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.