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

Long QT mutations impact intermediate states of the voltage sensor in human ERG channel

Long QT mutations impact intermediate states of the voltage sensor in human ERG channel

G604S-HERG mutation in LQT2 leads to autophagy via the UPR-related pathway

Congenital long QT syndrome (LQTS) is a heart channel disease associated with fatal ventricular arrhythmias or cardiac arrest. Human ether-a-go-go-related gene (HERG) mutation is one of the main causes in type 2 LQTS since it may lead to abundant immature HERG channel protein accumulate in the endoplasmic reticulum (ER). In our study, we have successfully constructed the G604S-HERG mutation in HEK293 cells and demonstrated that the immature HERG protein on ER via Western blot and immunofluorescence. Herein we found that unfolded protein reaction (UPR) process has been activated in order to counter this endoplasmic reticulum stress (ERS) since the main sensors got upregulated. Meanwhile, autophagy was also observed in this process and verified by Western blot and transmission electron microscopy. To explore the relationship underlying autophagy and UPR in the condition of ERS, we found that PERK-EIF2a-CHOP axis was activated. Our findings provides insight for G604S-HERG mutation in type 2 LQTS.

AKAP5 anchors PKA to enhance regulation of the HERG channel

The activation of the β-adrenergic receptor (β-AR) regulates the human ether a-go-go-related gene (HERG) channel via protein kinase A (PKA), which in turn induces lethal arrhythmia in patients with long QT syndromes (LQTS). However, the role of A-kinase anchoring proteins (AKAPs) in PKA's regulation of the HERG channel and its molecular mechanism are not clear. Here, HEK293 cells were transfected with the HERG gene alone or co-transfected with HERG and AKAP5 using Lipofectamine 2000. Western blotting was performed to determine HERG protein expression, and immunofluorescence and immunoprecipitation were used to assess the binding and cellular colocalization of HERG, AKAP5, and PKA. The HEK293-HERG and HEK293-HERG + AKAP5 cells were treated with forskolin at different concentrations and different time. HERG protein expression significantly increased under all treatment conditions (P < 0.001). The level of HERG protein expression in HEK293-HERG + AKAP5 cells was higher than that observed in HEK293-HERG cells (P < 0.001). Immunofluorescence and immunoprecipitation indicated that HERG bound to PKA and AKAP5 and was colocalized at the cell membrane. The HERG channel protein, AKAP5, and PKA interacted with each other and appeared to form intracellular complexes. These results provide evidence for a novel mechanism which AKAP5 anchors PKA to up-regulate the HERG channel protein.

The Drosophila ERG channel seizure plays a role in the neuronal homeostatic stress response.

Neuronal physiology is particularly sensitive to acute stressors that affect excitability, many of which can trigger seizures and epilepsies. Although intrinsic neuronal homeostasis plays an important role in maintaining overall nervous system robustness and its resistance to stressors, the specific genetic and molecular mechanisms that underlie these processes are not well understood. Here we used a reverse genetic approach in Drosophila to test the hypothesis that specific voltage-gated ion channels contribute to neuronal homeostasis, robustness, and stress resistance. We found that the activity of the voltage-gated potassium channel seizure (sei), an ortholog of the mammalian ERG channel family, is essential for protecting flies from acute heat-induced seizures. Although sei is broadly expressed in the nervous system, our data indicate that its impact on the organismal robustness to acute environmental stress is primarily mediated via its action in excitatory neurons, the octopaminergic system, as well as neuropile ensheathing and perineurial glia. Furthermore, our studies suggest that human mutations in the human ERG channel (hERG), which have been primarily implicated in the cardiac Long QT Syndrome (LQTS), may also contribute to the high incidence of seizures in LQTS patients via a cardiovascular-independent neurogenic pathway.

