Block of HERG channels by berberine: mechanisms of voltage- and state-dependence probed with site-directed mutant channels

Berberine prolongs the duration of cardiac action potentials without affecting resting membrane potential or action potential amplitude. Controversy exists regarding whether berberine exerts this action by preferential block of different components of the delayed rectifying potassium current, I(Kr) and I(Ks). Here we have studied the effects of berberine on hERG (I(Kr)) and KCNQ1/KCNE1 (I(Ks)) channels expressed in HEK-293 cells and Xenopus oocytes. In HEK-293 cells, the IC50 for berberine was 3.1 +/- 0.5 microM on hERG compared with 11 +/- 4% decreases on KCNQ1/KCNE1 channels by 100 microM berberine. Likewise in oocytes, hERG channels were more sensitive to block by berberine (IC50 = 80 +/- 5 microM) compared with KCNQ1/KCNE1 channels (approximately 20% block at 300 microM). hERG block was markedly increased by membrane depolarization. Mutation to Ala of Y652 or F656 located on the S6 domain, or V625 located at the base of the pore helix of hERG decreased sensitivity to block by berberine. An inactivation-deficient mutant hERG channel (G628C/S631C) was also blocked by berberine. Together these findings indicate that berberine preferentially blocks the open state of hERG channels by interacting with specific residues that were previously reported to be important for binding of more potent antagonists.     

Drug binding interactions in the inner cavity of HERG channels: molecular insights from structure-activity relationships of clofilium and ibutilide analogs

Block of human ether-a-go-go related gene (hERG) K(+) channels by otherwise useful drugs is the most common cause of long QT syndrome, a disorder of cardiac repolarization that predisposes patients to potentially fatal arrhythmias. This undesirable long QT side effect has been a major reason for the withdrawal of medications from the pharmaceutical market. Understanding the molecular basis of hERG block is therefore essential to facilitate the design of safe drugs. Binding sites for hERG blockers have been mapped within the inner cavity of the channel and include aromatic residues in the S6 helix (Tyr-652, Phe-656) and residues in the pore helix (Thr-623, Ser-624, Val-625). We used mutagenesis of these residues, combined with an investigation of hERG block by close analogs of clofilium and ibutilide, to assess how specific alterations in drug structure affected potency and binding interactions. Although changing the basic nitrogen from quaternary to tertiary accelerated the onset of block, the IC(50) and kinetics for recovery from block were similar. In contrast, analogs with different para-substituents on the phenyl ring had significantly different potencies for wild-type hERG block. The highest potency was achieved with polar or electronegative para-substituents, whereas neutral para-substituents had potencies more than 100-fold lower. Results from mutagenesis and molecular modeling studies suggest that phenyl ring para-substituents influence drug interactions with Thr-623, Ser-624, and Tyr-652 and strongly affect binding affinity. Together, these findings suggest that modifying the para-substituent could be a useful strategy for reducing hERG potency and increasing the safety margin of compounds in development.     

Blockade of HERG K+ channel by an antihistamine drug brompheniramine requires the channel binding within the S6 residue Y652 and F656

A number of clinically used drugs block delayed rectifier K+ channels and prolong the duration of cardiac action potentials associated with long QT syndrome. This study investigated the molecular mechanisms of voltage-dependent inhibition of human ether-a-go-go-related gene (HERG) delayed rectifier K+ channels expressed in HEK-293 cells by brompheniramine, an antihistamine. Brompheniramine inhibited HERG current in a concentration-dependent manner with the half-maximal inhibitory concentration (IC50) value of 1.7 microm at 0 mV. A block of HERG current by brompheniramine was enhanced by progressive membrane depolarization and showed significantly negative shift in voltage-dependence of channel activation. Inhibition of HERG current by brompheniramine showed time-dependence. The S6 residue HERG mutant Y652A and F656C largely reduced the blocking potency of HERG current. These results indicate that brompheniramine mainly inhibited the HERG potassium channel through the residue Y652 and F656 and these residues may be an obligatory determinant in inhibition of HERG current for brompheniramine.     

Inhibition of cardiac HERG potassium channels by antidepressant maprotiline

Many drugs block delayed rectifier K+ channels and prolong the cardiac action potential duration. Here we investigate the molecular mechanisms of voltage-dependent block of human ether-a-go-go-related gene (HERG) K+ channels expressed in cells HEK-293 and Xenopus oocytes by maprotiline. The IC50 determined at 0 mV on HERG expressed HEK-293 cell and oocytes was 5.2 and 23.7 microM, respectively. Block of HERG expressed in oocytes by maprotiline was enhanced by progressive membrane depolarization and accompanied by a negative shift in the voltage dependence of channel activation. The potency of maprotiline was reduced 7-fold by point mutation of a key aromatic residue (F656T) and 3-fold for Y652A, both located in the S6 domain. The mutation Y652A inverted the voltage dependence of HERG channel block by maprotiline. Together, these results suggest that voltage-dependent block of HERG results from gating dependent changes in the accessibility of Y652, a critical component of the drug binding site.     

