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.

项目概要/摘要该提案的重点是氯离子细胞内通道 6 (CLIC6) 的表征，这是最新的膜 CLIC 系列（FOA 中列出的频道：RFA-RM-21-012（调查待研究离子的试点项目）频道））。 CLIC6（也称为 Parchorin）是在兔胃壁细胞中发现的，它与具有泌酸机制。尽管它于 2003 年被发现，但有关其细胞和生理作用的信息尚不清楚。尚未建立。 CLIC 家族的成员，包括 CLIC6，被证明存在于可溶性和膜中形式。已知 CLIC1、CLIC2、CLIC3、CLIC4 和 CLIC5 的膜形式可形成功能离子渠道。在我们的初步实验中，我们发现CLIC6可以在平面上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）中强调其与 CLIC4 和 CLIC4 在染色体和线粒体内的基因位置的相似性本土化。根据我们的初步数据，我们将检验以下假设：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 通量发挥作用。我们有以下具体目标，1）建立生物物理特性 CLIC6 并鉴定成孔残基，2) 确定 CLIC6 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膜片钳方法来确定涉及的生物物理特性和调节机制 CLIC6 介导的电流的调制。在不存在任何 CLIC 蛋白晶体结构的情况下，我们的替代品使用镉进行半胱氨酸可及性方法（SCAM）筛查将提供毛孔内衬的重要信息 CLIC6 的残基、选择性和门控机制。在目标 2 中，我们将确定 CLIC6 如何本地化线粒体膜，其在天然线粒体膜中的生物物理特性由线粒体/ 线粒体膜片钳方法，以及 CLIC6 在线粒体功能中发挥的作用。该提案将通过将 CLIC6 蛋白建立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。线粒体功能障碍对多种病理状况（例如细胞凋亡、衰老和器官生理学）是有害的从心脏、肺、肾到大脑，并通过增加我们对 CLIC6 介导的了解线粒体功能障碍，我们将建立 CLIC6 作为新的治疗靶点。