Human Ether-a-go-go-related Gene Channel Research Articles

Absence of clinically important HERG channel blockade by three compounds that inhibit phosphodiesterase 5—sildenafil, tadalafil, and vardenafil

Compounds that inhibit phosphodiesterase 5 (PDE5) have been developed for the treatment of erectile dysfunction. Because men with erectile dysfunction frequently have comorbid cardiovascular disease, they may have limited cardiac repolarization reserve and be at risk of arrhythmia if treated with medications that prolong ventricular repolarization. The human ether-a-go-go related gene (HERG) channel is important for repolarization in human myocardium and is a common target for drugs that prolong the QT interval. We studied the ability of three compounds that inhibit PDE5—sildenafil, tadalafil, and vardenafil—to block the HERG channel. Using a whole cell variant of the patch-clamp method, the HERG current was measured in a stably transfected human embryonic kidney cell line expressing the HERG channel. The compounds produced dose-dependent reductions in HERG current amplitude over a concentration range of 0.1 to 100 μM. The IC 50 values were 12.8 μM for vardenafil and 33.3 μM for sildenafil. Because the maximum soluble concentration of tadalafil (100 μM) produced only a 50.9% inhibition of the HERG current amplitude, the IC 50 value for tadalafil could not be determined with the Hill equation. Tadalafil had the weakest capacity to block the HERG channel, producing a 50.9% blockade at the maximum soluble concentration (100 μM), compared with 86.2% for vardenafil (100 μM) and 75.2% for sildenafil (100 μM). In conclusion, the concentrations of the PDE5 inhibitors required to evoke a 50% inhibition of the HERG current were well above reported therapeutic plasma concentrations of free and total compound. None of the three compounds was a potent blocker of the HERG channel.

Functional interaction between extracellular sodium, potassium and inactivation gating in HERG channels.

We have studied the interaction between extracellular K(+) (K(+)(o)) and extracellular Na(+) (Na(+)(o)) in human ether-à-go-go related gene (HERG)-encoded K(+) channels expressed in Chinese hamster ovary (CHO-K1) cells, using the whole-cell voltage clamp technique. Prior studies indicate that Na(+)(o) potently inhibits HERG current (IC(50) 3 mm) by binding to an outer pore site, and also speeds recovery from inactivation. In this study, we sought to explore the relationship between the Na(+)(o) effect on recovery and Na(+)(o) inhibition of HERG current, and to determine whether inactivation gating plays a critical role in Na(+)(o) inhibition of HERG current. Na(+)(o) concentration-response relationships for current inhibition and speeding of recovery were different, with Na(+)(o) less potent at speeding recovery. Na(+)(o) inhibition of HERG current was relieved by physiological [K(+)](o), while Na(+)(o) speeded recovery from inactivation similarly in the absence or presence of physiological [K(+)](o). To examine the link between Na(+)(o) block and inactivation using an independent approach, we studied hyperpolarization-activated currents uncoupled from inactivation in the S4-S5 linker mutant D540K. Depolarization-activated D540K currents were inhibited by Na(+)(o), while hyperpolarization-activated currents were augmented by Na(+)(o). This result reveals a direct link between Na(+)(o) inhibition and a depolarization-induced conformational change, most likely inactivation. We attempted to simulate the disparate concentration-response relationships for the two effects of Na(+)(o) using a kinetic model that included Na(+)(o) site(s) affected by permeation and gating. While a model with only a single dynamic Na(+)(o) site was inadequate, a model with two distinct Na(+)(o) sites was sufficient to reproduce the data.

Molecular analysis of PIP2 regulation of HERG and IKr.

