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Byline: Dierk Thomas (1), Kezhong Wu (1), Anna-Britt Wimmer (1), Edgar Zitron (1), Bettina C. Hammerling (1), Sven Kathofer (1), Sonja Lueck (1), Ramona Bloehs (1), Volker A. W. Kreye (2), Johann Kiehn (1), Hugo A. Katus (1), Wolfgang Schoels (1), Christoph A. Karle (1) Keywords: Adrenergic (ant)agonists; Arrhythmia; K-channel; hERG channel; Long QT syndrome Abstract: Patients with cardiac disease typically develop life-threatening ventricular arrhythmias during physical or emotional stress, suggesting a link between adrenergic stimulation and regulation of the cardiac action potential. Human ether-a-go-go related gene (hERG) potassium channels conduct the rapid component of the repolarizing delayed rectifier potassium current, I.sub.Kr. Previous studies have revealed that hERG channel activation is modulated by activation of the [beta]-adrenergic system. In contrast, the influence of the [alpha]-adrenergic signal transduction cascade on hERG currents is less well understood. The present study examined the regulation of hERG currents by [alpha].sub.1A-adrenoceptors. hERG channels and human [alpha].sub.1A-adrenoceptors were heterologously coexpressed in Xenopus laevis oocytes, and currents were measured using the two-microelectrode voltage clamp technique. Stimulation of [alpha].sub.1A-receptors by applying 20 AuM phenylephrine caused hERG current reduction due to a 9.6-mV shift of the activation curve towards more positive potentials. Simultaneous application of the [alpha].sub.1-adrenoceptor antagonist prazosin (20 AuM) prevented the activation shift. Inhibition of PKC (3 AuM Ro-32-0432) or PKA (2.5 AuM KT 5720) abolished the [alpha]-adrenergic activation shift, suggesting that PKC and PKA are required within the regulatory mechanism. The effect was still present when the PKA- and PKC-dependent phosphorylation sites in hERG were deleted by mutagenesis. In summary, cardiac repolarizing hERG/I.sub.Kr potassium currents are modulated by [alpha].sub.1A-adrenoceptors via PKC and PKA independently of direct channel phosphorylation. This novel regulatory pathway of [alpha].sub.1-adrenergic hERG current regulation provides a link between stress and ventricular arrhythmias, in particular in patients with heart disease. Author Affiliation: (1) Department of Cardiology, Medical University Hospital, Im Neuenheimer Feld 410, 69120, Heidelberg, Germany (2) Department of Physiology and Pathophysiology, Medical School, University of Heidelberg, Im Neuenheimer Feld 326, 69120, Heidelberg, Germany Article History: Registration Date: 15/07/2004 Received Date: 29/04/2004 Accepted Date: 07/07/2004 Online Date: 08/09/2004 Article note: Electronic Supplementary Material Supplementary material is available for this article if you access the article at http://dx.doi.org/10.1007/s00109-004-0582-8. A link in the frame on the left on that page takes you directly to the supplementary material.

The single-channel and macropatch currents expressed by the human ether-a-go-go-related gene (HERG) in Xenopus oocytes were measured to examine how HERG produces rectification and a peak transient current. One of the two main findings revealed that the single-channel current-voltage relation displays an inward rectification. The other finding showed that single channels often exhibit no openings during depolarization, but still display bursts openings during repolarization.

To elucidate the ionic mechanism of endothelin-1 (ET-1)-induced focal ventricular tachyarrhythmias, the regulation of I K1 and its main molecular correlates, Kir2.1, Kir2.2 and Kir2.3 channels, by ET-1 was investigated. Native I K1 in human atrial cardiomyocytes was studied with whole-cell patch clamp. Human endothelin receptors were coexpressed with human Kir2.1, Kir2.2 and Kir2.3 channels in Xenopus oocytes. Currents were measured with a two-microelectrode voltage clamp. In human cardiomyocytes, ET-1 induced a marked inhibition of I K1 that could be suppressed by the protein kinase C (PKC) inhibitor staurosporine. To investigate the molecular mechanisms underlying this regulation, we studied the coupling of ETA receptors to homomeric and heteromeric Kir2.1, Kir2.2 and Kir2.3 channels in the Xenopus oocyte expression system. ETA receptors coupled functionally to Kir2.2 and Kir2.3 channels but not to Kir2.1 channels. In Kir2.2 channels lacking functional PKC phosphorylation sites, the inhibitory effect was abolished. The inhibition of Kir2.3 currents could be suppressed by the PKC inhibitors staurosporine and chelerythrine. The coupling of ETA receptors to heteromeric Kir2.1/Kir2.2 and Kir2.2/Kir2.3 channels resulted in a strong inhibition of currents comparable with the effect observed in Kir2.2 homomers. Surprisingly, in heteromeric Kir2.1/Kir2.3 channels, no effect was observed. ET-1 inhibits human cardiac I K1 current via a PKC-mediated phosphorylation of Kir2.2 channel subunits and additional regulatory effects on Kir2.3 channels. This mechanism may contribute to the intrinsic arrhythmogenic potential of ET-1. [ABSTRACT FROM AUTHOR]

