Increased vulnerability of human ventricle to re-entrant excitation in hERG-linked variant 1 short QT syndrome.

The short QT syndrome (SQTS) is a genetically heterogeneous condition characterized by abbreviated QT intervals and an increased susceptibility to arrhythmia and sudden death. This simulation study identifies arrhythmogenic mechanisms in the rapid-delayed rectifier K(+) current (I(Kr))-linked SQT1 variant of the SQTS. Markov chain (MC) models were found to be superior to Hodgkin-Huxley (HH) models in reproducing experimental data regarding effects of the N588K mutation on KCNH2-encoded hERG. These ionic channel models were then incorporated into human ventricular action potential (AP) models and into 1D and 2D idealised and realistic transmural ventricular tissue simulations and into a 3D anatomical model. In single cell models, the N588K mutation abbreviated ventricular cell AP duration at 90% repolarization (APD(90)) and decreased the maximal transmural voltage heterogeneity (δV) during APs. This resulted in decreased transmural heterogeneity of APD(90) and of the effective refractory period (ERP): effects that are anticipated to be anti-arrhythmic rather than pro-arrhythmic. However, with consideration of transmural heterogeneity of I(Kr) density in the intact tissue model based on the ten Tusscher-Noble-Noble-Panfilov ventricular model, not only did the N588K mutation lead to QT-shortening and increases in T-wave amplitude, but δV was found to be augmented in some local regions of ventricle tissue, resulting in increased tissue vulnerability for uni-directional conduction block and predisposing to formation of re-entrant excitation waves. In 2D and 3D tissue models, the N588K mutation facilitated and maintained re-entrant excitation waves due to the reduced substrate size necessary for sustaining re-entry. Thus, in SQT1 the N588K-hERG mutation facilitates initiation and maintenance of ventricular re-entry, increasing the lifespan of re-entrant spiral waves and the stability of scroll waves in 3D tissue.

Drug-induced hERG block and long QT syndrome.

Drug-induced long QT syndrome is a cardiac safety issue that all drugs seeking approval must currently address, in part via in vitro electrophysiological testing of the drug's effects on the function of the human Ether-à-go-go Related Gene (hERG) potassium channel. This regulatory strategy has also been scientifically successful, in that these in vitro assays are cheaper and faster than are many other surrogates for arrhythmogenic risk, including QT prolongation in humans and action potential prolongation in cardiomyocytes. In some ways hERG assays are also more sensitive to the underlying repolarization anomalies that lead to the risk of the Torsades de pointes arrhythmia. In addition, the higher throughput of hERG assays combined with advances in our understanding of the molecular structures underlying this pathophysiology have led to new approaches in the medicinal chemistry of "designing out" hERG liability from lead compounds. While generally effectual, hERG screening produces some false positives: drugs with an apparent liability that are known not to be clinically arrhythmogenic. New technologies continue to be developed to improve hERG screening, while further insights into the molecular pharmacology of hERG and cardiac repolarization are providing avenues to mitigate and make sense of the lack of torsadogenic specificity in extant hERG assays.

Emerging trends in ion channel-based assays for predicting the cardiac safety of drugs.

Regulators require prospective new drugs to be tested for potential cardiac effects that are relevant to drug safety. As a result, numerous technologies have been developed to screen drugs for off-target effects that compromise the function of hERG potassium channels, which are associated with acquired long QT syndrome - a disorder with a risk of sudden death as a result of arrhythmia. Automated patch clamp, which is the most important and commonly used technology for the preclinical assessment of the risk of arrhythmia, continues to evolve toward greater sophistication, higher success rates and increased ease of use. Patch clamp advances include the development of population patch clamp and the ability to make more consistent measurements from freshly thawed prevalidated cells. Nevertheless, several discrepancies between hERG testing, QT prolongation and the risk of arrhythmia have been well-publicized. As a result, ion channel screening has been further developed to enable the assaying of currents from a wider variety of cardiac ion channels and the measurement of action potentials from stem cell-derived cardiomyocyte-like cells. Emerging measurement technologies beyond patch clamp include the measurement of field potentials with multi-electrode arrays.

