hERG Protein Trafficking Research Articles

Propofol inhibits hERG K+ channels and enhances the inhibition effects on its mutations in HEK293 cells

QT interval prolongation, a potential risk for arrhythmias, may result from gene polymorphisms relevant to cardiomyocyte repolarization. Another noted cause of QT interval prolongation is the administration of chemical compounds such as anesthetics, which may affect a specific type of cardiac K+ channel encoded by the human ether-a-go-go-related gene (hERG).hERG K+ current was recorded using whole-cell patch clamp in human embryonic kidney (HEK293) cells expressing wild type (WT) or mutated hERG channels. Expression of hERG K+ channel proteins was evaluated using western blot and confirmed by fluorescent staining and imaging. Computational modeling was adopted to identify the possible binding site(s) of propofol with hERG K+ channels. Propofol had a significant inhibitory effect on WT hERG K+ currents in a concentration-dependent manner, with a half-maximal inhibitory concentration (IC50) of 60.9±6.4μM. Mutations in drug-binding sites (Y652A or F656C) of the hERG channel were found to attenuate hERG current blockage by propofol. However, propofol did not inhibit the trafficking of hERG protein to the cell membrane. Meanwhile, for the three selective hERG K+ channel mutant heterozygotes WT/Q738X-hERG, WT/A422T-hERG, and WT/H562P-hERG, the IC50 of propofol was calculated as 14.2±2.8μM, 3.3±1.2μM, and 5.9±1.9μM, respectively, which were much lower than that for the wild type.These findings indicate that propofol may potentially increase QT interval prolongation risk in patients via direct inhibition of the hERG K+ channel, especially in those with other concurrent triggering factors such as hERG gene mutations.

Fluconazole-induced long QT syndrome via impaired human ether-a-go-go-related gene (hERG) protein trafficking in rabbits.

hERG protein trafficking deficiency has long been known in drug-induced long QT syndrome (LQTS). However, validated evidence from in vivo data kept scanty. Our goal was to investigate the proarrhythmic action of fluconazole and its underlying mechanism in an animal model. Twenty female Japanese long-eared white rabbits were randomly distributed into a control group and a fluconazole group for a chronic 2-week treatment. The control group was treated with 0.5% sodium carboxymethylcellulose (CMCNa), and the fluconazole group was treated with fluconazole. Electrocardiograms (ECGs) were recorded during the experimental period. Isolated arterially perfused left ventricular wedge preparations from the rabbits were made 2 weeks after treatment, and the arrhythmia events, the transmural ECG, and action potential from both the endocardium and epicardium were recorded. The changes in hERG protein expression were measured by western blot. The fluconazole group showed a longer QT interval 1 week after treatment (P < 0.05) and a higher arrhythmia occurrence 2 weeks after treatment (P < 0.05) than the control group. The fluconazole group also showed a longer transmural dispersion of repolarization and a higher occurrence of life-threatening torsades de pointes in arterially perfused left ventricular preparations. Furthermore, western blot analysis showed that the density of mature hERG protein was lower in the fluconazole group than that in the control group. Fluconazole can prolong the QT interval and possess proarrhythmic activity due to its inhibition of hERG protein trafficking in our experimental model. These findings may impact the clinical potential of fluconazole in humans.

The protease inhibitor atazanavir blocks hERG K(+) channels expressed in HEK293 cells and obstructs hERG protein transport to cell membrane.

