Biophysical properties and role of CLIC6 in cardiomyocyte mitochondria

项目摘要/摘要 该提案的重点是最新的膜--氯离子细胞内通道6(CLIC6)的特性 CLIC家族(FOA中列出的一个渠道：RFA-RM-21-012(调查未研究的离子的试点项目 频道))。在兔胃壁细胞中发现了CLIC6(也称为Parclin)，它与 具有排酸机制。虽然在2003年发现了它，但关于它的细胞和生理作用的信息是 还没有确立。CLIC家族的成员，包括CLIC6，被证明存在于可溶性和膜中 表格。已知CLIC1、CLIC2、CLIC3、CLIC4和CLIC5的膜形式形成功能离子 频道。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 在HEK-293细胞中异位表达。我们还将蛋白质定位到 线粒体膜。CLIC6存在于保守的基因簇ACD(类似AML/CLIC/DSCR-1)中 突出了它在染色体和线粒体中的基因位置与CLIC4和CLIC4的相似性 本地化。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)作为离子通道，3)CLIC6对线粒体有贡献 通过调节氯离子通量发挥作用。我们有以下具体目标：1)建立生物物理性质 2)确定CLIC6所涉及的信号机制 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，以确定生物物理特性和涉及的调控机制 CLIC6介导的电流的调制。在没有任何CLIC蛋白的晶体结构的情况下，我们的替代 利用镉的半胱氨酸可及性方法(SCAM)筛查将提供毛孔衬里的重要信息 CLIC6的残基、选择性和门控机制。在目标2中，我们将确定CLIC6如何本地化到 线粒体膜及其在天然线粒体膜中的生物物理特性 线粒体膜片钳技术的研究进展，以及CLIC6在线粒体功能中的作用。这项提议将 通过将CLIC6蛋白建立为一个功能性的线粒体离子通道来加强我们对它的理解， 阐明其生物物理性质，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态下是有害的，如细胞凋亡、衰老和器官生理学。 从心脏、肺、肾脏到大脑，并通过增加我们对CLIC6介导的 线粒体功能障碍我们将建立CLIC6作为新的治疗靶点。

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.