Investigating dynamic protocol-dependence of hERG potassium channel inhibition at 37 degrees C: Cisapride versus dofetilide.

INTRODUCTION: Pharmacological inhibition of cardiac potassium channels encoded by hERG (human ether-à-go-go-related gene) is associated with QT interval prolongation and torsades de pointes arrhythmia. Electrophysiological assays of hERG channel inhibition are integral to the safety testing of novel drug candidates. This study was conducted to compare, for the high affinity hERG inhibitors dofetilide and cisapride, hERG blockade between action potential (AP) and conventional (step and step-ramp) screening waveforms. Furthermore, it evaluated dynamic (pulse-by-pulse) protocol-dependence of hERG channel inhibition by these drugs. METHODS: Whole-cell patch-clamp recordings were made at 37 degrees C from hERG-expressing HEK 293 cells. Half-maximal inhibitory concentrations (IC(50) values) for I(hERG) blockade were obtained using conventional voltage clamp and action potential clamp, using previously digitised ventricular and Purkinje fibre (PF) AP waveforms. RESULTS: A more marked variation in IC(50) values with different command waveforms was observed for cisapride (ranging from 7 to 72 nM) than for dofetilide (ranging from 4 to 15 nM), with higher IC(50)s obtained with AP than step or step-ramp commands. The two drugs differed little from one another in effects on voltage-dependent activation; however, I(hERG) blockade by each drug was initially voltage-dependent, but at steady-state was only voltage-dependent for cisapride. There was comparatively little difference between the two drugs in effects on I(hERG) availability or time constants of development of inactivation. Features of time-dependence of blockade and the use of protocols employing varying rest periods in drug or commands of alternating duration highlighted a pronounced ability of cisapride, but not dofetilide, to dissociate and reassociate from hERG on a pulse-by-pulse basis. DISCUSSION: Protocols described here that demonstrated dynamic variation (drug dissociation/reassociation) in hERG channel current blockade at 37 degrees C for cisapride may have future value for investigating drug interactions with the hERG channel. Downloadable digitised ventricular and PF AP waveforms that can be used in AP clamp experiments also accompany this article.

Inhibition of ERK and JNK decreases both osmosensitive taurine release and cell proliferation in glioma cells.

Cell swelling is associated with the activation of an increase in the osmosensitive taurine release (OTR) rate, which serves to decrease cell volume as part of a process known as regulatory volume decrease. OTR, which is sensitive to many pharmacological agents including anion channel blockers and signalling pathway modulators, has also been suggested to play a role in cell cycle progression. At non-cytotoxic concentrations, the anion channel blocker NPPB (25 microM), the extra-cellular signal-regulated kinase inhibitor PD98059 (50 microM), and the c-Jun NH2-terminal kinase inhibitor SP 600125 (5 microM) each decreased the OTR rate by > or =50%, decreased cell proliferation, and increased G0/G1 cell cycle arrest.

The hERG potassium channel as a therapeutic target.

The hERG (human ether-à-go-go-related gene) potassium channel has elicited intense scientific interest due to its counter-intuitive kinetics and its association with arrhythmia and sudden death. hERG blockade is involved in both antiarrhythmic pharmacotherapy and the pathogenesis of familial and acquired long QT syndrome (LQTS). Short QT syndrome (SQTS), muscular atrophy and many forms of cancer have also been associated with hERG as a target. Molecular models of both the channel and its blocker pharmacophores exist, revealing methods to design hERG liability out of potential drug molecules. Future developments will synthesise preclinical data on hERG with other criteria to determine net arrhythmogenic risk. Also, the molecular actions of hERG and its genetics will be elucidated in detail to allow clinical risk reduction.

hERG K+ channel blockade by the antipsychotic drug thioridazine: An obligatory role for the S6 helix residue F656.