We previously reported that cloned human ether-a-go-go-related gene (HERG) K+ channels are regulated by changes in phosphatidylinositol 4,5-bisphosphate (PIP2) concentration. Here we investigated the molecular determinants of PIP2 interactions with HERG channel protein. To establish the molecular nature of the PIP2-HERG interaction, we examined a segment of the HERG COOH terminus with a high concentration of positively charged amino acids (nos. 883-894) as a possible site of interaction with negatively charged PIP2. When we excised deletion-HERG (D-HERG) or mutated methionine-substituted-HERG (M-HERG) this segment of HERG to neutralize the amino acid charge, the mutant channels produced current that was indistinguishable from wild-type HERG. Elevating internal PIP2, however, no longer accelerated the activation kinetics of the mutant HERG. Moreover, PIP2-dependent hyperpolarizing shifts in the voltage dependence of activation were abolished with both mutants. PIP2 effects on channel-inactivation kinetics remained intact, which suggests an uncoupling of inactivation and activation regulation by PIP2. The specific binding of radiolabeled PIP2 to both mutant channel proteins was nearly abolished. Stimulation of alpha1A-adrenergic receptors produced a reduction in current amplitude of the rapidly activating delayed rectifier K+ current (the current carried by ERG protein) from rabbit ventricular myocytes. The alpha-adrenergic-induced current reduction was accentuated by PKC blockers and also unmasked a depolarizing shift in the voltage dependence of activation, which supports the conclusion that receptor activation of PLC results in PIP2 consumption that alters channel activity. These results support a physiological role for PIP2 regulation of the rapidly activating delayed rectifier K+ current during autonomic stimulation and localize a site of interaction to the COOH-terminal tail of the HERG K+ channel.

Inhibition of cardiac HERG currents by the DNA topoisomerase II inhibitor amsacrine: mode of action.

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.

Structural determinants of HERG channel block by clofilium and ibutilide.

Block of human ether-a-go-go related gene (HERG) K(+) channels by a variety of medications has been linked to acquired long QT syndrome, a disorder of cardiac repolarization that predisposes to lethal arrhythmias. The drug-binding site is composed of residues that face into the central cavity of the channel. Two aromatic residues located on the S6 domain (Tyr652 and Phe656) are particularly important structural determinants of drug block. The role of pore helix residues (Thr623, Ser624, Val625) is less clear. In this study, we compared the pharmacological properties of two structurally related compounds, ibutilide and clofilium. Both compounds are charged amines with a single phenyl ring. Clofilium, a chlorobenzene derivative, is a potent blocker of HERG channels, but has a remarkably slower time course for recovery from block than ibutilide, a methanesulfonanilide. The difference in the rate of recovery from block can be explained simply by variation in drug trapping. There is little recovery from clofilium block with D540K HERG channels that permit untrapping at hyperpolarized potentials. Alanine-scanning mutagenesis of the S6 domain and a portion of the pore helix revealed that the binding site residues were the same for both compounds. However, S624A, located at the base of the pore helix, was the only HERG mutation that enabled rapid recovery from clofilium block. In summary, the pore helix residues are important components of the HERG drug binding site, and may be particularly important for drugs with polar substituents, such as a halogen (e.g., clofilium) or a methanesulfonamide (e.g., ibutilide).

Comparison of kinetic properties of quinidine and dofetilide block of HERG channels

Many drugs inhibit human ether-a-go-go related gene (HERG) current and prolong cardiac action potential duration. We examined the kinetic properties of quinidine block of HERG channels expressed in Xenopus oocytes in comparison with those of the block by a class III antiarrhythmic dofetilide. Both of the drugs inhibited HERG currents in a use-dependent and frequency-independent manner. However, the underlying mechanisms were different. Under the steady state, quinidine block was voltage- and time-dependent. At positive membrane potentials, the onset of block was very fast. Thus, quinidine caused frequency-independent block mainly through this fast blocking kinetic. In contrast, dofetilide blocked HERG currents in a voltage- and time-independent manner under the steady state because of very slow unblocking at negative potentials, which also caused frequency-independent block. Therefore, quinidine and dofetilide might cause the reverse frequency-dependent prolongation of action potential duration through distinct mechanisms with regard to blocking and unblocking kinetics.

Comparison of kinetic properties of quinidine and dofetilide block of HERG channels

Many drugs inhibit human ether-a-go-go related gene (HERG) current and prolong cardiac action potential duration. We examined the kinetic properties of quinidine block of HERG channels expressed in Xenopus oocytes in comparison with those of the block by a class III antiarrhythmic dofetilide. Both of the drugs inhibited HERG currents in a use-dependent and frequency-independent manner. However, the underlying mechanisms were different. Under the steady state, quinidine block was voltage- and time-dependent. At positive membrane potentials, the onset of block was very fast. Thus, quinidine caused frequency-independent block mainly through this fast blocking kinetic. In contrast, dofetilide blocked HERG currents in a voltage- and time-independent manner under the steady state because of very slow unblocking at negative potentials, which also caused frequency-independent block. Therefore, quinidine and dofetilide might cause the reverse frequency-dependent prolongation of action potential duration through distinct mechanisms with regard to blocking and unblocking kinetics.

Block of HERG current expressed in HEK293 cells by the Na+-channel blocker cibenzoline.