Atazanavir (ATV) is a HIV-1 protease inhibitor for the treatment of AIDS patients, which is recently reported to provoke excessive prolongation of the QT interval and torsades de pointes (TdP). In order to elucidate its arrhythmogenic mechanisms, we investigated the effects of ATV on the hERG K(+) channels expressed in HEK293 cells. hERG K(+) currents were detected using whole-cell patch clamp recording in HEK293 cells transfected with EGFP-hERG plasmids. The expression of hERG protein was measured with Western blotting. Two mutants (Y652A and F656C) were constructed in the S6 domain within the inner helices of hERG K(+) channels that were responsible for binding of various drugs. The trafficking of hERG protein was studied with confocal microscopy. Application of ATV (0.01-30 μmol/L) concentration-dependently decreased hERG K(+) currents with an IC50 of 5.7±1.8 μmol/L. ATV (10 μmol/L) did not affect the activation and steady-state inactivation of hERG K(+) currents. Compared with the wild type hERG K(+) channels, both Y652A and F656C mutants significantly reduced the inhibition of ATV on hERG K(+) currents. Overnight treatment with ATV (0.1-30 μmol/L) concentration-dependently reduced the amount of fully glycosylated 155 kDa hERG protein without significantly affecting the core-glycosylated 135 kDa hERG protein in the cells expressing the WT-hERG protein. Confocal microscopy studies confirmed that overnight treatment with ATV obstructed the trafficking of hERG protein to the cell membrane. ATV directly blocks hERG K(+) channels via binding to the residues Y652 and F656 in the S6 domain, and indirectly obstructs the transport of the hERG protein to the cell membrane.

Hypoxia induced hERG trafficking defect leads to apoptosis: involvement of NADPH oxidase generated ROS and Hsp90 (710.2)

The alpha subunit of the voltage gated human ether‐go‐go‐related (hERG) potassium channel regulates cell excitability in a broad range of cell lines. hERG channels are also expressed in a variety of cancer cells and control cell proliferation and apoptosis. Sustained hypoxia (SH) alters gating properties of hERG currents in neuroblastoma (SH‐SY5Y) cells. In the present study, we examined the molecular mechanisms underlying SH altered hERG currents in SH‐SY5Y cells. Patch‐clamp studies revealed that 4days of SH decreased hERG tail currents by 40‐50%. SH down regulated the surface expression of the fully glycosylated form (fg) of hERG protein with accumulation of the endoplasmic reticulum (cg) form. These effects were stimulus dependent and reversible. Accumulation of cg form by SH was due to impaired trafficking of hERG protein. Prolonged association with molecular chaperone Hsp90 resulting in complex oligomeric insoluble aggregates contribute to SH induced ER retention and defective trafficking. SH increased NADPHoxidase‐2 (Nox2)‐mediated ROS generation. Silencing Nox2 prevented SH‐induced degradation of fg and accumulation of cg forms. HERG downregulation by hypoxia resulted in reduced cell proliferation, altered cell cycle distribution and increase in apoptotic cells and all these effects were prevented by genetic silencing of Nox2.Grant Funding Source: HL‐90554

Blockage of hERG current and the disruption of trafficking as induced by roxithromycin

Roxithromycin is an oral macrolide antibiotic agent that has been repeatedly reported to provoke excessive prolongation of the Q-T interval and torsades de pointes in clinical settings. To investigate the mechanisms underlying the arrhythmogenic side effects of roxithromycin, we studied the molecular mechanisms of roxithromycin on human ether-à-go-go-related gene (hERG) K(+) channels expressed in human embryonic kidney (HEK293) cells. Roxithromycin was found to inhibit wild-type (WT) hERG currents in a concentration-dependent manner with a half-maximum block concentration (IC50) of 55.8 ± 9.1 μmol/L. S6 residue hERG mutants (Y652A and F656C) showed reduced levels of hERG current blockage attributable to roxithromycin. Roxithromycin also inhibited the trafficking of hERG protein to the cell membrane, as confirmed by Western blot analysis and confocal microscopy. These findings indicate that roxithromycin may cause acquired long-QT syndrome via direct inhibition of hERG current and by disruption of hERG protein trafficking. Mutations in drug-binding sites (Y652A or F656C) of the hERG channel were found to attenuate hERG current blockage by roxithromycin, but did not significantly alter the disruption of trafficking.

GW24-e1194 The proarrhythmic action of Fluconazole and the underlying mechanism

ObjectivesTo investigate the proarrhythmic action of fluconazole and the underlying mechanism.MethodsTwenty Japanese long-eared white rabbits were randomly distributed into Normal group and Fluconazole group, the Normal group were treated with...

