Structural and phosphorylation studies of cardiac ATP-sensitive potassium channels

To establish the localization of KATP channel subunits in rat isolated cardiac ventricular myocytes, characterized polyclonal antibodies (anti-Kir6.1, Kir6.2, SUR2A and SUR2B) were utillized in immunocytochemistry experiments and Western blots on subcellular fractions isolated from rat heart. Results from immunocytochemistry and Western blots showed the presence of all four subunits in cardiac myocytes. Kir6.1 was predominantly localized in mitochondria shown by immunocytochemistry and Western blots in isolated rat heart mitochondrial fractions. Kir6.2 was localized to the sarcolemma, with some present in mitochondria. Western blots on microsomal membrane fractions further confirmed localization of Kir6.2 in sarcolemma. Immunocytochemistry results revealed SUR2A was localized to sarcolemma and mitochondria, but was not detected in Western blots on mitochondrial fraction. However, [ 32P] Kir6.1 was co-immunoprecipitated by anti SUR2A from whole myocyte extracts after stimulation of PKA and PKC. SUR2B localized to T-tubules of the myocytes. Both the SUR2 polypeptides were detected in immunoblots of microsomal membrane fractions. In vitro phosphorylation of KATP channel inward rectifier subunits revealed Kir6.1 to be a substrate for cyclic-AMP dependent protein kinase (PKA) and PKC-mediated phosphorylation, which was also confirmed by in vivo phosphorylation in isolated cardiac myocytes stimulated with forskolin and adenosine, respectively. In vivo phosphorylation of the Kir6.1 subunit, when stimulated with A1 receptor agonist, 2-chloro-N6-cyclopentyladenosine (CCPA), was significantly reduced when cells were incubated with the PKC inhibitor (Chelerythrine) but was unaltered when cells were incubated with other kinase inhibitors. Phosphoamino acid analysis on in vivo phosphorylated Kir6.1 subunit revealed serine as the major phosphorylated amino acid residue. In vitro PKC-mediated phosphorylation of chimaeras between Kir6.1 and Kir6.2 revealed the location of the phosphoserine residue to be between serine354- serine397 of the Kir6.1 protein, where five potential serine phosphorylation sites are present. Out of the five potential serine phosphorylation sites, in vitro expressed Kir6.1 mutant S379A was not phosphorylated in the presence of PKC.