(−)‐Epicatechin rescues the As2O3‐induced HERG K+ channel deficiency possibly through upregulating transcription factor SP1 expression

(-)-Epicatechin (EPI) has beneficial effects on the cardiovascular disease. The human ether-a-go-go-related gene (HERG) potassium channel is crucial for repolarization of cardiac action potential. Dysfunction of the HERG channel can cause long QT syndrome type 2 (LQT2). Arsenic trioxide (As2 O3 ) has shown efficacy in the treatment of acute promyelocytic leukemia. However, As2 O3 can induce the deficiency of HERG channel and cause LQT2. In this study, we examined whether EPI could rescue the As2 O3 -induced HERG channel deficiency. We found that 3μM EPI obviously increased protein expression and current of HERG channel. EPI was able to recover the protein expression and current of HERG channel disrupted by As2 O3 . EPI was able to increase the expression of SP1 protein and recover the expression of SP1 protein disrupted by As2 O3 . In addition, EPI significantly shortened action potential duration prolonged by As2 O3 . Our data suggest that EPI rescues As2 O3 -induced HERG channel deficiency through upregulating SP1 expression.

Induction of autoimmune response to the extracellular loop of the HERG channel pore induces QTc prolongation in guinea-pigs.

Channelopathies of autoimmune origin are novel and are associated with corrected QT (QTc) prolongation and complex ventricular arrhythmias. We have recently demonstrated that anti-SSA/Ro antibodies from patients with autoimmune diseases and with QTc prolongation on the ECG target the human ether-à-go-go-related gene (HERG) K+ channel by inhibiting the corresponding current, IKr , at the pore region. Immunization of guinea-pigs with a peptide (E-pore peptide) corresponding to the extracellular loop region connecting the S5 and S6 segments of the HERG channel induces high titres of antibodies that inhibit IKr , lengthen the action potential and cause QTc prolongation on the surface ECG. In addition, anti-SSA/Ro-positive sera from patients with connective tissue diseases showed high reactivity to the E-pore peptide. The translational impact is the development of a peptide-based approach for the diagnosis and treatment of autoimmune-associated long QT syndrome. We recently demonstrated that anti-SSA/52kDa Ro antibodies (Abs) from patients with autoimmune diseases and corrected QT (QTc) prolongation directly target and inhibit the human ether-à-go-go-related gene (HERG) K+ channel at the extracellular pore (E-pore) region, where homology with SSA/52kDa Ro antigen was demonstrated. We tested the hypothesis that immunization of guinea-pigs with a peptide corresponding to the E-pore region (E-pore peptide) will generate pathogenic inhibitory Abs and cause QTc prolongation. Guinea-pigs were immunized with a 31-amino-acid peptide corresponding to the E-pore region of HERG. On days 10-62 after immunization, ECGs were recorded and blood was sampled for the detection of E-pore peptide Abs. Serum samples from patients with autoimmune diseases were evaluated for reactivity to E-pore peptide by enzyme-linked immunosorbent assay (ELISA), and histology was performed on hearts using Masson's Trichrome. Inhibition of the HERG channel was assessed by electrophysiology and by computational modelling of the human ventricular action potential. The ELISA results revealed the presence of high titres of E-pore peptide Abs and significant QTc prolongation after immunization. High reactivity to E-pore peptide was found using anti-SSA/Ro Ab-positive sera from patients with QTc prolongation. Histological data showed no evidence of fibrosis in immunized hearts. Simulations of simultaneous inhibition of repolarizing currents by anti-SSA/Ro Ab-positive sera showed the predominance of the HERG channel in controlling action potential duration and the QT interval. These results are the first to demonstrate that inhibitory Abs to the HERG E-pore region induce QTc prolongation in immunized guinea-pigs by targeting the HERG channel independently from fibrosis. The reactivity of anti-SSA/Ro Ab-positive sera from patients with connective tissue diseases with the E-pore peptide opens novel pharmacotherapeutic avenues in the diagnosis and management of autoimmune-associated QTc prolongation.

Characterization of the novel mutant A78T-HERG from a long QT syndrome type 2 patient: Instability of the mutant protein and stabilization by heat shock factor 1

