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Background and Purpose: Drug-induced reduction of the rapid delayed rectifier potassium current carried by the human Ether-à-go-go-Related Gene (hERG) channel is associated with increased risk of arrhythmias. Recent updates to drug safety regulatory guidelines attempt to capture each drug's hERG binding mechanism by combining in vitro assays with in silico simulations. In this study, we investigate the impact on in silico proarrhythmic risk predictions due to uncertainty in the hERG binding mechanism and physiological hERG current model., Experimental Approach: Possible pharmacological binding models were designed for the hERG channel to account for known and postulated small molecule binding mechanisms. After selecting a subset of plausible binding models for each compound through calibration to available voltage-clamp electrophysiology data, we assessed their effects, and the effects of different physiological models, on proarrhythmic risk predictions., Key Results: For some compounds, multiple binding mechanisms can explain the same data produced under the safety testing guidelines, which results in different inferred binding rates. This can result in substantial uncertainty in the predicted torsade risk, which often spans more than one risk category. By comparison, we found that the effect of a different hERG physiological current model on risk classification was subtle., Conclusion and Implications: The approach developed in this study assesses the impact of uncertainty in hERG binding mechanisms on predictions of drug-induced proarrhythmic risk. For some compounds, these results imply the need for additional binding data to decrease uncertainty in safety-critical applications., (© 2023 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

Background and Purpose: Atrial fibrillation (AF) is the most common cardiac arrhythmia and is associated with an increased risk for stroke, heart failure and cardiovascular-related mortality. Candidate targets for anti-AF drugs include a potassium channel K(v)1.5, and the ionic currents I(KACh) and late I(Na), along with increased oxidative stress and activation of NFAT-mediated gene transcription. As pharmacological management of AF is currently suboptimal, we have designed and characterized a multifunctional small molecule, compound 1 (C1), to target these ion channels and pathways., Experimental Approach: We made whole-cell patch-clamp recordings of recombinant ion channels, human atrial I(Kur), rat atrial I(KACh), cellular recordings of contractility and calcium transient measurements in tsA201 cells, human atrial samples and rat myocytes. We also used a model of inducible AF in dogs., Key Results: C1 inhibited human peak and late K(v)1.5 currents, frequency-dependently, with IC₅₀ of 0.36 and 0.11 μmol·L(-1) respectively. C1 inhibited I(KACh)(IC₅₀ of 1.9 μmol·L(-1)) and the Na(v)1.5 sodium channel current (IC₅₀s of 3 and 1 μmol·L(-1) for peak and late components respectively). C1 (1 μmol·L(-1)) significantly delayed contractile and calcium dysfunction in rat ventricular myocytes treated with 3 nmol·L(-1) sea anemone toxin (ATX-II). C1 weakly inhibited the hERG channel and maintained antioxidant and NFAT-inhibitory properties comparable to the parent molecule, resveratrol. In a model of inducible AF in conscious dogs, C1 (1 mg·kg(-1)) reduced the average and total AF duration., Conclusion and Implications: C1 behaved as a promising multifunctional small molecule targeting a number of key pathways involved in AF., (© 2013 The British Pharmacological Society.)

Background and Purpose: Drug interference with normal hERG protein trafficking substantially reduces the channel density in the plasma membrane and thereby poses an arrhythmic threat. The chemical substructures important for hERG trafficking inhibition were investigated using pentamidine as a model drug. Furthermore, the relationship between acute ion channel block and correction of trafficking by dofetilide was studied., Experimental Approach: hERG and K(IR)2.1 trafficking in HEK293 cells was evaluated by Western blot and immunofluorescence microscopy after treatment with pentamidine and six pentamidine analogues, and correction with dofetilide and four dofetilide analogues that displayed different abilities to inhibit IKr . Molecular dynamics simulations were used to address mode, number and type of interactions between hERG and dofetilide analogues., Key Results: Structural modifications of pentamidine differentially affected plasma membrane levels of hERG and K(IR)2.1. Modification of the phenyl ring or substituents directly attached to it had the largest effect, affirming the importance of these chemical residues in ion channel binding. PA-4 had the mildest effects on both ion channels. Dofetilide corrected pentamidine-induced hERG, but not K(IR)2.1 trafficking defects. Dofetilide analogues that displayed high channel affinity, mediated by pi-pi stacks and hydrophobic interactions, also restored hERG protein levels, whereas analogues with low affinity were ineffective., Conclusions and Implications: Drug-induced trafficking defects can be minimized if certain chemical features are avoided or 'synthesized out'; this could influence the design and development of future drugs. Further analysis of such features in hERG trafficking correctors may facilitate the design of a non-blocking corrector for trafficking defective hERG proteins in both congenital and acquired LQTS., (© 2013 The British Pharmacological Society.)

