Abstract 19990: Identification and Characterization of an ATP-sensitive Potassium Channel in the Inner Membrane of Cardiac Mitochondria
Opening of mitochondrial potassium channels is cardioprotective, yet, to date, the pore-forming subunits of these channels remain unidentified. We therefore undertook an in-depth proteomic analysis of the mitochondria employing repeated fractionation at the organellar, protein, and peptide levels. Briefly, density-purified inner membranes were extracted with 1% lauryl maltoside, and fractionated by sucrose gradient centrifugation. Each fraction was digested with trypsin and subjected to strong-cation exchange HPLC prior to reversed-phase LC-coupled tandem mass spectrometry. 964 proteins were identified, of which 684 were classified as mitochondrial in UniProtKB and/or MitoCarta databases. From the inner membrane fraction, the ROMK (renal outer-medullary potassium) channel, was identified by 6 spectra matching two overlapping peptides. Matches were statistically validated at >95%. Subsequent bioinformatic analysis detected a mitochondrial localization sequence near the N-terminus of ROMK. To confirm mitochondrial localization experimentally, neonatal rat ventricular myocytes (NRVM) were transiently transfected with truncation mutants of human ROMK isoforms, fused with green fluorescence protein (GFP) or V5-tag at the C-terminus of the channel, and subsequently imaged by 2-photon microscopy. When the predicted targeting presequence was fused to GFP, the construct co-localized with tetramethylrhodamine methyl ester (TMRM) staining of the mitochondria. Furthermore, to determine whether a ROMK isoform might mediate mitochondrial potassium uptake, we measured K+-dependent swelling in rat heart mitochondria. In preliminary studies, diazoxide-induced mitochondrial swelling was abrogated by Tertiapin-Q, a high-affinity ROMK channel toxin. Reverse-Transcription/Polymerase Chain Reaction (RT-PCR) identified 3 isoforms (ROMK1, ROMK2&ROMK6) in the adult rat hearts and NRVMs. The findings support an isoform of ROMK as a candidate for the pore-forming subunit of mitoKATP. Ongoing targeted knockdown studies using siRNA will address the role of ROMK in cytoprotection against ischemic or oxidative damage.
- © 2010 by American Heart Association, Inc.