BackgroundThe human ether-a-go-go-related gene (HERG) encodes the α-subunit of rapidly activating delayed-rectifier potassium channels. Mutations in this gene cause long QT syndrome type 2 (LQT2). In most cases, mutations reduce the stability of the channel protein, which can be restored by heat shock (HS). MethodsWe identified the novel mutant A78T-HERG in a patient with LQT2. The purpose of the current study was to characterize this mutant protein and test whether HS and heat shock factors (HSFs) could stabilize the mutant protein. A78T-HERG and wild-type HERG (WT-HERG) were expressed in HEK293 cells and analyzed by immunoblotting, immunoprecipitation, immunofluorescence, and whole-cell patch clamping. ResultsWhen expressed in HEK293 cells, WT-HERG gave rise to immature and mature forms of the protein at 135 and 155kDa, respectively. A78T-HERG gave rise only to the immature form, which was heavily ubiquitinated. The proteasome inhibitor MG132 increased the expression of immature A78T-HERG and increased both the immature and mature forms of WT-HERG. WT-HERG, but not A78T-HERG, was expressed on the plasma membrane. In whole-cell patch clamping experiments, depolarizing pulses evoked E4031-sensitive HERG channel currents in cells transfected with WT-HERG, but not in cells transfected with A78T-HERG. The A78V mutant, but not A78G mutant, remained in the immature form similarly to A78T. Maturation of the A78T-HERG protein was facilitated by HS, expression of HSF-1, or exposure to geranyl geranyl acetone. ConclusionsA78T-HERG was characterized by protein instability and reduced expression on the plasma membrane. The stability of the mutant was partially restored by HSF-1, indicating that HSF-1 is a target for the treatment for LQT2 caused by the A78T mutation in HERG.

Pathogenesis of the Novel Autoimmune-Associated Long-QT Syndrome.

Emerging clinical evidence demonstrates high prevalence of QTc prolongation and complex ventricular arrhythmias in patients with anti-Ro antibody (anti-Ro Ab)-positive autoimmune diseases. We tested the hypothesis that anti-Ro Abs target the HERG (human ether-a-go-go-related gene) K(+) channel, which conducts the rapidly activating delayed K(+) current, IKr, thereby causing delayed repolarization seen as QT interval prolongation on the ECG. Anti-Ro Ab-positive sera, purified IgG, and affinity-purified anti-52kDa Ro Abs from patients with autoimmune diseases and QTc prolongation were tested on IKr using HEK293 cells expressing HERG channel and native cardiac myocytes. Electrophysiological and biochemical data demonstrate that anti-Ro Abs inhibit IKr to prolong action potential duration by directly binding to the HERG channel protein. The 52-kDa Ro antigen-immunized guinea pigs showed QTc prolongation on ECG after developing high titers of anti-Ro Abs, which inhibited native IKr and cross-reacted with guinea pig ERG channel. The data establish that anti-Ro Abs from patients with autoimmune diseases inhibit IKr by cross-reacting with the HERG channel likely at the pore region where homology between anti-52-kDa Ro antigen and HERG channel is present. The animal model of autoimmune-associated QTc prolongation is the first to provide strong evidence for a pathogenic role of anti-Ro Abs in the development of QTc prolongation. It is proposed that adult patients with anti-Ro Abs may benefit from routine ECG screening and that those with QTc prolongation should receive counseling about drugs that may increase the risk for life-threatening arrhythmias.

Phosphatidylinositol4-phosphate 5-kinase prevents the decrease in the HERG potassium current induced by Gq protein-coupled receptor stimulation

The human ether-a-go-go-related gene (HERG) potassium current (IHERG) has been shown to decrease in amplitude following stimulation with Gq protein-coupled receptors (GqRs), such as α1-adrenergic and M1-muscarinic receptors (α1R and M1R, respectively), at least partly via the reduction of membrane phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). The present study was designed to investigate the modulation of HERG channels by PI(4,5)P2 and phosphatidylinositol4-phosphate 5-kinase (PI(4)P5-K), a synthetic enzyme of PI(4,5)P2. Whole-cell patch-clamp recordings were used to examine the activity of HERG channels expressed heterologously in Chinese Hamster Ovary cells. The stimulation of α1R with phenylephrine or M1R with acetylcholine decreased the amplitude of IHERG accompanied by a significant acceleration of deactivation kinetics and the effects on IHERG were significantly attenuated in cells expressing PI(4)P5-K. The density of IHERG in cells expressing GqRs alone was significantly increased by the coexpression of PI(4)P5-K without significant differences in the voltage dependence of activation and deactivation kinetics. The kinase-deficient substitution mutant, PI(4)P5-K-K138A did not have these counteracting effects on the change in IHERG by M1R stimulation. These results suggest that the current density of IHERG is closely dependent on the membrane PI(4,5)P2 level, which is regulated by PI(4)P5-K and GqRs and that replenishing PI(4,5)P2 by PI(4)P5-K recovers IHERG.