Abstract: Objective: Hereditary long QT syndrome (LQTS) is a genetically heterogeneous disease characterized by prolonged QT intervals and an increased risk for ventricular arrhythmias and sudden cardiac death. Mutations in the voltage-gated potassium channel subunit KCNQ1 induce the most common form of LQTS. KCNQ1 is associated with two different entities of LQTS, the autosomal-dominant Romano–Ward syndrome (RWS), and the autosomal-recessive Jervell and Lange–Nielsen syndrome (JLNS) characterized by bilateral deafness in addition to cardiac arrhythmias. In this study, we investigate and discuss dominant-negative I Ks current reduction by a KCNQ1 deletion mutation identified in a RWS family. Methods: Single-strand conformation polymorphism analysis and direct sequencing were used to screen LQTS genes for mutations. Mutant KCNQ1 channels were heterologously expressed in Xenopus oocytes, and potassium currents were recorded using the two-microelectrode voltage clamp technique. Results: A heterozygous deletion of three nucleotides (CTT) identified in the KCNQ1 gene caused the loss of a single phenylalanine residue at position 339 (KCNQ1-ΔF339). Electrophysiological measurements in the presence and absence of the regulatory β-subunit KCNE1 revealed that mutant and wild type forms of an N-terminal truncated KCNQ1 subunit (isoform 2) caused much stronger dominant-negative current reduction than the mutant form of the full-length KCNQ1 subunit (isoform 1). Conclusion: This study highlights the functional relevance of the truncated KCNQ1 splice variant (isoform 2) in establishment and mode of inheritance in long QT syndrome. In the RWS family presented here, the autosomal-dominant trait is caused by multiple dominant-negative effects provoked by heteromultimeric channels formed by wild type and mutant KCNQ1-isoforms in combination with KCNE1. [Copyright &y& Elsevier]

Patients with cardiac disease typically develop life-threatening ventricular arrhythmias during physical or emotional stress, suggesting a link between adrenergic stimulation and regulation of the cardiac action potential. Human ether-a-go-go related gene (hERG) potassium channels conduct the rapid component of the repolarizing delayed rectifier potassium current, IKr. Previous studies have revealed that hERG channel activation is modulated by activation of the ß-adrenergic system. In contrast, the influence of the a-adrenergic signal transduction cascade on hERG currents is less well understood. The present study examined the regulation of hERG currents by a1A-adrenoceptors. hERG channels and human a1A-adrenoceptors were heterologously coexpressed inXenopus laevisoocytes, and currents were measured using the two-microelectrode voltage clamp technique. Stimulation of a1A-receptors by applying 20 µM phenylephrine caused hERG current reduction due to a 9.6-mV shift of the activation curve towards more positive potentials. Simultaneous application of the a1-adrenoceptor antagonist prazosin (20 µM) prevented the activation shift. Inhibition of PKC (3 µM Ro-32-0432) or PKA (2.5 µM KT 5720) abolished the a-adrenergic activation shift, suggesting that PKC and PKA are required within the regulatory mechanism. The effect was still present when the PKA- and PKC-dependent phosphorylation sites in hERG were deleted by mutagenesis. In summary, cardiac repolarizing hERG/IKr potassium currents are modulated by a1A-adrenoceptors via PKC and PKA independently of direct channel phosphorylation. This novel regulatory pathway of a1-adrenergic hERG current regulation provides a link between stress and ventricular arrhythmias, in particular in patients with heart disease. [ABSTRACT FROM AUTHOR]

Abstract: Objective: The human ether-a-go-go-related gene (hERG) encodes the rapid component of the cardiac repolarizing delayed rectifier potassium current, IKr. The direct interaction of the commonly used protein kinase C (PKC) inhibitor bisindolylmaleimide I (BIM I) with hERG, KvLQT1/minK, and IKr currents was investigated in this study. Methods: hERG and KvLQT1/minK channels were heterologously expressed in Xenopus laevis oocytes, and currents were measured using the two-microelectrode voltage clamp technique. In addition, hERG currents in stably transfected human embryonic kidney (HEK 293) cells, native IKr currents and action potentials in isolated guinea pig ventricular cardiomyocytes were recorded using whole-cell patch clamp electrophysiology. Results: Bisindolylmaleimide I blocked hERG currents in HEK 293 cells and Xenopus oocytes in a concentration-dependent manner with IC50 values of 1.0 and 13.2 μM, respectively. hERG channels were primarily blocked in the open state in a frequency-independent manner. Analysis of the voltage-dependence of block revealed a reduction of inhibition at positive membrane potentials. BIM I caused a shift of −20.3 mV in the voltage-dependence of inactivation. The point mutations tyrosine 652 alanine (Y652A) and phenylalanine 656 alanine (F656A) attenuated hERG current blockade, indicating that BIM I binds to a common drug receptor within the pore region. KvLQT1/minK currents were not significantly altered by BIM I. Finally, 1 μM BIM I reduced native IKr currents by 69.2% and lead to action potential prolongation. Conclusion: In summary, PKC-independent effects have to be carefully considered when using BIM I as PKC inhibitor in experimental models involving hERG channels and IKr currents. [Copyright &y& Elsevier]