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

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

Marine algal toxin azaspiracid is an open-state blocker of HERG potassium channels

Azaspiracids (AZA) are polyether marine dinoflagellate toxins that accumulate in shellfish and represent an emerging human health risk. Although human exposure is primarily manifested by severe and protracted diarrhea, this toxin class has been shown to be highly cytotoxic, a teratogen to developing fish, and a possible carcinogen in mice. Until now, AZA's molecular target has not yet been determined. Using three independent methods (voltage clamp, channel binding assay, and thallium flux assay), we have for the first time demonstrated that AZA1, AZA2, and AZA3 each bind to and block the hERG (human ether-à-go-go related gene) potassium channel heterologously expressed in HEK-293 mammalian cells. Inhibition of K(+) current for each AZA analogue was concentration-dependent (IC(50) value range: 0.64-0.84 μM). The mechanism of hERG channel inhibition by AZA1 was investigated further in Xenopus oocytes where it was shown to be an open-state-dependent blocker and, using mutant channels, to interact with F656 but not with Y652 within the S6 transmembrane domain that forms the channel's central pore. AZA1, AZA2, and AZA3 were each shown to inhibit [(3)H]dofetilide binding to the hERG channel and thallium ion flux through the channel (IC(50) value range: 2.1-6.6 μM). AZA1 did not block the K(+) current of the closely related EAG1 channel. Collectively, these data suggest that the AZAs physically block the K(+) conductance pathway of hERG1 channels by occluding the cytoplasmic mouth of the open pore. Although the concentrations necessary to block hERG channels are relatively high, AZA-induced blockage may prove to contribute to the toxicological properties of the AZAs.     

The membrane permeable calcium chelator BAPTA-AM directly blocks human ether a-go-go-related gene potassium channels stably expressed in HEK 293 cells

BAPTA-AM is a well-known membrane permeable Ca(2+) chelator. The present study found that BAPTA-AM rapidly and reversibly suppressed human ether a-go-go-related gene (hERG or Kv11.1) K(+) current, human Kv1.3 and human Kv1.5 channel currents stably expressed in HEK 293 cells, and the effects were not related to Ca(2+) chelation. The externally applied BAPTA-AM inhibited hERG channels in a concentration-dependent manner (IC(50): 1.3 microM). Blockade of hERG channels was dependent on channel opening, and tonic block was minimal. Steady-state activation V(0.5) of hERG channels was negatively shifted by 8.5 mV (from -3.7+/-2.8 of control to -12.2+/-3.1 mV, P<0.01), while inactivation V(0.5) was negatively shifted by 6.1 mV (from -37.9+/-2.0 mV of control to -44.0+/-1.6 mV, P<0.05) with application of 3 microM BAPTA-AM. The S6 mutant Y652A and the pore helix mutant S631A significantly attenuated blockade by BAPTA-AM at 10 microM causing profound blockade of wild-type hERG channels. In addition, BAPTA-AM inhibited hKv1.3 and hKv1.5 channels in a concentration-dependent manner (IC(50): 1.45 and 1.23 microM, respectively), and the blockade of these two types of channels was also dependent on channel opening. Moreover, EGTA-AM was found to be an open channel blocker of hERG, hKv1.3, hKv1.5 channels, though its efficacy is weaker than that of BAPTA-AM. These results indicate that the membrane permeable Ca(2+) chelator BAPTA-AM (also EGTA-AM) exerts an open channel blocking effect on hERG, hKv1.3 and hKv1.5 channels.     

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.     

