp methodology and an innovative heterologous expression system, we recently performed a comprehensive structure–function analysis of the MCUcx in the inner mitochondrial membrane. This expression system consists of gene knockout of each individual subunit of the MCUcx (more than 6 different knockout lines were generated by CRISPR/cas9) and is readily amenable to different patch-clamp configurations with a high success rate. Using this system and the patch-clamp technique, we propose to answer central questions in the field: 1) Where is the MCU gate, and how is this gate regulated by Ca2+ and MCU accessory subunits? and 2) How do the unique features in the MCU macromolecular structures (as revealed by recent structural studies) relate to MCU gating and function? Accomplishment of the aims of this proposal will provide better understanding of the mechanisms that regulate Ca2+ entry via the MCU and create an essential framework for the development of pharmacological interventions that target MCU gating for therapeutic purposes.

项目摘要 线粒体钙单向转运体复合物（MCUcx）是一种高度选择性的四聚体钙通道， 在将Ca 2+从细胞质转运到线粒体基质中的主要作用。虽然Ca2+的吸收， 线粒体在刺激ATP产生中起着至关重要的作用，基质Ca 2+超载可以触发线粒体的开放， 线粒体通透性转换孔，导致细胞死亡。微控制器功能障碍与 几种病理状况如心力衰竭、缺血-再灌注损伤、神经变性、癌症 和骨骼肌营养不良。最近的生物化学和结构研究表明，MCUcx是一种 由成孔MCU亚基、必需亚基EMRE和EF-手组成的大分子复合物 包含MICU1 - 3亚基的结构域。MCU复合体的每个子单元如何聚集在一起并调节 线粒体Ca~（2+）内流是近年来研究的热点。采用直接线粒体膜片钳技术 方法和创新的异源表达系统，我们最近进行了全面的 线粒体内膜中MCUcx的结构-功能分析。该表达系统 由MCUcx的每个单独亚基的基因敲除组成（在MCUcx中， 由CRISPR/cas9产生），并且易于适用于不同的膜片钳配置，具有高的生物学活性。 成功率利用这一系统和膜片钳技术，我们建议回答中心问题， 领域：1）MCU门在哪里，这个门是如何被Ca2+和MCU附属亚基调节的？和2） MCU大分子结构中的独特特征（如最近的结构研究所揭示的） 与MCU门控和功能有关？实现本提案的目标将提供更好的 了解通过MCU调节Ca2+进入的机制，并为以下操作创建一个基本框架： 开发针对MCU门控的药物干预措施以达到治疗目的。