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3.1 potassium channels
European Journal of Pharmacology 854 (2019) 92–100
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Effects of cariprazine on hERG 1A and hERG 1A/3.1 potassium channels a
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Hong Joon Lee , Bok Hee Choi , Jin-Sung Choi , Sang June Hahn a b c
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a,∗
Department of Physiology, College of Medicine, The Catholic University of Korea, Seoul, 06591, South Korea Department of Pharmacology, Institute for Medical Science, Chonbuk National University Medical School, Jeonju, Jeonbuk, 54097, South Korea College of Pharmacy, Integrated Research Institute of Pharmaceutical, The Catholic University of Korea, Gyeonggi-do, 14662, South Korea
A R T I C LE I N FO
A B S T R A C T
Keywords: Cariprazine hERG 1A/3.1 heterotetramer Open channel block
Cariprazine is a novel atypical antipsychotic drug that is widely used for the treatment of schizophrenia and bipolar mania/mixed disorder. We used the whole-cell patch-clamp technique to investigate the effects of cariprazine on hERG channels that are stably expressed in HEK cells. Cariprazine inhibited the hERG 1A and hERG 1A/3.1 tail currents at −50 mV in a concentration-dependent manner with IC50 values of 4.1 and 12.2 μM, respectively. The block of hERG 1A currents by cariprazine was voltage-dependent, and increased over a range of voltage for channel activation. Cariprazine shifted the steady-state inactivation curve of the hERG 1A currents in a hyperpolarizing direction and produced a use-dependent block. A fast application of cariprazine inhibited the hERG 1A currents elicited by a 5 s depolarizing pulse to +60 mV to fully inactivate the hERG 1A currents. During a repolarizing pulse wherein the hERG 1A current was deactivated slowly, cariprazine rapidly and reversibly blocked the open state of the hERG 1A current. However, cariprazine did not affect hERG 1A and hERG 1A/3.1 channel trafficking to the cell membrane. Our results indicated that cariprazine concentration-dependently inhibited hERG 1A and hERG 1A/3.1 currents by preferentially interacting with the open states of the hERG 1A channel, but not by the disruption of hERG 1A and hERG 1A/3.1 channel protein trafficking. Our study examined cariprazine's mechanism of action provides a biophysical profile that is necessary to assess the potential therapeutic effects of this drug.
1. Introduction Cariprazine, a dopamine D3/D2 receptor partial agonist, is a novel atypical antipsychotic drug that was recently approved in the USA for the treatment of schizophrenia and bipolar mania/mixed disorder (Caccia et al., 2013; Citrome, 2013). Cariprazine caused fewer extrapyramidal side effects and was generally well tolerated with minimal adverse effects (European Medicines Agency, 2017; Scarff, 2016). Many drugs used to treat psychiatric patients, specifically antipsychotic drugs, have been implicated in QT interval prolongation that causes cardiac arrhythmia, such as torsades de pointes (TdP) (Pacher and Kecskemeti, 2004; Sicouri and Antzelevitch, 2008). For example, fluoxetine blocks hERG channels, which is essential to ventricular repolarization (Thomas et al., 2002). Accordingly, this drug can delay ventricular repolarization, thus inducing QT interval prolongation and triggering TdP (Rajamani et al., 2006). However, the most interesting advantage of cariprazine is its low incidence of cardiovascular side effects (Durgam et al., 2017). Cariprazine had no significant effect on either the QT or QTc intervals of the ECG and produced no arrhythmias in experimental animals and human beings (European Medicines Agency,
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2017; Durgam et al., 2017). The cardiotoxicity of these drugs can be mainly attributed to their interactions with the hERG 1A channel, which underlies the rapidly activating delayed rectifier K+ currents in the heart (De Bruin et al., 2005; Sanguinetti and Tristani-Firouzi, 2006). Indeed, cariprazine has shown low potential for inhibiting hERG 1A channel activity that is associated with a risk of QT interval prolongation (European Medicines Agency, 2017). However, previous study has focused only on the concentration-dependent effect that cariprazine exerts on hERG 1A currents. In addition, the state dependence of drug binding often affects the potency of the drug in blocking hERG channels. Thus, it is important to investigate the cellular mechanisms for cariprazine action on hERG 1A channels via two mechanisms: direct block of hERG 1A channels and disruption of hERG 1A channel protein trafficking on the cell membrane (Dennis et al., 2007; Hancox et al., 2008). hERG 3.1 is an alternatively spliced isoform of hERG 1A, and is considered a potential therapeutic target for antipsychotic drugs (Apud et al., 2012; Huffaker et al., 2009). As with other voltage-gated K+ channels, hERG 3.1 assembles as homotetramers or heterotetramers, but is well known for its poor expression and displays impaired trafficking and channel activity (Calcaterra et al., 2016; Ke et al., 2014).