Background and Purpose: Peripheral blockade of cannabinoid CB(1) receptors has been proposed as a safe and effective therapy against obesity, putatively devoid of the adverse psychiatric side effects of centrally acting CB(1) receptor antagonists. In this study we analysed the effects of LH-21, a peripherally acting neutral cannabinoid receptor antagonist with poor brain penetration, in an animal model of diet-induced obesity., Experimental Approach: To induce obesity, male Wistar rats were fed a high-fat diet (HFD; 60 kcal% fat) whereas controls received a standard diet (SD; 10 kcal% fat). Following 10 weeks of feeding, animals received a daily i.p. injection of vehicle or 3 mg·kg(-1) LH-21 for 10 days. Plasma and liver samples were used for biochemical analyses whereas visceral fat-pad samples were analysed for lipid metabolism gene expression using real-time RT-PCR. In addition, the potential of LH-21 to interact with hepatic cytochrome P450 isoforms and cardiac human Ether-à-go-go Related Gene (hERG) channels was evaluated., Key Results: LH-21 reduced feeding and body weight gain in HFD-fed animals compared with the control group fed SD. In adipose tissue, this effect was associated with decreased gene expression of: (i) leptin; (ii) lipogenic enzymes, including SCD-1; (iii) CB(1) receptors; and (iv) both PPARα and PPARγ. Although there were no significant differences in plasma parameters between HFD- and SD-fed rats, LH-21 did not seem to induce hepatic, cardiac or renal toxicity., Conclusions and Implications: These results support the hypothesis that treatment with the peripherally neutral acting CB(1) receptor antagonist, LH-21, may promote weight loss through modulation of visceral adipose tissue., (© 2011 The Authors. British Journal of Pharmacology © 2011 The British Pharmacological Society.)

Background and Purpose: KB-R7943 is an isothiourea derivative that is used widely as a pharmacological inhibitor of sodium-calcium exchange (NCX) in experiments on cardiac and other tissue types. This study investigated KB-R7943 inhibition of hERG (human ether-à-go-go-related gene) K(+) channels that underpin the cardiac rapid delayed rectifier potassium current, I(Kr) ., Experimental Approach: Whole-cell patch-clamp measurements were made of hERG current (I(hERG) ) carried by wild-type or mutant hERG channels and of native rabbit ventricular I(Kr) . Docking simulations utilized a hERG homology model built on a MthK-based template., Key Results: KB-R7943 inhibited both I(hERG) and native I(Kr) rapidly on membrane depolarization with IC(50) values of ∼89 and ∼120 nM, respectively, for current tails at -40 mV following depolarizing voltage commands to +20 mV. Marked I(hERG) inhibition also occurred under ventricular action potential voltage clamp. I(hERG) inhibition by KB-R7943 exhibited both time- and voltage-dependence but showed no preference for inactivated over activated channels. Results of alanine mutagenesis and docking simulations indicate that KB-R7943 can bind to a pocket formed of the side chains of aromatic residues Y652 and F656, with the compound's nitrobenzyl group orientated towards the cytoplasmic side of the channel pore. The structurally related NCX inhibitor SN-6 also inhibited I(hERG) , but with a markedly reduced potency., Conclusions and Implications: KB-R7943 inhibits I(hERG) /I(Kr) with a potency that exceeds that reported previously for acute cardiac NCX inhibition. Our results also support the feasibility of benzyloxyphenyl-containing NCX inhibitors with reduced potential, in comparison with KB-R7943, to inhibit hERG., (© 2011 The Authors. British Journal of Pharmacology © 2011 The British Pharmacological Society.)

Background and Purpose: Drug candidates must be thoroughly investigated for their potential cardiac side effects. During the course of routine toxicological assessment, the compound RO5657, a CCR5 antagonist, was discovered to have the rare liability of inducing torsades de pointes (polymorphic ventricular arrhythmia) in normal, healthy animals. Studies were conducted to determine the molecular mechanism of this arrhythmia., Experimental Approach: Toxicological effects of repeat dosing were assessed in naïve monkeys. Cardiovascular effects were determined in conscious telemetry-implanted monkeys (repeat dosing) and anaesthetized instrumented dogs (single doses). Mechanistic studies were performed in guinea-pig isolated hearts and in cells recombinantly expressing human cardiac channels., Key Results: In cynomolgus monkeys, RO5657 caused a low incidence of myocardial degeneration and a greater incidence of ECG abnormalities including prolonged QT/QTc intervals, QRS complex widening and supraventricular tachycardia. In telemetry-implanted monkeys, RO5657 induced arrhythmias, including torsades de pointes and in one instance, degeneration to fatal ventricular fibrillation. RO5657 also depressed both heart rate (HR) and blood pressure (BP), with no histological evidence of myocardial degeneration. In the anaesthetized dog and guinea-pig isolated heart studies, RO5657 induced similar cardiovascular effects. RO5657 also inhibited Kv11.1 and sodium channel currents., Conclusions and Implications: The molecular mechanism of RO5657 is hypothesized to be due to inhibition of cardiac sodium and Kv11.1 potassium channels. These results indicate that RO5657 is arrhythymogenic due to decreased haemodynamic function (HR/BP), decreased conduction and inhibition of multiple cardiac channels, which precede and are probably the causative factors in the observed myocardial degeneration., (© 2011 The Authors. British Journal of Pharmacology © 2011 The British Pharmacological Society.)

