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

Aim:

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.

Methods:

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.

Results:

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.

Conclusion:

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.     

Allocryptopine and benzyltetrahydropalmatine block hERG potassium channels expressed in HEK293 cells

Aim:

Allocryptopine (ALL) is an alkaloid extracted from Corydalis decumbens (Thunb) Pers. Papaveraceae, whereas benzyltetrahydropalmatine (BTHP) is a derivative of tetrahydropalmatine extracted from Corydalis ambigua (Pall) Cham et Schlecht. The aim of this study was to investigate the effects of ALL and BTHP on the human ether-a-go-go related gene (hERG) current expressed in HEK293 cells.

Methods:

Cultured HEK293 cells were transiently transfected with hERG channel cDNA plasmid pcDNA3.1 using Lipofectamine. The whole-cell current IHERG was evoked and recorded using Axon MultiClamp 700B amplifier. The drugs were applied via supserfusion.

Results:

Both ALL and BTHP reversibly suppressed the amplitude and density of I_HERG in concentration- and voltage-dependent manners (the respective IC₅₀ value was 49.65 and 22.38 μmol/L). BTHP (30 μmol/L) caused a significant negative shift of the steady-state inactivation curve of I_HERG, while ALL (30 μmol/L) did not affect the steady-state inactivation of I_HERG. Furthermore, BTHP, but not ALL, shortened the time constants of fast inactivation and slow time constants of deactivation of I_HERG. But both the drugs markedly lengthened the time constants for recovery of I_HERG from inactivation. Using action potential waveform pulses, it was found that both the drugs at 30 μmol/L significantly suppressed the current densities in the late phase of action potential, but did not significantly affect the current densities in the early phase of action potential.

Conclusion:

Both ALL and BTHP derived from Chinese herbs potently block hERG current.     

Inhibition of hERG K+ currents by antimalarial drugs in stably transfected HEK293 cells

Several antimalarial drugs are known to produce a QT interval prolongation via a blockade of the rapidly activating delayed rectifier K+ current (IKr), encoded by the human-ether-a-go-go-related gene (hERG). We investigated the influence of lumefantrine and its major metabolite desbutyl-lumefantrine, as well as halofantrine, chloroquine, and mefloquine, on wild type hERG K+ channels in stably transfected human embryonic kidney cells (HEK293) using the whole cell patch-clamp technique. All of the tested antimalarial drugs inhibited the hERG K+ channels in a concentration- and time-dependent manner. Only halofantrine blocked hERG tail currents voltage-dependently. The ranking of the half-maximal inhibitory concentrations (IC50) of the antimalarials was: halofantrine (0.04 microM)相似文献   

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.     

Block of wild-type and inactivation-deficient human ether-a-go-go-related gene K+ channels by halofantrine

Halofantrine is an antimalarial drug developed as a treatment of P. falciparum resistant to chloroquine. However, halofantrine can also induce long QT syndrome (LQTS) and torsades de pointes, a potentially life-threatening ventricular arrhythmia. Drug-induced LQTS is usually caused by block of the human ether-a-go-go-related gene (HERG) channels that conduct the rapid delayed rectifier K⁺ current, I_Kr, in the heart. Here we show that halofantrine preferentially blocks open and inactivated HERG channels heterologously expressed in Xenopus laevis oocytes. The half-maximal inhibitory concentration (IC₅₀) for block of wild-type (WT) HERG was 1.0 M. As we reported previously for other HERG channel blockers, the potency of halofantrine was reduced by mutation to Ala of aromatic residues (Y652, F656) located in the S6 domain, or a Val (V625) located in the pore helix. Halofantrine at a concentration 10 M did not affect the transient outward potassium channel, Kv4.3, the slow delayed rectifier potassium channel, KvLQT1+minK and inward rectifier potassium channel, Kir2.1. An inactivation deficient mutant (G628C/S631C HERG) was only slightly less sensitive (IC₅₀=2.0 M). The rate of block onset by halofantrine at 0 mV was used to estimate the apparent association (k_on) and dissociation (k_off) rate constants for drug binding. For WT and G628C/S631C HERG, k_on was similar (0.0114 and 0.0163 M^–1/s^–1 respectively). In contrast, k_off was significantly faster for G628C/S631C (0.357 s^–1) than WT (0.155 s^–1), and explains the observed decrease in drug potency for the inactivation-deficient mutant channel. We conclude that halofantrine requires channels to open before it can gain access to its binding site located in the central cavity of the HERG channel.     

Epidermal growth factor receptor tyrosine kinase regulates the human inward rectifier potassium K(IR)2.3 channel, stably expressed in HEK 293 cells

BACKGROUND AND PURPOSE

The detailed molecular modulation of inward rectifier potassium channels (including the K_IR2.3 channel) is not fully understood. The present study was designed to determine whether human K_IR2.3 (K_IR2.3) channels were regulated by protein tyrosine kinases (PTKs).

EXPERIMENTAL APPROACH

Whole-cell patch voltage-clamp, immunoprecipitation, Western blot analysis and site-directed mutagenesis were employed to determine the potential PTK phosphorylation of Kir2.3 current in HEK 293 cells stably expressing Kir2.3 gene.