Corresponding author. Department of Physiology College of Medicine The Catholic University of Korea 222 Banpo-daero, Seocho-gu, Seoul, 06591, South Korea. E-mail address: [email protected] (S.J. Hahn).
https://doi.org/10.1016/j.ejphar.2019.04.006 Received 27 August 2018; Received in revised form 1 April 2019; Accepted 2 April 2019 Available online 05 April 2019 0014-2999/ © 2019 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/BY/4.0/).
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polyacrylamide electrophoresis gel. The proteins were transferred onto PVDF membranes and blocked with 5% non-fat dry milk for 1 h at room temperature. The membranes were incubated with anti-HA, anti-hERG, anti-Na+/K+ ATPase (1:1000; Santa Cruz Biotechnology), and anti-βactin (1:5000; Sigma-Aldrich) overnight at 4 °C and then incubated for 1 h at room temperature with HRP-conjugated secondary antibody (Cell Signaling Technology). A chemiluminescent signal was detected by the SuperSignal West Dura Extended Duration Substrate (Thermo Scientific, Rockford, IL, USA) and visualized using a LAS-4000 image analyzer (Fujifilm, Tokyo, Japan). The band intensity was analyzed using Multi Gauge V3.0 software (Fujifilm, Tokyo, Japan).
For that reason, it is very difficult to study the pharmacological effects of the drugs on currents recorded from hERG 3.1 homotetramers. Thus, the present study was also focused on the effect of cariprazine on hERG 1A/3.1 heterotetramers, and its potency was compared with that of hERG 1A channels using a patch-clamp and an immunoblot assay. 2. Materials and methods 2.1. Cell culture The hERG 1A-HEK293 recombinant cell line (CYL3039, Millipore, Billerica, MA, USA) was used for electrophysiological measurements, as described previously (Chae et al., 2014). Cells were maintained in an environment that consisted of 95% humidified air and 5% CO2 at 37 °C in D-MEM/F-12 (Invitrogen, Grand Island, NY, USA) and were supplemented with 10% fetal bovine serum, 1% nonessential amino acids, and 400 μg/ml geneticin, according to the manufacturer's instructions. For cells expressing the heterotetrameric hERG 1A/3.1 channel, hERG 1A stable cells were plated on glass coverslips placed inside a 35-mm dish or in 6-well plates. The cells were transfected 24 h later using a vector carrying the hERG 3.1 cDNA and Fugene® HD (Promega, Madison WI, USA) according to the manufacturer's instructions. The HAtagged hERG 3.1 construct in pIRES2-eGFP was a gift from Prof. Jamie Vandenberg.
2.4. Cell surface biotinylation For the cell surface biotinylation experiments, we used a Cell Surface Protein Isolation Kit (Thermo Scientific, Waltham, MA, USA). In brief, hERG 1A and hERG 1A/3.1 cells were treated with cariprazine for 24 h, washed with ice-cold PBS, and incubated with PBS containing 0.5 mg/ml EZ-Link-NHS-SS-biotin (Thermo Scientific) for 1 h at 4 °C. After the biotinylation reaction was quenched by adding the Quenching Solution for 30 min at 4 °C, the cells were then harvested with a gentle scraping and lysed with lysis buffer containing a protease inhibitor mixture (Thermo Scientific). The lysates were centrifuged at 15,000 g for 10 min at 4 C. Biotinylated proteins were isolated by NeutrAvidin agarose beads (Thermo Scientific) for 1 h at room temperature. Beads were then washed five times with PBS plus 0.1% SDS. Biotinylated proteins were eluted in sample buffer and heated at 37 °C for 30 min. The eluted proteins were electrophoresed on 8% SDS polyacrylamide gel and analyzed by Western blotting.