Additive effects of ziprasidone and D,L-sotalol on the action potential in rabbit Purkinje fibres and on the hERG potassium current

INTRODUCTION: Ziprasidone, an atypical antipsychotic has been shown to be devoid of cardiac adverse effects in spite of its propensity to prolong the QT-interval via a hERG current inhibition. However, the effects of ziprasidone on the action potential (AP) parameters have not been published yet. Moreover, very little information is available concerning pharmacodynamic interactions between ziprasidone and other hERG channel blockers. Thus, we investigated the putative interaction between ziprasidone and D,L-sotalol on the hERG channels at therapeutic concentrations and their consequences on the action potential prolongation. METHODS: AP were recorded at 1 and 0.2 Hz. Increasing concentrations of ziprasidone (0.01-10 micromol/L) were successively superfused for 30 min alone or in D,L-sotalol 10 micromol/L pre-treated fibres. Moreover, the effects of ziprasidone, alone or in association with d,l-sotalol, were investigated on the hERG current. RESULTS: Ziprasidone (1-10 microM) induced a concentration and reverse frequency-dependent increase in APD(90) (APD(90): +27% and +36%, respectively at 1 Hz and +50% and +70%, respectively at 0.2 Hz) due to a hERG current blockade (IC50: 0.24 micromol/L). A pre-treatment with D,L-sotalol 10 micromol/L led to an increase in APD(90) of +23% at 1 Hz, stable at 66+/-4 min. In these pre-treated fibres, ziprasidone (1 and 10 micromol/L) induced an additional AP prolongation (APD(90): +16% and +18%, respectively at 1 Hz) as compared to D,L-sotalol pre-treatment. Moreover, D,L-sotalol did not interact with the pharmacological profile of ziprasidone on the hERG channel. CONCLUSION: The present study demonstrates that ziprasidone induces an AP prolongation due to its propensity to block the hERG channel. Moreover, ziprasidone and d,l-sotalol, superfused concomitantly exhibit additive effects on the AP duration since they do not interact as competitors for the hERG channel.     

Verapamil blocks HERG channel by the helix residue Y652 and F656 in the S6 transmembrane domain

Aim： The objectives of this study were to investigate the inhibitory action of verapamil on wild-type（WT） and mutation HERG K^＋ channel current （IHERG）, and to determine whether mutations in the S6 region ale important for the inhibition of IHERG by verapamil. Methods： HERG channels （WT, Y652A, and F656A） were expressed in oocytes of Xenopus laevis and studied using the 2-electrode voltage-clamp technique. Results： WT HERG is blocked in a concentration-dependent manner by verapamil （half-maximal inhibition concentration [IC50]=5.1 μmol/L）, and the steady state activation and inactivation parameters are shifted to more negative values. However, mutation to Ala of Y652 and F656 located on the S6 domain produced 16-fold and 20-fold increases in IC50 for IHERG blockade, respectively. Simultaneously, the steady state activation and inactivation parameters for Y652A are also shifted to more negative values in the presence of the blockers. Conclusion： Verapamil preferentially binds to and blocks open HERG channels. Tyr-652 and Phe-656, 2 aromatic amino-acid residues in the inner （S6） helix, are critical in the verapamil-binding site.     

氟康唑对豚鼠心室肌细胞I_K和在HEK-293细胞中表达的HERG钾通道的抑制作用

目的研究氟康唑对豚鼠心室肌细胞延迟整流钾电流(IK)和在HEK-293细胞中表达的HERG钾通道的抑制作用。方法应用酶解法消化豚鼠单个心室肌细胞,观察氟康唑对IK的影响;采用磷酸钙沉淀瞬时转染的方法将HERG基因表达于HEK-293细胞上,观察氟康唑对野生型HERG钾通道电流、激活和失活曲线的影响,以及氟康唑对Y652A和F656C突变型HERG钾通道的作用;IK和HERG电流的记录均采用全细胞膜片钳技术。结果氟康唑(0.01、0.1、1、3、10、30、100、300和1 000μmol.L-1)浓度依赖性地抑制IK和HERG钾电流,其IC50值分别为(68.1±21.6)μmol.L-1和(48.2±9.4)μmol.L-1,对HERG钾通道的电压依赖性激活和失活曲线无影响;与野生型(WT)比较,Y652A和F656C突变型可减弱氟康唑对HERG通道的阻断作用。结论氟康唑能阻断IK和HERG通道,Y652和F656是氟康唑与HERG通道结合的关键位点。     

