Search

The zebrafish is increasingly recognized as an animal model for the analysis of hERG-related diseases. However, functional properties of the zebrafish orthologue of hERG have not been analyzed yet. We heterologously expressed cloned ERG channels in Xenopus oocytes and analyzed biophysical properties using the voltage clamp technique. zERG channels conduct rapidly activating and inactivating potassium currents. However, compared to hERG, the half-maximal activation voltage of zERG current is shifted towards more positive potentials and the half maximal steady-state inactivation voltage is shifted towards more negative potentials. zERG channel activation is delayed and channel deactivation is accelerated significantly. However, time course of zERG conducted current under action potential clamp is highly similar to the human orthologue. In summary, we show that ERG channels in zebrafish exhibit biophysical properties similar to the human orthologue. Considering the conserved channel function, the zebrafish represents a valuable model to investigate human ERG channel related diseases.

The antidepressant mianserin exhibits a tetracyclic structure that is different from typical tricyclic antidepressants (TCA) and that of selective serotonin reuptake inhibitors. In comparison to the older TCA, mianserin has been shown to have a superior risk profile regarding proarrhythmic effects, both in vitro and in vivo. However, the underlying molecular electrophysiological basis has not been elucidated to date. Therefore, we studied the effects of mianserin on cardiac hERG potassium channels, the predominant target of drug-induced proarrhythmia. HERG channels were expressed in the Xenopus oocyte expression system and in human embryonic kidney (HEK) cells and currents were measured with two-microelectrode voltage-clamp and whole-cell patch-clamp, respectively. Mianserin inhibited hERG currents in a dose-dependent manner with an IC(50) of 3.2 micromol/l in HEK cells. Onset of blockade was slow and the inhibitory effect was not reversible upon wash-out of the drug. In hERG channel mutants, Y652A and F656A, lacking aromatic residues in the S6 domain, the effect of mianserin was significantly reduced in comparison to the wild type. Mianserin inhibited hERG currents in the open and inactivated state, but not in the closed states. HERG inactivation kinetics were significantly altered by mianserin without marked effects on channel activation kinetics. The inhibitory effect was not frequency dependent. In conclusion, mianserin is a low-affinity hERG-blocking agent. However, taken together with the lack of APD-prolongation shown in other studies, mianserin seems to have a good safety profile. Lack of consistent QT prolonging effects of mianserin in previous studies may therefore be linked to additional effects such as inhibition of other cardiac ion channels. However, as demonstrated by clinical case reports, mianserin can induce proarrhythmic effects in susceptible patients. Therefore, in patients with complex co-medication (i.e., additional hERG-blocking agents) and in patients with risk factors for acquired long QT syndrome as well as in cases of overdose, adequate monitoring should be recommended.

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.

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.

The anticholinergic antiparkinson drug orphenadrine is an antagonist at central and peripheral muscarinic receptors. Orphenadrine intake has recently been linked to QT prolongation and Torsade-de-Pointes tachycardia. So far, inhibitory effects on I (Kr) or cloned HERG channels have not been examined. HERG channels were heterologously expressed in a HEK 293 cell line and in Xenopus oocytes and HERG current was measured using the whole cell patch clamp and the double electrode voltage clamp technique. Orphenadrine inhibits cloned HERG channels in a concentration dependent manner, yielding an IC(50) of 0.85 microM in HEK cells. Onset of block is fast and reversible upon washout. Orphenadrine does not alter the half-maximal activation voltage of HERG channels. There is no shift of the half-maximal steady-state-inactivation voltage. Time constants of direct channel inactivation are not altered significantly and there is no use-dependence of block. HERG blockade is attenuated significantly in mutant channels lacking either of the aromatic pore residues Y652 and F656. In conclusion, we show that the anticholinergic agent orphenadrine is an antagonist at HERG channels. These results provide a novel molecular basis for the reported proarrhythmic side effects of orphenadrine.