The phenothiazine antipsychotic agent thioridazine has been linked with prolongation of the QT interval on the electrocardiogram, ventricular arrhythmias, and sudden death. Although thioridazine is known to inhibit cardiac hERG K(+) channels there is little mechanistic information on this action. We have investigated in detail hERG K(+) channel current (I(hERG)) blockade by thioridazine and identified a key molecular determinant of blockade. Whole-cell I(hERG) measurements were made at 37 degrees C from human embryonic kidney (HEK-293) cells expressing wild-type and mutant hERG channels. Thioridazine inhibited I(hERG) tails at -40mV following a 2s depolarization to +20mV with an IC(50) value of 80nM. Comparable levels of I(hERG) inhibition were seen with physiological command waveforms (ventricular and Purkinje fibre action potentials). Thioridazine block of I(hERG) was only weakly voltage-dependent, though the time dependence of I(hERG) inhibition indicated contingency of blockade upon channel gating. The S6 helix point mutation F656A almost completely abolished, and the Y652A mutation partially attenuated, I(hERG) inhibition by thioridazine. In summary, thioridazine is one of the most potent hERG K(+) channel blockers amongst antipsychotics, exhibiting characteristics of a preferential open/activated channel blocker and binding at a high affinity site in the hERG channel pore.

Disopyramide is an effective inhibitor of mutant HERG K+ channels involved in variant 1 short QT syndrome.

The recently identified idiopathic short QT syndrome (SQTS) is associated with an increased risk of arrhythmia and sudden death. The use of implantable cardioverter defibrillators helps to protect SQTS patients from ventricular fibrillation; however, pharmacological treatments to normalise the QT interval are limited: thus far only quinidine has been found to be effective in a subset of patients, with the SQT1 variant. SQT1 is associated with an amino acid substitution (N588K) in the KCNH2-encoded HERG K(+) channel that reduces HERG current (I(HERG)) inactivation and sensitivity to drug block. We demonstrate here that the N588K-HERG mutation only slightly attenuates I(HERG) blockade by the Class Ia antiarrhythmic drug disopyramide (1.5-fold elevation of IC(50)), compared to quinidine (3.5-fold elevation of IC(50)) and the Class III antiarrhythmic drug E-4031 (11.5-fold elevation of IC(50)). Thus, of the drugs studied to date, disopyramide is the one least affected by the SQT1 HERG mutation. Disopyramide is associated with QT prolongation in normal use and our findings provide a rational basis for its evaluation as a treatment for SQT1.

Inhibition of the HERG K+ channel by the antifungal drug ketoconazole depends on channel gating and involves the S6 residue F656.

The mechanism of human ether-à-go-go-related gene (HERG) K+ channel blockade by the antifungal agent ketoconazole was investigated using patch-clamp recording from mammalian cell lines. Ketoconazole inhibited whole-cell HERG current (IHERG) with a clinically relevant half-maximal inhibitory drug concentration (IC50) value of 1.7 microM. The voltage- and time-dependent characteristics of IHERG blockade by ketoconazole indicated dependence of block on channel gating, ruling out a significant role for closed-state channel inhibition. The S6 HERG mutations Y652A and F656A produced approximately 4-fold and approximately 21-fold increases in IC50 for IHERG blockade, respectively. Thus, ketoconazole accesses the HERG channel pore-cavity on channel gating, and the S6 residue F656 is an important determinant of ketoconazole binding.

Erythromycin block of the HERG K+ channel: accessibility to F656 and Y652.

The HERG potassium channel might have a non-canonical drug binding site, distinct from the channel's inner cavity, that could be responsible for elements of closed-state pharmacological inhibition of the channel. The macrolide antibiotic erythromycin is a drug that may block unconventionally because of its size. Here we used whole-cell patch-clamp recording at 37 degrees C from heterologously expressed HERG channels in a mammalian cell line to show that erythromycin either produces a rapid open-state-dependent HERG channel inhibition, or components of both open-state-dependent and closed-state-dependent inhibition. Alanine-substitution of HERG's canonical determinants of blockade revealed that Y652 was not important as a molecular determinant of blockade, and that mutation of F656 resulted in only weak attenuation of inhibition. In computer models of the channel, erythromycin could make several direct contacts with F656, but not with Y652, in the open-state model, and erythromycin was unable to fit into a closed-state channel model.

