Assessment of temperature‐induced hERG channel blockade variation by drugs

Drug‐induced QT prolongation has been reported in humans and animals. This potentially lethal effect can be induced by drugs interacting with a cardiac potassium channel, namely hERG (human ether‐a go‐go‐related gene) leading to arrhythmia or torsade de pointes (TdP). Hence, in vitro evaluation of therapeutics for their effects on the rapid delayed rectifier current (I_Kr) mediated by the K⁺ ion channel encoded by hERG is a valuable tool for identifying potential arrhythmic side effects during drug safety testing. Our objective was to evaluate the temperature‐induced hERG channel blockade variation by human and veterinary drugs using the IonFlux 16 system. A panel of eight drugs was tested for I_Kr inhibition at both ambient (23 °C) and physiological (37 °C) temperatures at various concentrations using IonFlux 16, an automated patch clamp system. Our results established that both amiodarone (IC₅₀ = 0.56 μM at 23 °C and 0.30 μM at 37 °C) and β‐estradiol (IC₅₀ = 24.72 μM at 23 °C and 8.17 μM at 37 °C) showed a dose‐dependent I_Kr blockade with a higher blockade at 37 °C. Whereas, blockade of I_Kr by both ivermectin (IC₅₀ = 12.52 μM at 23 °C and 24.41 μM at 37 °C) and frusemide (IC₅₀ = 12.58 μM at 23 °C and 25.55 μM at 37 °C) showed a dose‐dependent I_Kr blockade with a lower blockade at 37 °C. Gentamicin, enrofloxacin, xylazine and albendazole did not block I_Kr at both the assessed temperatures. Collectively, these results demonstrate that the effect of temperature variation should be taken into consideration during the evaluation of test drugs for their hERG channel blockade potential. Copyright © 2014 John Wiley & Sons, Ltd.     

Validation of visualized transgenic zebrafish as a high throughput model to assay bradycardia related cardio toxicity risk candidates

Drug‐induced QT prolongation usually leads to torsade de pointes (TdP), thus for drugs in the early phase of development this risk should be evaluated. In the present study, we demonstrated a visualized transgenic zebrafish as an in vivo high‐throughput model to assay the risk of drug‐induced QT prolongation. Zebrafish larvae 48 h post‐fertilization expressing green fluorescent protein in myocardium were incubated with compounds reported to induce QT prolongation or block the human ether‐a‐go‐go‐related gene (hERG) K⁺ current. The compounds sotalol, indapaminde, erythromycin, ofoxacin, levofloxacin, sparfloxacin and roxithromycin were additionally administrated by microinjection into the larvae yolk sac. The ventricle heart rate was recorded using the automatic monitoring system after incubation or microinjection. As a result, 14 out of 16 compounds inducing dog QT prolongation caused bradycardia in zebrafish. A similar result was observed with 21 out of 26 compounds which block hERG current. Among the 30 compounds which induced human QT prolongation, 25 caused bradycardia in this model. Thus, the risk of compounds causing bradycardia in this transgenic zebrafish correlated with that causing QT prolongation and hERG K⁺ current blockage in established models. The tendency that high logP values lead to high risk of QT prolongation in this model was indicated, and non‐sensitivity of this model to antibacterial agents was revealed. These data suggest application of this transgenic zebrafish as a high‐throughput model to screen QT prolongation‐related cardio toxicity of the drug candidates. Copyright © 2012 John Wiley & Sons, Ltd.     