Background and Purpose: Cardiac toxicity is a major concern in drug development and it is imperative that clinical candidates are thoroughly tested for adverse effects earlier in the drug discovery process. In this report, we investigate the utility of an impedance-based microelectronic detection system in conjunction with mouse embryonic stem cell-derived cardiomyocytes for assessment of compound risk in the drug discovery process., Experimental Approach: Beating of cardiomyocytes was measured by a recently developed microelectronic-based system using impedance readouts. We used mouse stem cell-derived cardiomyocytes to obtain dose-response profiles for over 60 compounds, including ion channel modulators, chronotropic/ionotropic agents, hERG trafficking inhibitors and drugs known to induce Torsades de Pointes arrhythmias., Key Results: This system sensitively and quantitatively detected effects of modulators of cardiac function, including some compounds missed by electrophysiology. Pro-arrhythmic compounds produced characteristic profiles reflecting arrhythmia, which can be used for identification of other pro-arrhythmic compounds. The time series data can be used to identify compounds that induce arrhythmia by complex mechanisms such as inhibition of hERG channels trafficking. Furthermore, the time resolution allows for assessment of compounds that simultaneously affect both beating and viability of cardiomyocytes., Conclusions and Implications: Microelectronic monitoring of stem cell-derived cardiomyocyte beating provides a high throughput, quantitative and predictive assay system that can be used for assessment of cardiac liability earlier in the drug discovery process. The convergence of stem cell technology with microelectronic monitoring should facilitate cardiac safety assessment., (© 2011 ACEA Biosciences Inc. British Journal of Pharmacology © 2011 The British Pharmacological Society.)

Background and Purpose: Human ether-a-go-go related gene (HERG) channel inhibitors may be subdivided into compounds that are trapped in the closed channel conformation and others that dissociate at rest. The structural peculiarities promoting resting state dissociation from HERG channels are currently unknown. A small molecule-like propafenone is efficiently trapped in the closed HERG channel conformation. The aim of this study was to identify structural moieties that would promote dissociation of propafenone derivatives., Experimental Approach: Human ether-a-go-go related gene channels were heterologously expressed in Xenopus oocytes and potassium currents were recorded using the two-microelectrode voltage clamp technique. Recovery from block by 10 propafenone derivatives with variable side chains, but a conserved putative pharmacophore, was analysed., Key Results: We have identified structural determinants of propafenone derivatives that enable drug dissociation from the closed channel state. Propafenone and four derivatives with 'short' side chains were trapped in the closed channel. Five out of six bulky derivatives efficiently dissociated from the channel at rest. One propafenone derivative with a similar bulk but lacking an H-bond acceptor in this region was trapped. Correlations were observed between molecular weight and onset of channel block as well as between pK(a) and recovery at rest., Conclusion and Implications: The data show that extending the size of a trapped HERG blocker-like propafenone by adding a bulky side chain may impede channel closure and thereby facilitate drug dissociation at rest. The presence of an H-bond acceptor in the bulky side chain is, however, essential., (© 2011 The Authors. British Journal of Pharmacology © 2011 The British Pharmacological Society.)

Background and Purpose: K(v)11.1 channels are involved in regulating cellular excitability in various tissues including brain, heart and smooth muscle. In these tissues, at least two isoforms, K(v)11.1a and K(v)11.1b, with different kinetics, are expressed. K(v)11.1 activators are potential therapeutic agents, but their effects have only been tested on the K(v)11.1a isoform. In this study, the effects of two different K(v)11.1 activators, NS1643 and RPR260243, were characterized on K(v)11.1a and K(v)11.1b channels., Experimental Approach: K(v)11.1a and K(v)11.1b channels were expressed in Xenopus laevis oocytes, and currents were measured using two-electrode voltage clamp. I/V curves and channel kinetics were measured before and after application of 30 µM NS1643 or 10 µM RPR260243., Key Results: NS1643 increased steady-state currents through Kv11.1b several fold more than through K(v)11.1a channels, without affecting EC(50) values. NS1643 increased activation rates and decreased rates of inactivation, recovery from inactivation and deactivation for both channels. Except for activation, where effect of NS1643 was comparable, relative changes were greater for Kv11.1b than for K(v)11.1a. RPR260243 increased steady-state currents only through Kv11.1a channels, but slowed the process of deactivation for both channels primarily by decreasing time constant of slow deactivation. This effect was greater on K(v)11.1b than on K(v)11.1a. Effects of both compounds on heteromeric K(v)11.1a/K(v)11.1b channels were similar to those on K(v)11.1a., Conclusions and Implications: Both NS1643 and RPR260243 displayed differential effects on K(v)11.1a and K(v)11.1b channels, the effects being relatively more pronounced on K(v)11.1b channels. This affirms the importance of testing the effect of K(v)11.1 activators on different channel isoforms.