2.2. Electrophysiology hERG currents were measured in a whole-cell configuration of the patch-clamp technique using an Axopatch 700B amplifier (Molecular Devices, Sunnyvale, CA, USA). All experiments were carried out at room temperature (22–24 °C). The recording chamber (RC-13; Warner Instruments, Hamden, CT, USA) was continually perfused with an extracellular bath solution. Patch pipettes were formed from borosilicate glass (PG10165-4, World Precision Instruments, Sarasota, Fl, USA) with a tip resistance of 2–4 MΩ when filled with the internal solution. Series resistance was in the range of 4–5 MΩ and the effective series resistance was usually compensated by 80% if the hERG current exceeded 2 nA. Series resistance was periodically checked and was stable during the recordings of the current. Cells with significant leak currents were rejected and on-line leak subtraction was not used in this study. To accomplish a fast application, cariprazine was applied with a superfusion system using a piezoelectric-driven micromanipulator (P-287.70, Physik Instrumente, Waldbronn, Germany), as described previously (Chae et al., 2014). The internal pipette solution contained (in mM) 140 KCl, 1 CaCl2, 1 MgCl2, 10 HEPES, and 10 EGTA and was adjusted to pH 7.3 using KOH. The external bath solution contained (in mM) 140 NaCl, 5 KCl, 1.3 CaCl2, 1 MgCl2, 20 HEPES, and 10 glucose and was adjusted to pH 7.3 using NaOH. Cariprazine (MedChem Express, Monmouth Junction, NJ, USA) was dissolved in dimethyl sulfoxide (DMSO, Sigma, St. Louis, MO, USA). Stock solutions were diluted to appropriate concentrations with external bath solution, which had no effect of its own on the hERG currents at the highest concentration (30 μM) used (0.05% vol/vol) (Chae et al., 2014).
2.5. Data analysis and statistics Analysis of the data was performed using pClamp 10.0 software (Molecular Devices) and Origin 8.0 software (Microcal Software, Inc., Northampton, MA, USA). The data are expressed as the means ± S.E.M. Statistical analyses were accomplished using a Student's t-test for comparison between two groups and an analysis of variance followed by a Bonferroni test for a comparison of multiple groups. A value of P < 0.05 was considered statistically significant. 3. Results 3.1. Concentration-dependent inhibition of hERG 1A and hERG 1A/3.1 currents by cariprazine Fig. 1A shows the effects of cariprazine on hERG 1A and hERG 1A/ 3.1 currents expressed in HEK cells. The cells were depolarized from a holding potential of −80 mV to +20 mV for 4 s, and the hERG tail currents were elicited by 6 s repolarizing pulses to −50 mV after each voltage step. The hERG 1A/3.1 heterotetramers had gating kinetics that were intermediate between those of the homotetrameric hERG 1A and hERG 3.1currents (Heide et al., 2012). Thus, the hERG 1A/3.1 showed faster deactivation and produced tail currents with smaller amplitude compared with homotetrameric hERG 1A. The peak tail current density of hERG 1A was 105.2 ± 9.4 pA/pF for a voltage step to −50 mV (n = 17). The peak tail current density of hERG 1A/3.1 was decreased to 20.9 ± 1.7 pA/pF for a voltage step to −50 mV (n = 12). To investigate the concentration-dependent inhibition of hERG currents, a range of concentrations (0.3–30 μM) of cariprazine was applied sequentially in the same cell. The peak amplitudes of hERG tail currents at different concentrations of cariprazine were normalized to the fractional block of the control and then plotted against the concentration of cariprazine (Fig. 1B). A nonlinear least-squares fit of the Hill equation to the concentration-response data yielded an IC50 value of 4.1 ± 0.2 μM with a Hill coefficient of 1.1 ± 0.1 for hERG 1A currents (n = 11) and an IC50 value of 12.2 ± 1.1 μM with a Hill coefficient of
2.3. Western blot analyses To measure the cell surface expression of hERG 1A and hERG 1A/ 3.1 channel protein, we performed Western blot analysis as described previously (Lee et al., 2017). Cells were treated with cariprazine for 24 h, washed in cold PBS, and solubilized on ice for 30 min in ice-cold RIPA buffer (Millipore) containing protease/phosphatase inhibitor cocktail (Thermo Scientific, Rockford, IL, USA). Whole cell lysates were pelleted by centrifugation at 15,000 g for 15 min, and the total protein concentrations of the supernatants were analyzed using Bio-Rad Protein Assay dye reagent (Bio-Rad, Hercules, CA, USA). Samples from each cell corresponding to 45 μg of total protein were separated on 8% SDS93
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Fig. 1. Concentration-dependence of the cariprazine inhibition of hERG 1A and hERG 1A/3.1 currents (A). Whole-cell hERG currents were elicited by the voltage protocol shown in the inset. The effects of cariprazine on hERG 1A/3.1 tail currents are shown in the inset. The dotted line marks zero current. (B) The concentrationresponse curves of cariprazine on hERG 1A and hERG 1A/3.1 currents. Data were fitted to the Hill equation. Data are expressed as the means ± S.E.M.
