hERG Tail Currents Research Articles

Multiple Mechanisms Contribute to Cardiotoxicity Observed with the Antidepressant Desipramine

The cardiotoxicity of antidepressants is well recognized. Common electrocardiographic changes precipitated in particular by an overdose include QRS widening as well as prolongation of the QT interval and torsade de pointes tachycardia. QT prolongation by antidepressants has usually been associated with acute block of hERG/IKr currents. This study has been designed to provide a more complete picture of the molecular mechanisms underlying cardiac side effects induced by the antidepressant desipramine. We have studied acute block in HEK/hERG WT cells using patch-clamp recordings and found that desipramine reduced hERG currents with an IC50 value of 11.9μM. In HEK/hERG F656V, a mutation that reduces drug binding, hERG currents were blocked half-maximally with 48.3μM. We used Western blots to monitor the effects of desipramine on hERG trafficking. In these experiments we found that the fully-glycosylated cell surface form of hERG was reduced with an IC50 of 5.1μM on overnight incubation. When long-term effects were studied using electrophysiological current recordings, hERG tail currents were decreased with an IC50 of 7.5μM. Accordingly, hERG surface expression was reduced by desipramine when monitored directly using a cell-based assay (IC50, 17.3μM) or confocal imaging. Importantly, the reduction in surface expression was not attenuated by mutation of residue F656 in the drug binding site of hERG. In guinea pig ventricular myocytes action potential duration was prolonged in a dose-dependent manner as expected on acute desipramine exposure. However, long-term exposure increased action potential duration only marginally. Finally, desipramine triggered apoptosis in cells expressing hERG channels. Taken together, desipramine exerts adverse cardiac effects by at least three different mechanisms: (1) direct hERG channel block, (2) disruption of hERG trafficking, and (3) induction of apoptosis.

Response of IKrand hERG Currents to the Antipsychotics Tiapride and Sulpiride

The human ether-a-go-go-related gene (hERG) channel is important for repolarization in human myocardium and is a common target for drugs that prolong the QT interval. We studied the effects of two antipsychotics, tiapride and sulpiride, on hERG channels expressed in Xenopus oocytes and also on delayed rectifier K(+) currents in guinea pig cardiomyocytes. Neither the amplitude of the hERG outward currents measured at the end of the voltage pulse, nor the amplitude of hERG tail currents, showed any concentration-dependent changes with either tiapride or sulpiride (3~300 µM). However, our findings did show that tiapride increased the potential for half-maximal activation (V(1/2)) of HERG at 10~300 µM, whereas sulpiride increased the maximum conductance (G(max)) at 3, 10 and 100 µM. In guinea pig ventricular myocytes, bath applications of 100 and 500 µM tiapride at 36℃ blocked rapidly activating delayed rectifier K(+) current (I(Kr)) by 40.3% and 70.0%, respectively. Also, sulpiride at 100 and 500 µM blocked I(Kr) by 38.9% and 76.5%, respectively. However, neither tiapride nor sulpiride significantly affected the slowly activating delayed rectifier K(+) current (I(Ks)) at the same concentrations. Our findings suggest that the concentrations of the antipsychotics required to evoke a 50% inhibition of I(Kr) are well above the reported therapeutic plasma concentrations of free and total compound.

The Novel Mechanism of Oxymatrine Affecting hERG Currents at Different Temperatures

Background/Aims: Human ether-à-go-go-related gene (hERG) has an important role in the repolarization of the cardiac action potential. Our studies were to investigate the effects of oxymatrine (one of the natural constituents extracted from Chinese herb Sophora flavescens Ait) on hERG-encoded K⁺ channels at different temperatures and its underlying mechanism. Methods: The effects of oxymatrine were examined on hERG channels stably expressed in HEK293 cells using a whole-cell patch clamp technique. Results: At the temperature 30°C, oxymatrine inhibited hERG current in a concentration-dependent manner and the IC₅₀ was ∼665 µM. However at the temperature of 20°C, low concentration oxymatrine C≤100 µM increased hERG current density. However, high concentration oxymatrine C>100 µM inhibited the hERG current density significantly. Oxymatrine only affected the activation kinetic of hERG channels at all temperatures and had a high binding affinity for open state of hERG channels except the 300 µM-20°C group which had a high binding affinity for inactive state of hERG channels. Conclusion: Oxymatrine is a low potency blocker of hERG K+ channels at 30°C, low concentration oxymatrine affect the hERG activation gating with accelerating hERG tail current at 20°C, oxymatrine is a potential hERG activator at low temperatures.

