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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 antidepressant amoxapine has been linked to cases of QT prolongation, acute heart failure, and sudden death. Inhibition of cardiac hERG (Kv11.1) potassium channels causes prolonged repolarization and is implicated in apoptosis. Apoptosis in association with amoxapine has not yet been reported. This study was designed to investigate amoxapine effects on hERG currents, hERG protein trafficking, and hERG-associated apoptosis in order to elucidate molecular mechanisms underlying cardiac side effects of the drug. hERG channels were expressed in Xenopus laevis oocytes and HEK 293 cells, and potassium currents were recorded using patch clamp and two-electrode voltage clamp electrophysiology. Protein trafficking was evaluated in HEK 293 cells by Western blot analysis, and cell viability was assessed in HEK cells by immunocytochemistry and colorimetric MTT assay. Amoxapine caused acute hERG blockade in oocytes (IC50 = 21.6 µM) and in HEK 293 cells (IC50 = 5.1 µM). Mutation of residues Y652 and F656 attenuated hERG blockade, suggesting drug binding to a receptor inside the channel pore. Channels were mainly blocked in open and inactivated states, and voltage dependence was observed with reduced inhibition at positive potentials. Amoxapine block was reverse frequency-dependent and caused accelerated and leftward-shifted inactivation. Furthermore, amoxapine application resulted in chronic reduction of hERG trafficking into the cell surface membrane (IC50 = 15.3 µM). Finally, the antidepressant drug triggered apoptosis in cells expressing hERG channels. We provide evidence for triple mechanisms of hERG liability associated with amoxapine: (1) direct hERG current inhibition, (2) disruption of hERG protein trafficking, and (3) induction of apoptosis. Further experiments are required to validate a specific pro-apoptotic effect mediated through blockade of hERG channels. [ABSTRACT FROM AUTHOR]

Abstract: The antihypertensive drug doxazosin has been associated with an increased risk for congestive heart failure and cardiomyocyte apoptosis. Human ether-a-go-go-related gene (hERG) K+ channels, previously shown to be blocked by doxazosin at therapeutically relevant concentrations, represent plasma membrane receptors for the antihypertensive drug. To elucidate the molecular basis for doxazosin-associated pro-apoptotic effects, cell death was studied in human embryonic kidney cells using three independent apoptosis assays. Doxazosin specifically induced apoptosis in hERG-expressing HEK cells, while untransfected control groups were insensitive to treatment with the antihypertensive agent. An unexpected biological mechanism has emerged: binding of doxazosin to its novel membrane receptor, hERG, triggers apoptosis, possibly representing a broader pathophysiological mechanism in drug-induced heart failure. [Copyright &y& Elsevier]

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]

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: Inhibition of IK1 currents by adrenergic α1 receptors has been observed in cardiomyocytes and has been linked to arrhythmogenesis in an animal model. Both PKC-dependent and PKC-independent pathways have been implied in this regulation. The underlying molecular mechanisms, however, have not been elucidated to date. The molecular basis of native IK1 current is mainly formed by Kir2.1 (KCNJ2), Kir2.2 (KCNJ12) and Kir2.3 (KCNJ4) channels that are differentially regulated by protein kinases. We therefore sought to investigate the role of those different Kir2.x channel subunits in this regulation and to identify the major signalling pathways involved.Adrenergic α1A receptors (the predominant cardiac isoform) were co-expressed with cloned Kir2.1, Kir2.2 and Kir2.3 channels in Xenopus oocytes and electrophysiological experiments were performed using two-microelectrode voltage clamp. Native IK1 currents were measured with the whole-cell patch clamp technique in isolated rat ventricular cardiomyocytes.Activation of co-expressed adrenergic α1A receptors by phenylephrine induced differential effects in Kir2.x channels. No effect was noticed in Kir2.1 channels. However, a marked inhibitory effect was observed in Kir2.2 channels. This regulation was not attenuated by inhibitors of PKC, CamKII and PKA (chelerythrine, KN-93, KT-5720), and mutated Kir2.2 channels lacking functional phosphorylation sites for PKC and PKA exhibited the same effect as Kir2.2 wild-type channels. By contrast, the regulation could be suppressed by the general tyrosine kinase inhibitor genistein and by the src tyrosine kinase inhibitor PP2 indicating an essential role of src kinases. This finding was validated in rat ventricular cardiomyocytes where co-application of PP2 strongly attenuated the inhibitory regulation of IK1 current by adrenergic α1 receptors. The inactive analogue PP3 was tested as negative control for PP2 and did not reproduce the effects of PP2. In Kir2.3 channels, a marked inhibitory effect of α1A receptor activation was observed. This regulation could be attenuated by inhibition of PKC with chelerythrine or with Ro-32-0432, but not by tyrosine kinase inhibition with genistein.In summary, on the molecular level the inhibitory regulation of IK1 currents by adrenergic α1A receptors is probably based on effects on Kir2.2 and Kir2.3 channels. Kir2.2 is regulated via src tyrosine kinase pathways independent of protein kinase C, whereas Kir2.3 is inhibited by protein kinase C-dependent pathways. Src tyrosine kinase pathways are essential for the inhibition of native IK1 current by adrenergic α1 receptors. This regulation may contribute to arrhythmogenesis under adrenergic stimulation. [Copyright &y& Elsevier]