Identification of quaternary ammonium compounds as potent inhibitors of hERG potassium channels

The human ether-a-go-go-related gene (hERG) channel, a member of a family of voltage-gated potassium (K⁺) channels, plays a critical role in the repolarization of the cardiac action potential. The reduction of hERG channel activity as a result of adverse drug effects or genetic mutations may cause QT interval prolongation and potentially leads to acquired long QT syndrome. Thus, screening for hERG channel activity is important in drug development. Cardiotoxicity associated with the inhibition of hERG channels by environmental chemicals is also a public health concern. To assess the inhibitory effects of environmental chemicals on hERG channel function, we screened the National Toxicology Program (NTP) collection of 1408 compounds by measuring thallium influx into cells through hERG channels. Seventeen compounds with hERG channel inhibition were identified with IC₅₀ potencies ranging from 0.26 to 22 μM. Twelve of these compounds were confirmed as hERG channel blockers in an automated whole cell patch clamp experiment. In addition, we investigated the structure-activity relationship of seven compounds belonging to the quaternary ammonium compound (QAC) series on hERG channel inhibition. Among four active QAC compounds, tetra-n-octylammonium bromide was the most potent with an IC₅₀ value of 260 nM in the thallium influx assay and 80 nM in the patch clamp assay. The potency of this class of hERG channel inhibitors appears to depend on the number and length of their aliphatic side-chains surrounding the charged nitrogen. Profiling environmental compound libraries for hERG channel inhibition provides information useful in prioritizing these compounds for cardiotoxicity assessment in vivo.     

Fluconazole inhibits hERG K(+) channel by direct block and disruption of protein trafficking

Fluconazole, a commonly used azole antifungal drug, can induce QT prolongation, which may lead to Torsades de Pointes and sudden death. To investigate the arrhythmogenic side effects of fluconazole, we studied the effect of fluconazole on human ether-a-go-go-related gene (hERG) K⁺ channels (wild type, Y652A and F656C) expressed in human embryonic kidney (HEK293) cells using a whole-cell patch clamp technique, Western blot analysis and confocal microscopy. Fluconazole inhibited wild type hERG currents in a concentration-dependent manner, with a half-maximum block concentration (IC₅₀) of 48.2 ± 9.4 μM. Fluconazole did not change other channel kinetics (activation and steady-state inactivation) of hERG channel. Mutations in drug- binding sites (Y652A or F656C) of the hERG channel significantly attenuated the hERG current blockade by fluconazole. In addition, fluconazole inhibited the trafficking of hERG protein by Western blot analysis and confocal microscopy, respectively. These findings indicate that fluconazole may cause acquired long QT syndrome (LQTS) via a direct inhibition of hERG current and by disrupting hERG protein trafficking, and the mutations Y652 and F656 may be obligatory determinants in inhibition of hERG current for fluconazole.     

Proarrhythmic mechanisms of the common anti-diarrheal medication loperamide: revelations from the opioid abuse epidemic

Loperamide is a μ-opioid receptor agonist commonly used to treat diarrhea and often available as an over-the-counter medication. Recently, numerous reports of QRS widening accompanied by dramatic QT interval prolongation, torsades de pointe arrhythmia, and death have been reported in opioid abusers consuming large amounts of the drug to produce euphoria or prevent opiate withdrawal. The present study was undertaken to determine the mechanisms of this cardiotoxicity. Using whole-cell patch clamp electrophysiology, we tested loperamide on the cloned human cardiac sodium channel (Na_v1.5) and the two main repolarizing cardiac K⁺ channels cloned from the human heart: K_vLQT1/minK and the human ether-a-go-go-related gene (hERG) channel. Loperamide inhibited Na_v1.5 with IC₅₀ values of 297 and 239 nM at holding potentials of ?90 and ?70 mV, respectively. Loperamide was weakly active on K_vLQT1/minK producing 17 and 65 % inhibition at concentrations of 1 and 10 μM, respectively. Conversely, loperamide was found to be a very high affinity inhibitor of the hERG channel with an IC₅₀ value of 89 nM at room temperature and 33 nM when measured at physiological temperature. The QRS and QT interval prolongation and the attending arrhythmias, produced by loperamide, derive from high affinity inhibition of Na_v1.5 and especially hERG. Since the drug has been widely available and safely used as directed for many years, we believe that the potent inhibition loperamide possesses for cardiac ion channels has only been uncovered because of the excessive misuse of the drug as a consequence of the recent opioid abuse epidemic.     

