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European Journal of Pharmacology 702 (2013) 165–173 Contents lists available at SciVerse ScienceDirect European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar Cardiovascular pharmacology Block of hERG K þ channel and prolongation of action potential duration by fluphenazine at submicromolar concentration Hee-Kyung Hong a,1, Byung Hoon Lee a,b,1, Mi-Hyeong Park a, Seung Ho Lee c, Daehyun Chu d, Woo Jin Kim e, Han Choe d,n, Bok Hee Choi c,nn, Su-Hyun Jo a,nnn a Department of Physiology, Institute of Bioscience and Biotechnology, Kangwon National University School of Medicine, Chuncheon 200-701, South Korea Department of Radiology, Ilsan Paik Hospital, Inje University School of Medicine, Goyang 411-706, South Korea c Department of Pharmacology, Institute for Medical Sciences, Chonbuk National University Medical School, Jeonju 561-180, South Korea d Department of Physiology and Bio-Medical Institute of Technology, University of Ulsan College of Medicine, Seoul 138-736, South Korea e Department of Internal Medicine, Kangwon National University School of Medicine, Chuncheon 200-701, South Korea b a r t i c l e i n f o abstract Article history: Received 26 October 2012 Received in revised form 16 January 2013 Accepted 29 January 2013 Available online 6 February 2013 Fluphenazine is a potent antipsychotic drug that can increase action potential duration and induce QT prolongation in several animal models and in humans. As the block of cardiac human ether-a-go-gorelated gene (hERG) channels is one of the leading causes of acquired long QT syndrome, we investigated the acute effects of fluphenazine on hERG channels to determine the electrophysiological basis for its proarrhythmic potential. Fluphenazine at concentrations of 0.1–1.0 mM increased the action potential duration at 90% of repolarization (APD90) and action potential duration at 50% of repolarization (APD50) in 5 min when action potentials were elicited under current-clamp conditions in guinea pig ventricular myocytes. We examined the effects of fluphenazine on hERG channels expressed in Xenopus oocytes and HEK293 cells using two-microelectrode voltage-clamp and patch-clamp techniques. The IC50 for the fluphenazine-induced block of hERG currents in HEK293 cells at 36 1C was 0.102 mM at þ 20 mV. Fluphenazine-induced a concentration-dependent decrease of the current amplitude at the end of the voltage steps and hERG tail currents. The fluphenazine-dependent hERG block in Xenopus oocytes increased progressively relative to the degree of depolarization. Fluphenazine affected the channels in the activated and inactivated states but not in the closed states, and the S6 domain mutation from tyrosine to alanine at amino acid 652 (Y652A) attenuated the hERG current block. These results suggest that the antipsychotic drug fluphenazine is a potent blocker of hERG channels, providing a molecular mechanism for the drug-induced arrhythmogenic side effects. & 2013 Elsevier B.V. All rights reserved. Keywords: Action potential duration Antipsychotic drug hERG channel Fluphenazine Rapidly-activating delayed rectifier K þ channel 1. Introduction A number of non-cardiac drugs have been withdrawn from the market (e.g. terfenadine, cisapride, sertindole, grepafloxacin, thioridazine) or have been labeled for restricted use (e.g. mesoridazine, ziprasidone, droperidol, astemizol, arsenic trioxide) because of their potential proarrhythmic effects. Therefore, screening compounds for hERG and/or QT interval liability is now routine in the pharmaceutical industry (Su et al., 2006). hERG channel blockade has recently become a major topic in pharmacological research (Redfern et al., 2003). Previous studies have indicated a higher n Corresponding author. Tel.: þ82 2 3010 4292. Corresponding author. Tel.: þ 82 63 270 4250. Corresponding author. Tel.: þ 82 33 250 8824; fax: þ 82 33 255 8809. E-mail addresses: [email protected] (H. Choe), [email protected] (B. Hee Choi), [email protected] (S.-H. Jo). 1 These authors contribute equally to this article. nn nnn 0014-2999/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ejphar.2013.01.039 mortality rate from cardiovascular causes in patients treated with antipsychotics than in the general population (Ozeki et al., 2010). Long QT syndrome is associated with syncope, polymorphic ventricular tachycardia, torsade de pointes, and sudden cardiac death (Ozeki et al., 2010). Many antipsychotic drugs have been associated with QT interval prolongation on the electrocardiogram, which may be a precursor of life-threatening arrhythmias (Chong et al., 2003). Antipsychotic drugs may cause serious cardiovascular side effects, including myocarditis, cardiomyopathy, and rhythm abnormalities (Stöllberger et al., 2005). Antipsychotic drugs prolong the QT interval by blocking the rapidly-activating delayed rectifier K þ current (IKr) (Sanguinetti et al., 1995). The QT interval is measured from the beginning of the QRS complex to the end of the T wave, which is defined as the point of return to the isoelectric line, in a standard 12-lead ECG, preferentially in lead II. Prolongation of the QT interval reflects an increase in action potential duration (APD) of ventricular cardiomyocytes. Delayed repolarization increases the risk of arrhythmias such as torsade de 166 H.