Background and Aims: Hesperetin belongs to the flavonoid subgroup classified as citrus flavonoids and is the main flavonoid in oranges. A high dietary intake of flavonoids has been associated with a significant reduction in cardiovascular mortality. HERG potassium channels play a major role in cardiac repolarisation and represent the most important pharmacologic target of both antiarrhythmic and proarrhythmic drugs., Methods and Results: We used the two-microelectrode voltage-clamp technique to analyse inhibitory effects of hesperetin on hERG potassium channels heterologously expressed in Xenopus oocytes. Hesperetin blocked hERG potassium channels in a concentration dependent manner. Onset of block was fast and completely reversible upon wash-out. There was no significant effect of hesperetin on channel kinetics. Affinity of hesperetin to mutant F656A hERG channel was significantly decreased compared to WT hERG, indicating a binding site in the channel pore cavity. In contrast, affinity of hesperetin to Y652A hERG was not different from the affinity to WT hERG., Conclusion: We found an antagonist of cardiac hERG channels that modulates hERG currents by accessing the aromatic pore binding site, particularly amino acid phe-656. Regarding high hesperetin concentrations found in oranges and the increasing consumption of oranges and orange juice in Europe, potential effects of hesperetin on cardiac electrophysiology in vivo deserve further investigation.

beta(3)-adrenoceptors have recently been shown to induce a complex modulation of intracellular signaling pathways including cyclic guanine monophosphate, cyclic adenosine monophosphate, nitric oxide, and protein kinases A and C. They are expressed in a broad variety of tissues including the myocardium, vascular smooth muscle, and endothelium. In those tissues, resting membrane potential is controlled mainly by inwardly rectifying potassium channels of the Kir2 family namely, Kir2.1 in the vascular smooth muscle, Kir2.1-2.3 in the myocardium, and Kir2.1-2.2 in the endothelium. In the present study, we investigated the possible modulation of Kir2 channel function by beta(3)-adrenoceptors in an expression system. Human-cloned beta(3)-adrenoceptors and Kir2.1 (KCNJ2), Kir2.2 (KCNJ12), and Kir2.3 (KCNJ4) channels were coexpressed in Xenopus oocytes, and currents were measured with double-microelectrode voltage clamp. Activation of beta(3)-adrenoceptors with isoproterenol resulted in markedly increased currents in Kir2.1 and in Kir2.2 potassium channels with EC50 values of 27 and 18 nM, respectively. In contrast, Kir2.3 currents were not modulated. Coapplication of specific inhibitors of protein kinase A (KT-5720) and calmodulin kinase II (KN-93) had no effects on the observed regulation in Kir2.1. However, coapplication of protein kinase C (PKC) inhibitors staurosporine and chelerythrine suppressed the observed effect. In Kir2.2, coapplication of KT-5720 reduced the effect of beta(3)-adrenoceptor activation. No differences in current increase after application of isoproterenol were observed between mutant Kir2.2 potassium channels lacking all functional PKC phosphorylation sites and Kir2.2 wild-type channels. In heteromeric Kir2.x channels, all types of heteromers were activated. The effect was most pronounced in Kir2.1/Kir2.2 and in Kir2.2/Kir2.3 channels. In summary, homomeric and heteromeric Kir2.x channels are activated by beta(3)-adrenoceptors via different protein kinase-dependent pathways: Kir2.1 subunits are modulated by PKC, whereas Kir2.2 is modulated by protein kinase A. In heteromeric composition, a marked activation of currents can be observed particularly with involvement of Kir2.2 subunits. This regulation may contribute to the hyperpolarizing effects of beta(3)-adrenoceptors in tissues that exhibit modulation by Kir2 channel function.