A Na(+)-channel blocker, cibenzoline, blocks the delayed rectifier potassium current ( I(k)), but its detailed action on the rapidly activating component ( I(kr)) of I(k) encoded by the human ether-a-go-go-related gene ( HERG) has not been clarified. We examined the effects of cibenzoline on stably expressed HERG current in HEK293 cells recorded by the patch-clamp technique of whole-cell configuration. Cibenzoline blocked HERG current expressed in HEK293 cells with IC(50) = 3.7 +/- 0.963 micro M and Hill coefficient = 0.74 +/- 0.12. Voltage-depended activation was shifted in a negative direction by cibenzoline. No block or minor block was induced at test depolarization of -40 to -30 mV, and the block increased with depolarization reaching a plateau at 0 mV without a further increase at positive voltages. Voltage-dependent activation of HERG currents became faster at negative test voltages but there were no changes at positive voltages after cibenzoline. No frequency-dependent block of HERG tail current by cibenzoline after equilibration was noted between 1.33 and 0.2 Hz. Steady-state inactivation of the HERG current was shifted in a negative direction by approximately 8 mV but the time constants of fast inactivation were little affected by cibenzoline. Cibenzoline blocks the I(kr)-like current reconstituted by HERG clone transfection with an IC(50) value comparable to therapeutic concentrations. Cibenzoline has a preferential affinity, at least, to the open state of the HERG channel with a rapid access to the binding site.

Inhibition of human ether-a-go-go-related gene potassium channels by alpha 1-adrenoceptor antagonists prazosin, doxazosin, and terazosin.

Human ether-a-go-go-related gene (HERG) potassium channels are expressed in multiple tissues including the heart and adenocarcinomas. In cardiomyocytes, HERG encodes the alpha-subunit underlying the rapid component of the delayed rectifier potassium current, I(Kr), and pharmacological reduction of HERG currents may cause acquired long QT syndrome. In addition, HERG currents have been shown to be involved in the regulation of cell proliferation and apoptosis. Selective alpha 1-adrenoceptor antagonists are commonly used in the treatment of hypertension and benign prostatic hyperplasia. Recently, doxazosin has been associated with an increased risk of heart failure. Moreover, quinazoline-derived alpha 1-inhibitors induce apoptosis in cardiomyocytes and prostate tumor cells independently of alpha1-adrenoceptor blockade. To assess the action of the effects of prazosin, doxazosin, and terazosin on HERG currents, we investigated their acute electrophysiological effects on cloned HERG potassium channels heterologously expressed in Xenopus oocytes and HEK 293 cells.Prazosin, doxazosin, and terazosin blocked HERG currents in Xenopus oocytes with IC(50) values of 10.1, 18.2, and 113.2 microM respectively, whereas the IC(50) values for HERG channel inhibition in human HEK 293 cells were 1.57 microM, 585.1 nM, and 17.7 microM. Detailed biophysical studies revealed that inhibition by the prototype alpha 1-blocker prazosin occurred in closed, open, and inactivated channels. Analysis of the voltage-dependence of block displayed a reduction of inhibition at positive membrane potentials. Frequency-dependence was not observed. Prazosin caused a negative shift in the voltage-dependence of both activation (-3.8 mV) and inactivation (-9.4 mV). The S6 mutations Y652A and F656A partially attenuated (Y652A) or abolished (F656A) HERG current blockade, indicating that prazosin binds to a common drug receptor within the pore-S6 region. In conclusion, this study demonstrates that HERG potassium channels are blocked by prazosin, doxazosin, and terazosin. These data may provide a hypothetical molecular explanation for the apoptotic effect of quinazoline-derived alpha1-adrenoceptor antagonists.

Pharmacological rescue of trafficking defective HERG channels formed by coassembly of wild-type and long QT mutant N470D subunits

Mutations in the human ether-a-go-go-related gene (HERG) cause long QT syndrome. We previously showed that the HERG N470D mutation expressed as homotetrameric channels causes a protein trafficking defect, and this can be corrected by the HERG channel blocking drug E-4031. The N470D mutant also has been reported to cause dominant negative suppression of HERG current when coexpressed with wild-type channel subunits. The aims of this study were 1). to investigate the molecular mechanism responsible for the dominant negative effect of the N470D mutant coexpressed with wild-type subunits and 2). to test whether the trafficking defective heteromeric channels could be pharmacologically rescued by E-4031. Using a combination of immunoprecipitation and Western blot methods, we showed that N470D mutant and wild-type HERG subunits were physically associated in the endoplasmic reticulum as heteromeric channels. The coassembly resulted in the retention of both wild-type and N470D subunits in the endoplasmic reticulum. Culturing cells in E-4031 increased the cell surface expression of these channels, although with an altered electrophysiological phenotype. These results suggest that the dominant negative effect of the N470D wild-type coassembled channels is caused by retention of heteromeric channels in the endoplasmic reticulum and that the trafficking defect of these channels can be corrected by specific pharmacological strategies.