Structure‐activity relationships of pentamidine‐affected ion channel trafficking and dofetilide mediated rescue

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

Mechanisms of zolpidem‐induced long QT syndrome: acute inhibition of recombinant hERG K+ channels and action potential prolongation in human cardiomyocytes derived from induced pluripotent stem cells

Zolpidem, a short-acting hypnotic drug prescribed to treat insomnia, has been clinically associated with acquired long QT syndrome (LQTS) and torsade de pointes (TdP) tachyarrhythmia. LQTS is primarily attributed to reduction of cardiac human ether-a-go-go-related gene (hERG)/I(Kr) currents. We hypothesized that zolpidem prolongs the cardiac action potential through inhibition of hERG K(+) channels. Two-electrode voltage clamp and whole-cell patch clamp electrophysiology was used to record hERG currents from Xenopus oocytes and from HEK 293 cells. In addition, hERG protein trafficking was evaluated in HEK 293 cells by Western blot analysis, and action potential duration (APD) was assessed in human-induced pluripotent stem cell (hiPSC)-derived cardiomyocytes. Zolpidem caused acute hERG channel blockade in oocytes (IC(50) = 61.5 μM) and in HEK 293 cells (IC(50) = 65.5 μM). Mutation of residues Y652 and F656 attenuated hERG inhibition, suggesting drug binding to a receptor site inside the channel pore. Channels were blocked in open and inactivated states in a voltage- and frequency-independent manner. Zolpidem accelerated hERG channel inactivation but did not affect I-V relationships of steady-state activation and inactivation. In contrast to the majority of hERG inhibitors, hERG cell surface trafficking was not impaired by zolpidem. Finally, acute zolpidem exposure resulted in APD prolongation in hiPSC-derived cardiomyocytes. Zolpidem inhibits cardiac hERG K(+) channels. Despite a relatively low affinity of zolpidem to hERG channels, APD prolongation may lead to acquired LQTS and TdP in cases of reduced repolarization reserve or zolpidem overdose.

Mission possible: RNA interference rescues the hERG current

The congenital long QT syndrome (LQTS) is an inherited disease that causes cardiac arrhythmias and sudden cardiac death in the absence of evident structural heart defects. It is mainly attributed to genetic mutations that affect the function of ion channels that regulate the action potential waveform. LQTS is characterized by an abnormal prolongation of cardiac repolarization, leading to an increased QT interval, notched or biphasic T waves, and T-wave alternans. Affected patients are at a higher risk for developing arrhythmias, including polymorphic ventricular tachycardia, torsades de pointes, and ventricular fibrillation. The estimated prevalence of LQTS (based on electrocardiographic-guided identification of disease-causing mutations among whites) is approximately 1:2500 in apparently healthy live births. LQTS was initially described in the Jervell and Lange-Nielsen syndrome, where QT prolongation and deafness were present. A few years later, an autosomal dominant form of LQTS, in the absence of deafness, was reported. During the last decade, 13 LQTS variants have been described. Mutations in KCNQ1, KCNH2, and SCN5A genes account for approximately 90% of the positivegenotype cases of LQTS and underlie LQTS1, LQTS2, and LQTS3. Those genes encode Kv7.1, Kv11.1, and Nav1.5, respectively. Genetic analyses have identified more than 200 LQTS2causing mutations in KCNH2. Missense mutations leading to single amino acid substitutions in hERG (human ethera-gogo-related gene) protein—the alpha subunit of the rapid delayed rectifier current (IKr)—are the predominant abnormality. At the functional level, these mutations result in a decreased IKr. 11 It has been shown that LQTS2 mutations reduce IKr through a variety of mechanisms including aberrant hERG protein folding, trafficking, or ion-channel gating. Although not all identified mutations have been investigated, the majority of the mutations produce failure of the hERG subunits to exit the endoplasmic

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 (IC 50) 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.

Multiple mechanisms of hERG liability: K+ current inhibition, disruption of protein trafficking, and apoptosis induced by amoxapine