Acute chloroform intoxication can cause depression of the central nervous system and may lead to death from lethal arrhythmias or respiratory arrest. Thus, the organic solvent is no longer in clinical use as an anaesthetic, but still plays a role in cases of suicide, homicide or inhalation for psychotropic effects. Several cases of lethal arrhythmia after intoxication with chloroform have been described. Pharmacological inhibition of cardiac "human ether-à-go-go-related gene" (HERG) potassium channels is linked to proarrhythmic effects of cardiac and noncardiac drugs. To further investigate the electrophysiological basis of the arrhythmogenic potential of chloroform, we analysed inhibitory effects of chloroform on cloned HERG potassium channels, heterologously expressed in Xenopus oocytes and in Human Embryonic Kidney (HEK 293) cells using the double-electrode voltage-clamp technique and the whole-cell patch-clamp technique, respectively. In HEK cells, chloroform blocked HERG tail currents with an IC(50) of 4.97mM. Biophysical properties were further investigated in the Xenopus oocyte expression system. Onset and wash-out of block was fast and inhibition was completely reversible. Chloroform did not alter channel activation, however, direct channel inactivation was accelerated significantly. Steady-state-inactivation of HERG was not affected. Chloroform dependent block of HERG channels was voltage dependent with a decrease of inhibition at more positive membrane potentials. No frequency-dependence of block could be observed. In summary, chloroform blocked HERG potassium channels probably in a toxicologically relevant concentration. These findings contribute to the pathophysiology of proarrhythmic effects in acute chloroform intoxication.

Maprotiline is an antidepressant compound with an atypical tetracyclic structure that is widely used in elderly patients due to its favourable side-effect profile. However, there have been reports of proarrhythmia associated with maprotiline and in vitro studies of its electrophysiological properties have been lacking. Therefore, we characterised the effects of maprotiline on cardiac hERG channels. hERG channels were expressed in HEK cells and in the Xenopus oocyte expression system. Currents were measured using a whole-cell patch clamp and a two-microelectrode voltage-clamp. Maprotiline inhibited hERG currents with an IC(50) of 8.2 micromol/l in HEK cells and 29.2 micromol/l in Xenopus oocytes. Onset of the effect was rather slow and took several minutes. No wash-out of effect was observed. Maprotiline blocked hERG channels in the open and inactivated states, but not in the closed states. In mutant hERG channels Y652A and F656A, the effect was markedly attenuated (hERG-F656A) or completely abolished (hERG-Y652A). Voltage dependence of hERG current activation and inactivation was not affected by maprotiline. hERG inactivation was accelerated at positive potentials. The effect of maprotiline on hERG currents was voltage-dependent with a marked reduction at a more positive potential. hERG blockade by maprotiline was not frequency-dependent. Maprotiline is an antagonist of cardiac hERG potassium channels that preferably accesses the putative pore binding site Y652/F656. Although the affinity of maprotiline to hERG channels is low, its use in patients with risk factors for acquired long QT syndrome should be monitored appropriately.

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 ET(A) receptors to homomeric and heteromeric Kir2.1, Kir2.2 and Kir2.3 channels in the Xenopus oocyte expression system. ET(A) 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 ET(A) 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.

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-deltaF339). Electrophysiological measurements in the presence and absence of the regulatory beta-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.

Flavonoids are naturally occurring food ingredients that have been associated with reduced cardiovascular mortality in epidemiological studies. In a previous study, we demonstrated for the first time that flavonoids are inhibitors of cardiac human ether-à-go-go-related gene (HERG) channels. Furthermore, we observed that grapefruit juice induced mild QTc prolongation in healthy subjects. HERG blockade by grapefruit flavonoid naringenin is most likely to be the mechanism underlying this effect. Therefore, the electrophysiological properties of HERG blockade by naringenin were analysed in detail. HERG potassium currents expressed in Xenopus oocytes were measured with a two-microelectrode voltage clamp. Naringenin blocked HERG potassium channels with an IC50 value of 102.6 microM in Xenopus oocytes. The onset of blockade was fast. The effect was completely reversible upon wash-out. Naringenin binding to HERG required aromatic residue F656 in the putative pore binding site. Channels were blocked in the open and inactivated states but not in the closed states. Naringenin did not affect HERG current activation. However, the half maximal inactivation voltage was shifted by 14.9 mV towards more negative potentials and current inactivation at negative potentials was accelerated. No frequency dependence of blockade was observed. Naringenin inhibits HERG channels with pharmacological characteristics similar to those of well-known HERG antagonists. From a clinical point of view, this effect could have both proarrhythmic and antiarrhythmic consequences. This may have important implications for phytotherapy and for dietary recommendations for cardiologic patients. Therefore, electrophysiological effects of flavonoids deserve further investigation.

