Biophysical properties and role of CLIC6 in cardiomyocyte mitochondria

项目总结/摘要 该提案的重点是表征氯细胞内通道6（CLIC 6），最新的膜 CLIC系列（FOA：RFA-RM-21-012（研究未充分研究离子的试点项目）中列出的通道 通道））。CLIC 6（也称为Parchorin）是在兔胃壁细胞中发现的，它与胃粘膜上皮细胞的增殖有关。 具有泌酸机制。虽然在2003年被发现，但有关其细胞和生理作用的信息仍然存在。 尚未建立。CLIC家族的成员，包括CLIC 6，被证明存在于可溶性和膜 forms.已知CLIC 1、CLIC 2、CLIC 3、CLIC 4和CLIC 5的膜形式形成功能离子 渠道在我们的初步实验中，我们已经发现CLIC 6可以在平面上形成功能通道， 双层以及在HEK-293细胞中异位表达。我们还将蛋白质定位于 线粒体膜CLIC 6存在于保守基因簇ACD（AML/CLIC/DSCR-1样）中 突出了其在染色体内以及线粒体内的基因位置与CLIC 4和CLIC 4的相似性 本地化基于我们的初步数据，我们将检验以下假设：1）CLIC 6存在于 CLIC 6是线粒体膜的一个重要组成部分，2）它作为离子通道发挥作用，3）CLIC 6有助于线粒体膜的形成。 通过调节Cl通量发挥作用。我们有以下具体目标，1）建立生物物理性质的 CLIC 6的信号转导机制，并确定孔形成残基，和2）确定CLIC 6参与的信号机制 线粒体靶向及其在线粒体功能中的作用。在Aim 1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调控机制参与的， CLIC 6介导的电流的调节。在不存在任何CLIC蛋白的晶体结构的情况下，我们的取代的 半胱氨酸可及性法（SCAM）筛选镉将提供重要的信息，孔隙内衬 CLIC 6的残基、选择性和门控机制。在目标2中，我们将确定CLIC 6如何本地化为 线粒体膜，其生物物理特性在天然线粒体膜的线粒体/ 线粒体膜片钳方法，以及CLIC 6在线粒体功能中发挥的作用。该提案将 通过将CLIC 6蛋白确立为功能性线粒体离子通道来增强我们对CLIC 6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态如细胞凋亡和衰老以及器官生理学中是有害的 从心脏，肺，肾脏到大脑，通过增加我们对CLIC 6介导的 我们将CLIC 6确立为新的治疗靶点。

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.