Mechanism of hERG K+ channel blockade by the fluoroquinolone antibiotic moxifloxacin.

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.

Clemastine, a conventional antihistamine, is a high potency inhibitor of the HERG K+ channel.

Human ether-à-go-go-related gene (HERG) encodes the alpha-subunit of channels carrying the cardiac rapid delayed K+ current (Ikr), which is a major determinant of the duration of ventricular action potentials (APs) and of the QT interval. This study investigated the effects on HERG channel current (IHERG) of clemastine, a "conventional" antihistamine that has been associated with delayed ventricular repolarization in vitro, but for which no adverse effects on the human QT interval have been reported. Whole-cell patch-clamp measurements of IHERG were made at 37 degrees C from human embryonic kidney (HEK 293) cells stably expressing HERG channels. IHERG tails at -40 mV following depolarizing pulses to +20 mV were inhibited by clemastine with an IC50 value of 12 nM; this drug concentration also produced a marked inhibition of peak IHERG elicited during an AP voltage-clamp command. Clemastine produced a reversible approximately -5 mV shift in the IHERG steady-state voltage-dependent activation curve, but voltage-dependence of inactivation was unaffected. Development of IHERG inhibition by clemastine showed strong time-dependence. The S6 point mutations Y652A and F656A greatly attenuated the inhibitory effect of clemastine. We conclude that clemastine is a high potency inhibitor of IHERG, that this action is contingent upon channel gating and that clemastine interacts with a high affinity drug-binding site in the HERG channel pore cavity. The disparity between clemastine's potent IHERG inhibition and a lack of QT-prolongation in normal clinical use underscores the need to interpret HERG IC50 data for novel compounds in the context of information from other safety assays.

The N588K-HERG K+ channel mutation in the 'short QT syndrome': mechanism of gain-in-function determined at 37 degrees C.

The idiopathic short QT syndrome (SQTS) is characterised by an abnormally short QT interval on the electrocardiogram and by an increased risk of arrhythmia and sudden death. One variant of the syndrome is linked to missense mutations that lead to a single amino-acid change (N588K; asparagine to lysine) in the S5-Pore linker region of the cardiac HERG K(+) channel. This study was performed in order to determine how the N588K mutation alters HERG channel current (I(HERG)) kinetics at mammalian physiological temperature. The whole-cell current-voltage (I-V) relation for wild-type (WT) I(HERG) measured from Chinese Hamster Ovary cells was maximal at approximately 0 mV and showed marked inward rectification positive to this. In contrast, N588K I(HERG) showed marked rectification only at +60 mV and at more positive voltages. The voltage dependence of activation of N588K-HERG did not differ significantly from that of WT-HERG. However, N588K I(HERG) had a significantly more positive inactivation V(0.5) (-8.14+/-0.82 mV) than did WT I(HERG) (-70.05+/-0.82 mV; P<0.001, unpaired t test; n=5 for each). Its P(Na)/P(K) ratio was also greater. The instantaneous I-V relation for N588K I(HERG) under action potential voltage clamp peaked at approximately +40 mV, compared to approximately -37 mV for WT-I(HERG). These findings underscore the importance of the S5-P linker in HERG channel function and indicate that N588K-HERG contributes increased repolarising current earlier in the ventricular action potential at physiological temperature due to a approximately +60 mV shift in voltage dependence of I(HERG) inactivation.

High affinity HERG K(+) channel blockade by the antiarrhythmic agent dronedarone: resistance to mutations of the S6 residues Y652 and F656.