Ajmaline is a class Ia anti-arrhythmic drug used in several European countries and Japan as first-line treatment for ventricular tachyarrhythmia. Ajmaline has been reported to induce cardiac output (QT) prolongation and to inhibit cardiac potassium currents in guinea pig cardiomyocytes. In order to elucidate the molecular basis of these effects, we examined effects of ajmaline on human ether a-go-go related gene HERG potassium channels. Electrophysiological experiments were performed with human embryonic kidney (HEK) cells (whole-cell patch clamp) and Xenopus oocytes (double-electrode voltage clamp) expressing wild-type and mutant HERG channels. Ajmaline blocked HERG currents with an IC(50) of 1.0 micromol/l in HEK cells and 42.3 micromol/l in Xenopus oocytes. The onset of block was fast and reached steady-state conditions after 180 s. The inhibitory effect was completely reversible upon wash-out. In HERG mutant channels Y652A and F656A lacking aromatic residues in the S6 domain, the inhibitory effect of ajmaline was completely abolished. Ajmaline induced a small shift in HERG current half-maximal activation voltage towards more negative potentials. Ajmaline did not markedly affect HERG inactivation. Inhibitory effects were not voltage-dependent. Ajmaline block exhibited positive frequency dependence. Ajmaline blocked HERG channels in the open, but not in the closed states. Binding of ajmaline to inactivated HERG channels may also be possible. In inactivation-deficient HERG S620T channels, the sensitivity to ajmaline was markedly reduced. The IC(50) of HERG channel blockade in HEK cells lies within the range of unbound therapeutic plasma concentrations of ajmaline. Therefore, inhibitory effects on HERG channels may contribute to both the high anti-arrhythmic efficacy of ajmaline and to its pro-arrhythmic potential.

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(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(1A)-adrenoceptors. hERG channels and human alpha(1A)-adrenoceptors were heterologously coexpressed in Xenopus laevis oocytes, and currents were measured using the two-microelectrode voltage clamp technique. Stimulation of alpha(1A)-receptors by applying 20 microM 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(1)-adrenoceptor antagonist prazosin (20 microM) prevented the activation shift. Inhibition of PKC (3 microM Ro-32-0432) or PKA (2.5 microM 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(Kr) potassium currents are modulated by alpha(1A)-adrenoceptors via PKC and PKA independently of direct channel phosphorylation. This novel regulatory pathway of alpha1-adrenergic hERG current regulation provides a link between stress and ventricular arrhythmias, in particular in patients with heart disease.

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 micromol/l) or Ro-20-1724 (100 micromol/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 micromol/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 micromol/l) and KT-5720 (2.5 micromol/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., (Copryright 2004 European Society of Cardiology)

Trazodone is an atypical antidepressant that is commonly used in the treatment of affective disorders. There have repeatedly been reports of cardiac arrhythmia associated with this drug and concerns have been raised regarding the cardiac safety of trazodone. However, interaction with HERG channels as a main factor of cardiac side effects has not been studied to date. Therefore, we investigated the effect of trazodone on HERG potassium channels expressed in human embryonic kidney (HEK) cells and in Xenopus oocytes. Trazodone inhibited HERG currents in a dose-dependent manner with an IC50 of 2.9 microM in HEK cells and 13.2 microM in Xenopus oocytes. The electrophysiological properties of HERG blockade were analysed in detail. In HERG channel mutants Y652A and F656A lacking aromatic residues in the S6 domain, the affinity of trazodone was reduced profoundly. Trazodone accelerated inactivation of HERG currents without markedly affecting activation. Blockade was voltage dependent with a small reduction of block at positive membrane potentials. Frequency dependence of block was not observed. Trazodone block of HERG channels was state dependent. Channels were affected in the activated and inactivated states, but not in the closed states. In summary, the atypical antidepressant trazodone blocks cardiac HERG channels at concentrations that are probably relevant in vivo, particularly in overdosage.

