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 mitoch...