Background and Purpose: N-(6-aminohexyl)-5-chloro-1-naphthalene sulphonamide (W-7) is a well-known calmodulin inhibitor used to study calmodulin regulation of intracellular Ca(2+) signalling-related process. Here, we have determined whether W-7 would inhibit human ether gene (hERG or K(v) 11.1) potassium channels, hK(v) 1.5 channels or hK(IR) 2.1 channels expressed in human embryonic kidney (HEK) 293 cells., Experimental Approach: The hERG channel current, hK(v) 1.5 channel current or hK(IR) 2.1 channel current was recorded with a whole-cell patch clamp technique., Key Results: It was found that the calmodulin inhibitor W-7 blocked hERG, hK(v) 1.5 and hK(IR) 2.1 channels. W-7 decreased the hERG current (I(hERG) ) in a concentration-dependent manner (IC(50) : 3.5 µM), and the inhibition was more significant at depolarization potentials between +10 and +60 mV. The hERG mutations in the S6 region Y652A and F656V, and in the pore helix S631A, had the IC(50) s of 5.5, 9.8 and 25.4 µM respectively. In addition, the compound inhibited hK(v) 1.5 and hK(IR) 2.1 channels with IC(50) s of 6.5 and 13.4 µM respectively., Conclusion and Implications: These results indicate that the calmodulin inhibitor W-7 exerts a direct channel-blocking effect on hERG, hK(v) 1.5 and hK(IR) 2.1 channels stably expressed in HEK 293 cells. Caution should be taken in the interpretation of calmodulin regulation of ion channels with W-7., (© 2010 The Authors. British Journal of Pharmacology © 2010 The British Pharmacological Society.)

Background and Purpose: Potencies of compounds blocking K(V)11.1 [human ether-ago-go-related gene (hERG)] are commonly assessed using cell lines expressing the Caucasian wild-type (WT) variant. Here we tested whether such potencies would be different for hERG single nucleotide polymorphisms (SNPs)., Experimental Approach: SNPs (R176W, R181Q, Del187-189, P347S, K897T, A915V, P917L, R1047L, A1116V) and a binding-site mutant (Y652A) were expressed in Tet-On CHO-K1 cells. Potencies [mean IC(50); lower/upper 95% confidence limit (CL)] of 48 hERG blockers was estimated by automated electrophysiology [IonWorks HT (IW)]. In phase one, rapid potency comparison of each WT-SNP combination was made for each compound. In phase two, any compound-SNP combinations from phase one where the WT upper/lower CL did not overlap with those of the SNPs were re-examined. Electrophysiological WT and SNP parameters were determined using conventional electrophysiology., Key Results: IW detected the expected sixfold potency decrease for propafenone in Y652A. In phase one, the WT lower/upper CL did not overlap with those of the SNPs for 77 compound-SNP combinations. In phase two, 62/77 cases no longer yielded IC(50) values with non-overlapping CLs. For seven of the remaining 15 cases, there were non-overlapping CLs but in the opposite direction. For the eight compound-SNP combinations with non-overlapping CLs in the same direction as for phase 1, potencies were never more than twofold apart. The only statistically significant electrophysiological difference was the voltage dependence of activation of R1047L., Conclusion and Implications: Potencies of hERG channel blockers defined using the Caucasian WT sequence, in this in vitro assay, were representative of potencies for common SNPs.

Background and Purpose: Piceatannol is more potent than resveratrol in free radical scavenging in association with antiarrhythmic and cardioprotective activities in ischaemic-reperfused rat hearts. The present study aimed to investigate the antiarrhythmic efficacy and the underlying ionic mechanisms of piceatannol in rat hearts., Experimental Approach: Action potentials and membrane currents were recorded by the whole-cell patch clamp techniques. Fluo-3 fluorimetry was used to measure cellular Ca2+ transients. Antiarrhythmic activity was examined from isolated Langendorff-perfused rat hearts., Key Results: In rat ventricular cells, piceatannol (3-30 micromol.L(-1)) prolonged the action potential durations (APDs) and decreased the maximal rate of upstroke (V(max)) without altering Ca2+ transients. Piceatannol decreased peak I(Na) and slowed I(Na) inactivation, rather than induced a persistent non-inactivating current, which could be reverted by lidocaine. Resveratrol (100 micromol.L(-1)) decreased peak I(Na) without slowing I(Na) inactivation. The inhibition of peak I(Na) or V(max) was associated with a negative shift of the voltage-dependent steady-state I(Na) inactivation curve without altering the activation threshold. At the concentrations more than 30 micromol.L(-1), piceatannol could inhibit I(Ca,L), I(to), I(Kr), Ca2+ transients and Na+-Ca2+ exchange except I(K1). Piceatannol (1-10 micromol.L(-1)) exerted antiarrhythmic activity in isolated rat hearts subjected to ischaemia-reperfusion injury., Conclusions and Implications: The additional hydroxyl group on resveratrol makes piceatannol possessing more potent in I(Na) inhibition and uniquely slowing I(Na) inactivation, which may contribute to its antiarrhythmic actions at low concentrations less than 10 micromol.L(-1).