1 The topoisomerase II inhibitor amsacrine is used in the treatment of acute myelogenous leukemia. Although most anticancer drugs are believed not to cause acquired long QT syndrome (LQTS), concerns have been raised by reports of QT interval prolongation, ventricular fibrillation and death associated with amsacrine treatment. Since blockade of cardiac human ether-a-go-go-related gene (HERG) potassium currents is an important cause of acquired LQTS, we investigated the acute effects of amsacrine on cloned HERG channels to determine the electrophysiological basis for its proarrhythmic potential. 2 HERG channels were heterologously expressed in human HEK 293 cells and Xenopus laevis oocytes, and the respective potassium currents were recorded using patch-clamp and two-microelectrode voltage-clamp electrophysiology. 3 Amsacrine blocked HERG currents in HEK 293 cells and Xenopus oocytes in a concentration-dependent manner, with IC50 values of 209.4 nm and 2.0 microm, respectively. 4 HERG channels were primarily blocked in the open and inactivated states, and no additional voltage dependence was observed. Amsacrine caused a negative shift in the voltage dependence of both activation (-7.6 mV) and inactivation (-7.6 mV). HERG current block by amsacrine was not frequency dependent. 5 The S6 domain mutations Y652A and F656A attenuated (Y652A) or abolished (F656A, Y652A/F656A) HERG current blockade, indicating that amsacrine binding requires a common drug receptor within the pore-S6 region. 6 In conclusion, these data demonstrate that the anticancer drug amsacrine is an antagonist of cloned HERG potassium channels, providing a molecular mechanism for the previously reported QTc interval prolongation during clinical administration of amsacrine.

Budipine is a non-dopaminergic antiparkinsonian drug causing acquired forms of Long QT syndrome (aLQTS). As a consequence, the manufacturer has restricted the use of budipine in patients who exhibit additional risk factors for the development of "Torsades-de-Pointes" tachycardias (TdP). The molecular basis of this serious side effect has not been elucidated yet. Human ether-a-go-go related gene (HERG) channel block being the main cause of drug induced QT prolongation, we investigated the effect of budipine on the rapid component of the delayed-rectifier potassium current (I(K(r))) in guinea pig cardiomyocytes and on HERG potassium channels heterologously expressed in Xenopus oocytes. In guinea pig cardiomyocytes, budipine (10 microM) inhibited I(K(r)) by 86% but was without any effect on calcium currents. In Xenopus oocytes, HERG potassium channels were blocked by budipine with an IC(50) of 10.2 microM. Onset of block was fast and block was only slowly and incompletely reversible upon washout. Budipine blocked HERG channels in the open and inactivated state, but not in the closed states. The half-maximal activation voltage was slightly shifted towards more negative potentials. Steady-state inactivation of HERG was also influenced by budipine. Budipine block was neither voltage- nor frequency-dependent. In HERG channel mutants Y652A and F656A, drug affinity was reduced dramatically. Therefore, these two aromatic residues in the channel pore are likely to form a main part of the binding site for budipine. In summary, this is the first study that provides a molecular basis for the budipine-associated aLQTS observed in clinical practice. Furthermore, these findings underline the importance of the aromatic residues Y652 and F656 in the binding of lipophilic drugs to HERG channels.

Modulation of the slow component of the delayed rectifier potassium current (IKs) in heart critically affects cardiac arrhythmogenesis. Its current amplitude is regulated by the sympathetic nervous system. However, the signal transduction from the beta-adrenergic system to the KvLQT1/MinK (KCNQ1/KCNE1) potassium channel, which is the molecular correlate of the IKs current in human cardiomyocytes, is not sufficiently understood. In the human heart, three subtypes of beta-adrenergic receptors (beta(1-3)-ARs) have been identified. Only beta(1)- and beta(3)-ARs have been shown so far to be involved in the regulation of IKs. Special interest has been paid to the regulation of IKs by the beta(3)-AR because of its potential importance in congestive heart failure. In heart failure beta(1)-ARs are known to be down regulated while the density of beta(3)-ARs is increased. Unfortunately, studies on the modulation of IKs by beta(3)-AR revealed conflicting results. We investigated the functional role of protein kinase C (PKC) in the signal transduction cascade between beta3-adrenergic receptors and IKs by expressing heterologously its molecular components, the KvLQT1/MinK potassium channel, together with human beta(3)-AR in Xenopus oocytes. Membrane currents were measured with the double electrode voltage-clamp technique. Using activators and inhibitors of PKC we demonstrated that PKC is involved in this regulatory process. Experiments in which the putative C-terminal PKC-phosphorylation sites in the KvLQT1 protein were destroyed by site directed mutagenesis reduced the isoproterenol-induced current to 27+/-3,5% compared to control. These results indicate that the amplitude of KvLQT1/MinK current is mainly increased by PKC activation. Our results suggest that the regulation of the KvLQT1/MinK potassium channel via beta(3)-AR is substantially mediated by PKC phosphorylation of the KvLQT1 protein at its four C-terminal PKC phosphorylation sites.