Derek Nexus predicts human skin sensitisation accurately. What is the rationale behind its predictive performance?

Domiphen bromide and didecyl dimethylammonium bromide were widely used environmental chemicals with potent activity on blockade of human ether-a-go-go related gene (HERG) channels. But the mechanism of their action is not clear. The kinetics of block of HERG channels by domiphen bromide and didecyl dimethylammonium bromide was studied in order to characterize the inhibition of HERG currents by these quaternary ammonium compounds (QACs). Domiphen bromide and didecyl dimethylammonium bromide inhibited HERG channel currents in a dose-dependent manner with IC50 values of 9 nM and 5 nM, respectively. Block of HERG channel by domiphen bromide and didecyl dimethylammonium bromide was voltage-dependent and use-dependent. Domiphen bromide and didecyl dimethylammonium bromide caused substantial negative shift of the activation curves, accelerated activated process, but had no effects on the deactivation and reactivation processes. The docking models implied that these two compounds bound to PAS domain of HERG channels and inhibited its function. Our data demonstrated that domiphen bromide and didecyl dimethylammonium bromide blocked the HERG channel with a preference for the activated channel state.

Inhibition of HERG potassium channels by domiphen bromide and didecyl dimethylammonium bromide

Domiphen bromide and didecyl dimethylammonium bromide were widely used environmental chemicals with potent activity on blockade of human ether-a-go-go related gene (HERG) channels. But the mechanism of their action is not clear. The kinetics of block of HERG channels by domiphen bromide and didecyl dimethylammonium bromide was studied in order to characterize the inhibition of HERG currents by these quaternary ammonium compounds (QACs). Domiphen bromide and didecyl dimethylammonium bromide inhibited HERG channel currents in a dose-dependent manner with IC50 values of 9nM and 5nM, respectively. Block of HERG channel by domiphen bromide and didecyl dimethylammonium bromide was voltage-dependent and use-dependent. Domiphen bromide and didecyl dimethylammonium bromide caused substantial negative shift of the activation curves, accelerated activated process, but had no effects on the deactivation and reactivation processes. The docking models implied that these two compounds bound to PAS domain of HERG channels and inhibited its function. Our data demonstrated that domiphen bromide and didecyl dimethylammonium bromide blocked the HERG channel with a preference for the activated channel state.

Blockade of HERG Human K&lt;sup&gt;+&lt;/sup&gt; Channels by the Antidepressant Drug Paroxetine

The effects of paroxetine, a selective serotonin reuptake inhibitor, on human ether-a-go-go-related gene (HERG) channels were investigated using the whole-cell patch-clamp technique. The HERG channels were stably expressed in human embryonic kidney cells. Paroxetine inhibited the peak tail currents of the HERG channel in a concentration-dependent manner, with an IC50 value of 0.45 µM and a Hill coefficient of 0.85. These effects were reversible after wash-out of the drug. The paroxetine-induced inhibition of the HERG channels was voltage-dependent. There was a steep increase in inhibition over the voltage range of the channel opening. Also, a shallow voltage-dependent inhibition was detected over the voltage range in which the channels were fully activated. The fractional electrical distance was estimated to be 0.11. Paroxetine induced a leftward shift in the voltage-dependence of the steady-state activation of the HERG channels. Before and after application of the 1 µM paroxetine, the half-maximum activation was -14.21 mV and -27.04 mV, respectively, with no shift in the slope value. The HERG channel block was not use-dependent. The characteristics of the block were dependent on open and inactivated channel states rather than closed state. Paroxetine had no effect on activation and deactivation kinetics, steady-state inactivation. These results suggest that paroxetine blocks the HERG channels by binding to these channels in the open and inactivated states.

