Molecular Identity and Functional Characterization of Chloride Intracellular Channel (CLIC) Proteins in Cardiac Mitochondria

Abstract

CLIC proteins have six different paralogs in mammals (CLIC1-6) and exist in both soluble and integral membrane forms. Preconditioning rabbit ventriculocytes with IAA-94, an inhibitor of CLICs, before ischemia, increased myocardial infarction implicating CLICs role in cardioprotection. However, the molecular identity of cardiac CLIC, and the mechanism of CLIC-mediated cardioprotection is unknown. In this study, we focused on establishing the molecular identity, localization and functional role of CLICs in cardiac tissue of R. novergicus. Relative qPCR analyses indicate CLIC4, CLIC5 and CLIC1 as the most abundant CLICs present in the ventricle. CLIC4 (50±0.1%) and CLIC5 (75±3%) localize to the mitochondria of adult cardiomyocytes (n=3). Cardiac CLIC4 and CLIC5 are present in 40±2% and 74±3% of Percoll-purified mitochondria (n=3), respectively. Also, the only CLIC present in D. melanogaster showed mitochondrial localization in cardiac tubes. Further, IAA-94 partially blocked the channel activity of Percoll-purified mitochondrial membrane proteins reconstituted in a planar lipid bilayer. We also investigated the role of CLICs in modulation of ROS production by ETC. In the presence of succinate (complex II/III) and glutamate/malate (complex I), addition of 100 μM IAA-94 resulted in a robust release of ROS from mitochondria. The EC50 was found to be 16.5 +¬ 0.15 μM (n=3) for succinate. Initial fast release was followed by a significant reduction (82.5±3% n=3, p<0.05) in rate of ROS production. The ROS release is specific to chloride conductance inhibition, as it also occurs with DIDS (ClC blocker). Blocking mPTP by cyclosporine A did not alter ROS release generated by CLIC inhibition indicating it is independent of mPTP (n=3). In conclusion, we have deciphered the molecular identity of a cardiac CLIC, established its mitochondrial localization and a role of Chloride conductance in modulation of mitochondrial ROS generation.