Electrophysiological Effects of the Anti‐Cancer Drug Lapatinib on Cardiac Repolarization

Abstract: Lapatinib is one of several tyrosine kinase inhibitors used against solid tumour cancers such as breast and lung cancer. Although lapatinib is associated with a risk of QT prolongation, the effects of the drug on cellular cardiac electrical properties and on action potential duration (APD) have not been studied. To evaluate the potential effects of lapatinib on cardiac repolarization, we investigated its electrophysiological effects using a whole‐cell patch–clamp technique in transiently transfected HEK293 cells expressing human ether‐à‐go‐go (hERG; to examine the rapidly activating delayed rectifier K⁺ current, I_Kr), KCNQ1/KCNE1 (to examine the slowly activating delayed rectifier K⁺ current, I_Ks), KCNJ2 (to examine the inwardly rectifying K⁺ current, I_K1), or SCN5A (to examine the inward Na⁺ current, I_Na) and in rat cardiac myocytes (to examine the inward Ca²⁺ current, I_Ca). We also examined its effects on the APD at 90% (APD₉₀) in isolated rabbit Purkinje fibres. In ion channel studies, lapatinib inhibited the hERG current in a concentration‐dependent manner, with a half‐maximum inhibition concentration (IC₅₀) of 0.8 ± 0.09 μm . In contrast, at concentrations up to 3 μm , lapatinib did not significantly reduce the I_Na, I_K1 or I_Ca amplitudes; at 3 μm , it did slightly inhibit the I_Ks amplitude (by 19.4 ± 4.7%; p < 0.05). At 5 μm , lapatinib induced prolongation of APD₉₀ by 16.1% (p < 0.05). These results suggest that the APD₉₀‐prolonging effect of lapatinib on rabbit Purkinje fibres is primarily a result of inhibition of the hERG current and I_Ks, but not I_Na, I_K1 or I_Ca.     

Pharmacological rescue of hERG currents carried out by G604S and wide type hERG co‐expression

Mutations in human ether‐a‐go‐go‐related gene (hERG) can lead to type 2 long‐QT syndrome (LQT2). The authors previously identified the hERG mutation G604S results in a loss of function and obviously decreased current amplitude and impaired channel protein trafficking when co‐expressed with WT‐hERG. The present study further investigates the biological and electrophysiological consequences of pharmacologic chaperones in HEK293 cells expressing G604S‐hERG or co‐expressing G604S‐hERG and WT‐hERG. It was found that a low temperature (27°C), thapsigargin, NS1643 and E‐4031 fail to rescue the G604S mutation. Interestingly, only E‐4031 treatment resulted in a significant increase in hERG currents in cells co‐expressing G604S‐hERG and WT‐hERG, correspondingly more mature protein band at 155 kDa by Western blotting and an increased membrane staining by confocal microscopy. In addition, E‐4031 treatment shifted the steady‐state half maximal activation voltage (V_1/2) of the inactivation curve by +8 mV in cells co‐expressing G604S‐hERG and WT‐hERG. The present experimental results suggest that a G604S mutation is resistant to pharmacological rescue. E‐4031 treatment resulted in a significant increase in hERG currents by promoting the hERG channel processing and trafficking in cells co‐expressing G604S‐hERG and WT‐hERG.     