Tamoxifen is a nonsteroidal antiestrogen that is commonly used in the treatment of breast cancer. Although antiestrogenic drugs are generally believed not to cause acquired long QT syndrome (LQTS), concerns have been raised by recent reports of QT interval prolongation associated with tamoxifen treatment. Since blockade of human ether-a-go-go-related gene (HERG) potassium channels is critical in the development of acquired LQTS, we investigated the effects of tamoxifen on cloned HERG potassium channels to determine the electrophysiological basis for the arrhythmogenic potential of this drug. HERG channels were heterologously expressed in Xenopus laevis oocytes, and currents were measured using the two-microelectrode voltage clamp technique. Tamoxifen blocked HERG potassium channels with an IC(50) value of 45.3 microM. Inhibition required channel opening and unblocking occurred very slowly. Analysis of the voltage-dependence of block revealed loss of inhibition at positive membrane potentials, indicating that strong channel inactivation prevented block by tamoxifen. No marked changes in electrophysiological parameters such as voltage-dependence of activation or inactivation, or inactivation time constant could be observed, and block was not frequency-dependent. This study demonstrates that HERG potassium channels are blocked by the antiestrogenic drug tamoxifen. We conclude that HERG current inhibition might be an explanation for the QT interval prolongation associated with this drug.

Objective: Patients with HERG-associated long QT syndrome typically develop tachyarrhythmias during physical or emotional stress. Previous studies have revealed that activation of the beta-adrenergic system and consecutive elevation of the intracellular cAMP concentration regulate HERG channels via protein kinase A-mediated phosphorylation of the channel protein and via direct interaction with the cAMP binding site of HERG. In contrast, the influence of the alpha-adrenergic signal transduction cascade on HERG currents as suggested by recent reports is less well understood. The aim of the present study was to elucidate the biochemical pathways of the protein kinase C (PKC)-dependent regulation of HERG currents., Methods: HERG channels were heterologously expressed in Xenopus laevis oocytes, and currents were measured using the two-microelectrode voltage clamp technique., Results: Application of the phorbol ester PMA, an unspecific protein kinase activator, shifted the voltage dependence of HERG activation towards more positive potentials. This effect could be mimicked by activation of conventional PKC isoforms with thymeleatoxin. Coexpression of HERG with the beta-subunits minK or hMiRP1 did not alter the effect of PMA. Specific inhibition of PKC abolished the PMA-induced activation shift, suggesting that PKC is required within the regulatory mechanism. The PMA-induced effect could still be observed when the PKC-dependent phosphorylation sites in HERG were deleted by mutagenesis. Cytoskeletal proteins such as actin filaments or microtubules did not affect the HERG activation shift., Conclusion: In addition to the known effects of PKA and cAMP, HERG channels are also modulated by PKC. The molecular mechanisms of this PKC-dependent process are not completely understood but do not depend on direct PKC-dependent phosphorylation of the channel.

(1) Acquired long QT syndrome (aLQTS) is caused by prolongation of the cardiac action potential because of blockade of cardiac ion channels and delayed repolarization of the heart. Patients with aLQTS carry an increased risk for torsade de pointes arrhythmias and sudden cardiac death. Several antipsychotic drugs may cause aLQTS. Recently, cases of QTc prolongation and torsade de pointes associated with chlorpromazine treatment have been reported. Blockade of human ether-a-go-go-related gene (HERG) potassium channels, which plays a central role in arrhythmogenesis, has previously been reported to occur with chlorpromazine, but information on the mechanism of block is currently not available. We investigated the effects of chlorpromazine on cloned HERG potassium channels to determine the biophysical mechanism of block. (2) HERG channels were heterologously expressed in Xenopus laevis oocytes, and ion currents were measured using the two-microelectrode voltage-clamp technique. (3) Chlorpromazine blocked HERG potassium channels with an IC(50) value of 21.6 micro M and a Hill coefficient of 1.11. (4) Analysis of the voltage dependence of block revealed a reduction of inhibition at positive membrane potentials. (5) Inhibition of HERG channels by chlorpromazine displayed reverse frequency dependence, that is, the amount of block was lower at higher stimulation rates. No marked changes in electrophysiological parameters such as voltage dependence of activation or inactivation, or changes of the inactivation time constant were observed. (6) In conclusion, HERG channels were blocked in the closed and activated states, and unblocking occurred very slowly.