Pharmacological inhibition of human-ether-a-go-go-related gene (HERG) K(+) channels by structurally and therapeutically diverse drugs is associated with the 'acquired' form of long QT syndrome and with potentially lethal cardiac arrhythmias. Two aromatic amino-acid residues (Y652 and F656) on the inner (S6) helices are considered to be key constituents of a high affinity drug binding site within the HERG channel pore cavity. Using wild-type (WT) and mutant HERG channels expressed in mammalian cell lines, we have investigated HERG channel current (I(HERG)) blockade at 37+/-1 degrees C by dronedarone (DRONED), a non-iodinated analogue of the Class III antiarrhythmic agent amiodarone (AMIOD). Under our conditions WT I(HERG) tails, measured at -40 mV following activating pulses to +30 mV, were blocked with IC(50) values of approximately 59 and 70 nM for DRONED and AMIOD, respectively. I(HERG) inhibition by DRONED was contingent upon channel gating, with block developing rapidly on membrane depolarization, but with no preference for activated over inactivated channels. High external [K(+)] (94 mM) reduced the potency of I(HERG) inhibition by both DRONED and AMIOD. Strikingly, mutagenesis to alanine of the S6 residue F656 (F656A) failed to eliminate blockade by both DRONED and AMIOD, whilst Y652A had comparatively little effect on DRONED but some effect on AMIOD. These findings demonstrate that high affinity drug blockade of I(HERG) can occur without a strong dependence on the Y652 and F656 aromatic amino-acid residues.

Acquired QT interval prolongation and HERG: implications for drug discovery and development.

Putative interactions between the Human Ether-a-go-go Related Gene (HERG), QT interval prolongation and Torsades de Pointes (TdP) are now integral components of any discussion on drug safety. HERG encodes for the inwardly rectifying potassium channel (I(Kr)), which is essential to the maintenance of normal cardiac function. HERG channel mutations are responsible for one form of familial long QT syndrome, a potentially deadly inherited cardiac disorder associated with TdP. Moreover, drug-induced (acquired) QT interval prolongation has been associated with an increase in the incidence of sudden unexplained deaths, with HERG inhibition implicated as the underlying cause. Subsequently, a number of non-cardiovascular drugs which induce QT interval prolongation and/or TdP have been withdrawn. However, a definitive link between HERG, QT interval prolongation and arrhythmogenesis has not been established. Nevertheless, this area is subject to ever increasing regulatory scrutiny. Here we review the relationship between HERG, long QT syndrome and TdP, together with a summary of the associated regulatory issues, and developments in pre-clinical screening.

The low-potency, voltage-dependent HERG blocker propafenone--molecular determinants and drug trapping.

The molecular determinants of high-affinity human ether-a-go-go-related gene (HERG) potassium channel blockade by methanesulfonanilides include two aromatic residues (Phe656 and Tyr652) on the inner helices (S6) and residues on the pore helices that face into the inner cavity, but determinants for lower-affinity HERG blockers may be different. In this study, alanine-substituted HERG channel mutants of inner cavity residues were expressed in Xenopus laevis oocytes and were used to characterize the HERG channel binding site of the antiarrhythmic propafenone. Propafenone's blockade of HERG was strongly dependent on residue Phe656 but was insensitive or weakly sensitive to mutation of Tyr652, Thr623, Ser624, Val625, Gly648, or Val659 and did not require functional inactivation. Homology models of HERG based on KcsA and MthK crystal structures, representing the closed and open forms of the channel, respectively, suggest propafenone is trapped in the inner cavity and is unable to interact exclusively with Phe656 in the closed state (whereas exclusive interactions between propafenone and Phe656 are found in the open-channel model). These findings are supported by very slow recovery of wild-type HERG channels from block at -120 mV, but extremely rapid recovery of D540K channels that reopen at this potential. The experiments and modeling suggest that the open-state propafenone binding-site may be formed by the Phe656 residues alone. The binding site for propafenone (which may involve pi-stacking interactions with two or more Phe656 side-chains) is either perturbed or becomes less accessible because of closed-channel gating. This provides further evidence for the existence of gating-induced changes in the spatial location of Phe656 side chains.