Molecular determinants of HERG channel block

Drug-induced block of cardiac hERG K+ channels causes acquired long QT syndrome. Here, we characterized the molecular mechanism of hERG block by two low-potency drugs (Nifekalant and bepridil) and two high-potency drugs 1-[2-(6-methyl-2pyridyl)ethyl]-4-(4-methylsulfonyl aminobenzoyl)piperidine (E-4031) and dofetilide). Channels were expressed in Xenopus laevis oocytes, and currents were measured using the two-microelectrode voltage-clamp technique. All four drugs progressively reduced hERG current during a 20-s depolarization to 0 mV after a 10-min pulse-free period, consistent with the preferential block of open channels. Recovery from block in response to pulses to -160 mV was observed for D540K hERG channels but not for wild-type hERG channels, suggesting that all four drugs are trapped in the central cavity by closure of the activation gate. The molecular determinants of hERG channel block were defined by using a site-directed mutagenesis approach. Mutation to alanine of three residues near the pore helix (Thr623, Ser624, and Val625) and four residues in Ser6 (Gly648, Tyr652, Phe656, and Val659) reduced channel sensitivity to block by dofetilide and E-4031, effects identical with those reported previously for two other methanesulfonanilides, (+)- N -[1' -(6-cyano-1,2,3,4-tetrahydro-2(R)-naphthalenyl)-3,4-dihydro-4(R)-hydroxyspiro(2H -1-benzopyran-2,4' -piperidin)-6-yl]-methanesulfonamide] monohydrochloride (MK-499) and ibutilide. The effect of nifekalant on mutant channels was similar, except that V659A retained normal sensitivity and I655A channels were less sensitive. Finally, mutation of the three residues near the pore helix and Phe656 in the Ser6 domain reduced channel block by bepridil. We conclude that the binding site is not identical for all drugs that preferentially block hERG in the open state.     

Coexistence of hERG current block and disruption of protein trafficking in ketoconazole-induced long QT syndrome

BACKGROUND AND PURPOSE: Many drugs associated with acquired long QT syndrome (LQTS) directly block human ether-a-go-go-related gene (hERG) K(+) channels. Recently, disrupted trafficking of the hERG channel protein was proposed as a new mechanism underlying LQTS, but whether this defect coexists with the hERG current block remains unclear. This study investigated how ketoconazole, a direct hERG current inhibitor, affects the trafficking of hERG channel protein. EXPERIMENTAL APPROACH: Wild-type hERG and SCN5A/hNa(v) 1.5 Na(+) channels or the Y652A and F656C mutated forms of the hERG were stably expressed in HEK293 cells. The K(+) and Na(+) currents were recorded in these cells by using the whole-cell patch-clamp technique (23 degrees C). Protein trafficking of the hERG was evaluated by Western blot analysis and flow cytometry. KEY RESULTS: Ketoconazole directly blocked the hERG channel current and reduced the amount of hERG channel protein trafficked to the cell surface in a concentration-dependent manner. Current density of the hERG channels but not of the hNa(v) 1.5 channels was reduced after 48 h of incubation with ketoconazole, with preservation of the acute direct effect on hERG current. Mutations in drug-binding sites (F656C or Y652A) of the hERG channel significantly attenuated the hERG current blockade by ketoconazole, but did not affect the disruption of trafficking. CONCLUSIONS AND IMPLICATIONS: Our findings indicate that ketoconazole might cause acquired LQTS via a direct inhibition of current through the hERG channel and by disrupting hERG protein trafficking within therapeutic concentrations. These findings should be considered when evaluating new drugs.     

Altering extracellular potassium concentration does not modulate drug block of human ether-a-go-go-related gene (hERG) channels

1. Drug-induced block of the rapidly activating delayed rectifier K+ current (I(Kr)), encoded by human ether-a-go-go-related gene (hERG), has been linked to acquired long QT syndrome (aLQTS). Hypokalaemia is a recognized risk factor in aLQTS. To further understand why hypokalaemia is a risk factor in aLQTS, we examined the effect of [K+]o on drug block of the hERG potassium channel stably expressed in human embryonic kidney (HEK-293) cells using whole-cell voltage-clamp techniques. 2. The effects of selected [K+]o (1-20 mmol/L) on hERG block with four structurally diverse compounds (dofetilide, mesoridazine, quinidine and terfenadine) from different therapeutic classes were evaluated. Reducing [K+]o from 20 to 1 mmol/L had little effect on IC50 values for hERG current block for all four compounds. For example, evaluating quinidine in external potassium concentrations of 20, 10, 5 and 1 mmol/L resulted in IC50 values of 1.82 +/- 0.33, 2.04 +/- 0.28, 1.57 +/- 0.52 and 1.14 +/- 0.21 mmol/L, respectively. No statistically significant difference (P > 0.35, anova) was observed between drug block of hERG in different external potassium concentrations. These data are in contrast with previously reported results examining hERG channel modulation expressed in AT-1 cells under similar experimental conditions. 3. These results demonstrate that [K+]o does not directly modulate drug block of hERG channels expressed in an HEK-293 cell line. The enhanced risk of Torsades de Pointes associated with hypokalaemia in aLQTS may be due to reduction of other (non-hERG) potassium currents, further reducing the repolarization reserve, and not due to direct modulation of hERG block by [K+]o.     