Acute and subacute effects of the selective serotonin–noradrenaline reuptake inhibitor duloxetine on cardiac hERG channels

Duloxetine is a selective serotonin–noradrenaline reuptake inhibitor approved for treatment of major depressive disorder. So far, duloxetine has been found to be well tolerated and reported cardiac side effects were negligible. However, pharmacological effects on cardiac hERG channels have not been properly addressed yet. hERG channels were expressed in Xenopus oocytes and a human embryonic kidney (HEK) cell line. Currents were measured using voltage clamp and patch clamp techniques. Channel surface expression was quantified using Western blot analysis. We found that duloxetine inhibits heterologously expressed hERG channels in a concentration-dependent manner, yielding an IC₅₀ of 142.8 μM in Xenopus oocytes. Inhibitory effects were even more pronounced when using a mammalian cell line resulting in a 34 or 59 % current decrease by 10 or 30 μM duloxetine, respectively. Duloxetine did not affect channel activation or inactivation kinetics. However, channel deactivation was accelerated by duloxetine. We further showed that inhibition occurs in the open and inactivated, but not closed, states. There was no frequency dependence of block. However, effects of duloxetine were significantly attenuated when using the hERG pore mutants Y652A and F656A. Subacute effects of duloxetine on hERG channel expression were analyzed using the Western blot technique. We found that incubation with duloxetine results in a concentration-dependent decrease of channel surface expression. Whereas inhibitory effects of duloxetine seem negligible under therapeutically relevant concentrations, hERG block should be considered in cases of duloxetine overdose and when administering duloxetine to patients susceptible to drug-induced QT prolongation.     

Effects of Antipsychotic Drugs on I to, I Na, I sus, I K1, and hERG: QT Prolongation, Structure Activity Relationship, and Network Analysis

Purpose To evaluate in vitro and computationally model the effects of selected antipsychotic drugs on several ionic currents that contribute to changes in the action potential in cardiac tissue. Methods Fourteen antipsychotic drugs or metabolites were examined to determine whether QT interval prolongation could be accounted for by an effect on one or more myocardial ion channels [I_to, I_Na, I_sus, I_K1, and human ether-a-go-go related gene (hERG)]. Using the patch clamp technique, drug effects on these human cardiac currents were tested. Results All molecules had little inhibitory effect on ion channels (blocking at concentrations >5 μM) other than hERG. A significant correlation was observed between the estimated hERG blockade and the increase in corrected QT for five of the antipsychotics. Molecular modeling identified hydrophobic features related to the interaction with hERG and correctly rank-ordered the test set molecules olanzapine and its metabolites. A network analysis of ligand and protein interactions around hERG using MetaCore™ (GeneGo Inc., St. Joseph, MI, USA) was used to visualize antipsychotics with affinity for this channel and their interactions with other proteins in this database. Conclusion The antipsychotics do not inhibit the ion channels I_to, I_Na, I_sus, I_K1 to any appreciable extent; however, blockade of hERG is a likely mechanism for the prolongation of the QT interval.     

Identification of human Ether-à-go-go related gene modulators by three screening platforms in an academic drug-discovery setting

The human Ether-à-go-go related gene (hERG) potassium channel is responsible for the rapid delayed rectifier potassium current that plays a critical role in the repolarization of cardiomyocytes during the cardiac action potential. In humans, inhibition of hERG by drugs can prolong the electrocardiographic QT interval, which, in rare instance, leads to ventricular arrhythmia and sudden cardiac death. As such, several medications that block hERG channels in vitro have been withdrawn from the market due to QT prolongation and arrhythmias. The current FDA guidelines recommend that drug candidates destined for human use be evaluated for potential hERG activity ( www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/ucm074963.pdf ). Here, we employed automated planar patch clamp (APPC), high-throughput fluorescent Tl(+) flux, and moderate-throughput [3H]dofetilide competition binding assays to characterize a panel of 49 drugs for their activities at the hERG channel. Notably, we used the same HEK293-hERG cell line for all assays, facilitating comparisons of hERG potencies across screening platforms. In general, hERG inhibitors were most potent in APPC assays, intermediate potent in [3H]dofetilide binding assays, and least potent in Tl(+) flux assays. Binding affinity constants (pK(i) values) and Tl(+) flux potencies (pEC?? values) correlated well with APPC pEC?? values. Further, the inhibitory potencies of many known hERG inhibitors in APPC matched literature values from manual and/or automated patch clamp systems. We also developed a novel fluorescent Tl(+) flux assays to measure the effects of drugs that modulate hERG trafficking and surface expression.     