Cardiac ion channel effects of tolterodine.

Tolterodine is a muscarinic antagonist widely used in the treatment of urinary incontinence. Although tolterodine has not been reported to alter cardiac repolarization, it is chemically related to other muscarinic antagonists known to prolong cardiac repolarization. For this reason, we studied the effects of tolterodine on cardiac ion channels and action potential recordings. Using patch-clamp electrophysiology, we found that tolterodine was a potent antagonist of the human ether-a-go-go-related gene (HERG) K(+) channel, displaying an IC(50) value of 17 nM. This potency was similar to that observed for the antiarrhythmic drug dofetilide (IC(50) of 11 nM). Tolterodine block of HERG displayed a positive voltage dependence, suggesting an interaction with an activated state. Tolterodine had little effect on the human cardiac Na(+) channel at concentrations of up to 1 microM. Inhibition of L-type Ca(2+) currents by tolterodine was frequency-dependent with IC(50) values measuring 143 and 1084 nM at 1 and 0.1 Hz, respectively. Both tolterodine and dofetilide prolonged action potential duration in single guinea pig myocytes over the concentration range of 3 to 100 nM. However, prolongation was significantly larger for dofetilide compared with tolterodine. Tolterodine seems to be an unusual drug in that it blocks HERG with high affinity, but produces little QT prolongation clinically. Low plasma levels after therapeutic doses combined with mixed ion channel effects, most notably Ca(2+) channel blockade, may serve to attenuate the QT prolonging effects of this potent HERG channel antagonist.

Modulation of HERG potassium channel function by drug action

Repolarization of cardiomyocytes is mainly performed by the rapid component of the delayed rectifier potassium current, IKr, which is encoded by the human ether‐a‐go‐go‐related gene (HERG). Inhibition of HERG potassium currents by class III antiarrhythmic drugs causes lengthening of the cardiac action potential, which produces a beneficial antiarrhythmic effect. Conversely, excessive prolongation of the action potential by a wide variety of antiarrhythmic and non‐antiarrhythmic drugs may lead to acquired long‐QT syndrome, which is associated with a risk for ‘torsade de pointes’‐arrhythmias and sudden cardiac death. As a result, this undesirable side effect has prompted the withdrawal of several drugs from the market. Recent studies on HERG channel inhibition provide significant insights into the molecular factors that determine state‐, voltage‐, and use‐dependency of HERG current block. In addition, crucial properties of the putative drug binding site in HERG have been identified. The broad diversity in response to pharmacologic treatment among individuals is likely to depend on a combination of multiple factors from the fields of arrhythmia genetics, physiology and pharmacology. In conclusion, the increasing understanding of the molecular mechanisms that underlie HERG channel block by antiarrhythmic and non‐antiarrhythmic drugs may improve prevention and treatment of drug‐induced long‐QT syndrome.

Lack of action potential-prolonging effect of terfenadine on rabbit myocardial tissue preparations.

The effects of terfenadine, an antiallergic drug also known for its QT-prolonging and arrhythmogenic activities, on the action potential of isolated myocardial tissue preparations from rabbits were examined with microelectrode techniques. In the Purkinje fibers and atrium, terfenadine concentration dependently decreased the maximum rate of rise (+.V(max)) without affecting other action potential parameters. In the ventricle, terfenadine had little effect on action potential configuration. In the sinoatrial node, terfenadine 20 microM prolonged cycle length mainly through inhibition of +.V(max). Terfenadine 1 microM completely inhibited the human ether a go-go-related gene (HERG) channel current expressed in HEK293 cells in the same experimental solution as in microelectrode experiments. The lack of terfenadine effect on the action potential duration suggests that there are drugs for which the HERG channel inhibitory action underlying in vivo QT prolongation cannot be evaluated based on their action potential-prolonging activity in isolated myocardial tissue preparations.