Lack of Clinically Important hERG Channel Block by the Antipsychotics Tiapride and Sulpiride

The human ether-a-go-go-related gene (hERG) channel is important for repolarization in human myocardium and is a common target for drugs that prolong the QT interval. We studied the effects of two antipsychotics, tiapride and sulpiride on hERG channels expressed in Xenopus oocytes and also in the delayed rectifier K+ current of guinea pig cardiomyocytes. The amplitude of the hERG outward currents measured at the end of the pulse showed no concentration-dependent change with increasing either tiapride or sulpiride concentration (3-300 μM). Also, the amplitude of hERG tail currents did not show concentration-dependent changes with increasing either tiapride or sulpiride concentration (3-300 μM). However, our findings showed that tiapride increased the values of the potential for half-maximal activation (V1/2) at 10 - 300 μM, on the contrary, sulpiride increased the maximum conductance (Gmax) at 3, 10, 100 μM. In guinea pig ventricular myocytes, bath applications of 100 and 500 μM tiapride at 36°C blocked rapidly activating delayed rectifier K+ current (IKr) by 40.3% and 70.0%, respectively. Also, sulpiride at 100 and 500 μM blocked IKr by 38.9% and 76.5%, respectively, but tiapride and sulpiride at the concentrations did not significantly block slowly activating delayed rectifier K+ current (IKs). Our findings suggest that the concentrations of the antipsychotics required to evoke a 50% inhibition of the IKr were well above reported therapeutic plasma concentrations of free and total compound. None of tiapride and sulpiride was a potent blocker of the hERG channel.

H 1 antihistamine drug promethazine directly blocks hERG K + channel

Promethazine is a phenothiazine derivative with antihistaminic (H 1), sedative, antiemetic, anticholinergic, and antimotion sickness properties that can induce QT prolongation, which may lead to torsades de pointes. Since block of cardiac human ether- a- go- go-related gene ( hERG) channels is one of the leading causes of acquired long QT syndrome, we investigated the acute effects of promethazine on hERG channels to determine the electrophysiological basis for its proarrhythmic potential. Promethazine increased the action potential duration at 90% of repolarization (APD 90) in a concentration-dependent manner, with an IC 50 of 0.73 μM when action potentials were elicited under current clamp in guinea pig ventricular myocytes. We examined the effects of promethazine on the hERG channels expressed in Xenopus oocytes and HEK293 cells using two-microelectrode voltage-clamp and patch-clamp techniques. Promethazine induced a concentration-dependent decrease of the current amplitude at the end of the voltage steps and hERG tail currents. The IC 50 of promethazine dependent hERG block in Xenopus oocytes decreased progressively relative to the degree of depolarization. The IC 50 for the promethazine-induced block of the hERG currents in HEK293 cells at 36 °C was 1.46 μM at +20 mV. Promethazine affected the channels in the activated and inactivated states but not in the closed states. The S6 domain mutations, Y652A and F656A partially attenuated (Y652A) or abolished (F656A) the hERG current block. These results suggest that promethazine is a blocker of the hERG channels, providing a molecular mechanism for the arrhythmogenic side effects during the clinical administration of promethazine.

Cellular Mechanism of the QT Prolongation Induced by Sulpiride

In this study, the authors investigated the electrophysiological effect of sulpiride on cardiac repolarization using conventional microelectrode recording techniques in isolated canine Purkinje fibers and a whole-cell patch clamp technique in transiently transfected cells with the hERG, KCNQ1/KCNE1, KCNJ2, and SCN5A cDNA and in rat cardiac myocytes for I(Ca). In studies of action potential duration, 10 microM, 100 microM, 300 microM, and 1 mM sulpiride prolonged action potential duration in a concentration-dependent manner. In studies of cardiac ion channels, sulpiride did not significantly affect I(Na), I(Ca), I(Ks), I(K1), except for I(Kr). Sulpiride dose-dependently decreased the hERG tail current. It is considered that the prolonged action potential duration by sulpiride was mainly the result of inhibition of the hERG channel. The data suggest that the clinical use of sulpiride is reasonable within therapeutic plasma concentrations, but all patients taking this drug should be cautiously monitored for clinical signs of long-QT syndrome and severe arrhythmia.