Objective: The cardiac inwardly rectifying potassium current IK1 and its molecular correlates Kir2.1 and Kir2.2 play an important role in cardiac repolarisation and in the pathogenesis of hereditary long-QT syndrome (LQTS-7). Protein kinases A (PKA) and C (PKC) are key enzymes in adrenergic signal transduction, inducing arrhythmias in heart disease. This study investigated the regulation of Kir2.2 (KCNJ12) by PKA. Methods: Cloned Kir2.2 channels were expressed heterologously in Xenopus oocytes and currents were measured with the double-electrode voltage–clamp technique. Results: After activation of PKA by forskolin (100 μmol/l) or Ro-20-1724 (100 μmol/l), wild type currents at -120 mV were increased by 93.7% and 79.0%, respectively. Coapplication of the PKA inhibitor KT-5720 (2.5 μmol/l) attenuated this effect. No significant changes were apparent after mutation of the single PKA consensus site S430. In addition, removal of all four PKC consensus sites in Kir2.2 induced a phorbolester-mediated current increase which could be suppressed by PKA inhibitors H-89 (50 μmol/l) and KT-5720 (2.5 μmol/l). Conclusions: This study demonstrates antagonistic effects of PKA and PKC in the regulation of Kir2.2. Phosphorylation by PKC has been shown to cause an inhibition of Kir2.2 currents, whereas activation of PKA leads to current upregulation. [Copyright &y& Elsevier]

Ventricular arrhythmias are often precipitated by physical or emotional stress, in particular in patients with ischemic heart disease or hereditary long QT syndrome. Stimulation of the sympathetic nervous system in response to exercise or emotional stress causes activation of cardiac α- and β-adrenoceptors. The rapid component of the delayed rectifier potassium current, IKr, and the underlying hERG potassium channel are critical for the regulation of heart rhythm. Recent experimental studies revealed that hERG/IKr currents are modulated by α- and β-adrenergic stimulation, providing a pathophysiological explanation for the increased incidence of arrhythmias during stress. This review summarizes the current knowledge on hERG/IKr channel modulation by adrenergic activity. In addition, therapeutic approaches to future effective, more genotype-specific antiarrhythmic therapies are discussed. [ABSTRACT FROM AUTHOR]

Human hereditary long QT syndrome is a cardiac disease characterized by prolongation of the QT interval and increased susceptibility to ventricular arrhythmias and sudden cardiac death. Mutations in the human-ether-a-go-go-related gene (hERG), encoding the protein underlying the repolarizing cardiac IKr potassium current, cause chromosome 7-linked long QT syndrome 2. Loss of function of mutant hERG channels may be caused by several mechanisms, including altered current kinetics, altered ion selectivity, or defective intracellular protein trafficking. Especially the latter category has become a focus of particular interest recently, because some of the mutant subunits display wild type current properties when normal trafficking is restored and channels are inserted in the cell membrane in vitro. This review summarizes the current knowledge on hERG channel trafficking under physiological and pathological conditions. In addition, therapeutic approaches to restore normal hERG trafficking in vitro and in vivo are discussed. [Copyright &y& Elsevier]

1 Dronedarone is a noniodinated benzofuran derivative that has been synthesized to overcome the limiting iodine-associated adverse effects of the potent antiarrhythmic drug amiodarone. In this study, the acute electrophysiological effects of dronedarone on repolarizing potassium channels were investigated to determine the class III antiarrhythmic action of this compound. HERG and KvLQT1/minK potassium channels conduct the delayed rectifier potassium current I[subk] in human heart, being a primary target for class III antirrhythmic therapy. 2 HERG and KvLQTI/minK were expressed heterologously in Xenopue laevis oocytes, and the respective potassium currents were recorded using the two-microelectrode voltage-clamp technique. 3 Dronedarone blocked HERG channels with an IC[sub50] value of 9.2 μM and a maximum tail current reduction of 85.2%. 4 HERG channels were blocked in the closed, open, and inactivated states. The half-maximal activation voltage was shifted by —6.1 mV, and HERG current block by dronedarone was voltage-dependent, but not use-dependent. 5 Dronedarone exhibited a weaker block of KvLQTI/minK currents (33.2% at l00 μM drug concentration), without causing significant changes in the corresponding current—voltage relationships. 6 In conclusion, these data demonstrate that dronedarone is an antagonist of cloned HERG potassium channels, with additional inhibitory effects on KvLQTI/minK currents at higher drug concentrations, providing a molecular mechanism for the class III antiarrhythmic action of the drug. [ABSTRACT FROM AUTHOR]