Automated electrophysiology in the preclinical evaluation of drugs for potential QT prolongation

INTRODUCTION: The human ether-a-go-go-related gene (hERG) potassium channel plays a major role in the electrical conductances involved in human heart repolarization. Drugs that decrease hERG K(+) currents are at risk to produce a prolongation of the cardiac action potential, resulting in an increase of the QT interval. Drug-induced QT prolongation or acquired long QT (aLQT) can lead to a fatal arrhythmia known as Torsade de Pointes (TdP). Electrophysiological methods are the best approach to evaluate potential drug candidates for hERG current inhibition. Here we identify limitations with the PatchXpress 7000A automated electrophysiology instrument and describe hERG protocol optimizations necessary for reliable preclinical assessment. METHODS: The PatchXpress 7000A automated electrophysiology system was used to evaluate a group of drugs with known hERG activity under voltage clamp conditions. We used a recombinant cell line expressing hERG, and assessed the inhibition of hERG K(+) currents at different drug concentrations. These data were used to determine hERG IC(50) values and compare assay parameters under different recording conditions. RESULTS: We found that due to limitations with the PatchXpress 7000A instrument, repeated compound additions were critical for achieving steady state drug concentrations that generated data comparable to standard patch clamp methods, particularly when similar voltage pulse protocols were implemented. Some discrepancies were observed between the PatchXpress 7000A and standard patch clamp techniques including shifts in IC(50) values for very hydrophobic compounds. Most hERG IC(50) values were within 3-fold of standard patch clamp IC(50) values. DISCUSSION: Automation of electrophysiology technologies has greatly improved the throughput of assessing lead drug candidates for hERG liability. To maintain hERG data quality comparable to standard patch clamp techniques, the PatchXpress 7000A instrument limitations should be recognized and protocols optimized accordingly to ensure accuracy.     

Mechanisms of zolpidem-induced long QT syndrome: acute inhibition of recombinant hERG K+ channels and action potential prolongation in human cardiomyocytes derived from induced pluripotent stem cells

Background and Purpose

Zolpidem, a short-acting hypnotic drug prescribed to treat insomnia, has been clinically associated with acquired long QT syndrome (LQTS) and torsade de pointes (TdP) tachyarrhythmia. LQTS is primarily attributed to reduction of cardiac human ether-a-go-go-related gene (hERG)/I_Kr currents. We hypothesized that zolpidem prolongs the cardiac action potential through inhibition of hERG K⁺ channels.

Experimental Approach

Two-electrode voltage clamp and whole-cell patch clamp electrophysiology was used to record hERG currents from Xenopus oocytes and from HEK 293 cells. In addition, hERG protein trafficking was evaluated in HEK 293 cells by Western blot analysis, and action potential duration (APD) was assessed in human-induced pluripotent stem cell (hiPSC)-derived cardiomyocytes.

Key Results

Zolpidem caused acute hERG channel blockade in oocytes (IC₅₀ = 61.5 μM) and in HEK 293 cells (IC₅₀ = 65.5 μM). Mutation of residues Y652 and F656 attenuated hERG inhibition, suggesting drug binding to a receptor site inside the channel pore. Channels were blocked in open and inactivated states in a voltage- and frequency-independent manner. Zolpidem accelerated hERG channel inactivation but did not affect I–V relationships of steady-state activation and inactivation. In contrast to the majority of hERG inhibitors, hERG cell surface trafficking was not impaired by zolpidem. Finally, acute zolpidem exposure resulted in APD prolongation in hiPSC-derived cardiomyocytes.

Conclusions and Implications

Zolpidem inhibits cardiac hERG K⁺ channels. Despite a relatively low affinity of zolpidem to hERG channels, APD prolongation may lead to acquired LQTS and TdP in cases of reduced repolarization reserve or zolpidem overdose.     