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

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

Comparison of HERG channel blocking effects of various beta-blockers-- implication for clinical strategy

beta-Blockers are widely used in the treatment of cardiovascular diseases. However, their effects on HERG channels at comparable conditions remain to be defined. We investigated the direct acute effects of beta-blockers on HERG current and the molecular basis of drug binding to HERG channels with mutations of putative common binding site (Y652A and F656C). beta-Blockers were selected based on the receptor subtype. Wild-type, Y652A and F656C mutants of HERG channel were stably expressed in HEK293 cells, and the current was recorded by using whole-cell patch-clamp technique (23 degrees C). Carvedilol (nonselective), propranolol (nonselective) and ICI 118551 (beta(2)-selective) inhibited HERG current in a concentration-dependent manner (IC(50) 0.51, 3.9 and 9.2 microM, respectively). The IC(50) value for carvedilol was a clinically relevant concentration. High metoprolol (beta(1)-selective) concentrations were required for blockade (IC(50) 145 microM), and atenolol (beta(1)-selective) did not inhibit the HERG current.Inhibition of HERG current by carvedilol, propranolol and ICI 118551 was partially but significantly attenuated in Y652A and F656C mutant channels. Affinities of metoprolol to Y652A and F656C mutant channels were not different compared with the wild-type. HERG current block by all beta-blockers was not frequency-dependent. Drug affinities to HERG channels were different in beta-blockers. Our results provide additional strategies for clinical usage of beta-blockers. Atenolol and metoprolol may be preferable for patients with type 1 and 2 long QT syndrome. Carvedilol has a class III antiarrhythmic effect, which may provide the rationale for a favourable clinical outcome compared with other beta-blockers as suggested in the recent COMET (Carvedilol Or Metoprolol European Trial) substudy.     

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.     

H1 antihistamine drug promethazine directly blocks hERG K channel

Promethazine is a phenothiazine derivative with antihistaminic (H₁), sedative, antiemetic, anticholinergic, and antimotion sickness properties that can induce QT prolongation, which may lead to torsades de pointes. Since block of cardiac human ether-a-go-go-related gene (hERG) channels is one of the leading causes of acquired long QT syndrome, we investigated the acute effects of promethazine on hERG channels to determine the electrophysiological basis for its proarrhythmic potential. Promethazine increased the action potential duration at 90% of repolarization (APD₉₀) in a concentration-dependent manner, with an IC₅₀ of 0.73 μM when action potentials were elicited under current clamp in guinea pig ventricular myocytes. We examined the effects of promethazine on the hERG channels expressed in Xenopus oocytes and HEK293 cells using two-microelectrode voltage-clamp and patch-clamp techniques. Promethazine induced a concentration-dependent decrease of the current amplitude at the end of the voltage steps and hERG tail currents. The IC₅₀ of promethazine dependent hERG block in Xenopus oocytes decreased progressively relative to the degree of depolarization. The IC₅₀ for the promethazine-induced block of the hERG currents in HEK293 cells at 36 °C was 1.46 μM at +20 mV. Promethazine affected the channels in the activated and inactivated states but not in the closed states. The S6 domain mutations, Y652A and F656A partially attenuated (Y652A) or abolished (F656A) the hERG current block. These results suggest that promethazine is a blocker of the hERG channels, providing a molecular mechanism for the arrhythmogenic side effects during the clinical administration of promethazine.     

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.     

Mechanism of action of a novel human ether-a-go-go-related gene channel activator

1,3-Bis-(2-hydroxy-5-trifluoromethyl-phenyl)-urea (NS1643) is a newly discovered activator of human ether-a-go-go-related gene (hERG) K(+) channels. Here, we characterize the effects of this compound on cloned hERG channels heterologously expressed in Xenopus laevis oocytes. When assessed with 2-s depolarizations, NS1643 enhanced the magnitude of wild-type hERG current in a concentration- and voltage-dependent manner with an EC(50) of 10.4 microM at -10 mV. The fully activated current-voltage relationship revealed that the drug increased outward but not inward currents, consistent with altered inactivation gating. NS1643 shifted the voltage dependence of inactivation by +21 mV at 10 microM and +35 mV at 30 microM, but it did not alter the voltage dependence of activation of hERG channels. The effects of the drug on three inactivation-deficient hERG mutant channels (S620T, S631A, and G628C/S631C) were determined. In the absence of channel inactivation, NS1643 did not enhance hERG current magnitude. The agonist activity of NS1643 was facilitated by mutations (F656 to Val, Met, or Thr) that are known to greatly attenuate channel inhibition by hERG blockers. We conclude that NS1643 is a partial agonist of hERG channels and that the mechanism of activation is reduced channel inactivation.