KEY RESULTS

The broad-spectrum PTK inhibitor genistein (10 µM) and the selective epidermal growth factor (EGF) kinase inhibitor AG556 (10 µM) reversibly decreased K_IR2.3 current and the effect was reversed by the protein tyrosine phosphatase inhibitor, orthovanadate (1 mM). Although EGF (100 ng·mL⁻¹) and orthovanadate enhanced K_IR2.3 current, this effect was antagonized by AG556. However, the Src-family tyrosine kinase inhibitor PP2 (10 µM) did not inhibit K_IR2.3 current. Tyrosine phosphorylation of K_IR2.3 channels was decreased by genistein or AG556, and was increased by EGF or orthovanadate. The decrease of tyrosine phosphorylation of K_IR2.3 channels by genistein or AG556 was reversed by orthovanadate or EGF. Interestingly, the response of K_IR2.3 channels to EGF or AG556 was lost in the K_IR2.3 Y234A mutant channel.

CONCLUSION AND IMPLICATIONS

These results demonstrate that the EGF receptor tyrosine kinase up-regulates the K_IR2.3 channel via phosphorylation of the Y234 residue of the WT protein. This effect may be involved in the endogenous regulation of cellular electrical activity.     

Effector coupling of stably transfected human A3 adenosine receptors in CHO cells

CHO cells stably transfected with adenosine receptors are widely utilized models for binding and functional studies. The effector coupling of human A3 adenosine receptors expressed in such a cellular model was characterized. Inhibition of adenylyl cyclase via a pertussis toxin-sensitive G protein was confirmed and exhibited a pharmacological profile in accordance with agonist binding data. The agonist potency was dependent on the assay system utilized to measure cyclase inhibition. Agonists were more potent in a cell-based assay than in experiments where cyclase inhibition was measured in a membrane preparation suggesting that receptor-effector coupling might be more efficient in intact cells. In addition to the modulation of cyclase activity, stimulation of A3 receptors elicited a Ca2+ response in CHO cells with agonist potencies corresponding to the values for the whole cell cAMP assay. The Ca2+ signal was completely eliminated by pertussis toxin treatment suggesting that it is mediated via betagamma release from a heterotrimeric G protein of the Gi/o family. These results show that cAMP and Ca2+ signaling characteristics of the A3 adenosine receptor are comparable to the ones found for the A1 subtype.     

Chemical modification of the human ether-a-go-go-related gene (HERG) K+ current by the amino-group reagent trinitrobenzene sulfonic acid

We investigated the effects of trinitrobenzene sulfonic acid (TNBS), an amino-group reagent, on the human ether-a-go-go-related gene (HERG) K+ channels expressed in Xenopus oocytes. TNBS neutralizes the positively charged amino-groups of peptide N-terminal and lysine residues. External application of TNBS at 10 mM for 5 min irreversibly shifted the curves for currents at the end of the pulse and tail currents of HERG to a more negative potential and decreased the maximal amplitude of the I(tail) curve (I(tail,max)). TNBS had little effect on either the activated current-voltage relationship or the reversal potential of HERG current, indicating that TNBS did not change ion selectivity properties. TNBS shifted the time constant curves of both activation and deactivation of the HERG current to a more hyperpolarized potential; TNBS's effect was greater on channel opening than channel closing. External H+ is known to inhibit HERG current by shifting V(1/2) to the right and decreasing I(tail,max). TNBS enhanced the blockade of external H+ by exaggerating the effect of H+ on I(tail,max), not on V(1/2). Our data provide evidence for the presence of essential amino-groups that are associated with the normal functioning of the HERG channel and evidence that these groups modify the blocking effect of external H+ on the current.     

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

Background and Purpose

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.

Experimental Approach

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.

Key Results

Zolpidem caused acute hERG channel blockade in oocytes (IC₅₀ = 61.5 μM) and in HEK 293 cells (IC₅₀ = 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.

Conclusions and Implications

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.     

Inhibition of delayed rectifier K+ channels by phenytoin in rat neuroblastoma cells

1. The action of the anticonvulsant drug phenytoin on K⁺ currents was investigated in neuroblastoma cells by whole-cell voltage-clamp recording.
2. Neuroblastoma cells expressed an outward K⁺ current with a voltage- and time-dependence which resembled the delayed-rectifier K⁺ current found in other cells. When added to the standard external solution at concentrations ranging between 1 and 200 μM, phenytoin reduced the current (n=65). Inhibition was concentration-dependent with a half-maximal inhibitory concentration of 30.9±0.8 μM.
3. The K⁺ current inhibition by phenytoin was voltage-dependent with block by phenytoin being relieved by depolarization.
4. The times taken to reach steady-state inhibition and complete recovery from inhibition were about 20 s. Neither the activation and inactivation rates of the K⁺ current nor the K⁺ channel availability were significantly altered by the blocking drug. A use-dependent block was observed at phenytoin concentrations of 10, 25 and 50 μM.
5. These results suggest that phenytoin affects K⁺ currents and that this effect might lead to a reduction in neuronal excitability.