The antidepressant amoxapine has been linked to cases of QT prolongation, acute heart failure, and sudden death. Inhibition of cardiac hERG (Kv11.1) potassium channels causes prolonged repolarization and is implicated in apoptosis. Apoptosis in association with amoxapine has not yet been reported. This study was designed to investigate amoxapine effects on hERG currents, hERG protein trafficking, and hERG-associated apoptosis in order to elucidate molecular mechanisms underlying cardiac side effects of the drug. hERG channels were expressed in Xenopus laevis oocytes and HEK 293 cells, and potassium currents were recorded using patch clamp and two-electrode voltage clamp electrophysiology. Protein trafficking was evaluated in HEK 293 cells by Western blot analysis, and cell viability was assessed in HEK cells by immunocytochemistry and colorimetric MTT assay. Amoxapine caused acute hERG blockade in oocytes (IC(50) = 21.6 microM) and in HEK 293 cells (IC(50) = 5.1 microM). Mutation of residues Y652 and F656 attenuated hERG blockade, suggesting drug binding to a receptor inside the channel pore. Channels were mainly blocked in open and inactivated states, and voltage dependence was observed with reduced inhibition at positive potentials. Amoxapine block was reverse frequency-dependent and caused accelerated and leftward-shifted inactivation. Furthermore, amoxapine application resulted in chronic reduction of hERG trafficking into the cell surface membrane (IC(50) = 15.3 microM). Finally, the antidepressant drug triggered apoptosis in cells expressing hERG channels. We provide evidence for triple mechanisms of hERG liability associated with amoxapine: (1) direct hERG current inhibition, (2) disruption of hERG protein trafficking, and (3) induction of apoptosis. Further experiments are required to validate a specific pro-apoptotic effect mediated through blockade of hERG channels.

Multiple Mechanisms of hERG Liability: K+ Current Inhibition, Disruption of Protein Trafficking, and Apoptosis Induced by Amoxapine

The antidepressant amoxapine has been linked to QT prolongation, acute heart failure, and sudden death. Drug binding to cardiac hERG (Kv11.1) potassium channels causes prolonged repolarization and induces apoptosis. This study was designed to investigate amoxapine effects on hERG currents, hERG protein trafficking, and hERG-associated apoptosis in order to elucidate molecular mechanisms underlying cardiac side effects of the drug. hERG channels were expressed in Xenopuslaevis oocytes and HEK 293 cells, and potassium currents were recorded using patch clamp and two-electrode voltage clamp electrophysiology.

Effect of Imipramine, a tricyclic antidepressant, on hERG current and protein trafficking

Effect of Imipramine, a tricyclic antidepressant, on hERG current and protein trafficking

Comparative effects of sophocarpine and sophoridine on hERG K + channel

Human ether-à-go-go-related gene (hERG) has an important role in the repolarization of the cardiac action potential. Sophocarpine and sophoridine are quinolizidine alkaloids and their structures are similar. Our aim was to investigate the effects of sophocarpine or sophoridine on hERG-encoded K + channels and the underlying structure–activity relationships. The effects of sophocarpine and sophoridine were examined on stably expressed hERG channels in HEK293 cells using a whole-cell patch clamp technique and Western blot analysis. The oil–water partition coefficients of sophocarpine and sophoridine were determined by a validated RP-HPLC method. At 300 µM, fractional block was 60.9 ± 1.4% for sophocarpine versus 41.9 ± 2.0% for sophoridine. Compared with sophocarpine, voltage-dependence of hERG channels inhibition by sophoridine was more notable. Sophoridine altered the activation properties, but not sophocarpine. Sophocarpine shifted the inactivation curve in a negative direction, but not sophoridine. Both drugs had no significant effect on the expression of hERG protein. The partition coefficients for the n-octanol/water system of sophocarpine and sophoridine at 37 °C were 16.03 ± 0.42 and 1.94 ± 0.03, respectively. In summary, sophocarpine and sophoridine are low potency blockers of hERG channels that functions by changing the channel kinetics, and sophocarpine is a more potent blocker of hERG K + channels than sophoridine, which may be due to higher hydrophobic nature of sophocarpine compared with sophoridine. Sophocarpine may have a higher binding affinity for the inactivate state. In contrast, sophoridine has a higher binding affinity for the open state. Both drugs have no effect on the generation and trafficking of hERG protein.