but did not change the slope factor (control: V1/2 = −32.5 ± 1.7 mV, k = 7.2 ± 0.4 mV; cariprazine: V1/2 = −38.4 ± 1.8 mV, k = 6.9 ± 0.8 mV, n = 7, P < 0.05). When quantified as the relative tail current for voltage ranging from −30 to −10 mV (Fig. 2C), the percentage block gradually increased (32.7 ± 4.0% of block at −30 mV, 43.4 ± 3.0% at −20 mV and 48.5 ± 1.9% at −10 mV, n = 7, P < 0.05) and reached a steady state at 0 mV, indicating a voltagedependent block of hERG 1A tail currents by cariprazine. For greater depolarized potentials where the channels are fully activated (between 0 and 60 mV), there was no voltage-dependent block of the hERG 1A currents.
0.7 ± 0.1 for hERG 1A/3.1 currents (n = 10). Thus, cariprazine inhibits both hERG 1A and hERG 1A/3.1 with its effects on hERG 1A being approximately 3-fold more potent. 3.2. Voltage-dependent block of the hERG 1A current by cariprazine Fig. 2A shows the representative whole-cell hERG 1A currents elicited by applying 4 s depolarizing pulses at holding potentials ranging from −80 mV to +60 mV in 10 mV steps in the absence and presence of cariprazine on the same cell. Fig. 2B shows the average normalized current-voltage relationships for the hERG 1A currents measured at the end of depolarizing pulses and the peak hERG 1A tail currents before and after the addition of cariprazine. Cariprazine inhibited the amplitude of hERG 1A currents at all membrane potentials between −50 and + 60 mV. To investigate the effects of cariprazine on the voltage dependency of activation curves, the peak hERG 1A tail currents were normalized and were fitted to a Boltzmann function. Cariprazine shifted the activation curve of hERG 1A currents in a hyperpolarizing direction,
3.3. Effects of cariprazine on the steady-state inactivation of the hERG 1A current Fig. 3A illustrates the effects of cariprazine on the voltage dependence of the steady-state inactivation of hERG 1A currents. The steadystate inactivation curves for hERG 1A under control conditions had a 94
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Fig. 2. Voltage-dependent block of hERG 1A currents. (A) Whole-cell currents were elicited by the voltage protocol shown in the inset under control conditions and in the presence of cariprazine. The dotted line marks zero current. (B) Current-voltage relationships of steady-state and peak hERG 1A tail currents under control conditions and in the presence of cariprazine. The peak amplitudes of hERG 1A tail currents in the presence of cariprazine were normalized to those at each level of voltage under control conditions. Data were fitted to a Boltzmann equation. (C) Voltage dependence of a fractional block, defined as normalized current (ICariprazine/ IControl). * Significant difference from data obtained at −30 mV (P < 0.05). The dotted line represents the activation curve of hERG 1A currents under control conditions. Data are expressed as the means ± S.E.M.
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Fig. 3. Effects of cariprazine on the steady-state inactivation of hERG 1A currents. (A) Representative currents elicited by a double pulse protocol shown in the inset in the absence and presence of cariprazine. (B) The normalized currents are plotted against the membrane potential and fitted with a Boltzmann equation. Data are expressed as the means ± S.E.M.
half-inactivation point (V1/2) of 62.1 ± 3.0 and a slope factor (k) of 21.5 ± 0.6 mV (n = 8). In the presence of cariprazine, V1/2 of the inactivation curves and k values were −68.9 ± 1.8 and 20.5 ± 0.6 mV (n = 8), respectively (Fig. 3B). Thus, cariprazine shifted the steadystate inactivation curve of hERG 1A currents in a hyperpolarizing direction.
Fig. 4. Effects of cariprazine on use-dependent block of hERG 1A currents. (A) hERG 1A currents obtained from depolarizing pulses as shown in the inset at 0.2 and 1 Hz under control conditions and in the presence of cariprazine. (B) The peak amplitudes of hERG 1A currents at each pulse were normalized by the peak amplitude measured at the first pulse, and then plotted against the pulse number. Data are expressed as the means ± S.E.M.
3.4. Effects of cariprazine on the use-dependence of the hERG 1A current 3.7 ± 0.6% at 1 Hz, n = 9) (Fig. 4B). In the presence of cariprazine, the inhibition was significantly increased to 4.4 ± 0.3% at 0.2 Hz; 8.5 ± 1.2% at 1 Hz during the 15 depolarizing pulses, indicating that the inhibition of hERG 1A tail currents by cariprazine was use-dependent.
