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  • We further verified the presence of CLIC


    We further verified the presence of CLIC1, CLIC4 and CLIC5 in purified mitochondria following protocols described earlier (Singh et al., 2012). As shown in Fig. 4, CLIC4 and CLIC5 were present in the ultra-pure mitochondrial fraction (M3). All the three fractions obtained from 30% (v/v) Percoll gradient along with the crude mitochondria were analyzed for the presence of all other organelle markers. Percoll-purified mitochondrial M3 fraction was devoid of other organelles such as plasma membrane (pan-cadherin), nucleus (laminin b1), endoplasmic reticulum (GRP78 BiP), and Golgi apparatus (GM130), but showed the presence of mitochondrial markers (VDAC1 and Cox2) (Fig. 4). Although M1 and M2 fractions showed the presence of lamin B1 and pan cadherin, the ultrapure M3 fraction was devoid of any other cellular organelle contamination. Further in immuno organelle chemistry, CLIC4 and CLIC5 showed high degree of colocalization to ultra-pure mitochondrial fraction loaded with mitotracker (48.85±6.9% and 69.13±12.2%, respectively, n=3) but not CLIC1 (29.5±11.2%, n=3) (Fig. 5). These results in tandem with biochemical analysis strongly support that mammalian CLIC5 and CLIC4 are cardiac mitochondrial proteins. To further understand the localization and geographical distribution of CLIC4 and CLIC5 in the cardiac mitochondria, the Percoll-purified cardiac mitochondria were further separated into IMM and OMM fractions. As shown in the Western blot (Fig. 6A), CLIC4 showed higher levels of expression in OMM like VDAC1, and CLIC5-specific signals at ~30kDa and ~50kDa were enriched more in the IMM. To further analyze the distribution of CLIC4 and CLIC5, isolated mitochondria were imaged with the custom-built STED nanomicroscope. Confocal images were acquired before obtaining the STED images. As shown in Fig. 6II A–C and F–H, CLIC4 and CLIC5 showed differential distribution in isolated mitochondrion. CLIC4 had unique cluster distribution as reported for VDAC1 in isolated mitochondrion (Singh et al., 2012) but CLIC5 followed a continuous distribution. As CLICs play a role in ROS generation (Jiang et al., 2012, Milton et al., 2008, Wang et al., 2014), and mitochondrial ion Dihydro-β-erythroidine hydrobromide are also known to regulate ROS generation (O'Rourke et al., 2005), we set on to determine the role of CLICs in cardiac mitochondrial ROS generation. Mitochondria were isolated from clic1, clic4 and clic5 mice to study the effect of CLICs on ROS generation. In the presence of succinate, we observed that disruption of CLIC5 significantly increased (p<0.05, n=3) the rate of ROS production in KO mice in the mitochondria as compared to WT mice. There was no significant change in the rate of ROS production by clic1 and clic4 mice suggesting a potential specific role of CLIC5 in modulating mitochondrial function. We measured the total ROS generated by mitochondria isolated from clic1, clic4 and clic5 mice; only mitochondria isolated from clic5 mice showed a significant increase (p<0.05, n=3) in total ROS generation (Fig. 7). However, in clic4 mice, total mitochondrial ROS generation was not significantly different (p=0.11) than wild type as only 50% mice showed higher levels of ROS production.
    Discussion Mitochondria are the key bioenergetic cellular organelles and are known to have spectrum of cation and anion channels (Jentsch et al., 2002, O'Rourke, 2007, Szabo and Zoratti, 2014). Despite significant advancement made in the field of mitochondrial cation channels, majority of Cl channels remained uncharacterized. In the outer membrane of mitochondria, VDAC is the well-recognized Cl channel (Jentsch et al., 2002, O'Rourke, 2007, Szabo and Zoratti, 2014). On functional and electrophysiological basis, several Cl channels including inner membrane anion channel (IMAC) have been shown to be present in the mitochondria (Szabo and Zoratti, 2014), and are known to play a role in arrhythmia, ischemic pre-conditioning (IPC) and adaptive remodeling of heart during heart failure (Duan et al., 2005). Even though several pharmacological and physiological evidences indicate the presence of Cl channels in mitochondrial membranes, the molecular identity of the majority of mitochondrial Cl channels is yet to be established. In our studies we have investigated the identity and localization of a unique class of Cl channels, i.e., CLICs in cardiac mitochondria.