Pre-clinical QT Risk Assessment in Pharmaceutical Companies - Issues of Current QT Risk Assessment -

Since the Committee for Proprietary Medicinal Products (CPMP) of the European Union issued in 1997 a "points to consider" document for the assessment of the potential for QT interval prolongation by non-cardiovascular agents to predict drug-induced torsades de pointes (TdP), the QT liability has become the critical safety issue in the development of pharmaceuticals. As TdP is usually linked to delayed cardiac repolarization, international guideline (ICH S7B) has advocated the standard repolarization assays such as in vitro IKr (hERG current) and in vivo QT interval, or in vitro APD (as a follow up) as the best biomarkers for predicting the TdP risk. However, the recent increasing evidence suggests that the currently used above biomarkers and/or assays are not fully predictive for TdP, but also does not address potential new druginduced TdP due to the selective disruption of hERG protein trafficking to the cell membrane or VT and/or VF with QT shortening. There is, therefore, an urgent need for other surrogate markers or assays that can predict the proarrhythmic potential of drug candidate. In this review, we provide an ideal pre-clinical strategy to predict the potentials of QT liability and lethal arrhythmia of the drug candidates with recent issues in this field in mind, not at the expense of discarding therapeutically innovative drugs.

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

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. 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. 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. 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.

Improved functional expression of recombinant human ether-a-go-go (hERG) K+ channels by cultivation at reduced temperature

BackgroundHERG potassium channel blockade is the major cause for drug-induced long QT syndrome, which sometimes cause cardiac disrhythmias and sudden death. There is a strong interest in the pharmaceutical industry to develop high quality medium to high-throughput assays for detecting compounds with potential cardiac liability at the earliest stages of drug development. Cultivation of cells at lower temperature has been used to improve the folding and membrane localization of trafficking defective hERG mutant proteins. The objective of this study was to investigate the effect of lower temperature maintenance on wild type hERG expression and assay performance.ResultsWild type hERG was stably expressed in CHO-K1 cells, with the majority of channel protein being located in the cytoplasm, but relatively little on the cell surface. Expression at both locations was increased several-fold by cultivation at lower growth temperatures. Intracellular hERG protein levels were highest at 27°C and this correlated with maximal 3H-dofetilide binding activity. In contrast, the expression of functionally active cell surface-associated hERG measured by patch clamp electrophysiology was optimal at 30°C. The majority of the cytoplasmic hERG protein was associated with the membranes of cytoplasmic vesicles, which markedly increased in quantity and size at lower temperatures or in the presence of the Ca2+-ATPase inhibitor, thapsigargin. Incubation with the endocytic trafficking blocker, nocodazole, led to an increase in hERG activity at 37°C, but not at 30°C.ConclusionOur results are consistent with the concept that maintenance of cells at reduced temperature can be used to boost the functional expression of difficult-to-express membrane proteins and improve the quality of assays for medium to high-throughput compound screening. In addition, these results shed some light on the trafficking of hERG protein under these growth conditions.

Combined hERG channel inhibition and disruption of trafficking in drug‐induced long QT syndrome by fluoxetine: a case‐study in cardiac safety pharmacology

Drug-induced prolongation of the rate-corrected QT interval (QTCI) on the electrocardiogram occurs as an unwanted effect of diverse clinical and investigational drugs and carries a risk of potentially fatal cardiac arrhythmias. hERG (human ether-à-go-go-related gene) is the gene encoding the alpha-subunit of channels mediating the rapid delayed rectifier K+ current, which plays a vital role in repolarising the ventricles of the heart. Most QTCI prolonging drugs can inhibit the function of recombinant hERG K+ channels, consequently in vitro hERG assays are used widely as front-line screens in cardiac safety-testing of novel chemical entities. In this issue, Rajamani and colleagues report a case of QTCI prolongation with the antidepressant fluoxetine and correlate this with a dual effect of the drug and of its major metabolite norfluoxetine on hERG channels. Both compounds were found to produce an acute inhibition of the hERG channel by pharmacological blockade, but in addition they also were able to disrupt the normal trafficking of hERG protein to the cell membrane. Mutations to a key component of the drug binding site in the S6 region of the channel greatly attenuated channel block, but did not impair disruption of trafficking; this suggests that channel block and drug effects on trafficking were mediated by different mechanisms. These findings add to growing evidence for disruption of hERG channel trafficking as a mechanism for drug-induced long QT syndrome and raise questions as to possible limitations of acute screening methods in the assessment of QTcI prolonging liability of drugs in development.