Molecular and Cellular Pharmacology
Fluconazole inhibits hERG K+ channel by direct block and disruption of protein trafficking

https://doi.org/10.1016/j.ejphar.2010.10.010Get rights and content

Abstract

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.

Introduction

The human ether-a-go-go-related gene (hERG) encodes the pore-forming subunit of the cardiac rapidly activating delayed rectifier K+ current (IKr). IKr is a major determinant of ventricular action potential repolarization and, thereby, of the QTc interval (Vandenberg et al., 2001). Drug-induced long QT syndrome (LQTS) is more common than the inherited form and is a side effect of both cardiac and noncardiac medications (Roden et al., 1996). hERG channels appear to have a larger pore cavity than other (Kv) six transmembrane domain K+ channels and possess particular aromatic amino acid residues in the S6 region of the channel (Mitcheson and Perry, 2003). These features combine to confer a higher susceptibility of the hERG channel to pharmacological blockade. The drug-induced diseases can result from direct block of channel conduction (Recanatini et al., 2005), disruption of channel trafficking to the surface (Eckhardt et al., 2005), or indirect mechanisms such as inhibition of cytochrome P450 and a reduction in hepatic metabolism of other QT-prolonging agents (Dumaine et al., 1998). Although the incidence of life-threatening arrhythmias is rare, it became evident that medications for even the most benign conditions could cause sudden death (Viskin, 1999). Several drugs have been withdrawn from the market because of this adverse effect (e.g. cisapride, terfenadine, and thioridazine) (Thomas et al., 2003, Roden, 2004). The association between drug-induced QTc interval prolongation and pharmacological blockade of hERG channels is very strong, and therefore drug-screening against recombinant hERG channels is currently an important component of cardiac safety pharmacology during drug development.

Fluconazole is a widely prescribed azole antifungal drug available in topical, vaginal and parenteral formulae. Azole antifungals (for example, fluconazole, ketoconazole and miconazole) are associated with acquired LQTS and ventricular arrhythmias (Khazan and Mathis, 2002, Roden, 2001, Kikuchi et al., 2005). These agents inhibit multiple cytochrome P450 enzymes in the liver and gastrointestinal tract (Dresser et al., 2000, Zhang et al., 2002), thus their arrhythmogenic mechanism is likely to involve a rise in the plasma concentrations of QT interval-prolonging drugs that use the same metabolic pathway (Tonini et al., 1999). However, ventricular arrhythmia can occur in the absence of other QT-interval prolonging drugs (Wassmann et al., 1999), suggesting that azole antifungal drugs may have direct effects on the heart. In fact, ketoconazole and miconazole have been reported to directly block hERG currents (Kikuchi et al., 2005, Takemasa et al., 2008). Moreover, one report has shown that ketoconazole directly inhibits the hERG current and reduces the surface membrane expression of hERG channels by disrupting channel protein trafficking (Takemasa et al., 2008). However, to date, there is a lack of information regarding the inhibitory action of fluconazole on recombinant hERG channels which had lead us to hypothesize that fluoconazole can act as an inhibitor of the hERG channel. Therefore, the goal of the present study was to investigate the effects of fluconazole on cloned hERG potassium channels expressed heterologously in human embryonic kidney (HEK293) cells.

Section snippets

HEK293 cells culture and transfection

The hERG wild type cDNA subcloned into the pcgi-EGFP vector was kindly provided by Dr. Zhao Zhang (College of Life Science in Nanjing Normal University, China). Y652A (tyrosine to alanine substitution at position 652) and F656C (phenylalanine to cysteine substitution at position 656) mutations were constructed by site-directed mutagenesis of wild type hERG cDNA in pcgi-EGFP vector using the QuickChange site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA), and the integrity of the

Concentration-dependent inhibition of hERG currents by fluconazole

To examine the effects of fluconazole on hERG currents expressed in HEK293 cells, hERG step currents were elicited from a holding potential of −80 mV by 3 s long depolarizing steps between −50 and + 60 mV applied in 10-mV increments. Tail current was recorded with a step to −40 mV for 2 s (Fig. 1A, upper panel). Representative currents recorded under control conditions and 5 min after application of fluconazole (10 μM) in the same cell are shown in Fig. 1A. Fluconazole reduced hERG step currents

Discussion

Fluconazole is a systemic antifungal medication that has been reported to cause prolonged QT interval and Torsade de Pointes (TdP) ventricular tachycardia in adults and children (Gandhi et al., 2003, Khazan and Mathis, 2002, Esch and Kantoch, 2008). Although fluconazole is associated with cardiac arrhythmias, the underlying mechanism(s) remains speculative. Since fluconazole is known to inhibit multiple cytochrome P450 enzymes in the liver and gastrointestinal tract (Nivoix et al., 2008),

Acknowledgements

This work was supported by grants from the Key Technologies R&D Program of Henan Province (No. 92102310225).

We are grateful to Jeremy Springer (Department of Physiology and Biophysics, Dalhousie University, Canada) for the careful reading of this paper and participation in discussion.

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