-K. Hong et al. / European Journal of Pharmacology 702 (2013) 165–173 pointes (Sanguinetti and Tristani-Firouzi, 2006). Drug-induced prolongation of cellular APD and torsades de pointes ventricular arrhythmia are often caused by high affinity block of the IKr (Tamargo, 2000), which is one of the most important membrane currents responsible for ventricular action potential repolarization. The human ether-a-go-go-related gene (hERG) encodes the pore-forming subunits of the rapidly-activating delayed rectifier K þ channel in the heart (Sanguinetti et al., 1995), and several antipsychotics have been known to block the hERG channel (Osypenko et al., 2001). Fluphenazine, a phenothiazine derivative, is a neuroleptic drug used to treat psychoses such as schizophrenia and manic disorders (Iqbal et al., 2005). Fluphenazine has been significantly associated with myocarditis and cardiomyopathy (Coulter et al., 2001). As for the effect of the drug on cardiac rhythmicity, fluphenazine was identified as significant predictor for QT prolongation (Chong et al., 2003; Turbott et al., 1987) and is known to induce torsades de pointes (Crouch et al., 2003). However, the biophysical properties and molecular determinants for the fluphenazine block of the hERG channel have not been reported. In this study, we examined the possible fluphenazine block of hERG channels expressed in Xenopus oocytes and HEK cells as well as the biophysical properties and molecular determinants of the fluphenazine block using a mutant channel. Finally, a virtual docking simulation was carried out to understand the blocking mode of the hERG channel by fluphenazine using the KvAP channel structure as a template. 2. Materials and methods 2.1. Ventricular myocyte isolation Single ventricular myocytes were isolated from each guinea pig heart using a method described previously (Jo et al., 2009). Guinea pigs (300–500 g) were anesthetized with pentobarbital ( 50 mg/kg, i.p.). The heart was quickly excised and retrogradely perfused at 37 1C with solution A containing 750 mM Ca2 þ and a Ca2 þ -free solution A followed by an enzyme solution. The enzyme solution contained solution A, 150 mM Ca2 þ , collagenase type II, and protease type XIV. Solution A contained (mM): 130 NaCl, 4.5 KCl, 21 glucose, 2.5 MgCl2, 1 NaH2PO4, 20 taurine, 5 creatine, and 23 HEPES (pH 7.2). The heart was then flushed with a 150 mM Ca2 þ solution. The ventricles were removed and chopped into small pieces, which were then shaken in a flask containing a 150 mM Ca2 þ solution. The cell suspension was left to sediment. The supernatant was replaced with a 500 mM Ca2 þ solution. The cells were kept at room temperature. This study was performed according to the Research Guidelines of Kangwon National University IACUC. 2.2. Solutions, action potential recording from ventricular myocytes Isolated ventricular myocytes in the experimental chamber were continuously superfused and their action potentials were elicited at 0.33 Hz by current clamping and recorded by a method previously described (Jo et al., 2009). We analyzed the parameters of AP including resting membrane potential (RMP), action potential amplitude (APA), AP duration at 20% (APD20), 50% (APD50) and 90% (APD90) of repolarization. The antipsychotic drug fluphenazine and all reagents were purchased from Sigma (St. Louis, MO, USA). A stock solution of fluphenazine was prepared in distilled water and added to the external solutions at suitable concentrations shortly before each experiment. Data acquisition was performed with a digital computer, analog data acquisition equipment (National Instruments, Austin, TX, USA), and the online software WCP (written and supplied by John Dempster of Strathclyde University). 2.3. Expression of hERG in oocytes hERG (accession no. U04270) cRNA was synthesized by in vitro transcription from 1 mg of linearized cDNA using T7 message machine kits (Ambion, Austin, TX, USA) and stored in 10 mM Tris–HCl (pH 7.4) at 80 1C. Stage V–VI oocytes were surgically removed from female Xenopus laevis (Nasco, Modesto, CA, USA) and anesthetized with 0.17% tricane methanesulfonate (Sigma, St. Louis, MO, USA). Using fine forceps, the theca and follicle layers were manually removed from the oocytes, and then each oocyte was injected with 40 nL of cRNA (0.1–0.5 mg/ml). The injected oocytes were maintained in a modified Barth’s Solution. The modified Barth’s Solution contained (mM): 88 NaCl, 1 KCl, 0.4 CaCl2, 0.33 Ca(NO3)2, 1 MgSO4, 2.4 NaHCO3, 10 HEPES (pH 7.4), and 50 mg/ml gentamicin sulfonate. Currents were studied two to seven days after injection. This study was performed according to the Research Guidelines of Kangwon National University IACUC. 2.4. Solutions and voltage-clamp recordings from oocytes Normal Ringer’s Solution contained 96 mM NaCl, 2 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, and 10 mM HEPES (pH adjusted to 7.4 with NaOH). A stock solution of fluphenazine was prepared in distilled water and added to the external solutions at suitable concentrations shortly before each experiment. Solutions were applied to oocytes by continuous perfusion of the chamber while recording. Solution exchanges were completed within 3 min, and the hERG currents were recorded 5 min after the solution exchange. Currents were measured at room temperature (20– 23 1C) with a two-microelectrode voltage-clamp amplifier (Warner Instruments, Hamden, CT, USA). Electrodes were filled with 3 M KCl and had a resistance of 2–4 MO for voltage-recording electrodes and 0.6–1 MO for current-passing electrodes. Stimulation and data acquisition were controlled with an AD–DA converter (Digidata 1200, Axon Instruments) and a pCLAMP software (v 5.1, Axon Instruments). 