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  • IAA sensitive Cl channels were


    IAA-94-sensitive Cl channels were shown to mediate cardioprotection due to IPC (Diaz et al., 1999) and cyclosporine A (Diaz et al., 2013). CLIC-like channel activity was also observed in cardiac mitoplast (Misak et al., 2013) but the molecular identity of these intracellular Cl channels and their presence in cardiac mitochondria and functional roles are not completely understood. CLICs were first identified by IAA-94-affinity purification and shown to exhibit reconstitutable Cl channel activity that can be inhibited by IAA-94 and specific SB 265610 australia (Landry et al., 1990, Singh, 2010). CLICs are conserved from bacteria to mammals and are present in all the prominent tissues and organs (Littler et al., 2010). In our studies, we have established that CLICs are present in the mammalian and Drosophila hearts. These results were corroborated by Western blot as well as immunohistochemistry, and also previous studies, where some of these CLICs were studied individually at transcript and/or at protein levels (Berryman and Bretscher, 2000, Padmakumar et al., 2014, Qian et al., 1999, Takano et al., 2012). CLICs are known to be present in intracellular organelles, specifically CLIC4 (also known as mtCLIC) was shown to be present in the mitochondria of keratinocytes (Fernandez-Salas et al., 2002), and CLIC1 to the nucleus of CHO-K1 cells where IAA-94 arrested these cells in G2/M phase (Valenzuela et al., 1997, 2000). CLIC4 was also reported to be overexpressed in the mitochondria of mtDNA depleted L929 cells (mouse fibroblasts) wherein it was involved in maintaining the mitochondrial membrane potential (Arnould et al., 2003), thereby protecting the cells from undergoing apoptosis. In our comprehensive study, for the first time we show that CLIC5, CLIC4, and DmCLIC preferentially localize to cardiac mitochondria, whereas CLIC1 and a subfraction of CLIC4 localize to the cardiac ER. Interestingly, in all the fractions of mitochondria, a higher molecular protein band of size ~50kDa was observed when probed with anti-CLIC5 antibodies (Figs. 1C, and Fig. 4, Fig. 6I). This could be an isoform of CLIC5B reported in humans (Berryman and Bretscher, 2000), which may also be present in the rodent heart. Even though CLIC1 is reported to be present in the nucleus of CHO-K1 (Valenzuela et al., 1997) and Panc1 (Ulmasov et al., 2007) cells, none of the cardiac CLIC proteins (CLIC1, CLIC4 and CLIC5) studied here localize to the nucleus, which may be due to the highly-differentiated terminal state of adult cardiomyocytes. Percentage localization of CLIC1 to the mitochondria of neonatal and adult cardiomyocytes was comparable to CLIC4 but the localization reduced upon purification of mitochondria. This suggests the association of CLIC1 to mitochondrial associated membranes as reported for ABCC6 transporter protein (Martin et al., 2012). Even though we have shown cardiac mitochondrial localization of CLICs across species (R. norvegicus and D. melanogaster), in silico analyses showed no conserved mitochondrial targeting sequence, as also reported for other mitochondrial ion channels such as mitoBKCa (Singh et al., 2013). Although most of the analyses predicted high probability of VDAC2 (Table 1) in mitochondria, the probability score for CLICs being in mitochondria was very low. Therefore the mechanism of mitochondrial transport of these ion channels would be interesting to evaluate. Further, CLICs are intracellular ion channel proteins present in soluble and membrane forms, and they can also be used as model proteins to study mitochondrial translocation of soluble proteins. IAA-94-sensitive Cl currents of ~129±3pS (Misak et al., 2013) in cardiac mitoplast are similar to the reported single channel conductance of CLIC5 (Singh et al., 2007). Our results further indicate that CLIC4 and/or CLIC5 could be the potential components of this channel activity. Functionally, mtCLIC4, like VDAC1 is established in mediating apoptosis (Fernandez-Salas et al., 2002, Suh et al., 2005). The presence of CLIC4 in the OMM of cardiomyocytes probably suggests their role in regulating cell death pathways under stress conditions in cardiomyocytes as well. Since CLIC4 is known to play an active role in apoptosis, it will also be of interest to see if it interacts with mitochondrial permeability transition pore (mPTP) or is a part of mPTP in the outer membrane. Although CLIC4 is enriched in the outer membrane, we do detect a subfraction of CLIC4 in the IMM which could explain its possible role in the modulation of membrane potential (Arnould et al., 2003). Interestingly, CLIC5 was highly enriched in IMM, representing it as a probable contributor for multiple inner membrane anion channel (iMAC) conductance observed in earlier studies (Szabo and Zoratti, 2014). The presence of CLIC5 in IMM further speculates its functional role in modulating oxidative phosphorylation or its interaction with electron transport chain (ETC) complex proteins.