Cardiac inwardly rectifying IK1 current is regulated by protein kinase A via AKAP15 and AKAP79

Background: Inwardly rectifying potassium current IK1 stabilizes the diastolic resting membrane potential. On the molecular level it is composed by heterotetrameric assembly of Kir2.1, Kir2.2 and Kir2.3 channels. Dysfunction of IK1 current strongly predisposes to focal ventricular ectopy. Protein kinase A (PKA) is a key enzyme in adrenergic signal transduction that is involved in catecholaminergic arrhythmogenesis. Targeted protein phosphorylation through PKA is mediated by A Kinase Anchoring Proteins (AKAPs). To date, however, only little is known about the AKAPs that are of functional relevance for the regulation of cardiac IK1 potassium current. Methods: Cardiac IK1 current was measured in isolated rat ventricular cardiomyocytes using whole-cell patch clamp. Kir2.x channels and AKAPs were expressed in Xenopus oocytes and current recordings were performed using the double-electrode voltage-clamp technique. Association of channels and AKAPs was examined with the use of co-immunoprecipitation and immunofluorescent confocal microscopy in isolated cardiomyocytes and in mammalian cell lines. Results: IK1 current in isolated rat ventricular cardiomyocytes was inhibited by activation of protein kinase A. This regulation was markedly attenuated by disrupting PKA-binding to AKAPs with the peptide Ht31 in the expression system. We observed functional coupling of AKAP15 and AKAP79 to Kir2.1 and Kir2.2 channels, but not to Kir2.3 channels. Kir2.1 channels were only sensitive to PKA regulation if membrane-associated AKAPs were co-expressed. Kir2.2 channels exhibited the strongest sensitivity to PKA regulation that was even further increased after co-expression of AKAPs. By contrast, Kir2.3 channels were insensitive to PKA regulation even after co- expression of AKAPs. When co-expressing the AKAP-IS related inhibitor peptide Ht31 together with AKAPs and Kir2.1 and Kir2.2 channels, PKA- dependent effects were almost completely suppressed. Both AKAP79 and AKAP15 co-immunoprecipitate and co-localize with Kir2.1 and Kir2.2 in cardiomyocytes indicating co-assembly in membrane-bound signalling complexes. Conclusion: In this project, we provide evidence for the functional and biochemical coupling of cardiac Kir2.1 and Kir2.2 channels to the membrane-associated anchoring proteins AKAP15 and AKAP79. Membrane-associated AKAPs may provide novel molecular targets for antiarrhythmic therapy downstream from the cardiac beta1-adrenoreceptor.