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Background: Cardiac inwardly rectifying IK1 current drives terminal repolarisation. Inhibition of IK1 current can suppress re-entry based arrhythmias and has been demonstrated to exert anti-fibrillatory effects. Human IK1 current is mainly formed by heterotetrameric assembly of three subunits: Kir2.1, Kir2.2 and Kir2.3 channels. Amiodarone and dronedarone have been shown to inhibit cardiac IK1 current, but the molecular basis of these effects has not been elucidated to date. Methods: Human Kir2.1 - Kir2.3 channels were expressed in Xenopus oocytes and current recordings were performed using the double-electrode voltage-clamp technique. Results: Amiodarone and dronedarone exerted differential inhibitory effects on Kir2.1, Kir2.2 and Kir2.3 channels, respectively. Both drugs inhibited only Kir2.1 channels without an effect on Kir2.2 and Kir2.3 channels. We analyzed effects of dronedarone in detail. Onset of inhibition was slow, but the effect was completely reversible upon washout. Biophysical current characteristics such as inwardly-rectifying properties and reversal potential of Kir2.1 currents were not modified. The inhibitory effect did not exhibit frequency dependence. Interestingly, dronedarone inhibited Kir2.1 currents mainly at negative potentials. Conclusions: Amiodarone and dronedarone both inhibit only Kir2.1 channels without affecting Kir2.2 and Kir2.3 channels. In view of the functional significance of Kir2.1 channels, this effect probably underlies the observed inhibition of IK1 current by both compounds and may contribute to their anti-fibrillatory efficacy.

Background: The cardiac inwardly rectifying potassium current IK1 stabilises the diastolic resting membrane potential of ventricular cardiomyocytes. Protein kinase A (PKA) induces an inhibition of IK1 current which strongly promotes focal arrhythmogenesis under increased catecholamine levels. The molecular mechanisms underlying this regulation have only partially been elucidated to date. Furthermore, the role of A Kinase Anchoring Proteins (AKAPs) in this regulation has not been examined yet. The objective of this project was to elucidate the molecular mechanisms underlying the inhibition of IK1 by PKA and to identify novel molecular targets for antiarrhythmic therapy downstream β-adrenoreceptors. 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: In patch clamp experiments, activation of PKA inhibited IK1 current in rat ventricular cardiomyocytes. This regulation was markedly attenuated by disrupting PKA-binding to AKAPs with the peptide inhibitor AKAP-IS. In expression systems, we observed functional and spatial coupling of the plasma membrane-associated AKAP15/18 and AKAP79 to Kir2.1 and Kir2.2 channel subunits, but not to Kir2.3 channels, which underly IK1 currents. In contrast, AKAPyotiao had no functional effect on the PKA regulation of Kir channels. AKAP15/18 and AKAP79 co-immunoprecipitated with and co-localized to Kir2.1 and Kir2.2 channel subunits in ventricular cardiomyocytes. Conclusions: In this study, we provide evidence for the functional and spatial coupling of cardiac Kir2.1 and Kir2.2 subunits as molecular components of IK1 current with the plasma membrane-bound AKAPs 15/18 and 79. Cardiac membrane-associated AKAPs are a functionally essential part of the regulatory cascade determining IK1 current function and may be novel molecular targets for antiarrhythmic therapy downstream from β-adrenoreceptors.

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.