Background and Purpose: There is increasing evidence that angiotensin II (Ang II) is associated with the occurrence of ventricular arrhythmias. However, little is known about the electrophysiological effects of Ang II on ventricular repolarization. The rapid component of the delayed rectifier K(+) current (I(Kr)) plays a critical role in cardiac repolarization. Hence, the aim of this study was to assess the effect of Ang II on I(Kr) in guinea-pig ventricular myocytes., Experimental Approach: The whole-cell patch-clamp technique was used to record I(Kr) in native cardiocytes and in human embryonic kidney (HEK) 293 cells, co-transfected with human ether-a-go-go-related gene (hERG) encoding the alpha-subunit of I(Kr) and the human Ang II type 1 (AT(1)) receptor gene., Key Results: Ang II decreased the amplitude of I(Kr) in a concentration-dependent manner with an IC(50) of 8.9 nM. Action potential durations at 50% (APD(50)) and 90% (APD(90)) repolarization were prolonged 20% and 16%, respectively by Ang II (100 nM). Ang II-induced inhibition of the I(Kr) was abolished by the AT(1) receptor blocker, losartan (1 muM). Ang II decreased hERG current in HEK293 cells and significantly delayed channel activation, deactivation and recovery from inactivation. Moreover, PKC inhibitors, stausporine and Bis-1, significantly attenuated Ang II-induced inhibition of I(Kr)., Conclusions and Implications: Ang II produces an inhibitory effect on I(Kr)/hERG currents via AT(1) receptors linked to the PKC pathway in ventricular myocytes. This is a potential mechanism by which elevated levels of Ang II are involved in the occurrence of arrhythmias in cardiac hypertrophy and failure.

Background and Purpose: Chlorthalidone is used for the treatment of hypertension as it produces a lengthening of the cardiac action potential. However, there is no experimental evidence that chlorthalidone has electrophysiological effects on the potassium currents involved in cardiac repolarization., Experimental Approach: Ventricular myocytes and oocytes, transfected with human ionic channels that produce IK current, were exposed to different concentrations of chlorthalidone. Action potentials and potassium currents were recorded using a patch clamp technique. To determine which component of the current was affected by chlorthalidone, human channel proteins (hERG, minK and KvLQT1) were used., Key Results: Chlorthalidone prolonged the ventricular action potential at 50 and 90% by 13 and 14%, respectively. The cardiac potassium currents I(to) and IK(1) were not affected by chlorthalidone at any concentration, whereas the delayed rectifier potassium current, IK, was blocked in a dose-response, voltage-independent fashion. In our preparation, 100 microM chlorthalidone blocked the two components of the delayed rectifier potassium current with the same potency (50.1+/-5% for IK(r) and 54.6+/-6% for IK(s)) (n=7, P<0.05). The chlorthalidone-sensitive current was slow and saturated at potentials greater than +30 mV. In our conditions only the KvLQT1 potassium current was affected by the drug, by 14%., Conclusions and Implications: Chlorthalidone was demonstrated to have a direct effect on cardiac ventricular myocytes; it blocked the delayed rectifier potassium current (IK), specifically the KvLQT1 component of the potassium current. These results indicate that it has potential for use as an antiarrhythmic but further studies are needed.

Background and Purpose: The rabbit isolated Langendorff heart model (SCREENIT) was used to investigate the proarrhythmic potential of a range of marketed drugs or drugs intended for market. These data were used to validate the SCREENIT model against clinical outcomes., Experimental Approach: Fifty-five drugs, 3 replicates and 2 controls were tested in a blinded manner. Proarrhythmia variables included a 10% change in MAPD(60), triangulation, instability and reverse frequency-dependence of the MAP. Early after-depolarisations, ventricular tachycardia, TdP and ventricular fibrillation were noted. Data are reported at nominal concentrations relative to EFTPC(max). Proarrhythmic scores were assigned to each drug and each drug category., Key Results: Category 1 and 2 drugs have the highest number of proarrhythmia variables and overt proarrhythmia while Category 5 drugs have the lowest, at every margin. At 30-fold the EFTPC(max), the mean proarrhythmic scores are: Category 1, 101+/-24; Category 2, 101+/-14; Category 3, 72+/-20; Category 4, 59+/-16 and Category 5, 22+/-9 points. Only drugs in Category 5 have mean proarrhythmic scores, below 30-fold, that remain within the Safety Zone., Conclusions and Implications: A 30-fold margin between effects and EFTPC(max) is sufficiently stringent to provide confidence to proceed with a new chemical entity, without incurring the risk of eliminating potentially beneficial drugs. The model is particularly useful where compounds have small margins between the hERG IC(50) and predicted EFTPC(max). These data suggest this is a robust and reliable assay that can add value to an integrated QT/TdP risk assessment.