2.5. HEK cell culture and whole-cell patch recording HEK293 cells stably expressing hERG channels, a kind gift from Dr. C. January (Zhou et al., 1998) were used for electrophysiological recordings. The method for establishing hERG channels expression in HEK293 cells is briefly described as follows: hERG cDNA was transferred into the plasmid expression vector pCDNA3 vector (Invitrogen Corporation, San Diego, CA, USA). HEK293 cells were stably transfected with hERG cDNA using the calcium phosphate precipitate method (Invitrogen) or lipofectamine (Invitrogen). The transfected cells were cultured in minimum essential medium (MEM) supplemented with 10% fetal bovine serum, 1 mM sodium pyruvate, 0.1 mM non-essential amino-acid solution, 100 units/ml penicillin, 100 mg/ml streptomycin sulfate. The cultures were passaged every 4–5 days with a brief trypsin-EDTA treatment followed by seeding onto glass coverslips (diameter: 12 mm, Fisher Scientific, Pittsburgh, PA, USA) in a Petri dish. After 12–24 h, the cell-attached coverslips were used for electrophysiological recordings. hERG currents were recorded from HEK293 cells, with the wholecell patch-clamp technique (Hamill et al., 1981) at room temperature (22–23 1C). The micropipettes fabricated from glass capillary tubing (PG10165-4; World Precision Instruments, Sarasota, FL, USA) with a double-stage vertical puller (PC-10; Narishige, Tokyo) had a tip resistance of 2–3 MO when filled with the pipette solution. Wholecell currents were amplified with Axopatch 1D amplifier (Molecular H.-K. Hong et al. / European Journal of Pharmacology 702 (2013) 165–173 Devices, Sunnyvale, CA, USA), digitized with Digidata 1200 A (Molecular Devices) at 5 kHz and low-pass filtered with a four-pole Bessel filter at 2 kHz. Capacitive currents were canceled and series resistance was compensated at 80% with the amplifier, while leak subtraction was not used. The generation of voltage commands and acquisition of data were controlled with pClamp 6.05 software (Molecular Devices) running on an IBM-compatible Pentium computer. The recording chamber (RC-13, Warner Instrument Corporation, Hamden, CT, USA) was continuously perfused with a bath solution (see below for composition) at a rate of 1 ml/min. The external solution contained 137 mM NaCl, 4 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, 10 mM glucose and 10 mM HEPES (adjusted to pH 7.4 with NaOH). The intracellular solution contained 130 mM KCl, 1 mM MgCl2, 5 mM EGTA, 5 mM MgATP and 10 mM HEPES (adjusted to pH 7.4 with KOH). Azelastine (Enzo Life Sciences) was dissolved in distilled water to make a 30 mM stock solution and further diluted in the bath solution. 2.6. Pulse protocols and analysis To obtain concentration–response curves in the presence of fluphenazine, dose–dependent inhibition was fitted with the equation: h n i Itail ¼ Itailmax = 1 þ IC50 =D , where Itail indicates peak tail currents, Itailmax is the maximum peak tail current, D is the concentration of the small molecule, n is the Hill coefficient, and IC50 is the concentration at which the half-maximal peak tail currents were inhibited. 2.7. Virtual docking The three-dimensional structure of fluphenazine was built + using LigPrep (Schrodinger, LLC, New York, NY). The virtual docking of fluphenazine to the hERG channel was performed by + Glide (Schrodinger, LLC, New York, NY). PoseView (BioSolveIT GmbH, Sankt Augustin, Germany) was used to investigate the interactions of the fluphenazine molecule with the hERG channel. All structural figures were prepared using PyMOL v1.2 (DeLano Scientific LLC, San Francisco, CA) and ChemDraw Ultra 11.0 (CambridgeSoft, Cambridge, MA). 2.8. Statistical evaluations All data are expressed as mean7S.E.M. Unpaired or paired Student t tests, or ANOVA were used for statistical comparisons when appropriate, and differences were considered significant at Po0.05. 3. Results 3.1. Effects of fluphenazine on action potentials in Guinea pig ventricular myocytes We examined the effect of fluphenazine on the action potential in isolated guinea pig ventricular myocytes. Fig. 1A shows superimposed traces of the action potentials recorded before and during exposure to different concentrations of fluphenazine for 10 min. Fluphenazine at concentrations of 0.1–1.0 mM increased the APD90 and APD50 of guinea pig ventricular myocytes (Fig. 1B, C) within 5 min. The APD90 and APD50 increased by 15.173.4 and 16.574.6% after 0.1 mM fluphenazine treatment for 5 min (n¼ 4–5, Po0.05). However, fluphenazine at the same concentrations did not change APD20, APA, and the RMP even after exposure of 10 mM fluphenazine for 10 min (n¼4–5, Po0.05; Fig. 2D, E, and F, respectively). 167 3.2. Concentration dependence of WT hERG channel block by fluphenazine in Xenopus oocytes Next, we examined the effect of fluphenazine on the hERG currents using a Xenopus oocyte expression system. Throughout these experiments, the holding potential was maintained at 70 mV, and tail currents (Itail) were recorded at 60 mV after depolarizing pulses from 50 to þ40 mV. Fig. 2A gives an example of a voltage-clamp recording from a Xenopus oocyte and the representative current traces both under the control conditions and after exposure to 30 mM fluphenazine. The amplitude of the outward currents measured at the end of the pulse (IhERG) increased with increasing positive voltage steps, reaching a maximum at 10 mV. The amplitude of IhERG was normalized to the maximum amplitude of the IhERG obtained under control conditions and was plotted against the potential of the step depolarization (IhERG, nor, Fig. 2B). The amplitude of IhERG, nor showed a concentrationdependent decrease with increasing fluphenazine concentration. After the depolarizing steps, repolarization to 60 mV induced an outward Itail, which had an amplitude even greater than that of IhERG during depolarization, which is due to rapid recovery from inactivation and a slow deactivation mechanism (Zhou et al., 1998). When 10 mM fluphenazine was added to the perfusate, both IhERG and