Functional Analysis of Regulatory Phosphorylation Events in Cardiac KATP Channel Subunits in Ischaemia

Protein kinase C (PKC)–mediated phosphorylation of ATP sensitive potassium channels (KATP) is believed to be involved in ischaemic preconditioning. Five consensus PKC phosphorylation sites in the Kir6.1 subunit, S354, S379, S385, S391 and S397, have been proposed as possible regulatory sites. The objective of this study was to investigate the role of these residues in regulating the functional properties of Kir6.1-containing KATP channels. HEK cells expressing Kir6.1/SUR2A or Kir6.1/SUR2B channels were used to assess electrical activity of the channels responding to PKC using the whole cell patch clamp technique. It was found that PKC activator, Phorbol 12-Myristate 13-Acetate (PMA), inhibited pinacidil-activated Kir6.1/SUR2A and Kir6.1/SUR2B currents. Mutation of serine 379 and serine 385 resulted in a significant reduction of the inhibitory effect of PKC in both Kir6.1/SUR2A and kir6.1/SUR2B channels. The inhibitory effect of PKC was also reduced in S397 mutant in Kir6.1/SUR2A channel. An endosome targeting Kir6.1/SUR2A mutant was assessed for its functional response to PKC. The inhibitory effect of PKC was abolished in this mutant. Further investigation by immunofluorescence microscopy showed that incubating HEK cells expressing Kir6.1-HA tag/SUR2A with PMA for 30 minutes resulted in internalization of the Kir6.1 protein, but not in non-treated cells. In the presence of the dynamin GTPase inhibitor, dynasore, PMA-induced internalization was blocked indicating that internalisation was via a dynamin dependent mechanism. To clarify which PKC isoform was responsible for KATP channel phosphorylation, Kir6.1 phosphorylation in rat ventricular myocytes was labelled with ³²P and stimulated with Adenosine A1 receptor agonist, 2-chloro-N6-cyclopentyl-adenosine (CCPA). The phosphorylation was investigated in the absence and presence of permeable Tat-linked PKC inhibitor peptides. Kir6.1 phosphorylation was stimulated by CCPA and inhibited in the presence of Tat-coupled PKCε inhibitor peptide but not Tat- PKCα, β, γ or δ inhibitor peptides.