Download Block of hERG K+ channel and prolongation of action potential

European Journal of Pharmacology 702 (2013) 165–173
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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
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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
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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
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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.