Mitochondrial membrane isolation. Kir6.1 immunoreactivity (anti-Kir6.1 (SC)), was detected in bovine (panel A), rat (panel B) mitochondrial membranes. This immunoreactivity paralleled that of CoxIV, an inner membrane marker, even as markers of the plasma membrane (Na⁺/K⁺ ATPase) and sarcoplasmic reticulum (SERCA) declined. This trend was also observed in rat tissues, including liver and brain (Panel B). Immuno-electron microscopy. The Kir6.1 (SC) antibody labels mitochondria specifically. Magnification: 40,000 × (Panel C); 100,000 × (Panel D).

Kir6.1 immunoreactivity, assessed with an antibody from Alomone Labs, was detected in bovine mitoplasts (Panel A). Immunoreactivity to Kir6.1 paralleled that of ATP synthase, (beta subunit) through mitochondrial isolation, even as myofilament (cTnI) plasma membrane (Na⁺/K⁺ ATPase) and sarcoplasmic reticulum (SERCA) markers declined. Mitochondrial localization of the Kir6.1 signal was confirmed by immunofluorescence microscopy (Panel B). Sections from rat heart ventricles were fixed, prepared and probed as described in the methods section. Kir6.1-immunoreactivity coregisters with that of ATP synthase in sections from bovine ventricle.

Mitochondrial membranes were solubilized separated by density gradient centrifugation. Kir6.1(SC) antibody recognized a 51kDa protein in fractions containing 30% and 27.5% (w/v) sucrose (Panel A). The fractions were pooled and subjected to FPLC on a Resource Q column eluted with a linear gradient of KCl (Panel B). Fractions containing the 51kDa band were pooled for further analysis. Native gel electrophoresis (Panel C) revealed that the proteins within the fraction migrate as a macromolecular complex with a MW of approx 800 kDa. 2-D gels (IEF/SDS-PAGE; Panel D) show that Kir6.1 immunoreactivity migrates to isoelectric points between 7.5 and 8.4. The 51-kDa Coomassie-stained band that co-migrated with the Kir6.1-immunoreactive band was resolved by SDS-PAGE, excised and processed for MS/MS analysis (Panel E), which identified the predominant protein as NADH-dehydrogenase flavoprotein I. Panel F depicts the MS/MS spectrum of a unique peptide, NACGSGYDFDVFVVR (Mascot ion score: 113, Peptide confidence: 95%, Protein confidence 100%).

Mitochondrial membranes were solubilized and separated by density gradient centrifugation (Panel A). Kir6.1 antibody from Alomone Labs recognized a 48-kDa protein predominantly in fractions containing 17.5% and 15% (w/v) sucrose. The fractions were pooled and subjected to FPLC on a Mono S column eluted with a linear gradient of KCl. (Panel B). Fractions containing the 48-kDa band were pooled for further characterization. The Coomassie-stained band at 48 kDa was resolved, excised and prepared for MS/MS analysis (Panel C). The MS/MS spectrum (Panel D) corresponding to the sequence, DQTNDQVTIDSALATQK (Mascot Score: 135, Peptide confidence: 95%, Protein confidence: 100%) is one of the unique peptides that identifies mitochondrial isocitrate dehydrogenase (NADP-binding form) as the preponderant 48kDa band. The isoelectric point of the Kir6.1 immunoreactivity (pI≈9; Panel E) was consistent with IDH2. Finally, sonication of mitochondria and subsequent centrifugation to separate membrane proteins from those of the matrix (Panel E) revealed that Kir6.1 immunoreactivity was found in the supernatant (matrix) fraction, inconsistent with a bona fide Kir6.1 channel.