Clozapine, a commonly used antipsychotic drug, can induce QT prolongation, which may lead to torsades de pointes and sudden death. To investigate the arrhythmogenic side effects of clozapine, we studied the impact of clozapine on human ether-a-go-go-related gene (HERG) channels expressed in Xenopus oocytes and HEK293 cells, and on the delayed rectifier K(+) currents of guinea-pig cardiomyocytes. Clozapine dose-dependently decreased the amplitudes of the currents at the end of voltage steps, and the tail currents of HERG. The IC(50) for the clozapine blockade of HERG currents in Xenopus oocytes progressively decreased relative to depolarization (39.9 microM at -40 mV, 28.3 microM at 0 mV and 22.9 microM at +40 mV), whereas the IC(50) for the clozapine-induced blockade of HERG currents in HEK293 cells at 36 degrees C was 2.5 microM at +20 mV. The clozapine-induced blockade of HERG currents was time dependent: the fractional current was 0.903 of the control at the beginning of the pulse, but declined to 0.412 after 4 s at a test potential of 0 mV. The clozapine-induced blockade of HERG currents was use-dependent, exhibiting more rapid onset and greater steady state blockade at higher frequencies of activation, with a partial relief of blockade observed when the frequency of activation was decreased. In guinea-pig ventricular myocytes held at 36 degrees C, treatment with 1 and 5 microM clozapine blocked the rapidly activating delayed rectifier K(+) current (I(Kr)) by 24.7 and 79.6%, respectively, but did not significantly block the slowly activating delayed rectifier K(+) current (I(Ks)). Our findings collectively suggest that blockade of HERG currents and I(Kr), but not I(Ks), may contribute to the arrhythmogenic side effects of clozapine.

The fluoroquinolone antibiotic moxifloxacin has been associated with the acquired long QT syndrome and is used as a positive control in the evaluation of the QT-interval prolonging potential of new drugs. In common with other QT-prolonging agents, moxifloxacin is known to inhibit the hERG potassium K+ channel, but at present there is little mechanistic information available on this action. This study was conducted in order to characterise the inhibition of hERG current (I(hERG)) by moxifloxacin, and to determine the role in drug binding of the S6 aromatic amino-acid residues Tyr652 and Phe656. hERG currents were studied using whole-cell patch clamp (at room temperature and at 35-37 degrees C) in an HEK293 cell line stably expressing hERG channels. Moxifloxacin reversibly inhibited currents in a dose-dependent manner. We investigated the effects of different voltage commands to elicit hERG currents on moxifloxacin potency. Using a 'step-ramp' protocol, the IC50 was 65 microM at room temperature and 29 microM at 35 degrees C. When a ventricular action potential waveform was used to elicit currents, the IC50 was 114 microM. Block of hERG by moxifloxacin was found to be voltage-dependent, occurred rapidly and was independent of stimulation frequency. Mutagenesis of the S6 helix residue Phe656 to Ala failed to eliminate or reduce the moxifloxacin-mediated block whereas mutation of Tyr652 to Ala reduced moxifloxacin block by approximately 66%. Our data demonstrate that moxifloxacin blocks the hERG channel with a preference for the activated channel state. The Tyr652 but not Phe656 S6 residue is involved in moxifloxacin block of hERG, concordant with an interaction in the channel inner cavity.

The antifungal antibiotic clotrimazole (CLT) shows therapeutic effects on cancer, sickle cell disease, malaria, etc. by inhibiting membrane intermediate-conductance Ca2+ -activated K+ channels (IKCa). However, it is unclear whether this drug would affect human cardiac K+ currents. The present study was therefore designed to investigate the effects of CLT on transient outward K+ current (Ito1), and ultra-rapid delayed rectifier K+ current (IKur) in isolated human atrial myocytes, and cloned hERG channel current (IhERG) and recombinant human cardiac KCNQ1/KCNE1 channel current (IKs) expressed in HEK 293 cells. It was found that CLT inhibited Ito1 with an IC50 of 29.5 microM, accelerated Ito1 inactivation, and decreased recovery of Ito1 from inactivation. In addition, CLT inhibited human atrial I(Kur) in a concentration-dependent manner (IC50 = 7.6 microM). CLT substantially suppressed IhERG (IC50 = 3.6 microM), and negatively shifted the activation conductance of IhERG. Moreover, CLT inhibited IKs (IC50 = 15.1 microM), and positively shifted the activation conductance of the current. These results indicate that the antifungal antibiotic CLT substantially inhibits human cardiac repolarization K+ currents including Ito1, IKur, IhERG, and IKs. However, caution is recommended when correlating the observed in vitro effects on cardiac ion currents to the clinical relevance.

