Biophysical properties and role of CLIC6 in cardiomyocyte mitochondria

CLIC6 在心肌细胞线粒体中的生物物理特性和作用

基本信息

批准号：
10451254
负责人：
Harpreet Singh
金额：
$ 15.75万
依托单位：
OHIO STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-06-01 至 2024-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10451254
关键词：
Acids Address Aging Antibodies Apoptosis Brain CLIC4 gene Cadmium Cardiac Myocytes Cell physiology Cells Chlorides Chromosome 21 Chromosome 4 Chromosome 6 Chromosomes Crystallization Custom Cysteine DSCR1 protein Data Ectopic Expression Electrophysiology (science)Endoplasmic Reticulum Environment Family Family member Gastric Parietal Cells Gene Cluster Gene Order Genes Genetic Diseases Heart Homeostasis Homologous Protein Intervention Intracellular Membranes Ion Channel Ions Kidney Knowledge Link Location Lung Mediating Membrane Methods Mitochondria Molecular Names Neonatal Oocytes Organ Oryctolagus cuniculus Oxidation-Reduction Pathologic Pharmaceutical Preparations Physiological Physiology Pilot Projects Play Protein Family Proteins Regulation Resources Role Signal Transduction Structure Testing Therapeutic Time Transmembrane Domain apical membrane base biophysical properties experimental study human disease interdisciplinary approach member mitochondrial dysfunction mitochondrial membrane new therapeutic target novel patch clamp public health relevance screening therapeutic target

项目摘要

Project Summary / Abstract The proposal is focused on characterization of Chloride intracellular channel 6 (CLIC6), the newest membrane of the CLIC family (a channel listed in FOA: RFA-RM-21-012 (Pilot Projects Investigating Understudied Ion Channels)). CLIC6 (also known as Parchorin) was discovered in rabbit gastric parietal cells, and it is associated with acid secretion mechanism. Though discovered in 2003, information on its cellular and physiological roles is not yet established. Members of the CLIC family including CLIC6, are shown to exist in soluble and membrane forms. The membrane forms of CLIC1, CLIC2, CLIC3, CLIC4, and CLIC5 are known to form functional ion channels. In our preliminary experiments, we have discovered that CLIC6 can form functional channels in planar bilayers as well as on ectopic expression in HEK-293 cells. We have also localized the protein to the mitochondrial membranes. CLIC6 is present in the conserved gene cluster ACD (AML/CLIC/DSCR-1-like) highlighting its similarity to CLIC4 and CLIC4 in its gene position within the chromosome as well as mitochondrial localization. Based on our preliminary data, we will test the hypothesis that: 1) CLIC6 is present in mitochondrial membranes, 2) it functions as an ion channel, and 3) CLIC6 contributes to mitochondrial function by regulating Cl fluxes. We have the following specific aim, 1) establish biophysical properties of CLIC6 and identify the pore-forming residues, and 2) determine the signal mechanism involved in CLIC6 mitochondrial targeting and its role in mitochondrial function. In Aim1, we will use a combination of planar bilayers and patch-clamp approaches to determine biophysical properties and regulatory mechanisms involved in the modulation of the CLIC6-mediated currents. In absence of crystal structure of any CLIC protein, our Substituted Cysteine Accessibility Method (SCAM) screening using cadmium will provide vital information of pore-lining residues, selectivity, and gating mechanisms of CLIC6. In Aim 2, we will determine how CLIC6 localizes to mitochondrial membranes, its biophysical properties in native mitochondrial membranes by mitochondria/ mitoplast patch-clamp approaches, and the role played by CLIC6 in mitochondrial function. The proposal will enhance our understanding of CLIC6 protein by establishing it as a functional mitochondrial ion channel, elucidating its biophysical properties, and determining its role in mitochondrial function. Mitochondrial dysfunction is detrimental in several pathological conditions such as apoptosis and aging and organ physiology ranging from heart, lungs, kidneys to the brain, and by increasing our understanding of CLIC6-mediated mitochondrial dysfunction we will establish CLIC6 as a novel therapeutic target.

项目摘要 /摘要该提案的重点是氯化物内通道6（CLIC6）的表征，最新的膜 CLIC家族（FOA中列出的渠道：RFA-RM-21-012（试点项目调查了研究研究的离子频道））。在兔胃顶细胞中发现了clic6（也称为parchorin），它与之相关具有酸分泌机制。尽管在2003年发现了有关其细胞和生理作用的信息是尚未确定。 Clic家族的成员包括Clic6，在可溶性和膜上存在表格。已知CLIC1，CLIC2，CLIC3，CLIC4和CLIC5的膜形式形成功能性离子频道。在我们的初步实验中，我们发现CLIC6可以在平面中形成功能通道双层以及HEK-293细胞中的异位表达。我们还将蛋白质定位到线粒体膜。 Clic6存在于保守基因簇ACD（AML/Clic/DSCR-1样）中突出显示其与Clic4和Clic4在染色体中的基因位置以及线粒体的相似性本土化。根据我们的初步数据，我们将测试以下假设：1）Clic6存在于线粒体膜，2）它充当离子通道，3）Clic6有助于线粒体通过调节CL通量来功能。我们有以下特定目的，1）建立生物物理特性 clic6并确定孔形成残基，2）确定clic6中涉及的信号机制线粒体靶向及其在线粒体功能中的作用。在AIM1中，我们将使用平面双层的组合和贴片钳方法来确定与该生物物理特性和调节机制有关 Clic6介导的电流的调节。在没有任何Clic蛋白的晶体结构的情况下，我们取代半胱氨酸可及性法（SCAM）使用镉筛选将提供孔衬的重要信息 Clic6的残基，选择性和门控机制。在AIM 2中，我们将确定CLIC6如何本地化线粒体膜，线粒体/线粒体的天然线粒体膜中的生物物理特性线粒体斑块钳接近，以及Clic6在线粒体功能中所起的作用。提案将通过将其确定为功能性线粒体离子通道来增强我们对CLIC6蛋白的理解，阐明其生物物理特性，并确定其在线粒体功能中的作用。线粒体功能障碍在几种病理状况（例如凋亡和衰老和器官生理学）下有害从心脏，肺，肾脏到大脑，并通过增加对Clic6介导的理解线粒体功能障碍我们将建立CLIC6作为一种新型的治疗靶点。