To assess whether the inhibition of hERG 1A currents by cariprazine shows use dependence, cells were depolarized to +20 mV for 300 ms, followed by a repolarizing pulse to −40 mV for 300 ms to record the hERG tail currents at frequencies of 0.2 and 1 Hz (Stork et al., 2007) in the absence and presence of cariprazine (Fig. 4A). Under control conditions, the currents were slightly decreased over the course of 15 repetitive depolarizing pulses for either frequency (0.9 ± 0.4% at 0.2 Hz; 96
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dependent block of hERG 1A currents by cariprazine. A 5 s depolarizing prepulse to +60 mV served to inactivate the channels, followed by a 5 s repolarizing pulse to −40 mV that induced a recovery from inactivation and an open state of the channels (Fig. 5A). Cariprazine inhibited the hERG 1A currents by 16.6 ± 1.6% at the end of the depolarizing pulse to +60 mV, which increased to 36.0 ± 1.2% (n = 10, P < 0.05) during the repolarization to −40 mV. To further investigate the interaction of cariprazine with the open state of hERG 1A channels, hERG 1A tail currents were elicited by a long repolarizing pulse for 10 s, during which the hERG 1A currents recovered from inactivation rapidly and deactivated slowly, which allowed the outward currents (Fig. 5B). Cariprazine reversibly blocked the hERG 1A tail currents to a similar extent at 38.6% ± 1.5 (n = 11), as shown in Fig. 5A. These results suggest that cariprazine mainly interacts with hERG 1A channels in the open state rather than interacting with the inactivated state of the hERG 1A channel. 3.6. Effects of cariprazine on the hERG channel trafficking and surface expression We performed Western blot analysis to examine that disruption of hERG trafficking to the cell membrane inhibited hERG 1A and hERG 1A/3.1 channel currents. In Western blots, hERG 1A channel protein exists as two bands, a core glycosylated (CG) immature ER-resident form (135 kDa) and a fully glycosylated (FG) mature cell surface form (155 kDa) (Fig. 6A). Cariprazine caused no significant changes to either of the bands, when HEK293 cells stably expressing hERG 1A channel was exposed for 24 h to increasing concentrations of cariprazine. As
Fig. 5. Effects of the fast application of cariprazine on hERG 1A currents. (A) To investigate the interaction of cariprazine with the inactivated state of hERG 1A channels, the currents were elicited by a 5 s depolarizing pulse to +60 mV followed by 5 s of repolarization to −40 mV. (B) To investigate the interaction of cariprazine with the open state of hERG 1A channels, hERG 1A tail currents were elicited by a long repolarizing pulse for 10 s, which induced recovery from inactivation and the open state of channels. Cariprazine was rapidly applied during the pulses as indicated by the stippled bars. The hERG 1A current measured at the end of the drug application (●) was used to estimate the potency of the drug.
Fig. 6. Effects of cariprazine on hERG 1A and hERG 1A/3.1 channel trafficking. Western blot analyses of hERG 1A (A) and hERG 1A/3.1 (B) protein under control conditions and after 24 h incubation with increasing concentrations of cariprazine (0.3–30 μM). Cell lysates extracted from HEK 293 cells that expressed hERG 1A and hERG 1A/3.1 channels were probed with anti-hERG and anti-HA. β–Actin was used as a loading control and fluoxetine was used as a positive control. Molecular weight marker is indicated in kDa. Data shown are representative of four independent experiments. FLX, fluoxetine; FG, a fully glycosylated mature cell surface form; CG, a core glycosylated immature ERresident form.
3.5. Inhibition of the inactivated and open state of the hERG 1A current by cariprazine We used a fast drug application system to investigate the state97
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Fig. 7. Effects of cariprazine on the surface membrane expression of hERG 1A and hERG 1A/3.1 channels. The HEK 293 cells expressing hERG 1A (A) and hERG 1A/ 3.1 (B) channels were treated by the indicated concentrations of cariprazine for 24 h. Whole cell lysates and surface membrane proteins were probed with anti-hERG and anti-HA antibody to detect hERG 1A and hERG 1A/3.1 channels, respectively. Na+/K+ ATPase was used as a loading control for membrane fraction. Molecular weight marker is indicated in kDa. Data shown are representative of three independent experiments. FG, a fully glycosylated mature cell surface form; CG, a core glycosylated immature ER-resident form.
