Novel Mitochondrial Ion Transporters

新型线粒体离子转运蛋白

• 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
• 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

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）：质子转运穿过线粒体内膜为ATP合成提供质子动力，但近年来，其他离子（例如K+、Na+、Ca 2+和阴离子）的转运在生物能量学调节中的重要性变得越来越明显。在过去的几年里，我们的重点一直在三个主要领域，i）表征K+摄取途径参与保护缺血性损伤，ii）了解Na+和Ca 2+动态如何影响能量供需平衡，iii）表征内膜氧化剂敏感的能量耗散通道如何被应激激活。虽然已经学到了很多使用药理学工具和操纵离子梯度在分离的线粒体，细胞，和完整的心脏，一个主要的限制是缺乏分子信息的蛋白质介导的离子转运在内膜。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进行的旨在充分表征线粒体蛋白质组及其在缺血-再灌注期间的修饰的团队方法，我们已经成功地鉴定了许多新的、高置信度的候选物，我们认为这些候选物是线粒体内膜中重要的K+、Na+和Ca 2+转运途径的基础。有趣的是，我们还确定了新的线粒体阴离子转运蛋白，可以证明参与线粒体功能的调节。该项目将联合收割机分子技术相结合，用于操纵通过质谱鉴定的线粒体蛋白质的表达水平，并在分离的线粒体和细胞中进行功能测定，以将特定的离子转运途径与其相应的蛋白质相关联。基于已经通过质谱法获得的证据，我们相信我们正在明确地解析包括线粒体ATP敏感性（mitoKATP）和钙激活（mitoKCa）钾通道的孔形成亚基的分子实体，并且目前正在寻找能够介导线粒体钠钙交换（mNCE）和单价阳离子-氢交换（KHE或NHE）的强有力候选物。我们还将调查可能的功能作用，确定的，但不确定的，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。 公共卫生关系：缺血和再灌注期间的心律失常和组织损伤与线粒体维持用于肌肉收缩和细胞离子稳态的能量产生的能力受损有关。线粒体内膜离子正常转运的中断是细胞死亡的起始因素，但负责这一过程的蛋白质尚未被确定。本计画将鉴定参与线粒体能量调节的关键离子转运蛋白，并研究它们在缺血再灌注损伤易感性中的作用。