S641 contributes HERG K+ channel inactivation.

The kinetics of voltage-dependent inactivation of the rapidly activating delayed rectifier, IKr, are unique among K+ channels. The human ether-a-gogo-related gene (HERG) encodes the pore-forming subunit of IKr and shares a high degree of homology with ether-a-gogo (EAG) channels that do not inactivate. Within those segments thought to contribute to the channel pore, HERG possesses several serine residues that are not present in EAG channels. Two of these serines, S620 and S631, are known to be required for inactivation. We now show that a third serine, S641, which resides in the outer portion of the sixth transmembrane segment, is also critical for normal inactivation. As with the other serines, S641 is also involved in maintaining ion selectivity of the HERG channel and alters sensitivity to block by E4031. Larger charged or polar substitutions (S641D and S641T) disrupted C-type inactivation in HERG. Smaller aliphatic and more conservative substitutions (S641A and S641C) facilitated C-type inactivation. Our data show that, like S620 and S631, S641 is another key residue for the rapid inactivation. The altered inactivation of mutations at S620, S631, and S641 were dominant, suggesting that a network of hydroxyl side chains is required for the unique inactivation, permeation, and rectification of HERG channels.

Mesoridazine: an open-channel blocker of human ether-a-go-go-related gene K + channel

Mesoridazine, a phenothiazine antipsychotic agent, prolongs the QT interval of the cardiac electrocardiogram and is associated with Torsade de pointes-type arrhythmias. In this study, we examined the effects of mesoridazine on human ether-a-go-go-related gene (HERG) K + currents. HERG channels were stably expressed in human embryonic kidney 293 cells and studied using standard whole-cell patch-clamp technique (37 °C). Mesoridazine blocked HERG currents in a concentration-dependent manner (IC 50 550 nM at 0 mV); block increased significantly over the voltage range where HERG activates and saturated at voltages eliciting maximal HERG channel activation. Tonic block of HERG current by mesoridazine (1.8 μM) was minimal (<2–4%). The rate of the onset of HERG channel block was rapid and dose dependent ( τ = 54 ± 7 ms at 0 mV and 1.8 μM mesoridazine), but not significantly affected by test potentials ranging from –30 to +30 mV. The V 1/2 for steady-state activation was shifted from –31.2 ± 1.0 to –39.2 ± 0.5 mV ( P < 0.01). The apparent rate of HERG channel deactivation was significantly reduced (fast τ = 153 ± 8 vs. 102 ± 6 ms at –50 mV, P < 0.01; slow τ = 1113 ± 63 vs. 508 ± 27 ms, P < 0.01). The inactivation kinetics and voltage dependence of steady-state inactivation of the HERG channel were not significantly altered by mesoridazine. These findings demonstrate that mesoridazine is a potent and rapid open-channel blocker of HERG channels. This block would explain the QT prolongation seen clinically at therapeutic concentrations (0.3–3.6 μM).

Drug binding to aromatic residues in the HERG channel pore cavity as possible explanation for acquired Long QT syndrome by antiparkinsonian drug budipine.

Budipine is a non-dopaminergic antiparkinsonian drug causing acquired forms of Long QT syndrome (aLQTS). As a consequence, the manufacturer has restricted the use of budipine in patients who exhibit additional risk factors for the development of "Torsades-de-Pointes" tachycardias (TdP). The molecular basis of this serious side effect has not been elucidated yet. Human ether-a-go-go related gene (HERG) channel block being the main cause of drug induced QT prolongation, we investigated the effect of budipine on the rapid component of the delayed-rectifier potassium current (I(K(r))) in guinea pig cardiomyocytes and on HERG potassium channels heterologously expressed in Xenopus oocytes. In guinea pig cardiomyocytes, budipine (10 microM) inhibited I(K(r)) by 86% but was without any effect on calcium currents. In Xenopus oocytes, HERG potassium channels were blocked by budipine with an IC(50) of 10.2 microM. Onset of block was fast and block was only slowly and incompletely reversible upon washout. Budipine blocked HERG channels in the open and inactivated state, but not in the closed states. The half-maximal activation voltage was slightly shifted towards more negative potentials. Steady-state inactivation of HERG was also influenced by budipine. Budipine block was neither voltage- nor frequency-dependent. In HERG channel mutants Y652A and F656A, drug affinity was reduced dramatically. Therefore, these two aromatic residues in the channel pore are likely to form a main part of the binding site for budipine. In summary, this is the first study that provides a molecular basis for the budipine-associated aLQTS observed in clinical practice. Furthermore, these findings underline the importance of the aromatic residues Y652 and F656 in the binding of lipophilic drugs to HERG channels.