Cellular impedance assay for prediction of QT prolongation induced by hERG and non-hERG channel modulators using human stem cell derived cardiomyocytes

Torsades de pointes (TdP) arrhythmia is a potentially fatal form of ventricular arrhythmia that occurs under conditions where cardiac repolarization is delayed (as indicated by prolonged QT intervals from electrocardiographic recordings). A likely mechanism for QT interval prolongation and TdP arrhythmias is blockade of the rapid component of the cardiac delayed rectifier K+ current (IKr), which is encoded by human ether-a-go-go-related gene (HERG). Over 100 non-cardiovascular drugs have the potential to induce QT interval prolongations in the electrocardiogram (ECG) or TdP arrhythmias. The binding site of most HERG channel blockers is located inside the central cavity of the channel. An evaluation of possible effects on HERG channels during the development of novel drugs is recommended by international guidelines. During cardiac ischaemia activation of ATP-sensitive K+ (KATP) channels contributes to action potential (AP) shortening which is either cardiotoxic by inducing re-entrant ventricular arrhythmias or cardioprotective by inducing energy-sparing effects or ischaemic preconditioning (IPC). KATP channels are formed by an inward-rectifier K+ channel (Kir6.0) and a sulfonylurea receptor (SUR) subunit: Kir6.2 and SUR2A in cardiac myocytes, Kir6.2 and SUR1 in pancreatic β-cells. Sulfonylureas and glinides stimulate insulin secretion via blockade of the pancreatic β-cell KATP channel. Clinical studies about cardiotoxic effects of sulfonylureas are contradictory. Sulfonylureas and glinides differ in their selectivity for pancreatic over cardiovascular KATP channels, being either selective (tolbutamide, glibenclamide) or non-selective (repaglinide). The possibility exists that non-selective KATP channel inhibitors might have cardiovascular side effects. Blockers of the pore-forming Kir6.2 subunit are insulin secretagogues and might have cardioprotective or cardiotoxic effects during cardiac ischaemia.

Cholesterol regulates HERG K+ channel activation by increasing phospholipase C β1 expression

Human ether-a-go-go-related gene (HERG) K+ channel underlies the rapidly activating delayed rectifier K+ conductance (IKr) during normal cardiac repolarization. Also, it may regulate excitability in many neuronal cells. Recently, we showed that enrichment of cell membrane with cholesterol inhibits HERG channels by reducing the levels of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] due to the activation of phospholipase C (PLC). In this study, we further explored the effect of cholesterol enrichment on HERG channel kinetics. When membrane cholesterol level was mildly increased in human embryonic kidney (HEK) 293 cells expressing HERG channel, the inactivation and deactivation kinetics of HERG current were not affected, but the activation rate was significantly decelerated at all voltages tested. The application of PtdIns(4,5)P2 or inhibitor for PLC prevented the effect of cholesterol enrichment, while the presence of antibody against PtdIns(4,5)P2 in pipette solution mimicked the effect of cholesterol enrichment. These results indicate that the effect of cholesterol enrichment on HERG channel is due to the depletion of PtdIns(4,5)P2. We also found that cholesterol enrichment significantly increases the expression of β1 and β3 isoforms of PLC (PLCβ1, PLCβ3) in the membrane. Since the effects of cholesterol enrichment on HERG channel were prevented by inhibiting transcription or by inhibiting PLCβ1 expression, we conclude that increased PLCβ1 expression leads to the deceleration of HERG channel activation rate via downregulation of PtdIns(4,5)P2. These results confirm a crosstalk between two plasma membrane-enriched lipids, cholesterol and PtdIns(4,5)P2, in the regulation of HERG channels.

Blocking of the Human Ether-à-go-go-Related Gene Channel by Imatinib Mesylate

Imatinib mesylate (IM), a widely prescribed powerful tyrosine kinase inhibitor, has been associated with increased risk of heart failure and is known to induce cell apoptosis and death in isolated cardiomyocytes. In addition to acquired long QT syndrome, pharmacological inhibition of human ether-à-go-go-related gene (HERG) channel has been reported to involve in apoptosis. The present study was undertaken to characterize the biophysical properties of IM on HERG and the molecular determinants of HERG blockade using mutant channels (Y652A and F656A). Wild type (WT) and mutant HERG channels were expressed in HEK-293 cells and Xenopus oocytes and the currents (I(HERG)) were measured using patch-clamp and two-microelectrode voltage-clamp techniques. IM inhibited WT I(HERG) in a concentration-dependent manner with an IC(50) of 19.51±2.50 µmol/L and 44.76±1.54 µmol/L in HEK-293 cells and Xenopus oocytes, respectively. The IM-induced inhibition of WT I(HERG) followed a voltage- and time-dependent manner. The blockade was enhanced by further activation of currents, which were in accordance with an open-channel blockade. The V(1/2) for steady-state activation shifted from -15.48±1.21 to -26.66±2.98 mV (p<0.05, n=6). The inactivation kinetics and voltage dependence of steady-state inactivation of the WT HERG channel were not significantly altered by IM. Two S6 domain mutants, F652A and Y656A, attenuated IM-induced inhibition of WT I(HERG). Therefore, IM preferentially blocked the open HERG channel through F652 and Y656, providing a molecular mechanism for the cardiac side effects during the clinical administration of IM.