Background: Protein kinases A (PKA) and C (PKC) are activated in ischemic preconditioning and heart failure, conditions in which patients develop arrhythmias. The native inward rectifier potassium current (IK1) plays a central role in the stabilization of the resting membrane potential and the process of arrhythmogenesis. This study investigates the functional relationship between PKC and IK1., Methods and Results: In whole-cell patch-clamp experiments with isolated human atrial cardiomyocytes, the IK1 was reduced by 41% when the nonspecific activator of PKC phorbol 12 myristate 13-acetate (PMA; 100 nmol/L) was applied. To investigate the effects of PKC on cloned channel underlying parts of the native IK1, we expressed Kir(2.1b) heterologously in Xenopus oocytes and measured currents with the double-electrode voltage-clamp technique. PMA decreased the current by an average of 68%, with an IC50 of 0.68 nmol/L. The inactive compound 4-alpha-PMA was ineffective. Thymeleatoxin and 1-oleolyl-2-acetyl-sn-glycerol, 2 specific activators of PKC, produced effects similar to those of PMA. Inhibitors of PKC, ie, staurosporine and chelerytrine, could inhibit the PMA effect (1 nmol/L) significantly. After mutation of the PKC phosphorylation sites (especially S64A and T353A), PMA became ineffective., Conclusions: The human IK1 in atrial cardiomyocytes and one of its underlying ion channels, the Kir(2.1b) channel, is inhibited by PKC-dependent signal transduction pathways, possibly contributing to arrhythmogenesis in patients with structural heart disease in which PKC is activated.

1. Bertosamil is chemically related to the class-III anti-arrhythmic drug tedisamil and has been developed as a bradycardic, anti-ischemic and anti-arrhythmic drug. Its anti-arrhythmic properties might in part be attributed to its block of voltage-dependent potassium channels Kv(1.2), Kv(1.4). and Kv(1.5). However, HERG-potassium channel block as an important target for class-III drugs has not yet been investigated. 2. We investigated the effect of bertosamil on the HERG potassium channel heterologously expressed in Xenopus oocytes with the two-electrode voltage-clamp technique. 3. Bertosamil (70 microM) inhibited HERG tail currrent after a test pulse to 30 mV by 49.3+/-8.4% (n=5) and the IC(50) was 62.7 microM. Onset of block was fast, i.e. 90% of inhibition developed within 180+/-8.22 s (n=5), and block was totally reversible upon washout within 294+/-38.7 s (n=5). 4. Bertosamil-induced block of HERG potassium channels was state-dependent with block mainly to open- and inactivated channels. Half-maximal activation voltage was slightly shifted towards more negative potentials. 5. Steady-state inactivation of HERG was not influenced by bertosamil. Bertosamil block elicited voltage-but no frequency-dependent effects. 6. In summary, bertosamil blocked the HERG potassium channel. These blocking properties may contribute to the anti-arrhythmic effects of bertosamil in the treatment of atrial and particular ventricular arrhythmias.