Itail were reduced (Fig. 2A, bottom panel). The amplitude of Itail was normalized to the peak amplitude obtained under the control conditions at the maximum depolarization and was plotted against the potential of the step depolarization (Fig. 2C). The data obtained under the control conditions were well-fitted by the Boltzmann Equation with a half-maximal activation (V1/2) at 19.2 mV. The peak Itail amplitude decreased with increasing fluphenazine concentration (41 mM), which indicates that the maximum conductance of the hERG channels is decreased by fluphenazine. In addition, in the presence of fluphenazine, Itail does not reach the steady-state level but decreases at more positive potentials, indicating that the blockade is more pronounced at the positive potentials. The values shown in Fig. 2C were normalized to the respective maximum values at each concentration to determine if fluphenazine shifts the activation curve (Fig. 2D). The V1/2 calculations are consistent with this finding, yielding values of 19.270.11, 20.370.36, 20.770.58, 21.770.71, 22.470.95 and 24.872.55 mV in the control and 0.3, 1, 3, 10 and 30 mM fluphenazine-treated groups, respectively (n¼10, P40.05). Therefore, the V1/2 values in the presence of 0.3–30 mM fluphenazine were similar, indicating that fluphenazine does not alter the activation gating at this concentration range. 3.3. Voltage-dependent block of WT hERG channel by fluphenazine The fluphenazine-induced decrease in Itail at different potentials was compared in order to determine if the effect of fluphenazine was voltage-dependent (Fig. 3A). Dose–response relationships were constructed at þ40, þ10 and –20 mV. The percentage inhibition in the hERG current by 10 mM fluphenazine at 20, 0, þ20, and þ40 mV was 36.3%713.1%, 51.7%710.4%, 56.2%7 10.7%, and 58.4%710.6%, respectively (n¼5; Fig. 3B). This suggests that the fluphenazine-induced blockade of the hERG currents progressively increases with increasing depolarization. 3.4. Time-dependence of WT hERG channel block by fluphenazine The currents were activated using a protocol containing a single depolarizing step to 0 mV for 8 s to determine if the channel was blocked in the closed or activated (i.e. open and/or inactivated) state (Fig. 4A). After obtaining the control measurement, 10 mM fluphenazine was applied and the recordings were made. Fig. 4B shows the degree of inhibition (i.e. (1-fluphenazine current/control current) 100 (in %)). Analysis of the test pulse after the application 168 H.-K. Hong et al. / European Journal of Pharmacology 702 (2013) 165–173 Fig. 1. Effects of fluphenazine exposure on action potential of guinea pig ventricular myocytes. (A) Representative traces of ECG in the absence of fluphenazine and in the presence of 0.1 and 1 mM fluphenazine for 10 min. (B)–(F) The effects of fluphenazine on action potential duration at 90% of repolarization (APD90), action potential duration at 50% of repolarization (APD50), action potential duration at 20% of repolarization (APD20), action potential amplitude (APA), and resting membrane potential (RMP), respectively. Bars with error bars represent mean 7 S.E.M. (n¼4–5). nPo 0.05. of fluphenazine revealed a time-dependent increase in blockage in this representative cell to 40% at 2 s (Fig. 4B). At the beginning of the pulse, the fractional sustained current, which was obtained by normalizing the currents with fluphenazine relative to control currents, was 0.9470.01 of the control (n¼ 9). This indicates that the hERG channels were only slightly blocked by fluphenazine while remaining at the holding potential. In this series of experiments, 10 mM fluphenazine reduced the hERG outward currents at the end of the 0 mV pulse by 39.3%74.9% (n¼9). In order to address whether hERG channels are also blocked by fluphenazine in their inactivated state, a long test pulse to þ80 mV (4 s) was applied to inactivate the channels, which was followed by a second voltage step (0 mV, 3.5 s) to open the hERG channels (n¼9). Fig. 4C shows typical current traces under the control conditions and after the application of 10 mM fluphenazine. Fig. 4D shows the normalized relative blockage upon channel opening during the second voltage pulse (0 mV), indicating that the pronounced inhibition of the hERG channels had already been reached during the previous inactivating þ80 mV pulse. No additional time-dependent blockage of the open channels was observed during the 0 mV pulse. The currents at the end of the second voltage step (0 mV) were decreased by 40.9%75.6% (n¼9). Overall, fluphenazine inhibits the hERG channels mainly in the open and inactivated state rather than in the closed state. 3.5. Fluphenazine block of WT hERG currents expressed in HEK cells The IC50 values of many hERG channel blockers have been shown to differ depending on whether the hERG channels are expressed in Xenopus oocytes or mammalian cells, an effect probably due to the sequestration of blockers in the large ooplasm of oocytes. We therefore tested the effects of fluphenazine in H.