Modulation of HERG K+ Channels by Chronic Exposure to Activators and Inhibitors of PKA and PKC: Actions Independent of PKA and PKC Phosphorylation

Background: Human ether-a-go-go-related gene (HERG) channel is the major molecular component of the native rapid delayed rectifier K⁺ current (I_Kr) that is a crucial determinant of cardiac repolarization. Impairment of I_Kr/HERG function is commonly believed to be a mechanism causing long QT syndromes (LQTS), a lethal ventricular tachyarrhythmia. The cAMP-dependent protein kinase A (PKA) and PKC activities are markedly increased in some pathological conditions of the heart such as heart failure. This study was designed to investigate the effects of acute and chronic exposure to PKA or PKC activators and inhibitors on HERG channel activities and to provide insight into the mechanisms for the modulations. Methods: Channel activity was measured in HEK293 cells stably expressing HERG using whole-cell patch-clamp techniques. Intracellular reactive oxygen species (ROS) were measured by CM-H2DFDA. Mitochondrial membrane potential (ΔΨ_m) was measured using JC-1 dye. HERG channel phosphorylation was assayed by [³²P]orthophosphate methods. Results: Acute exposure of cells to PKA or PKC activators by bath superfusion minimally affected I_HERG, and so did the PKA or PKC inhibitor. By comparison, prolonged exposure (chronic incubation) of cells to PKA or PKC activators significantly impaired HERG K⁺ channel function as reflected by reduced I_HERG density and positive shift of the steady-state activation curve. Antioxidants vitamin E and MnTBAP both abolished the depressive effects of PKA or PKC activators on HERG function. Further, both PKA and PKC activators stimulated production of intracellular reactive oxygen species (ROS), an effect efficiently prevented by antioxidants or by PKA and PKC inhibitors. Conclusions: HERG function is insensitive to PKA or PKC phosphorylation modulation per se, but can be impaired by the activators of PKA or PKC with long exposure likely via generation of ROS. In view of the critical role of HERG K⁺ channel in regulating cardiac repolarization and the sustained activation of both PKA and PKC in many pathological conditions of the heart such as heart failure, it is conceivable that HERG impairment by ROS accumulation induced by PKA and PKC contributes to the impaired cardiac repolarization.

Role of the activation gate in determining the extracellular potassium dependency of block of HERG by trapped drugs

Drug induced long QT syndrome (diLQTS) results primarily from block of the cardiac potassium channel HERG (human-ether-a-go-go related gene). In some cases long QT syndrome can result in the lethal arrhythmia torsade de pointes, an arrhythmia characterized by a rapid heart rate and severely compromised cardiac output. Many patients requiring medication present with serum potassium abnormalities due to a variety of conditions including gastrointestinal dysfunction, renal and endocrine disorders, diuretic use, and aging. Extracellular potassium influences HERG channel inactivation and can alter block of HERG by some drugs. However, block of HERG by a number of drugs is not sensitive to extracellular potassium. In this study, we show that block of WT HERG by bepridil and terfenadine, two drugs previously shown to be trapped inside the HERG channel after the channel closes, is insensitive to extracellular potassium over the range of 0 mM to 20 mM. We also show that bepridil block of the HERG mutant D540K, a mutant channel that is unable to trap drugs, is dependent on extracellular potassium, correlates with the permeant ion, and is independent of HERG inactivation. These results suggest that the lack of extracellular potassium dependency of block of HERG by some drugs may in part be related to the ability of these drugs to be trapped inside the channel after the channel closes.