Inhibition of I(K1) currents by adrenergic alpha(1) receptors has been observed in cardiomyocytes and has been linked to arrhythmogenesis in an animal model. Both PKC-dependent and PKC-independent pathways have been implied in this regulation. The underlying molecular mechanisms, however, have not been elucidated to date. The molecular basis of native I(K1) current is mainly formed by Kir2.1 (KCNJ2), Kir2.2 (KCNJ12) and Kir2.3 (KCNJ4) channels that are differentially regulated by protein kinases. We therefore sought to investigate the role of those different Kir2.x channel subunits in this regulation and to identify the major signalling pathways involved. Adrenergic alpha(1A) receptors (the predominant cardiac isoform) were co-expressed with cloned Kir2.1, Kir2.2 and Kir2.3 channels in Xenopus oocytes and electrophysiological experiments were performed using two-microelectrode voltage clamp. Native I(K1) currents were measured with the whole-cell patch clamp technique in isolated rat ventricular cardiomyocytes. Activation of co-expressed adrenergic alpha(1A) receptors by phenylephrine induced differential effects in Kir2.x channels. No effect was noticed in Kir2.1 channels. However, a marked inhibitory effect was observed in Kir2.2 channels. This regulation was not attenuated by inhibitors of PKC, CamKII and PKA (chelerythrine, KN-93, KT-5720), and mutated Kir2.2 channels lacking functional phosphorylation sites for PKC and PKA exhibited the same effect as Kir2.2 wild-type channels. By contrast, the regulation could be suppressed by the general tyrosine kinase inhibitor genistein and by the src tyrosine kinase inhibitor PP2 indicating an essential role of src kinases. This finding was validated in rat ventricular cardiomyocytes where co-application of PP2 strongly attenuated the inhibitory regulation of I(K1) current by adrenergic alpha(1) receptors. The inactive analogue PP3 was tested as negative control for PP2 and did not reproduce the effects of PP2. In Kir2.3 channels, a marked inhibitory effect of alpha(1A) receptor activation was observed. This regulation could be attenuated by inhibition of PKC with chelerythrine or with Ro-32-0432, but not by tyrosine kinase inhibition with genistein. In summary, on the molecular level the inhibitory regulation of I(K1) currents by adrenergic alpha(1A) receptors is probably based on effects on Kir2.2 and Kir2.3 channels. Kir2.2 is regulated via src tyrosine kinase pathways independent of protein kinase C, whereas Kir2.3 is inhibited by protein kinase C-dependent pathways. Src tyrosine kinase pathways are essential for the inhibition of native I(K1) current by adrenergic alpha(1) receptors. This regulation may contribute to arrhythmogenesis under adrenergic stimulation.