Molecular Physiology of Mitochondrial Calcium Uniporter

线粒体钙单向转运蛋白的分子生理学

基本信息

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
项目类别：
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

项目摘要

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Uniporter复合物（MCUCX）是高度选择的四聚体CA2+通道。将Ca2+从细胞质转运到线粒体基质中的主要作用。线粒体在刺激ATP的产生中起着至关重要的作用，矩阵Ca2+超负荷可以触发开放线粒体通透性过渡孔，导致细胞死亡。多种病理状况，例如心力衰竭。和骨骼肌营养不良。最近的生化和结构研究表明大分子由形成孔的MCU亚基，必需的亚基EMRE和EF手组成识别域的MICU1-3亚基。线粒体Ca2+进入是许多近期研究的重点。方法论和一个动力的异源表达系统，我们最近进行了汇编结构 - 内部膜膜中MCUCX的功能分析由MCUCX的每个单个亚基的基因敲除（超过6种不同的敲除线由CRISPR/CAS9生成），可清楚地适用于不同的贴片钳配置成功率。字段：1）MCU门在哪里，而GATE是如何通过Ca2+和MCU附件子来定制的？ MCU大分子结构中的独特特征如何（如最近的结构研究所引起的）与MCU门控和功能有关？理解通过MCU定期进入CA2+进入的机制，并创建一个必需的框架叉用于治疗目的的药理学国际干预目标MCU门控。