Identification of a COOH-terminal Segment Involved in Maturation and Stability of Human Ether-a-go-go-related Gene Potassium Channels

Mutations in the potassium channel encoded by the human ether-a-go-go-related gene (HERG) have been linked to the congenital long QT syndrome (LQTS), a cardiac disease associated with an increased preponderance of ventricular arrhythmias and sudden death. The COOH terminus of HERG harbors a large number of LQTS mutations and its removal prevents functional expression for reasons that remain unknown. In this study, we show that the COOH terminus of HERG is required for normal trafficking of the ion channel. We have identified a region critical for trafficking between residues 860 and 899 that includes a novel missense mutation at amino acid 861 (HERGN861I). Truncations or deletion of residues 860-899, characterized in six different expression systems including a cardiac cell line, resulted in decreased expression levels and an absence of the mature glycosylated form of the HERG protein. Deletion of this region did not interfere with the formation of tetramers but caused retention of the assembled ion channels within the endoplasmic reticulum. Consequently, removal of residues 860-899 resulted in the absence of the ion channels from the cell surface and a more rapid turnover rate than the wild type channels, which was evident very early in biogenesis. This study reveals a novel role of the COOH terminus in the normal biogenesis of HERG channels and suggests defective trafficking as a common mechanism for abnormal channel function resulting from mutations of critical COOH-terminal residues, including the LQTS mutant HERGN861I.

Effects of Na+ channel blocker, pilsicainide, on HERG current expressed in HEK-293 cells.

Pilsicainide, classified as a relatively pure Na+ channel blocker, occasionally causes QT prolongation, suggesting inhibitory actions on K+ currents. We studied effects of pilsicainide on the K+ channel current of the human ether-a-go-go-related gene (HERG) in heterologous expression system. The Patch-clamp technique in whole-cell configuration was used to record the channel current of HERG stably expressed in HEK293 cells. Pilsicainide suppressed peak currents of HERG channel during depolarizing pulses and tail currents upon repolarization. Pilsicainide blocked HERG current with IC50 = 20.4 microM and Hill coefficient = 0.98. Voltage-dependent activation was shifted in a negative direction by approximately 10 mV by 10 to 20 microM pilsicainide. Block increased with depolarization to voltages between -20 and 0 mV and reached the maximum level at positive voltages to 0 mV without further increase. Following drug equilibration for 10 minutes (holding potential at -100 mV), the peak outward current upon the first depolarization showed time-dependent block; tail current block was maximal. Frequency-dependent block evaluated from tail current was absent with pulse frequencies of 1.33, 0.5, and 0.2 Hz. After a steady state block was achieved, time course of current activation and deactivation was slowed by pilsicainide, and steady-state inactivation and time course of fast inactivation were mildly affected. Pilsicainide blocks HERG current with a preferential affinity, at least, to the open state of the channels with a fast access to binding sites.

Gating currents associated with intramembrane charge displacement in HERG potassium channels.

HERG (human ether-a-go-go-related gene) encodes a delayed rectifier K+ channel vital to normal repolarization of cardiac action potentials. Attenuation of repolarizing K+ current caused by mutations in HERG or channel block by common medications prolongs ventricular action potentials and increases the risk of arrhythmia and sudden death. The critical role of HERG in maintenance of normal cardiac electrical activity derives from its unusual gating properties. Opposite to other voltage-gated K+ channels, the rate of HERG channel inactivation is faster than activation and appears to be intrinsically voltage dependent. To investigate voltage sensor movement associated with slow activation and fast inactivation, we characterized HERG gating currents. When the cut-open oocyte voltage clamp technique was used, membrane depolarization elicited gating current with fast and slow components that differed 100-fold in their kinetics. Unlike previously studied voltage-gated K+ channels, the bulk of charge movement in HERG was protracted, consistent with the slow rate of ionic current activation. Despite similar kinetic features, fast inactivation was not derived from the fast gating component. Analysis of an inactivation-deficient mutant HERG channel and a Markov kinetic model suggest that HERG inactivation is coupled to activation.

Drug binding to HERG channels: evidence for a 'non-aromatic' binding site for fluvoxamine.

British Journal of Pharmacology (2003) 139, 883–884. doi:10.1038/sj.bjp.0705336