Moderately elevated plasma homocysteine impairs functional endothelial recovery following denudation of mouse carotid arteries.

Increased total plasma homocysteine is an independent risk factor for cardiovascular disease. This study was designed to determine whether it can impair endothelial function, by examining the recovery of acetylcholine-evoked relaxation following mechanical denudation of the endothelium in the arteries of cystathionine beta-synthase knockout (CbetaS(+/-)) mice. Heterozygous CbetaS(+/-) mice had total plasma homocysteine concentrations significantly higher (8.9 +/- 1.1 micromol/L, n = 12) than strain-matched wild-types (4.6 +/- 0.4 micromol/L, n = 5; P =.003). Left common carotid arteries were denuded of endothelium using a 250-microm polytetrafluoroethylene filament. After 10 days, when the endothelium had completely regrown, relaxation to acetylcholine was measured in precontracted segments of artery. Uninjured right carotid arteries from the same animals served as internal controls. Relaxation to acetylcholine was significantly attenuated in the injured arteries of the CbetaS(+/-) mice, compared to wild-types (P =.017); furthermore, there was a significant negative correlation between sensitivity to acetylcholine and total plasma homocysteine concentration measured in the same animal (r = -0.69, P <.003). These data suggest that even modest homocysteinemia has a deleterious effect on the function of healed endothelium in mouse arteries. This may account for its adverse influence on chronic cardiovascular disease.

Lidoflazine is a high affinity blocker of the HERG K(+)channel.

Lidoflazine is an antianginal calcium channel blocker that carries a significant risk of QT interval prolongation and ventricular arrhythmia. We investigated whether or not lidoflazine inhibits current through the rapid delayed rectifier K(+) channel alpha subunit (encoded by HERG - human ether-a-go-go-related gene), since this channel has been widely linked to drug-induced QT-prolongation. Lidoflazine inhibited potently HERG current (I(HERG)) recorded from HEK 293 cells stably expressing wild-type HERG (IC(50) of approximately 16 nM). It was approximately 13-fold more potent against HERG than was verapamil under similar conditions. On membrane depolarization, I(HERG) inhibition developed gradually, ruling out closed-channel state dependent inhibition. The effect of command voltage on the drug's action suggested that lidoflazine preferentially inhibits activated/open HERG channels. The S6 mutation Y652A largely eliminated the inhibitory action of lidoflazine, whilst the F656A mutation also reduced blocking potency. We conclude: first, that lidoflazine produces high affinity blockade of the alpha subunit of the HERG channel by binding to aromatic amino acid residues within the channel pore and, second, that this is likely to represent the molecular mechanism of QT interval prolongation by this drug.

Preferential closed channel blockade of HERG potassium currents by chemically synthesised BeKm-1 scorpion toxin.

The scorpion toxin peptide BeKm-1 was synthesised by fluorenylmethoxycarbonyl solid phase chemistry and folded by air oxidation. The peptide's effects on heterologous human ether-a-go-go-related gene potassium current (I(HERG)) in HEK293 cells were assessed using 'whole-cell' patch clamp. Blockade of I(HERG) by BeKm-1 was concentration-dependent, temperature-dependent, and rapid in onset and reversibility. Blockade also exhibited inverse voltage dependence, inverse dependence on duration of depolarisation, and reverse use- and frequency-dependence. Blockade by BeKm-1 and recombinant ergtoxin, another scorpion toxin known to block HERG, differed in their recovery from HERG current inactivation elicited by strong depolarisation and in their ability to block HERG when the channels were already activated. We conclude that synthetic BeKm-1 toxin blocks HERG preferentially through a closed (resting) state channel blockade mechanism, although some open channel blockade also occurs.

Blockade of HERG potassium currents by fluvoxamine: incomplete attenuation by S6 mutations at F656 or Y652.

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.