Pentamidine, an antiprotozoal agent, has been traditionally known to cause QT prolongation and arrhythmias; however, its ionic mechanism has not been illustrated. In a stable HEK-293 cell line, we observed a concentration-dependent inhibition of the hERG current with an IC50 of 252 microM. In freshly isolated guinea-pig ventricular myocytes, pentamidine showed no effect on the L-type calcium current at concentrations up to 300 microM, with a slight prolongation of the action potential duration at this concentration. Since the effective concentrations of pentamidine on the hERG channel and APD were much higher than clinically relevant exposures (approximately 1 microM free or lower), we speculated that this drug might not prolong the QT interval through direct inhibition of I(Kr) channel. We therefore incubated hERG-HEK cells in 1 and 10 microM pentamidine-containing media (supplemented with 10% serum) for 48 h, and examined the hERG current densities in the vehicle control and pentamidine-treated cells. In all, 36 and 85% reductions of the current densities were caused by 1- and 10-microM pentamidine treatment (P<0.001 vs control), respectively. A similar level of reduction of the hERG polypeptides and a reduced intensity of the hERG protein on the surface membrane in treated cells were observed by Western blot analysis and laser-scanning confocal microscopy, respectively. Taken together, our data imply that chronic administration of pentamidine at clinically relevant exposure reduces the membrane expression of the hERG channel, which may most likely be the major mechanism of QT prolongation and torsade de pointes reported in man.

1. Miconazole, an imidazole antifungal agent, is associated with acquired long QT syndrome and ventricular arrhythmias. Miconazole increases the plasma concentration of QT-prolonging drugs by inhibiting the hepatic cytochrome P450 metabolic pathway, but whether it has direct effects on cardiac ion channels has not been elucidated. 2. To determine the mechanism underlying these clinical findings, we investigated the effect of miconazole on human ether-a-go-go-related gene (HERG) K+ channels. 3. HERG channels were heterologously expressed in human embryonic kidney 293 (HEK293) cells and whole-cell currents were recorded using a patch-clamp technique (23 degrees C). 4. Miconazole inhibited HERG peak tail current in a concentration-dependent manner (0.4-40 microM) with an IC50 of 2.1 microM (n=3-5 cells at each concentration, Hill coefficient 1.2). HERG block was not frequency-dependent. It required channel activation, occurred rapidly, and had very slow dissociation properties. 5. The activation curve was shifted in a negative direction (V(1/2): -9.5+/-2.3 mV in controls and -15.3+/-2.4 mV after 4 microM miconazole, P<0.05, n=6). Miconazole did not change other channel kinetics (activation, deactivation, onset of inactivation, recovery from inactivation, steady-state inactivation). 6. The S6 domain mutation, F656C, abolished the inhibitory action of miconazole on HERG current indicating that miconazole preferentially binds to an aromatic amino-acid residue within the pore-S6 region. 7. Our findings indicate that miconazole causes HERG channel block by binding to a common drug receptor, and this involves preferential binding to activated channels. Thus, miconazole prolongs the QT interval by direct inhibition of HERG channels.

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.

1. Phospholipid metabolites lysophospholipids cause extracellular K(+) accumulation and action potential shortening with increased risk of arrhythmias during myocardial ischemia. Here we studied effects of several lysophospholipids with different lengths of hydrocarbon chains and charged headgroups on HERG K(+) currents (I(HERG)) expressed in HEK293 cells and the potential mechanisms using whole-cell patch-clamp techniques. 2. Only the lipids with 16 hydrocarbons such as 1-palmitoyl-lysophosphatidylcholine (LPC-16) and 1-palmitoyl-lysophosphatidylglycerol (LPG-16) were found to produce significant enhancement of I(HERG) and negative shifts of HERG activation, although the voltage dependence of the effects was different between LPC-16 and LPG-16 which have differently charged headgroups. The lipid with 18 hydrocarbons modestly increased I(HERG). The lipids with 6 or 24 hydrocarbons had no effect or slightly decreased I(HERG). 3. Inhibition or activation of protein kinase C did not alter the effects of LPC-16 and LPG-16. Participation of phosphatidylinositol-4,5-bisphosphate in I(HERG) enhancement by LPC-16/LPG-16 was also excluded. 4. Vitamin E augmented the effects of LPC-16/LPG-16 whereas xanthine/xanthine oxidase reduced I(HERG): indicating that LPC-16/LPG-16 produced dual effects on I(HERG): direct enhancement of I(HERG) and indirect suppression via production of superoxide anion. 5. We conclude that enhancement of HERG function by lysophospholipids is specific to the lipids with 16-hydrocarbon chain structure and the pattern of voltage dependence is determined by the polar headgroups. The increase in I(HERG) is best described by direct interactions between lipid molecules and HERG proteins, which is consistent with lack of effects via membrane destabilization or modulation by intracellular signaling pathways.

