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  • br Introduction Al Awqati and


    Introduction Al Awqati and his colleagues using R(+)-methylindazone, R(+)-[(6,7-dichloro-2-cyclopentyl-2,3-dihydro-2-methyl-1-oxo-1H-inden-5-yl)-oxy] acetic Pyrrolidinedithiocarbamate ammonium (IAA-94) (Landry et al., 1987) isolated the first chloride intracellular ion channel (CLIC) proteins (p64) from bovine kidney cortex microsomal membrane fractions and tracheal apical epithelium (Landry et al., 1989, Redhead et al., 1992). So far six paralogs of CLIC are identified in mammals (CLIC1–CLIC6) (Ashley, 2003, Berryman and Bretscher, 2000, Duncan et al., 1997, Edwards, 1999, Friedli et al., 2003, Heiss and Poustka, 1997, Littler et al., 2010, Nishizawa et al., 2000, Qian et al., 1999, Tulk and Edwards, 1998), four in plants (AtDHAR1–AtDHAR4) (Elter et al., 2007, Littler et al., 2010) and three in invertebrates (EXC-4, EXL-1 and DmCLIC) (Berry et al., 2003, Littler et al., 2008). CLICs exist in both soluble and integral membrane forms and exhibit trans-redox regulated channel activity (Singh, 2010, Singh and Ashley, 2006) in artificial bilayers or upon overexpression in mammalian cells (Tulk et al., 2002, Valenzuela et al., 2013, Warton et al., 2002). They exhibit differential tissue specific distribution and as the name specifies are localized to the intracellular organelles (Singh, 2010) specifically nuclear membrane (Ulmasov et al., 2007, Valenzuela et al., 1997), secretory vesicles of hippocampal neurons (Chuang et al., 1999), caveolae (Edwards and Kahl, 2010), trans-Golgi network (Edwards and Kahl, 2010), endoplasmic reticulum (Duncan et al., 1997) and mitochondria (Arnould et al., 2003, Edwards and Kahl, 2010, Fernandez-Salas et al., 1999). CLICs are multifunctional proteins playing a role in membrane trafficking, cytoskeletal function (Berryman et al., 2004), apoptosis (Fernandez-Salas et al., 2002, Suh et al., 2004), cell cycle control (Valenzuela et al., 2000), tubulogenesis (Berry et al., 2003), VEGF-mediated angiogenesis of endothelial cells (Tung et al., 2009), modulation of ryanodine receptors (Board et al., 2004, Takano et al., 2012) and cell differentiation (Suh et al., 2007). CLICs are also implicated in modulating cardiovascular physiology, specifically, CLIC4 regulates vascular endothelial growth factor (VEGF)-mediated tubulogenesis in mammalian endothelial cells (Ulmasov et al., 2009). Recently, it was reported that inhibition of CLIC4 attenuates the development of pulmonary hypertension in chronically hypoxic mice (Wojciak-Stothard et al., 2014). Additionally, pharmacological pretreatment of heart using IAA-94 abrogated the cardioprotective effects of ischemic pre-conditioning (IPC) (Batthish et al., 2002, Diaz et al., 1999), and also cyclosporine A-mediated cardioprotection against ischemia–reperfusion injury (IR) (Diaz et al., 2013). Although these studies suggest the prominence of chloride (Cl) channel-mediated cell volume regulation in cardioprotection, the molecular identity of CLIC channels in cardiac mitochondria has not been deciphered. As CLIC4 (also known as mtCLIC4) localizes to the mitochondria of both keratinocytes and L929 cells (Arnould et al., 2003, Fernandez-Salas et al., 1999, Suh et al., 2007), and IAA-94-sensitive CLIC-like currents were observed in cardiac mitoplast (Misak et al., 2013), we set on to determine the presence and localization of CLICs in cardiac cells as well as isolated cardiac mitochondria. We demonstrate that CLIC1, CLIC4 and CLIC5 are the most abundant CLIC transcripts present in the heart. CLIC4 and CLIC5 but not CLIC1 localize to adult cardiac mitochondria. CLIC localization in the mitochondria was also observed in cardiac tubes of Drosophila melanogaster. Further, we show that CLIC4 is enriched in the outer mitochondrial membrane (OMM) whereas CLIC5 localizes to inner mitochondrial membrane (IMM). Also, cardiac mitochondria from CLIC5 knockout (KO) mice showed increased reactive oxygen species (ROS) generation, thus implicating a direct role of CLIC5 in modulating mitochondrial ROS generation.