High-throughput screening of drug-binding dynamics to HERG improves early drug safety assessment

The use of computational models to predict drug-induced changes in the action potential (AP) is a promising approach to reduce drug safety attrition but requires a better representation of more complex drug-target interactions to improve the quantitative prediction. The blockade of the human ether-a-go-go-related gene (HERG) channel is a major concern for QT prolongation and Torsade de Pointes risk. We aim to develop quantitative in-silico AP predictions based on a new electrophysiological protocol (suitable for high-throughput HERG screening) and mathematical modeling of ionic currents. Electrophysiological recordings using the IonWorks device were made from HERG channels stably expressed in Chinese hamster ovary cells. A new protocol that delineates inhibition over time was applied to assess dofetilide, cisapride, and almokalant effects. Dynamic effects displayed distinct profiles for these drugs compared with concentration-effects curves. Binding kinetics to specific states were identified using a new HERG Markov model. The model was then modified to represent the canine rapid delayed rectifier K(+) current at 37°C and carry out AP predictions. Predictions were compared with a simpler model based on conductance reduction and were found to be much closer to experimental data. Improved sensitivity to concentration and pacing frequency variables was obtained when including binding kinetics. Our new electrophysiological protocol is suitable for high-throughput screening and is able to distinguish drug-binding kinetics. The association of this protocol with our modeling approach indicates that quantitative predictions of AP modulation can be obtained, which is a significant improvement compared with traditional conductance reduction methods.

Identification and Characterization of a Compound That Protects Cardiac Tissue from Human Ether-à-go-go-related Gene (hERG)-related Drug-induced Arrhythmias

The human Ether-à-go-go-related gene (hERG)-encoded K(+) current, I(Kr) is essential for cardiac repolarization but is also a source of cardiotoxicity because unintended hERG inhibition by diverse pharmaceuticals can cause arrhythmias and sudden cardiac death. We hypothesized that a small molecule that diminishes I(Kr) block by a known hERG antagonist would constitute a first step toward preventing hERG-related arrhythmias and facilitating drug discovery. Using a high-throughput assay, we screened a library of compounds for agents that increase the IC(70) of dofetilide, a well characterized hERG blocker. One compound, VU0405601, with the desired activity was further characterized. In isolated, Langendorff-perfused rabbit hearts, optical mapping revealed that dofetilide-induced arrhythmias were reduced after pretreatment with VU0405601. Patch clamp analysis in stable hERG-HEK cells showed effects on current amplitude, inactivation, and deactivation. VU0405601 increased the IC(50) of dofetilide from 38.7 to 76.3 nM. VU0405601 mitigates the effects of hERG blockers from the extracellular aspect primarily by reducing inactivation, whereas most clinically relevant hERG inhibitors act at an inner pore site. Structure-activity relationships surrounding VU0405601 identified a 3-pyridiyl and a naphthyridine ring system as key structural components important for preventing hERG inhibition by multiple inhibitors. These findings indicate that small molecules can be designed to reduce the sensitivity of hERG to inhibitors.

Blockade of human HERG K+ channels by rosiglitazone, an antidiabetic drug

This study examined the effect of rosiglitazone, an oral antidiabetic drug, on human ether-a-gogo-related gene (HERG) channels expressed in human embryonic kidney (HEK293) cells. Using the whole-cell patch-clamp technique, interaction between rosiglitazone and HERG in HEK293 cells was studied. Rosiglitazone inhibited HERG channels in a concentration-dependent manner, with an IC₅₀ value of 18.8 μM and a Hill coefficient of 1.0. These effects were reversible after wash-out of the drug. The rosiglitazone-induced inhibition of HERG channels was voltagedependent, with a steep increase in inhibition over the voltage range of channel opening. However, inhibition was voltage-independent over the voltage range in which channels are fully activated. Rosiglitazone did not change the steady-state activation or inactivation curves or the activation or deactivation kinetics, implying that rosiglitazone blocks HERG channels predominantly in the open and inactivated state rather than in the closed state. The present study suggests that rosiglitazone blocks HERG channels by binding to activated and inactivated channels, and rosiglitazone use should thus be carefully monitored in patients with pre-existing QT prolongation.