Block of hERG K+ Channel by Classic Histamine H1 Receptor Antagonist Chlorpheniramine

Chlorpheniramine is a potent first-generation histamine H(1) receptor antagonist that can increase action potential duration and induce QT prolongation in several animal models. Since block of cardiac human ether-a-go-go-related gene (hERG) channels is one of leading causes of acquired long QT syndrome, we investigated the acute effects of chlorpheniramine on hERG channels to determine the electrophysiological basis for its proarrhythmic potential. We examined the effects of chlorpheniramine on the hERG channels expressed in Xenopus oocytes using two-microelectrode voltage-clamp techniques. Chlorpheniramine induced a concentration-dependent decrease of the current amplitude at the end of the voltage steps and hERG tail currents. The IC(50) of chlorpheniramine-dependent hERG block in Xenopus oocytes decreased progressively relative to the degree of depolarization. Chlorpheniramine affected the channels in the activated and inactivated states but not in the closed states. The S6 domain mutations Y652A and F656A partially attenuated (Y652A) or abolished (F656A) the hERG current block. These results suggest that the H(1) antihistamine, chlorpheniramine is a blocker of the hERG channels, providing a molecular mechanism for the drug-induced arrhythmogenic side effects.

Clomipramine block of the hERG K + channel: Accessibility to F656 and Y652

Clomipramine is a tricyclic antidepressant for psychiatric disorders that can induce QT prolongation, which may lead to torsades de pointes. Since blockade of cardiac human ether- a- go- go-related gene (hERG) channels is an important cause of acquired long QT syndrome, we investigated the acute effects of clomipramine on hERG channels to determine the electrophysiological basis for its proarrhythmic potential. We examined the effects of clomipramine on the hERG channels expressed in Xenopus oocytes and HEK293 cells using two-microelectrode voltage-clamp and patch-clamp techniques. Clomipramine induced a concentration-dependent decrease in the current amplitude at the end of the voltage steps and hERG tail currents. The IC50 for clomipramine needed to block the hERG current in Xenopus oocytes decreased progressively relative to the degree of depolarization. The fractional electrical distance was estimated to be δ = 0.83. The IC50 for the clomipramine-induced blockade of the hERG currents in HEK293 cells at 36 °C was 0.13 μM at + 20 mV. Clomipramine affected the channels in the activated and inactivated states but not in the closed states. The clomipramine-induced blockade of hERG was found to be use-dependent, exhibiting a more rapid onset and a greater steady-state block at the higher frequencies of activation. The S6 domain mutations, Y652A and F656A partially attenuated (Y652A) or abolished (F656A) the hERG-current blockade. These results suggest that clomipramine is a blocker of the hERG channels, providing a molecular mechanism for the arrhythmogenic side effects during the clinical administration of clomipramine.

2-[2-(3,4-Dichloro-phenyl)-2,3-dihydro-1H-isoindol-5-ylamino]-nicotinic Acid (PD-307243) Causes Instantaneous Current through HumanEther-a-go-go-Related Gene Potassium Channels

Long and short QT syndromes associated with loss and gain of human ether-a-go-go-related gene (hERG) channel activity, respectively, can cause life-threatening arrhythmias. As such, modulation of hERG channel activity is an important consideration in the development of all new therapeutic agents. In the present study, we investigated the mechanisms of action of 2-[2-(3,4-dichloro-phenyl)-2,3-dihydro-1H-isoindol-5-ylamino]-nicotinic acid (PD-307243), a known hERG channel activator, on hERG channels stably expressed in Chinese hamster ovary (CHO) cells using the patch-clamp technique. In the whole-cell recordings, the extracellular application of PD-307243 concentration-dependently increased the hERG current and markedly slowed hERG channel deactivation and inactivation. PD-307243 had no effect on the selectivity filter of hERG channels. The activity of PD-307243 was use-dependent. PD-307243 (3 and 10 muM) induced instantaneous hERG current with little decay at membrane potentials from -120 to -40 mV. At more positive voltages, PD-307243 induced an I(to)-like upstroke of hERG current. The actions of PD-307243 on the rapid component of delayed rectifier K(+) current (I(Kr)) in rabbit ventricular myocytes were similar to those observed in hERG channel-transfected CHO cells. Inside-out patch experiments revealed that PD-307243 increased hERG tail currents by 2.1 +/- 0.6 (n = 7) and 3.4 +/- 0.3-fold (n = 4) at 3 and 10 muM, respectively, by slowing the channel deactivation but had no effect on channel activation. During a voltage-clamp protocol using a prerecorded cardiac action potential, 3 muM PD-307243 increased the total potassium ions passed through hERG channels by 8.8 +/- 1.0-fold (n = 5). Docking studies suggest that PD-307243 interacts with residues in the S5-P region of the channel.