-K. Hong et al. / European Journal of Pharmacology 702 (2013) 165–173 +40 1.0 -60mV -70 0.8 0.6 Control 169 [Fluphenazine] ( M) Control 0.3 1 3 10 30 0.4 1 A 0.2 0.0 10 M Fluphenazine 1 A 1.2 0.9 0.6 -60 [Fluphenazine] ( M) Control 0.3 1 3 10 30 1.0 0.8 0.6 0.4 0.3 -20 0 20 40 [Fluphenazine] ( M) Control 0.3 1 3 10 30 0.2 0.0 0.0 -60 -40 -0.2 -40 -20 0 20 40 -60 -40 -20 0 20 40 Fig. 2. The effect of fluphenazine on human-ether-a-go-go-related gene (hERG) currents (IhERG) elicited by depolarizing voltage pulses. (A) Superimposed current traces elicited by depolarizing voltage pulses (4 s) in 10 mV steps (upper panel) from a holding potential of 70 mV in the absence of fluphenazine (control, center panel) and in the presence of 10 mM fluphenazine (lower panel). (B) Plot of the normalized hERG current measured at the end of depolarizing pulses (IhERG, nor) against the pulse potential in the control and fluphenazine conditions. The maximal amplitude of the IhERG in the control was given a value of 1. (C) Plot of the normalized tail current measured at its peak just after repolarization. The peak amplitude of the tail current in the absence of the drug was set as 1. Control data were fitted to the Boltzmann Equation, y¼ 1/{1þ exp[( V þV1/2)/dx]}, with V1/2 of 19.2 mV. (D) Activation curves with values normalized to the respective maximum value at each concentration of fluphenazine. Symbols with error bars represent mean 7S.E.M. (n¼ 10). HEK293 cells expressing hERG channels at 36 1C (Fig. 5). As shown in Fig. 5A, whole-cell currents were elicited with 4 s depolarization to þ20 mV from a holding potential of 80 mV, and the tail current was recorded at 60 mV for 6 s. Bath-applied fluphenazine reduced the IhERG in a concentration-dependent manner. As shown in Fig. 5B, dose dependency of the steady-state currents measured at the end-pulse of þ20 mV or peak tail currents was analyzed quantitatively. A nonlinear least-squares fit of dose– response plots with the Hill equation yielded an IC50 value of 0.102 mM for the peak tail currents (n ¼3). These results indicate that the fluphenazine-induced inhibition of hERG channels stably expressed in HEK293 cells occurred at a concentration 100 times lower concentration than that required by hERG channels expressed in Xenopus oocytes. mutant channels. This indicates that the Tyr-652 mutant failed to attenuate the levels of block compared with that of wild-type hERG. 3.6. S6 domain mutation, Y652A, attenuate hERG channel block by fluphenazine 4. Discussion Tyr-652 is located in S6 domain, faces the pore cavity of the hERG channel, and is an important component of the binding site of a number of compounds (Mitcheson et al., 2000). The potency of a channel block for the wild type and mutant hERG channel (Y652A) were compared in order to determine if the key residue is also important in the fluphenazine-induced blocking of the hERG channel using Xenopus oocytes injected with cRNA of Y652A. As shown in Fig. 6, the IC50 values for WT hERG channels were 13.375.6 mM (n¼4), and the value increased up to 89.07152 mM (n¼4) in Y652A 3.7. Virtual docking simulation for the binding of fluphenazine to the hERG channel To understand the binding mode of fluphenazine to the hERG channel, we performed virtual docking of the drug in the binding pocket of the hERG channel, using the Glide program. Our docking result is shown in Fig. 7A detailed analysis of the receptor–ligand interaction using the PoseView program showed that fluphenazine interacts with Y652 and F656, and also that the protonated nitrogen of fluphenazine forms a hydrogen bond with the carbonyl oxygen of residue T623. Fluphenazine is a potent phenothiazine antipsychotic that was introduced into clinical practice in the 1950s (Darling, 1959). In addition to its neuroleptic action, it has a high affinity for both D1 and D2 dopamine receptor subtypes (Morgan and Finch, 1986), which provides a mechanistic basis for the antipsychotic effects of the drug but also confers extrapyramidal symptoms (Levinson et al., 1990). In addition, fluphenazine has been shown to increase the incidence of dilated ventricles of the CNS, skeletal defects, reduction in fetal weight (Shepard, 1992), and diabetes (Vucicevic et al., 2007). In cardiac side effects, fluphenazine is significantly 170 H.-K. Hong et al. / European Journal of Pharmacology 702 (2013) 165–173 0.5 A 0.5 A 0.5 A Control 10 M Fluphenazine 2s -70 2s -20 2s +40 +10 -60 mV -70 -60 mV -70 -60 mV 80 60 40 20 +40 +20 0 -20 0 Fig. 3. Voltage dependence of fluphenazine-induced hERG current blockade. (A) Current traces from a cell depolarized to 20 mV (left panel), þ 10 mV (middle panel) and þ40 mV (right panel), before and after exposure to 10 mM fluphenazine, showing increased blockade of hERG current at the more positive potential. The protocol consisted of 4 s depolarizing steps to 20, þ10 or þ40 mV from a holding potential of 70 mV, followed by repolarization to 60 mV. (B) Fluphenazine-induced hERG current inhibition at different voltages. At each depolarizing voltage step ( 20, 0, þ 20 or þ 40 mV), the tail currents in the presence of 10 mM fluphenazine were normalized to the tail current obtained in the absence of drug. Bars with error bars represent mean 7 S.E.M. (n¼ 5). Fluphenazine 100 nA 1s 0 Current inhibition (%) 1.2 Control -70 1.0 0.8 0.6 0.4 0.2 0.0 0 20000 40000 60000 80000 Time (ms) Control Fluphenazine 0.5 A 1S +80 0 Current inhibition (%) 100 -70 80 60 40 20 0 0 1000 2000 3000 4000 Time (ms) Fig. 4. Blocking of activated hERG channels by fluphenazine. (A) An original recording of currents under control conditions (control) and after exposure to 10 mM fluphenazine (for 7 min, without any intermittent test pulse). (B) The degree of hERG current inhibition in percentages (%). Current inhibition increased time-dependently to 40% at 2 s in this representative cell, indicating that mostly open and/or inactivated channels were blocked. (C) Inhibition of inactivated channels by 10 mM fluphenazine. hERG channels were inactivated by a first voltage-step to þ 80 mV, followed by channel opening at 0 mV. (D) The corresponding relative block during the 0 mV step is displayed. Maximum inhibition was achieved in the inactivated state during the first step, and no further time-dependent blockage occurred upon channel opening during the second voltage step. associated with myocarditis and cardiomyopathy (Coulter et al., 2001), and the drug inhibits the pyruvate dehydrogenase complex from bovine hearts (Sacks et al., 1991). As for the effect of the drug on cardiac rhythmicity, the drug was identified as significant predictor for QT prolongation (Chong et al., 2003; Turbott et al., 1987) and is known to induce the life-threatening torsades de pointes (Crouch et al., 2003); however, the underlying mechanisms have not been examined. H.