Novel Mitochondrial Ion Transporters

新型线粒体离子转运蛋白

基本信息

批准号：
8155013
负责人：
Brian O'Rourke
金额：
$ 46.44万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-08-15 至 2016-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8155013
关键词：
ATP Synthesis Pathway Anions Area Arrhythmia Bioenergetics Biological Assay CLIC4 gene Calcium Calcium-Activated Potassium Channel Cardiac Carrier Proteins Cations Cell Death Cell model Cells Cystic Fibrosis Transmembrane Conductance Regulator Equilibrium Fractionation Functional disorder Funding GABA Receptor Goals Heart Homeostasis Hydrogen Inner mitochondrial membrane Ion Channel Ion Transport Ions Ischemia KCNJ1 gene Learning Link Mass Spectrum Analysis Mediating Membrane Messenger RNA Mitochondria Mitochondrial Proteins Mitochondrial Swelling Modification Molecular Molecular Biology Monovalent Cations Movement Muscle Contraction Mutagenesis Oxidants Oxidation-Reduction Oxidative Stress Pathway interactions Permeability Physiological Potassium Channel Predisposition Principal Investigator Process Production Protein Family Proteins Proteome Proteomics Protons RNA Splicing Regulation Reperfusion Injury Reperfusion Therapy Reporting Research Role Simulate Sodium Stress Techniques Tissues Translations Variant Water Workload antiporter base design gamma-Aminobutyric Acid genetic manipulation inhibitor/antagonist inward rectifier potassium channel mitochondrial K(ATP) channel mitochondrial membrane novel overexpression programs protein purification response tool trafficking uptake

项目摘要

DESCRIPTION (provided by applicant): Proton transport across the mitochondrial inner membrane provides the protonmotive force for ATP synthesis, but the importance of the transport of other ions (e.g. K+, Na+, Ca2+, and anions) in the regulation of bioenergetics has become increasingly evident in recent years. Over the past several years, our focus has been on three main areas, i) characterizing the K+ uptake pathways involved in protection against ischemic damage, ii) understanding how Na+ and Ca2+ dynamics impact energy supply and demand balance, and iii) characterizing how inner membrane oxidant-sensitive energy dissipating channels are activated by stress. While much has been learned using pharmacological tools and manipulation of ion gradients in isolated mitochondria, cells, and intact hearts, a major limitation has been the lack of molecular information about the proteins mediating ion transport in the inner membrane. Based on an exhaustive protein purification and fractionation strategy, and the team approach undertaken during the prior funding period designed to fully characterize the mitochondrial proteome and its modifications during ischemia- reperfusion, we have been successful in identifying a number of novel, high confidence candidates that we believe underlie important K+, Na+ and Ca2+ transport pathways in the mitochondrial inner membrane. Intriguingly, we have also identified novel mitochondrial anion transporters that could prove to be involved in the regulation of mitochondrial function. This project will combine molecular techniques for manipulating the expression levels of mitochondrial proteins identified by mass spectrometry with functional assays in isolated mitochondria and cells to correlate a particular ion transport pathway with its corresponding protein. Based on evidence already obtained by mass spectrometry, we believe we are on track to unequivocally resolve the molecular entities comprising the pore forming subunits of the mitochondrial ATP-sensitive (mitoKATP) and calcium-activated (mitoKCa) potassium channels, and are currently pursuing strong candidates that could mediate mitochondrial sodium calcium exchange (mNCE) and monovalent cation-hydrogen exchange (KHE or NHE). We will also investigate the possible functional role of identified, but uncharacterized, anion transporters that may be involved in mitochondrial volume regulation and the response to oxidative stress. The ultimate goal of the project is to overcome a significant roadblock to progress in the area of mitochondrial biology - the molecular identification of ion transport proteins that are critical to normal function and to the pathophysiology of ischemia-reperfusion. PUBLIC HEALTH RELEVANCE: Cardiac arrhythmias and tissue damage during ischemia and reperfusion have been linked to an impaired ability of mitochondria to maintain energy production for muscle contraction and cellular ion homeostasis. Disruption of the normal transport of ions across the mitochondrial inner membrane is the initiating factor in cell death, yet the proteins responsible for this process have not been identified. This project will identify key ion transport proteins involved in the regulation of mitochondrial energetics and examine their role in the susceptibility to ischemia- reperfusion injury.

描述（由申请人提供）：穿过线粒体内膜的质子运输为 ATP 合成提供质子动力，但其他离子（例如 K+、Na+、Ca2+ 和阴离子）运输在生物能调节中的重要性已变得越来越重要近年来明显。在过去的几年里，我们的重点一直在三个主要领域，i) 描述参与预防缺血性损伤的 K+ 吸收途径，ii) 了解 Na+ 和 Ca2+ 动态如何影响能量供需平衡，以及 iii) 描述内部如何影响能量供应和需求平衡。膜上的氧化敏感能量耗散通道被压力激活。虽然使用药理学工具和操纵分离的线粒体、细胞和完整心脏中的离子梯度已经了解了很多，但主要的限制是缺乏有关介导内膜离子转运的蛋白质的分子信息。基于详尽的蛋白质纯化和分级分离策略，以及在先前资助期间采取的旨在充分表征线粒体蛋白质组及其在缺血再灌注过程中的修饰的团队方法，我们已经成功地鉴定了许多新颖的、高可信度的候选物，我们相信这是线粒体内膜中重要的 K+、Na+ 和 Ca2+ 转运途径的基础。有趣的是，我们还发现了新型线粒体阴离子转运蛋白，它们可能参与线粒体功能的调节。该项目将把通过质谱鉴定的用于操纵线粒体蛋白表达水平的分子技术与分离的线粒体和细胞中的功能测定相结合，以将特定的离子转运途径与其相应的蛋白质相关联。基于质谱已获得的证据，我们相信我们正在明确解析包含线粒体 ATP 敏感 (mitoKATP) 和钙激活 (mitoKCa) 钾通道的成孔亚基的分子实体，并且目前正在寻求强有力的方法。可以介导线粒体钠钙交换（mNCE）和单价阳离子氢交换（KHE 或 NHE）的候选者。我们还将研究已识别但未表征的阴离子转运蛋白的可能功能作用，这些阴离子转运蛋白可能参与线粒体体积调节和氧化应激反应。该项目的最终目标是克服线粒体生物学领域进展的一个重大障碍——对正常功能和缺血再灌注病理生理学至关重要的离子转运蛋白的分子鉴定。公众健康相关性：缺血和再灌注期间的心律失常和组织损伤与线粒体维持肌肉收缩和细胞离子稳态的能量产生能力受损有关。离子穿过线粒体内膜的正常运输的破坏是细胞死亡的起始因素，但负责该过程的蛋白质尚未确定。该项目将鉴定参与线粒体能量调节的关键离子转运蛋白，并检查它们在缺血再灌注损伤易感性中的作用。