reported previously, fluoxetine (FLX) was used as a positive control to inhibit hERG 1A channel trafficking (Rajamani et al., 2006). On the other hand, hERG 1A/3.1 channel exists in only 1 band (a core glycosylated (CG) immature ER-resident form (about 135 kDa)), as described previously (Calcaterra et al., 2016; Ke et al., 2014; Lee et al., 2017) (Fig. 6B). In a similar manner, cariprazine had no effect on hERG 1A stable cells transiently transfected with HA-tagged hERG 3.1 to express heteromeric hERG 1A/3.1 channel protein. To examine whether cariprazine affects the surface membrane expression of hERG 1A and hERG 1A/3.1 channel, we isolated surface membrane protein using a biotinylation method. The mature hERG 1A protein on the plasma membrane was isolated from hERG 1A cells under control conditions or treated with indicated concentrations of cariprazine (0.3–30 μM) for 24 h and was analyzed by Western blot with anti-hERG antibody. Treatment with cariprazine had no effect on whole cell lysate and surface membrane expression of hERG 1A channels (Fig. 7A). The only immature form of hERG 1A/3.1 protein (CG, approximately 135 kDa) was detected in the whole cell proteins and was not changed by treatment of cariprazine compared with control (Fig. 7B). As shown in Fig. 6B, when probed with anti-HA antibody, the mature form of hERG 1A/3.1 protein was not seen by Western blot analysis. However, we detected visible bands (FG, approximately 155 kDa) for the mature hERG 1A/3.1 in the biotinylated surface proteins collected from hERG 1A/3.1 cells. Likewise, the expression levels
of the mature hERG 1A/3.1 protein were not altered in cells treated with cariprazine compared with control cells (Fig. 7B). Although this hERG 1A/3.1 protein is poorly expressed at the plasma membrane, our data provide supportive evidence for recordings of heterotetrameric hERG 1A/3.1 currents. These results suggest that cariprazine do not affect the disruption of hERG 1A and hERG 1A/3.1 channel trafficking and surface membrane expression. 4. Discussion Our data can be summarized as follows: 1) cariprazine inhibited hERG 1A and hERG 1A/3.1 currents in a concentration-dependent manner, 2) a fast application study showed that cariprazine blocked hERG 1A currents mainly in the open state, 3) cariprazine did not affect hERG 1A and hERG 1A/3.1 channel protein trafficking to the cell membrane. In the present study, cariprazine produced voltage-dependent block; the block was steeply increased in the activation range (−30 mV to −10 mV) of the hERG 1A currents. Cariprazine also induced a shift of the steady-state inactivation curve in the hyperpolarizing direction, and produced use-dependent block. These results are consistent with the interaction of cariprazine with activated (open and inactivated states) states of the channels (Paul et al., 2002; Wang et al., 1995). hERG 1A channels are found mainly in one of three states: closed, open and 98
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produced smaller amplitude currents than homotetrameric hERG 1A, and had a level of gating kinetics that fell between those of the homotetrameric hERG 1A and hERG 3.1 currents (Heide et al., 2012). Cariprazine blocked hERG 1A/3.1 at concentrations much higher than the therapeutic plasma concentration (IC50 of 12.2 μM). However, the physiochemical properties of cariprazine are as follows (Caccia et al., 2013; Citrome, 2013): a highly lipophilic antipsychotic that is rapidly absorbed and extensively distributed in tissues; a drug that is metabolized very slowly, with an elimination half-life of 2–5 days in schizophrenic patients; a drug that readily crosses the blood-brain barrier after oral administration in rats reaching brain concentrations that are approximately eight times those in plasma (Caccia et al., 2013). Thus, the potential clinical relevance of our findings remains to be confirmed. In conclusion, cariprazine inhibited hERG 1A and hERG 1A/3.1 currents in a concentration-dependent manner. At substantially supratherapeutic concentrations, cariprazine blocked hERG 1A currents by preferentially interacting with the open state of hERG 1A channels. Cariprazine did not affect membrane trafficking of either hERG 1A or hERG 1A/3.1 channels to the cell surface. Our study provides a biophysical profile that is necessary to assess the potential therapeutic effects of this drug.
