Role of Splice Variant Clic5B in Cardiac Mitochondrial Localization and Function

Blocking chloride channels (Cl - ) with indanyloxyacetic acid (IAA-94) abrogated the cardio-protective effects of ischemic preconditioning (IPC) and increased myocardial infarction suggesting the possible contribution of IAA-94 sensitive Cl - channels in IPC mediated cardio-protection. Unlike cation channels, insights into the role of Cl - channels in cardioprotection is limited due to lack of information on their molecular identity. Chloride intracellular channel (CLIC) proteins are one of the known targets of IAA-94. They exist in both soluble and integral membrane form. Amongst the six known mammalian paralogs, CLIC1, CLIC4 and CLIC5 are most abundant in heart. Previously, we showed differential localization of CLIC4 and CLIC5 to outer and inner mitochondrial membrane respectively where as CLIC1 is present in the endoplasmic reticulum. Both CLIC4 and CLIC5 are important for mitochondrial functional integrity as well as cardioprotection from ischemia-reperfusion injury. Interestingly, none of the CLICs possesses conventional mitochondrial targeting sequence, which intrigued us to understand the mechanism of their localization to mitochondria. In this study we demonstrate that the mitochondrial localization of CLIC5 is related with the presence of splice variant CLIC5B (~50 kDa) in heart but not in lung lysates. Two different bands of CLIC5 at ~30 kDa (CLIC5A) and ~50 kDa, were observed in the cardiac mitochondrial lysates. Lung lysates showed the presence of only CLIC5A. In addition, mass spectrometric analysis revealed a peptide coverage of ~30% corresponding to CLIC5B at 50 kDa in heart but not in lung lysates further confirming its presence in heart. Moreover CLIC5 localized to mitochondria in p3 neonatal cardiomyocytes isolated from mouse but not in mouse lung epithelial (11 ± 2.1 %) cells which lacked CLIC5B indicating that the splice variant CLIC5B has a role in IMM localization. Interestingly, cardiac mitochondria from clic5 -/- mice exhibited increased ROS (p< 0.05, n=3) production whereas the clic5 -/- lung mitochondria did not show any change, suggesting the role of CLIC5B in regulating the mitochondrial function as well. Our study establishes splice variation as a mechanism for targeting ion channels to mitochondria.