Objective: To investigate the molecular mechanism of human ether-a-go-go-related gene (HERG) potassium channels regulated by protein kinase A (PKA) in a human cell line., Methods: HERG channels were stably expressed in human embryonic kidney (HEK) 293 cells, and currents were measured with the patch clamp technique. The direct phosphorylation of HERG channel proteins expressed heterologously in Xenopus laevis oocytes was examined by (32)P labeling and immunoprecipitation with an anti-HERG antibody., Results: Elevation of the intracellular cAMP-concentration by incubation with the adenylate cyclase activator, forskolin (10 micromol/L), and the broad range phosphodiesterase inhibitor, IBMX (100 micromol/L), caused a HERG tail current reduction of 83.2%. In addition, direct application of the membrane permeable cAMP analog, 8-Br-cAMP (500 micromol/L), reduced the tail current amplitude by 29.3%. Intracellular application of the catalytic subunit of protein kinase A (200 U/ml) led to a tail current decrease by 56.9% and shifted the activation curve by 15.4 mV towards more positive potentials. HERG WT proteins showed two phosphorylated bands, an upper band with a molecular mass of approximately 155 kDa and a lower band with a molecular mass of approximately 135 kDa, indicating that both the core- and the fully glycosylated forms of the protein were phosphorylated., Conclusions: PKA-mediated phosphorylation of HERG channels causes current reduction in a human cell line. The coupling between the repolarizing cardiac HERG potassium current and the protein kinase A system could contribute to arrhythmogenesis under pathophysiological conditions.

Objective: The antiarrhythmic potential of betablockers contributes to their beneficial effects in the treatment of cardiac diseases, although the molecular basis of their class II antiarrhythmic action has not been clarified yet., Methods: To investigate a putative functional link between beta-adrenoreceptors and the fast component of cardiac delayed rectifier K(+) channels (I(Kr)), whole-cell patch-clamp experiments were performed with isolated guinea-pig ventricular myocytes. Tail currents of I(Kr) were measured at -40 mV after short (200 ms) test pulses to +40 mV., Results: After application of the unspecific beta-receptor agonist isoproterenol (10 microM) for 12 min, the I(Kr) tail current was decreased by 72%, with an IC(50) of 1.4 microM. The specific beta(1)-blocker CGP207120A (10 microM) significantly attenuated the isoproterenol effect (net 24% decrease). The specific beta(1)-agonist xamoterol (10 microM), could mimic the isoproterenol effect (58% decrease). Modulators of beta(2)- or beta(3)-adrenoreceptors were far less effective. When isoproterenol or xamoterol were combined with KT5720 (2.5 microM), a specific inhibitor of protein kinase A (PKA), their effects were drastically reduced, indicating that PKA presumably mediates the beta(1)-adrenergic inhibition of I(Kr). Tail current reductions by cAMP, forskolin, PKA catalytic subunit and a combination of PKA holoenzyme and cAMP support an involvement of PKA in the regulation of I(Kr)., Conclusions: The functional link between I(Kr) and the beta(1)-adrenergic receptor involving PKA may play an important role in arrhythmogenesis and contribute to the antiarrhythmic action of clinically used beta(1)-blockers.

Objective: The aryloxypropanolamine carvedilol is a multiple action cardiovascular drug with blocking effects on alpha-receptors, beta-receptors, Ca(2+)-channels, Na(+)-channels and various native cardiac K(+) channels, thereby prolonging the cardiac action potential. In a number of clinical trials with patients suffering from congestive heart failure, carvedilol appeared to be superior to other beta-blocking agents in reducing total mortality. Given the multiple pharmacological actions of carvedilol, this may be due to specific channel blockade rather than beta-antagonistic activity. Since human ether-a-go-go related gene (HERG) K(+)channels play a critical role in the pathogenesis of cardiac arrhythmias and sudden cardiac death, the effects of carvedilol on HERG K(+)channels were investigated., Methods: Double-electrode voltage-clamp experiments were performed on HERG potassium channels which were expressed heterologously in Xenopus oocytes., Results: Carvedilol at a concentration of 10 microM blocked HERG potassium tail currents by 47%. The electrophysiological characteristics of HERG, i.e. activation, steady-state inactivation and recovery from inactivation were not affected by carvedilol. Inhibition of current gradually increased from 0% immediately after the test pulse to about 80% at 600 ms with subsequent marginal changes of current kinetics during the resting 29 s, indicating a very fast open channel block by carvedilol as the major blocking mechanism., Conclusion: This is the first study demonstrating that carvedilol blocks HERG potassium channels. The biophysical data presented in this study with a potentially antiarrhythmic effect may contribute to the positive outcome of clinical trials with carvedilol.