inactivated (Kiehn et al., 1999). In this study, the fast application system was used to investigate the state-dependent block of hERG 1A currents by cariprazine (Chae et al., 2014; Ganapathi et al., 2009). As shown in Fig. 5, cariprazine more potently blocked hERG 1A currents during repolarization (in open state) than at the end of depolarization (in inactivated state), which suggests a preferential interaction with the open state. Furthermore, cariprazine produced a rapid and reversible block of hERG 1A tail currents during repolarization, wherein the channels recover from inactivation and are in an open state. Therefore, these results indicate that cariprazine blocks hERG 1A currents by preferentially binding to the open states of the channels. Another mechanism for inhibition of hERG current is the disruption of hERG channel protein trafficking to the cell surface (Rajamani et al., 2006). In our Western blot experiments, cariprazine had no effect on either hERG 1A or hERG 1A/3.1 channel trafficking to the cell surface membrane. hERG 1A channels are a key determinant in the repolarization of cardiac action potentials, and the inhibition of hERG 1A current can result in a QT interval prolongation of an ECG that is a surrogate marker for predicting the risk of potential cardiac arrhythmia, as in the case of TdP (Finlayson et al., 2004; Hancox et al., 2008). In patients receiving the maximum recommended human dose of 6 mg/day, maximal plasma concentrations of cariprazine were about 54 nM (European Medicines Agency, 2017). In the present study, the concentration at which cariprazine inhibited hERG 1A currents was 55–75-fold higher than the therapeutic plasma concentration. In addition, cariprazine had no effect on hERG 1A channel trafficking to the cell membrane. In dogs, cariprazine had no significant effect on the QT interval of the ECG, and induced no arrhythmogenesis (European Medicines Agency, 2017). Even at supratherapeutic doses in humans, an ECG showed no signs of QT interval prolongation (Caccia et al., 2013). Thus, in terms of the cardiovascular side effects, cariprazine can be considered a relatively safe drug. Because the hERG gene is widely expressed in many regions of the central nervous system, in addition to its well-defined expression in the heart, hERG channels play critical roles in regulating the resting membrane potentials, membrane excitability and spike frequency adaptation in neuronal cells (Pessia et al., 2008; Sacco et al., 2003). Recently, SNPs within the intron 2 of hERG 1A have been significantly associated with a high incidence of schizophrenic disorders, and hERG 1A mRNA is known to be decreased in individuals with schizophrenia, so that the ratio of hERG 3.1 to hERG 1A is 2.5-fold higher in hippocampal formation (Huffaker et al., 2009). Since these changes in expression patterns may influence the electrical properties of these neurons, it is likely that inhibition of hERG 1A and/or hERG 3.1 contributes in some way to the therapeutic action or to the neurological side effects of the drugs. Alterations in the biophysical properties of dopaminergic neurons by antipsychotics have been implicated in the therapeutic action of these drugs (Grace et al., 1997; White and Wang, 1983). Indeed, the expression of hERG 3.1 influences the treatment response to antipsychotic drugs, such as an improvement in the ratings of a positive syndrome and in general psychopathology, suggesting that the therapeutic effects of antipsychotics, particularly risperidone, may be in part associated with their actions on hERG 3.1 (Apud et al., 2012; Heide et al., 2016; Huffaker et al., 2009). Although it is unknown whether hERG 1A coassembles with hERG 3.1 in native neuronal tissues, the subunits of the erg subfamily are able to form heteromultimers with their subfamily in native tissues and heterologous cells, which is indicated by in situ hybridization and immunoprecipitation (Ke et al., 2014; Wimmers et al., 2001, 2002). Moreover, co-immunoprecipitation of hERG 1A and hERG 3.1 channels from transfected HEK293 cells had been demonstrated (Ke et al., 2014). Since the hERG 3.1 channel is well known for its poor expression and channel activity due to impaired trafficking (Calcaterra et al., 2016), we used hERG 1A/3.1 heterotetramers to study the pharmacological effects of the drugs on these currents. Although the stoichiometry of hERG 1A/3.1 heterotetramers is unknown, in the present study the hERG 1A/3.1 heterotetramers