The 5-HT2 antagonist ketanserin is an open channel blocker of human cardiac ether-à-go-go-related gene (hERG) potassium channels

Background and purpose:

Ketanserin, a selective 5-HT receptor antagonist, prolongs the QT interval of ECG in patients. The purpose of the present study was to determine whether ketanserin would block human cardiac ether-à-go-go-related gene (hERG) potassium channels.

Experimental approach:

Whole-cell patch voltage-clamp technique was used to record membrane currents in HEK 293 cells expressing wild type or mutant hERG channel genes.

Key results:

Ketanserin blocked hERG current (I_hERG) in a concentration-dependent manner (IC₅₀=0.11 μM). The drug showed an open channel blocking property, the block increasing significantly at depolarizing voltages between +10 to +60 mV. Voltage-dependence for inactivation of hERG channels was negatively shifted by 0.3 μM ketanserin. A 2.8 fold attenuation of inhibition by elevation of external K⁺ concentration (from 5.0 to 20 mM) was observed, whereas the inactivation-deficient mutants S620T and S631A had the IC₅₀s of 0.84±0.2 and 1.7±0.4 μM (7.6 and 15.4 fold attenuation of block). In addition, the hERG mutants in pore helix and S6 also significantly reduced the channel block (2–59 fold) by ketanserin.

Conclusions and implications:

These results suggest that ketanserin binds to and blocks the open hERG channels in the pore helix and the S6 domain; channel inactivation is also involved in the blockade of hERG channels. Blockade of hERG channels most likely contributes to the prolongation of QT intervals in ECG observed clinically at therapeutic concentrations of ketanserin.     

Effects of an hERG Activator,ICA-105574, on Electrophysiological Properties of Canine Hearts

In short QT syndrome, inherited gain-of-function mutations in the human ether a-go-go-related gene (hERG) K⁺ channel have been associated with development of fatal arrhythmias. This implies that drugs that activate hERG as a side effect may likewise pose significant arrhythmia risk. hERG activators have been found to have diverse mechanisms of activation, which may reflect their distinct binding sites. Recently, the new hERG activator ICA-105574 was introduced, which disables inactivation of the hERG channel with very high potency. We explored characteristics of this new drug in several experimental models. Patch clamp experiments were used to verify activation of hERG channels by ICA-105574 in human embryonic kidney cells stably-expressing hERG channels. ICA-105574 significantly shortened QT and QTc intervals and monophasic action potential duration (MAP₉₀) in Langendorff-perfused guinea-pig hearts. We also administered ICA-105574 to anesthetized dogs while recording ECG and drug plasma concentrations. ICA-105574 (10 mg/kg) significantly shortened QT and QTc intervals, with a free plasma concentration of approximately 1.7 μM at the point of maximal effect. Our data showed that unbound ICA-105574 caused QT shortening in dogs at concentrations comparable to the half maximal effective concentration (EC₅₀, 0.42 μM) of hERG activation in the patch clamp studies.     

The Action of a Novel Fluoroquinolone Antibiotic Agent Antofloxacin Hydrochloride on Human‐Ether‐à‐go‐go‐Related Gene Potassium Channel

Abstract: The administration of certain fluoroquinolone antibacterials has recently been linked to QT interval prolongation, raising the clinical concerns over the cardiotoxicity of these agents. In this study, the effects of a novel fluoroquinolone, antofloxacin hydrochloride (AX) on human‐ether‐à‐go‐go‐related gene (HERG) encoding potassium channels and the biophysical mechanisms of drug action were performed with whole‐cell patch‐clamp technique in transiently transfected HEK293 cells. The administration of AX caused voltage‐ and time‐dependent inhibition of HERG K⁺ current (I_HERG/MiRP1) in a concentration‐dependent manner but did not markedly modify the properties of channel kinetics, including activation, inactivation, deactivation and recovery from inactivation as well. In comparison with sparfloxacin (SPX), levofloxacin lactate (LVFX), the potency of AX to inhibit HERG tail currents was the least one, with an IC₅₀ value of 460.37 μM. By contrast, SPX was the most potent compound, displaying an IC₅₀ value of 2.69 μM whereas LVFX showed modest potency, with an IC₅₀ value of 43.86 μM, respectively. Taken together, our data suggest that AX only causes a minor reduction of I_HERG/MiRP1 at the estimated free plasma level.     

