Distribution of Kir6.0 and SUR2 ATP-sensitive potassium channel subunits in isolated ventricular myocytes.
The subcellular distribution of ATP-sensitive potassium (K(ATP)) channel subunits in rat-isolated ventricular myocytes was investigated using a panel of subunit-specific antisera. Kir6.1 subunits were associated predominantly with myofibril structures and were co-localized with the mitochondrial marker MitoFluor red (correlation coefficient (cc) = 0.63 +/- 0.05). Anti-Kir6.1 antibodies specifically recognized a polypeptide of 48 kDa in mitochondrial membrane fractions consistent with the presence of Kir6.1 subunits in this organelle. Both Kir6.2 and SUR2A subunits were distributed universally over the sarcolemma. Lower-intensity antibody-associated immunofluorescence was detected intracellularly, which was correlated with the distribution of MitoFluor red in both cases (cc, Kir6.2, 0.56 +/- 0.05; SUR2A, 0.61 +/- 0.06). A polypeptide of 40 kDa was recognized by anti-Kir6.2-subunit antibodies in western blots of both microsomal and mitochondrial membrane fractions consistent with the presence of this subunit in the sarcolemma and mitochondria. Similarly, SUR2A and SUR2B subunits were detected in western blots of microsomal membrane proteins consistent with a sarcolemmal localization for these polypeptides. SUR2B subunits were shown in confocal microscopy to co-localize strongly with t-tubules (cc, 0.81 +/- 0.05). Together, the results indicate that Kir6.2 and SUR2A subunits predominate in the sarcolemma of ventricular myocytes consistent with a Kir6.2/SUR2A-subunit combination in the sarcolemmal K(ATP)channel. Kir6.1, Kir6.2 and SUR2A subunits were demonstrated in mitochondria. Combinations of these subunits would not explain the reported pharmacology of the mitochondrial K(ATP) channel (Mol Pharmacol 59 (2001) 225) suggesting the possibility of further unidentified components of this channel.
Singh
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Division of Medicine and Therapeutics, University of Leicester, Robert Kilpatrick Building, Leicester Royal Infirmary, LE2 7LX, Leicester, UK.
Hudman
D
D
Lawrence
C L
CL
Rainbow
R D
RD
Lodwick
D
D
Norman
R I
RI
D018528:ATP-Binding Cassette Transporters / Q000276:immunology / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D006352:Heart Ventricles / Q000276:immunology / Q000378:metabolism; D032383:Myocytes, Cardiac / Q000276:immunology / Q000378:metabolism*; D011188:Potassium / Q000378:metabolism*; D015221:Potassium Channels / Q000276:immunology / Q000378:metabolism*; D024661:Potassium Channels, Inwardly Rectifying; D051381:Rats; D011955:Receptors, Drug / Q000276:immunology / Q000378:metabolism*; D064233:Sulfonylurea Receptors
D018528:ATP-Binding Cassette Transporters; C577512:Abcc9 protein, rat; D015221:Potassium Channels; D024661:Potassium Channels, Inwardly Rectifying; D011955:Receptors, Drug; D064233:Sulfonylurea Receptors; D000255:Adenosine Triphosphate; D011188:Potassium