-K. Hong et al. / European Journal of Pharmacology 702 (2013) 165–173 171 0 -70 4S -14 0 mV Y652A Wild Type 30 M Fluphenazine 30 M Fluphenazine Control 2 A 1 A 0.2 S Control 0.2 S 0.6 Wild Type Y652A 0.4 0.2 0.0 0.1 Fig. 5. Concentration dependence of fluphenazine-induced inhibition of hERG channels stably expressed in HEK293 cells. (A) Superimposed IhERG traces were elicited with 4-s depolarizations to þ 20 mV from a holding potential of 80 mV, and the tail current was recorded at 60 mV for 6 s in the absence and presence of 0.1, 0.3, 1, and 3 mM fluphenazine, as indicated. The protocol was applied every 15 s. (B) Concentration-dependent curve of inhibition by fluphenazine for peak tail currents (closed circles). The respective normalized currents were plotted against various concentrations of fluphenazine. The solid line is fitted to the data points by the Hill equation. Our results show that fluphenazine blocked hERG channels, which mediates the important hyperpolarizing current during action potential, IKr, with an IC50 of 0.102 mM (Fig. 5). Concomitantly, fluphenazine at 0.1 mM prolonged the APD90 and APD50 by 15.1% and 16.5%, respectively, without changing APD20 after a 5 min treatment (Fig. 1). The mutation Y652A in the S6 domain of the hERG channel attenuated the hERG current block by 7 folds (Fig. 6). Therefore the present study suggests that fluphenazineinduced cardiac arrhythmia, such as QT prolongation and torsades de pointes, could be caused by the drug-induced block of the hERG channel and that fluphenazine predominantly binds to drug binding site within the pore-S6 domain. The effective systemic doses of fluphenazine for the treatment of schizophrenia and manic disorders range from 2 to 40 mg daily and the elimination half-life was estimated to be 10–20 h (Iqbal et al., 2005). Responders showed the greatest improvement at fluphenazine plasma levels above 1.0 ng/ml ( 2 nM) and doses above 0.20–0.25 mg/kg per day (Levinson et al., 1990). However, all formulations of fluphenazine show a very high volume of distribution (Hubbard et al., 1999) with significant tissue accumulation; the concentration of fluphenazine is 20 to 54-fold higher in peripheral tissues than in plasma (Aravagiri et al., 1995; Baldessarini and Tarazi, 2001). Therefore, the possible accumulated concentration of fluphenazine may be assumed to be in the range from 40 to 108 nM in the heart, which is 1 10 100 Fig. 6. Concentration-dependent inhibition of WT and mutant hERG channels expressed in oocytes. (A) Representative traces for WT and mutant hERG channel currents in the presence and absence of indicated concentrations of fluphenazine. The effect of the drug on WT and Y652A was tail currents were recorded at 140 mV instead of 60 mV after the 4 s activating pulses. (B) The concentration–response curves were fitted with a logistic dose–response equation to obtain the IC50 values of 13.3 75.6 mM (n¼ 4), and 89.0 7152 mM (n ¼4) in WT (obtained using protocol of panel A), and Y652A hERG channels, respectively. Data were expressed as mean 7 S.E.M. comparable with our findings that fluphenazine at 0.1 mM prolonged APD90 and blocked hERG channels effectively. In this context, fluphenazine at the clinically relevant concentrations could induce QT prolongation and torsades de pointes in ECGs by blocking hERG channels in these patients (Chong et al., 2003; Crouch et al., 2003; Turbott et al., 1987). Fluphenazine has been shown to modulate various kinds of ion channels, particularly in neuronal cells. In sympathetic neurons, fluphenazine blocked N-type Ca2 þ channels (Sah and Bean, 1994). Fluphenazine is a state- and use-dependent Na þ channel blocker in neuronal cell line (Zhou et al., 2006) and is able to inhibit native Na þ current in peripheral sensory neurons (Dong et al., 2008). In pancreatic b-cell line, fluphenazine inhibited the ATP-sensitive K þ (KATP) channel with an IC50 value of 6 mM, which could affect insulin secretion (Müller et al., 1991). Also, fluphenazine has been known to block the human small conductance calcium-activated K þ channel (hSK3) channels, which contribute to the after hyperpolarization that follows action potential and neuronal cell firing, with an IC50 value of 13 mM (Terstappen et al., 2001). As for the effect of fluphenazine on the electrophysiology in cardiomyocytes, Kremers et al. (1985) showed that fluphenazine at 10 mM shifted the action potential plateau to a more negative voltage and increased the rate of phase 2 repolarization without changing APD in rabbit ventricular myocytes, possibly by drug-induced Ca2 þ -calmodulin inhibition. However, fluphenazine at 10 mM, which is a concentration 100 times higher than our experimental conditions, can cause nonspecific effects because phenothiazines are not specific for 172 H.-K. Hong et al. / European Journal of Pharmacology 702 (2013) 165–173 Fig. 7. Docking of fluphenazine within the inner cavity of a hERG channel homology model. (A) Inner view of the fluphenazine docked to the ligand binding site in hERG channel. Fluphenazine is shown in stick form and dotted line means a hydrogen bond between the blocker and receptor. The hERG channel inhibitor, fluphenazine, shows interactions with THR623 (subunit B), TYR652 and PHE656. (B) PoseView analysis for protein–ligand interactions. Hydrogen bonding is denoted as a dotted line between the participating atoms. The green lines with residue name and number indicate hydrophobic interactions between the ligand and receptor. Note the hydrogen bond between the hydrogen atom of the protonated nitrogen (fluphenazine) and the carbonyl oxygen atom of THR623B (hERG). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) calmodulin and have stabilizing effects on cell membranes due to their hydrophobic properties (Silver and Stull, 1983; Seeman, 1977). Our present results support that the direct inhibition of hERG by fluphenazine would be a major cause of APD prolongation because the pore mutation (Y652A) of the channel protein significantly attenuated the drug-induced hERG block (Fig. 6). Many of the drugs that block hERG channels do so in a voltagedependent manner, which suggests that these drugs bind to the open or inactivated state of hERG channels (Osypenko et al., 2001; Rampe et al., 1997; Suessbrich et al., 1996). Our result shows that fluphenazine decreased the amplitudes of the maximum outward current and maximum peak tail current (Fig. 2). The magnitude of the block increased with increasing positive voltage, which increased the open probability and enhanced inactivation (Fig. 3). In addition, fluphenazine block may be state-dependent: hERG channels are blocked mainly in the open and inactivated states but not in the closed state (Fig. 4). Finally, fluphenazine did not significantly alter the V1/2 values of the activation curve, which suggests that the drug blocks hERG channels without altering the activation properties (Fig. 2). The properties of open channel block and voltage dependence of fluphenazine could be comparable to other antipsychotics’ action on hERG channel such as haloperidol, pimozide, and fluspirilen (Osypenko et al., 2001). Also, the properties might result in increased block at higher heart rates although our results are limited in that the drug increased APD90 at an unphysiologically low stimulation rate of 0.33 Hz (Fig. 1). Another view of the voltage dependence of a fluphenazine block of the hERG channels is that the drug may increase the APD and make the heart more prone to arrhythmia, particularly under the pathological conditions associated with rapid heart actions and partially depolarized membranes. Two amino acids, Y652 and F656, were identified to be important for the blocking of the hERG channel using an alanine-scanning mutagenesis study (Mitcheson et al., 2000). It was suggested that a protonated nitrogen of hERG channel inhibitors would form a cation–p interaction with the aromatic ring of hERG residue Y652 and a hydrophobic interaction with hERG residue F656 (Sanguinetti et al., 2005). However we have proposed that that the protonated nitrogen of a blocker makes a hydrogen bond with the carbonyl oxygen of residue T623 or with the hydroxyl oxygen of S624, an aromatic moiety of the blocker makes a p–p interaction with the aromatic ring of residue Y652, and a hydrophobic group of the blocker makes a hydrophobic interaction with the benzene ring of residue F656 (Choe et al., 2006). Therefore the docking result of fluphenazine to the hERG channel is consistent with the model that we have proposed. In conclusion, we found that the antipsychotic drug fluphenazine directly prolongs APD and inhibits hERG channels in voltageand time-dependent manner at submicromolar concentration, which can make cardiac cells prone to be arrhythmogenic. The block was shown to occur at the pore of the channel using a pore mutant channel and molecular simulation. Therefore, the present study provides the molecular mechanism for fluphenazineinduced QT prolongation and fatal cardiac arrhythmias such as torsades de pointes during treatment. Acknowledgments This study was supported by a grant (08172KFDA465) from the Korea Food & Drug Administration and a grant (20110013171) from Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology. References Aravagiri, M., Marder, S.R., Yuwiler, A., Midha, K.K., Kula, N.S., Baldessarini, R.J., 1995. Distribution of fluphenazine and its metabolites in brain regions and other tissues of the rat. Neuropsychopharmacology 13, 235–247. Balsessarini, R.J., Tarazi, F.I., 2001. Drugs and the treatment of psychiatric disorders. In: Hardman, J.G., Limbird, L.E., Gilman, A.G. (Eds.), Goodman and Gilman’s the Pharmacological Basis of Therapeutics, McGraw-Hill, NY, 2001, pp. 485–520. Choe, H., Nah, K.H., Lee, S.N., Lee, H.S., Jo, S.H., Leem, C.H., Jang, Y.J., 2006. A novel hypothesis for the binding mode of hERG channel blockers. Biochem. Biophys. Res. Commun. 344, 72–78. Chong, S.A., Mythily, Lum A., Goh, H.Y., Chan, Y.H., 2003. Prolonged QTc intervals in medicated patients with schizophrenia. Hum. Psychopharmacol. 18, 647–649. Coulter, D.M., Bate, A., Meyboom, R.H., Lindquist, M., Edwards, I.R., 2001. Antipsychotic drugs and heart muscle disorder in international pharmacovigilance: data mining study. BMJ 322, 1207–1209. Crouch, M.A., Limon, L., Cassano, A.T., 2003. Clinical relevance and management of drug-related QT interval prolongation. Pharmacotherapy, 881–908. H.-K. Hong et al. / European Journal of Pharmacology 702 (2013) 165–173 Darling, H.F., 1959. Fluphenazine: a preliminary study. Dis. Nerv. Syst. 20, 167–170. Dong, X.W., Jia, Y., Lu, S.X., Zhou, X., Cohen-Williams, M., Hodgson, R., Li, H., Priestley, T., 2008. The antipsychotic drug, fluphenazine, effectively reverses mechanical allodynia in rat models of neuropathic pain. Psychopharmacology (Berl) 195, 559–568. Hamill, O.P., Marty, A., Neher, E., Sakmann, B., Sigworth, F.J., 1981. Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflugers Arch. 391, 85–100. Hubbard, J.W., Hadad, S., Luo, J.P., McKay, G., Midha, K.K., 1999. Pharmacokinetics of fluphenazine, a highly lipophilic drug, estimated from a pulse dose of a stable isotopomer in dogs at steady state. J. Pharm. Sci. 88, 918–921. Iqbal, M.M., Aneja, A., Rahman, A., Megna, J., Freemont, W., Shiplo, M., Nihilani, N., Lee, K., 2005. The potential risks of commonly prescribed antipsychotics: during pregnancy and lactation. Psychiatry (Edgmont) 2, 36–44. Jo, S.H., Hong, H.K., Chong, S.H., Lee, H.S., Choe, H., 2009. H(1) antihistamine drug promethazine directly blocks hERG K( þ) channel. Pharmacol. Res. 60, 429–437. Kremers, M.S., Kenyon, J.L., Ito, K., Sutko, J.L., 1985. Phenothiazine suppression of transient depolarizations in rabbit ventricular cells. Am. J. Physiol. 248, H291–H296. Levinson, D.F., Simpson, G.M., Singh, H., Yadalam, K., Jain, A., Stephanos, M.J., Silver, P., 1990. Fluphenazine dose, clinical response, and extrapyramidal symptoms during acute treatment. Arch. Gen. Psychiatry 47, 761–768. Mitcheson, J.S., Chen, J., Lin, M., Culberson, C., Sanguinetti, M.C., 2000. A structural basis for drug-induced long QT syndrome. Proc. Natl. Acad. Sci. USA 97, 12329–12333. Morgan, D.G., Finch, C.E., 1986. [3H]Fluphenazine binding to brain membranes: simultaneous measurement of D-1 and D-2 receptor sites. J. Neurochem. 46, 1623–1631. Müller, M., De Weille, J.R., Lazdunski, M., 1991. Chlorpromazine and related phenothiazines inhibit the ATP-sensitive K þ channel. Eur. J. Pharmacol. 198, 101–104. Osypenko, V.M., Degtiar, Vie., Naid’onov, V.G., Shuba, IaM., 2001. Blockade of HERG K þ channels expressed in Xenopus oocytes by antipsychotic agents. Fiziol. Zh. 47, 17–25. Ozeki, Y., Fujii, K., Kurimoto, N., Yamada, N., Okawa, M., Aoki, T., Takahashi, J., Ishida, N., Horie, M., Kunugi, H., 2010. QTc prolongation and antipsychotic medications in a sample of 1017 patients with schizophrenia. Prog. Neuropsychopharmacol. Biol. Psychiatry 34, 401–405. Rampe, D., Roy, M.L., Dennis, A., Brown, A.M., 1997. A mechanism for the proarrhythmic effects of cisapride (Propulsid): high affinity blockade of the human cardiac potassium channel hERG. FEBS Lett. 417, 28–32. Redfern, W.S., Carlsson, L., Davis, A.S., Lynch, W.G., MacKenzie, I., Palethorpe, S., Siegl, P.K., Strang, I., Sullivan, A.T., Wallis, R., Camm, A.J., Hammond, T.G., 2003. Relationships between preclinical cardiac electrophysiology, clinical QT interval prolongation and torsade de pointes for a broad range of drugs: evidence 173 for a provisional safety margin in drug development. Cardiovasc. Res. 58, 32–45. Sacks, W., Esser, A.H., Sacks, S., 1991. Inhibition of pyruvate dehydrogenase complex (PDHC) by antipsychotic drugs. Biol. Psychiatry 29, 176–182. Sah, D.W., Bean, B.P., 1994. Inhibition of P-type and N-type calcium channels by dopamine receptor antagonists. Mol. Pharmacol. 45, 84–92. Sanguinetti, M.C., Tristani-Firouzi, M., 2006. hERG potassium channels and cardiac arrhythmia. Nature 440, 463–469. Sanguinetti, M.C., Jiang, C., Curran, M.E., Keating, M.T., 1995. A mechanistic link between an inherited and an acquired cardiac arrhythmia: hERG encodes the IKr potassium channel. Cell 81, 299–307. Sanguinetti, M.C., Chen, J., Fernandez, D., Kamiya, K., Mitcheson, J., SanchezChapula, J.A., 2005. Physicochemical basis for binding and voltagedependent block of hERG channels by structurally diverse drugs. Novartis Found. Symp. 266, 159–166. Seeman, P., 1977. Anti-schizophrenic drugs—membrane receptor sites of action. Biochem. Pharmacol. 26, 1741–1748. Shepard, T.H., 1992. Catalog of Teratogenic Agents, 7th Ed. The John Hopkins University Press, Maryland. Silver, P.J., Stull, J.T., 1983. Effects of the calmodulin antagonist, fluphenazine, on phosphorylation of myosin and phosphorylase in intact smooth muscle. Mol. Pharmacol. 23, 665–670. Stöllberger, C., Huber, J.O., Finsterer, J., 2005. Antipsychotic drugs and QT prolongation. Int. Clin. Psychopharmacol. 20, 243–251. Su, Z., Chen, J., Martin, R.L., McDermott, J.S., Cox, B.F., Gopalakrishnan, M., Gintant, G.A., 2006. Block of hERG channel by ziprasidone: biophysical properties and molecular determinants. Biochem. Pharmacol. 71, 278–286. Suessbrich, H., Waldegger, S., Lang, F., Busch, A.E., 1996. Blockade of hERG channels expressed in Xenopus oocytes by the histamine receptor antagonists terfenadine and astemizole. FEBS. Lett. 385, 77–80. Tamargo, J., 2000. Drug-induced torsade de pointes: from molecular biology to bedside. Jpn. J. Pharmacol. 83, 1–19. Terstappen, G.C., Pula, G., Carignani, C., Chen, M.X., Roncarati, R., 2001. Pharmacological characterisation of the human small conductance calcium-activated potassium channel hSK3 reveals sensitivity to tricyclic antidepressants and antipsychotic phenothiazines. Neuropharmacology 40, 772–783. Turbott, J., Villiger, J., Hunter, L., 1987. Depot neuroleptic medication and serum levels by radioreceptor assay: prolactin concentration, electrocardiogram abnormalities and six-month outcome. Aust. NZ J. Psychiatry 21, 327–338. Vucicevic, Z., Degoricija, V., Alfirevic, Z., Vukicevic-Badouin, D., 2007. Fatal hyponatremia and other metabolic disturbances associated with psychotropic drug polypharmacy. Int. J. Clin. Pharmacol. Ther. 45, 289–292. Zhou, X., Dong, X.W., Priestley, T., 2006. The neuroleptic drug, fluphenazine, blocks neuronal voltage-gated sodium channels. Brain Res. 1106, 72–81. Zhou, Z., Gong, Q., Ye, B., Fan, Z., Makielski, J.C., Robertson, G.A., January, C.T., 1998. Properties of hERG channels stably expressed in HEK 293 cells studied at physiological temperature. Biophys. J. 74, 230–241.