In vitro modulation of HERG channels by organochlorine solvent trichlormethane as potential explanation for proarrhythmic effects of chloroform

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 4.97 mM. 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.

Inhibition of hERG K + currents by antimalarial drugs in stably transfected HEK293 cells

Several antimalarial drugs are known to produce a QT interval prolongation via a blockade of the rapidly activating delayed rectifier K + current ( I Kr), encoded by the human-ether-a-go-go-related gene (hERG). We investigated the influence of lumefantrine and its major metabolite desbutyl-lumefantrine, as well as halofantrine, chloroquine, and mefloquine, on wild type hERG K + channels in stably transfected human embryonic kidney cells (HEK293) using the whole cell patch-clamp technique. All of the tested antimalarial drugs inhibited the hERG K + channels in a concentration- and time-dependent manner. Only halofantrine blocked hERG tail currents voltage-dependently. The ranking of the half-maximal inhibitory concentrations (IC 50) of the antimalarials was: halofantrine (0.04 μM)<chloroquine (2.5 μM)<mefloquine (2.6 μM)<desbutyl-lumefantrine (5.5 μM)<lumefantrine (8.1 μM). Lumefantrine and desbutyl-lumefantrine showed a slower inhibition of I Kr than the other tested antimalarials. In conclusion, lumefantrine and desbutyl-lumefantrine inhibited significantly the hERG tail current with a higher IC 50-value than mefloquine, chloroquine and halofantrine. This, together with the calculated cardiac safety indices, suggests that lumefantrine and desbutyl-lumefantrine have a weaker proarrhythmic potential than their comparator compounds.

Single-walled Carbon Nanotubes Are a New Class of Ion Channel Blockers

Here we identify a novel class of biological membrane ion channel blockers called single-walled carbon nanotubes (SWNTs). SWNTs with diameter distributions peaked at approximately 0.9 and 1.3 nm, C60 fullerenes, multi wall nanotubes (MWNTs), and hyperfullerenes (nano-"onions") were synthesized by several techniques and applied to diverse channel types heterologously expressed in mammalian cells. External as-fabricated and purified SWNTs blocked K+ channel subunits in a dose-dependent manner. Blockage was dependent on the shape and dimensions of the nanoparticles used and did not require any electrochemical interaction. SWNTs were more effective than the spherical fullerenes and, for both, diameter was the determining factor. These findings postulate new uses for SWNTs in biological applications and provide unexpected insights into the current view of mechanisms governing the interaction of ion channels with blocking molecules.

Kinetic modulation of HERG potassium channels by the volatile anesthetic halothane.

(human ether-a-gogo related gene) encodes the cardiac rapidly activating delayed rectifier potassium currents (I(kr)), which play an important role in cardiac action potential repolarization. General anesthetics, like halothane, can prolong Q-T interval, suggesting that they act on myocellular repolarization, possibly involving HERG channels. Evidence for direct modulation of HERG channels by halothane is still lacking. To gain insight on HERG channel modulation by halothane the authors recorded macroscopic currents expressed in Xenopus oocytes and conducted non-stationary noise analysis to evaluate single channel parameters modified by the anesthetic. Macroscopic currents were recorded in 120 mM K(+) internal-5 mM K(+) external solutions with the cut open oocyte technique. Macropatch recordings for non-stationary noise analysis of HERG tail currents were made in symmetrical 120 mM K(+) solutions. Pulse protocols designed for HERG current recording were elicited from a holding potential of -80 mV. Halothane was delivered via gravity-fed perfusion. Halothane (0.7%, 1.5%, and 3%) decreased macroscopic currents in a concentration-dependent manner (average reduction by 14%, 22%, and 35% in the range of -40 mV to 40 mV) irrespective of potential. HERG currents had slower activation and accelerated deactivation and inactivation. Non-stationary noise analysis revealed that halothane, 1.5%, decreased channel P(o) by 27%, whereas single-channel current amplitudes and number of channels in the patch remained unchanged. Halothane inhibits HERG currents expressed in oocytes in a concentration-dependent manner. It slowed down activation and accelerated deactivation and inactivation of HERG channels. The authors' results demonstrate that halothane decreased HERG currents by modulating kinetic properties of HERG channels, decreasing their open probability. Partial block of I(kr) currents could contribute to delayed myocellular repolarization and altered cardiac electrophysiology.