1. Pharmacological blockade of the Human ether-a-go-go related gene (HERG) potassium channel is commonly linked with acquired long QT syndrome and associated proarrhythmia. The objectives of this study were (i) to identify and characterise any inhibitory action on HERG of the selective-serotonin re-uptake inhibitor fluvoxamine, (ii) to then determine whether fluvoxamine shared the consensus molecular determinants of HERG blockade of those drugs so far tested. 2. Heterologous HERG potassium current (I(HERG)) was measured at 37 degrees C, using the whole-cell patch-clamp technique, from a mammalian cell line (Human embryonic kidney 293) expressing HERG channels. I(HERG) tails, following repolarisation from +20 to -40 mV, were blocked by fluvoxamine with an IC(50) of 3.8 micro M. 3. Blockade of wild-type HERG was of extremely rapid onset (within 10 ms) and showed voltage dependence, with fluvoxamine also inducing a leftward shift in voltage-dependent activation of I(HERG). Characteristics of block were consistent with a component of closed channel (or extremely rapidly developing open channel) blockade and dependence on open and inactivated channel states. The attenuated-inactivation mutation S631A partially reduced the blocking effect of fluvoxamine. 4. The S6 mutations, Y652A and F656A, and the pore helix mutant S631A only partially attenuated blockade by fluvoxamine at concentrations causing profound blockade of wild-type HERG. 5. All HERG-blocking pharmaceuticals studied to date have been shown to block F656 mutant channels with over 100-fold reduced potency compared to their blockade of the wild-type channel. Fluvoxamine is therefore quite distinct in this regard from previously studied agents.

1 We investigated the inhibition of hEAG1 potassium channels, expressed in mammalian cells and Xenopus oocytes, by several blockers that have previously been reported to be blockers of hERG1 channels. 2 In the whole-cell mode of mammalian cells, LY97241 was shown to be a potent inhibitor of both hEAG1 and hERG1 channels (IC(50) of 4.9 and 2.2 nM, respectively). Clofilium, E4031, and haloperidol apparently inhibited hEAG1 channels with lower potency than hERG1 channels, but they cannot be considered hERG1-specific. 3 The block of hEAG1 channels by LY97241 and clofilium was time-, use-, and voltage-dependent, best explained by an open-channel block mechanism. 4 Both drugs apparently bind from the intracellular side of the membrane at (a) specific site(s) within the central cavity of the channel pore. They can be trapped by closure of the activation gate. 5 In inside-out patches from Xenopus oocytes, hEAG1 block by clofilium was stronger than by LY97241 (IC(50) of 0.8 and 1.9 nM, respectively). In addition, hEAG1 block by clofilium was much faster than by LY97241 although there was no difference in the voltage dependence of the on-rate of block. 6 Physico-chemical differences of clofilium and the weak base LY97241 determine the access of the drugs to the binding site and thereby the influence of the recording mode on the apparent block potencies. This phenomenon must be considered when assessing the inhibitory action of drugs on ion channels.

1 Levobupivacaine and ropivacaine are the pure S(-) enantiomers of N-butyl- and N-propyl-2',6'-pipecoloxylidide, developed as less cardiotoxic alternatives to bupivacaine. In the present study, we have analysed the effects of levobupivacaine, ropivacaine and bupivacaine on HERG channels stably expressed in CHO cells. 2 The three drugs blocked HERG channels in a concentration-, time- and state-dependent manner. Block measured at the end of 5 s pulses to -10 mV induced by 20 microM bupivacaine (52.7+/-2.0%, n=15) and ropivacaine (55.5+/-2.7%, n=13) was similar (P>0.05) and both lower than that induced by levobupivacaine (67.5+/-4.2%, n=11) (P<0.05). 3 Dextrobupivacaine (20 microM) was less potent (47.2+/-5.2%, n=10) than levobupivacaine (P<0.05), indicating stereoselective HERG channel block. 4. Block induced by the three local anaesthetics exhibited a steep voltage dependence in the range of channel activation. In all cases, block measured at the maximum peak current at a test potential of 0 mV after promoting recovery from inactivation (I-->O) was lower than that observed at the end of 5-s pulses (I+O). 5. Levobupivacaine, ropivacaine and bupivacaine accelerated HERG inactivation kinetics, slowed the recovery from inactivation and shifted the inactivation curve towards more negative membrane potentials. The three local anaesthetics induced a rapid time-dependent decline after using a protocol that quickly activates HERG channels. 6. All these results suggest that: (1) these drugs bind to the open and the inactivated states of HERG channels, (2) they stabilize HERG channels in the inactivated state, and (3) block induced by bupivacaine enantiomers is stereoselective.