Multiple mechanisms of hERG liability: K+ current inhibition, disruption of protein trafficking, and apoptosis induced by amoxapine.

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 (IC(50) = 21.6 microM) and in HEK 293 cells (IC(50) = 5.1 microM). 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 (IC(50) = 15.3 microM). 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.

The human cardiac K2P3.1 (TASK-1) potassium leak channel is a molecular target for the class III antiarrhythmic drug amiodarone.

Two-pore-domain (K(2P)) potassium channels mediate background potassium currents, stabilizing resting membrane potential and expediting action potential repolarization. In the heart, K(2P)3.1 (TASK-1) channels are implicated in the cardiac plateau current, I ( KP ). Class III antiarrhythmic drugs target cardiac K(+) currents, resulting in action potential prolongation and suppression of atrial and ventricular arrhythmias. The objective of this study was to investigate acute effects of the class III antiarrhythmic drug amiodarone on human K(2P)3.1 channels. Potassium currents were recorded from Xenopus oocytes using the two-microelectrode voltage clamp technique. Amiodarone produced concentration-dependent inhibition of hK(2P)3.1 currents (IC(50) = 0.40 microM) with maximum current reduction of 58.1%. Open rectification properties that are characteristic to hK(2P)3.1 currents were not altered by amiodarone. Channels were blocked in open and closed states in reverse frequency-dependent manner. hK(2P)3.1 channel inhibition was voltage-independent at voltages between -40 and +60 mV. Modulation of protein kinase C activity by amiodarone does not contribute to hK(2P)3.1 current reduction, as pre-treatment with the protein kinase C inhibitor, staurosporine, did not affect amiodarone block. Amiodarone is an inhibitor of cardiac hK(2P)3.1 background channels. Amiodarone blockade of hK(2P)3.1 may cause prolongation of cardiac repolarization and action potential duration in patients with high individual plasma concentrations, possibly contributing to the antiarrhythmic efficacy of the class III drug.

Biophysical properties of zebrafish ether-à-go-go related gene potassium channels.

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.

Selective noradrenaline reuptake inhibitor atomoxetine directly blocks hERG currents.

BACKGROUND AND PURPOSE: Atomoxetine is a selective noradrenaline reuptake inhibitor, recently approved for the treatment of attention-deficit/hyperactivity disorder. So far, atomoxetine has been shown to be well tolerated, and cardiovascular effects were found to be negligible. However, two independent cases of QT interval prolongation, associated with atomoxetine overdose, have been reported recently. We therefore analysed acute and subacute effects of atomoxetine on cloned human Ether-à-Go-Go-Related Gene (hERG) channels. EXPERIMENTAL APPROACH: hERG channels were heterologously expressed in Xenopus oocytes and in a human embryonic kidney cell line and hERG currents were measured using voltage clamp and patch clamp techniques. Action potential recordings were made in isolated guinea-pig cardiomyocytes. Gene expression and channel surface expression were analysed using quantitative reverse transcriptase polymerase chain reaction, Western blot and the patch clamp techniques. KEY RESULTS: In human embryonic kidney cells, atomoxetine inhibited hERG current with an IC(50) of 6.3 micromol.L(-1). Development of block and washout were fast. Channel activation and inactivation were not affected. Inhibition was state-dependent, suggesting an open channel block. No use-dependence was observed. Inhibitory effects of atomoxetine were attenuated in the pore mutants Y652A and F656A. In guinea-pig cardiomyocytes, atomoxetine lengthened action potential duration without inducing action potential triangulation. Overnight incubation with high atomoxetine concentrations resulted in a decrease of channel surface expression. CONCLUSIONS AND IMPLICATIONS: Whereas subacute effects of atomoxetine seem negligible under therapeutically relevant concentrations, hERG channel block should be considered in cases of atomoxetine overdose and when administering atomoxetine to patients at increased risk for the development of acquired long-QT syndrome.

Dual regulation of renal Kir7.1 potassium channels by protein Kinase A and protein Kinase C.

The renal inward rectifier potassium channel Kir7.1 has been proposed to be functionally important for tubular K(+) recycling and secretion. This study investigated the regulation of Kir7.1 by PKA and PKC. Cloned human Kir7.1 channels were expressed heterologously in Xenopus oocytes. After pharmacological PKC activation, Kir7.1 currents were strongly inhibited. Co-application of PKC inhibitors attenuated this effect. Inactivation of PKC consensus sites also strongly attenuated the effect with a single site ((201)S) being essential for almost the total PKC sensitivity. In contrast, PKA activation induced an increase of Kir7.1 currents. This effect was absent in mutant Kir7.1 channels lacking PKA consensus site (287)S. In summary, this study demonstrates the dual regulation of Kir7.1 channel function by PKA and PKC. Structurally, these regulations depend on two key residues in the C-terminal channel domain ((Ser)201 for PKC and (Ser)287 for PKA).

Inhibition of cardiac hERG potassium channels by tetracyclic antidepressant mianserin.

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.

Doxazosin induces apoptosis of cells expressing hERG K+ channels.

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.

Kir2.x inward rectifier potassium channels are differentially regulated by adrenergic alpha1A receptors.

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.

Green tea flavonoid epigallocatechin-3-gallate (EGCG) inhibits cardiac hERG potassium channels.

The catechin EGCG is the main flavonoid compound of green tea and has received enormous pharmacological attention because of its putative beneficial health effects. This study investigated for the first time the effect of EGCG on hERG channels, the main pharmacological target of drugs that cause acquired long QT syndrome. Cloned hERG channels were expressed in Xenopus oocytes and in HEK293 cells. Heterologous hERG currents were inhibited by EGCG with an IC50 of 6.0 micromol/l in HEK293 cells and an IC50 of 20.5 micromol/l in Xenopus laevis oocytes. Onset of effect was slow and only little recovery from inhibition was observed upon washout. In X. laevis oocytes EGCG inhibited hERG channels in the open and inactivated states, but not in the closed states. The half-maximal activation voltage of hERG currents was shifted by EGCG towards more positive potentials. In conclusion, EGCG is a low-affinity inhibitor of hERG sharing major electrophysiological features with pharmaceutical hERG antagonists.

Anticholinergic antiparkinson drug orphenadrine inhibits HERG channels: block attenuation by mutations of the pore residues Y652 or F656.

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.

Regulation of cardiac inwardly rectifying potassium current IK1 and Kir2.x channels by endothelin-1.

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.

Class Ia anti-arrhythmic drug ajmaline blocks HERG potassium channels: mode of action.

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.

Activation of cardiac human ether-a-go-go related gene potassium currents is regulated by alpha(1A)-adrenoceptors.

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.

Inhibition of cardiac HERG potassium channels by the atypical antidepressant trazodone.

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.