Inhibition of HERG Potassium Channel Current by the Class 1a Antiarrhythmic Agent Disopyramide

The Class 1a antiarrhythmic drug disopyramide (DISO) is associated with ‘acquired’ prolongation of the QT interval of the electrocardiogram (ECG). This potentially proarrhythmic effect is likely to reflect drug actions on ion channels involved in ventricular action potential repolarisation. In this study, we examined the effects of DISO on potassium channels encoded by HERG, as this K channel type has been implicated in both congenital and acquired long-QT syndromes (LQTS). Chinese hamster ovary cells were transiently transfected with HERG cDNA for subsequent whole cell patch clamp recording. HERG tail currents recorded at −40 mV following test pulses to +30 mV were inhibited in a dose-dependent fashion by DISO concentrations within the clinical range (IC50 = 7.23 ± 0.72 μM; mean ± SEM). Experiments with 10 μM DISO indicated that the degree of HERG blockade showed some voltage dependence. Further data obtained using an ‘envelope of tails’ protocol (pulse potential +40 mV) were consistent with a significant role for open-channel blockade at lower drug concentrations. At higher concentrations it is possible that blockade may have involved drug binding to both resting and open channels. Inhibition of the inactivation-deficient mutant HERG-S631A was comparable to that seen for wild-type HERG. Therefore, channel inactivation was not obligatory for DISO to exert its effect. Native delayed rectifier tail currents from rabbit isolated ventricular myocytes were also inhibited by DISO. We conclude (a) that DISO inhibits HERG encoded potassium channels at clinically relevant concentrations and (b) that this action may constitute the molecular basis for acquired LQTS associated with this drug.

Inhibition of the current of heterologously expressed HERG potassium channels by imipramine and amitriptyline.

1 Tricyclic antidepressants (TCAs) are associated with cardiovascular side effects including prolongation of the QT interval of the ECG. In this report we studied the effects of two TCAs (imipramine and amitriptyline) on ionic current mediated by cloned HERG potassium channels. 2 Voltage clamp measurements of HERG currents were made from CHO cells transiently transfected with HERG cDNA. HERG-encoded potassium channels were inhibited in a reversible manner by both imipramine and amitriptyline. HERG tail currents (IHERG) following test pulses to +20 mV were inhibited by imipramine with an IC50 of 3.4+/-0.4 microM (mean+/-s.e.mean) and a Hill coefficient of 1.17+/-0.03 (n = 5). 3 microM amitriptyline inhibited IHERG by 34+/-6% (n = 3). The inhibition showed only weak voltage dependence. 3 Using an 'envelope of tails' comprised of pulses to +20 mV of varying durations, the tau of activation was found to be 155+/-30 ms for control and 132+/-26 ms for 3 microM imipramine (n = 5). Once maximal channel activation was achieved after 320 ms (as demonstrated by maximal tail currents), further prolongation of depolarization did not increase imipramine-mediated HERG channel inhibition. 4 Taking current measurements every second during a 10 s depolarizing pulse from -80 mV to 0 mV, block was observed during the first pulse in the presence of imipramine and the level of IHERG block was similar throughout the pulse (n=5). 5 A three pulse protocol (two depolarizing pulses to +20 mV separated by 20 ms at -80 mV) revealed that imipramine did not significantly alter the kinetics of IHERG inactivation. The tau of inactivation was 8+/-2 ms and 5.6+/-0.4 ms (n = 5) in the absence and presence of 3 microM imipramine, respectively, and currents inactivated to a similar extent. 6 Our data are consistent with TCAs causing components of block of the HERG channel in both the closed and open states. Any component of open channel block occurs rapidly upon depolarization. Inhibition of IHERG by the prototype TCAs imipramine and amitriptyline may suggest a mechanism for QT prolongation associated with risks of arrhythmia and sudden death that accompany high concentrations of TCAs following overdose.