Combined hERG channel inhibition and disruption of trafficking in drug-induced long QT syndrome by fluoxetine: a case-study in cardiac safety pharmacology

Drug-induced prolongation of the rate-corrected QT interval (QTCI) on the electrocardiogram occurs as an unwanted effect of diverse clinical and investigational drugs and carries a risk of potentially fatal cardiac arrhythmias. hERG (human ether-à-go-go-related gene) is the gene encoding the alpha-subunit of channels mediating the rapid delayed rectifier K+ current, which plays a vital role in repolarising the ventricles of the heart. Most QTCI prolonging drugs can inhibit the function of recombinant hERG K+ channels, consequently in vitro hERG assays are used widely as front-line screens in cardiac safety-testing of novel chemical entities. In this issue, Rajamani and colleagues report a case of QTCI prolongation with the antidepressant fluoxetine and correlate this with a dual effect of the drug and of its major metabolite norfluoxetine on hERG channels. Both compounds were found to produce an acute inhibition of the hERG channel by pharmacological blockade, but in addition they also were able to disrupt the normal trafficking of hERG protein to the cell membrane. Mutations to a key component of the drug binding site in the S6 region of the channel greatly attenuated channel block, but did not impair disruption of trafficking; this suggests that channel block and drug effects on trafficking were mediated by different mechanisms. These findings add to growing evidence for disruption of hERG channel trafficking as a mechanism for drug-induced long QT syndrome and raise questions as to possible limitations of acute screening methods in the assessment of QTcI prolonging liability of drugs in development.     

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

Chlorpheniramine is a potent first-generation histamine H₁ 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 IC₅₀ 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 H₁ antihistamine, chlorpheniramine is a blocker of the hERG channels, providing a molecular mechanism for the drug-induced arrhythmogenic side effects.     

The Effects of a Novel Anti‐arrhythmic Drug,Acehytisine Hydrochloride,on the Human Ether‐a‐go‐go Related Gene K+ Channel and Its Trafficking

Abstract: Many drugs cause severe side‐effects such as long QT syndromes by blocking the human ether‐a‐go‐go related gene (HERG) K⁺ channels and/or disrupting HERG protein trafficking. Acehytisine hydrochloride is an anti‐arrhythmic drug in phase IV clinical trial. To study whether acehytisine hydrochloride affects HERG channel activity and protein trafficking, we expressed HERG in human embryonic kidney 293 (HEK293) cells and recorded HERG channel currents with a whole‐cell patch clamp technique. We also measured the protein levels by Western blot analysis. We found that acehytisine hydrochloride inhibited HERG step current (I_HERG) in a concentration‐dependent manner. However, it had little effect on the tail current (I_tail). In addition, acehytisine hydrochloride accelerated channel inactivation and slowed recovery from inactivation. In contrast, it did not inhibit HERG protein (135 and 155 kD) trafficking, although it reduced the 155 kD band density at 2500 µM. Moreover, the F656C mutation in the S6 domain abolished acehytisine hydrochloride inhibition on the I_HERG and enhanced the inhibitive effects on the trafficking of the 155 kD band. Acehytisine hydrochloride interrupted HERG protein trafficking at 1000 and 2500 µM. Our data showed that acehytisine hydrochloride could inhibit I_HERG, but had no effects on I_tail until the concentration was above 1000 µM. Therefore, acehytisine hydrochloride may not induce QT interval prolongation and could be a promising anti‐arrhythmic drug without severe side‐effects.