Conflicts of interest The authors declare no conflicts of interest. Authorship contributions Participated in research design: BH Choi and SJ Hahn. Conducted experiments: HJ Lee. Performed data analysis and interpretation: HJ Lee, JS Choi. Wrote or contributed to the writing of the manuscript: SJ Hahn. Acknowledgements We thank Professor Jamie Vanderberg (Victor Chang Cardiac Research Institute, Darlinghurst, NSW, Australia) for the hERG 3.1 plasmid. This research was supported by the Basic Science Research Program through the National Research Foundation of Korea funded by the Ministry of Education (2017R1D1A1B03028108). References Apud, J.A., Zhang, F., Decot, H., Bigos, K.L., Weinberger, D.R., 2012. Genetic variation in KCNH2 associated with expression in the brain of a unique hERG isoform modulates treatment response in patients with schizophrenia. Am. J. Psychiatry 169, 725–734. Caccia, S., Invernizzi, R.W., Nobili, A., Pasina, L., 2013. A new generation of antipsychotics: pharmacology and clinical utility of cariprazine in schizophrenia. Therapeut. Clin. Risk Manag. 9, 319–328. Calcaterra, N.E., Hoeppner, D.J., Wei, H., Jaffe, A.E., Maher, B.J., Barrow, J.C., 2016. Schizophrenia-associated hERG channel Kv11.1-3.1 exhibits a unique trafficking deficit that is rescued through proteasome inhibition for high throughput screening. Sci. Rep. 6, 19976. Chae, Y.J., Jeon, J.H., Lee, H.J., Kim, I.B., Choi, J.S., Sung, K.W., Hahn, S.J., 2014. Escitalopram block of hERG potassium channels. Naunyn-Schmiedeberg’s Arch. Pharmacol. 387, 23–32. Citrome, L., 2013. Cariprazine: chemistry, pharmacodynamics, pharmacokinetics, and metabolism, clinical efficacy, safety, and tolerability. Expert Opin. Drug Metabol. Toxicol. 9, 193–206. De Bruin, M.L., Pettersson, M., Meyboom, R.H., Hoes, A.W., Leufkens, H.G., 2005. AntiHERG activity and the risk of drug-induced arrhythmias and sudden death. Eur. Heart J. 26, 590–597. Dennis, A., Wang, L., Wan, X., Ficker, E., 2007. hERG channel trafficking: novel targets in drug-induced long QT syndrome. Biochem. Soc. Trans. 35, 1060–1063. Durgam, S., Greenberg, W.M., Li, D., Lu, K., Laszlovszky, I., Nemeth, G., Migliore, R., Volk, S., 2017. Safety and tolerability of cariprazine in the long-term treatment of schizophrenia: results from a 48-week, single-arm, open-label extension study. Psychopharmacology (Berlin) 234, 199–209. European Medicines Agency, 2017. Assessment Report. Reagila. . http://www.ema. europa.eu/docs/en_GB/document_library/EPAR_-_Public_assessment_report/human/ 002770/WC500234926.pdf. Finlayson, K., Witchel, H.J., McCulloch, J., Sharkey, J., 2004. Acquired QT interval
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prolongation and HERG: implications for drug discovery and development. Eur. J. Pharmacol. 500, 129–142. Ganapathi, S.B., Kester, M., Elmslie, K.S., 2009. State-dependent block of HERG potassium channels by R-roscovitine: implications for cancer therapy. Am. J. Physiol. Cell Physiol. 296, C701–C710. Grace, A.A., Bunney, B.S., Moore, H., Todd, C.L., 1997. Dopamine-cell depolarization block as a model for the therapeutic actions of antipsychotic drugs. Trends Neurosci. 20, 31–37. Hancox, J.C., McPate, M.J., El Harchi, A., Zhang, Y.H., 2008. The hERG potassium channel and hERG screening for drug-induced torsades de pointes. Pharmacol. Ther. 119, 118–132. Heide, J., Mann, S.A., Vandenberg, J.I., 2012. The schizophrenia-associated Kv11.1-3.1 isoform results in reduced current accumulation during repetitive brief depolarizations. PLoS One 7, e45624. Heide, J., Zhang, F., Bigos, K.L., Mann, S.A., Carr, V.J., Shannon Weickert, C., Green, M.J., Weinberger, D.R., Vandenberg, J.I., 2016. Differential response to risperidone in schizophrenia patients by KCNH2 genotype and drug metabolizer status. Am. J. Psychiatry 173, 53–59. Huffaker, S.J., Chen, J., Nicodemus, K.K., Sambataro, F., Yang, F., Mattay, V., Lipska, B.K., Hyde, T.M., Song, J., Rujescu, D., Giegling, I., Mayilyan, K., Proust, M.J., Soghoyan, A., Caforio, G., Callicott, J.H., Bertolino, A., Meyer-Lindenberg, A., Chang, J., Ji, Y., Egan, M.F., Goldberg, T.E., Kleinman, J.E., Lu, B., Weinberger, D.R., 2009. A primate-specific, brain isoform of KCNH2 affects cortical physiology, cognition, neuronal repolarization and risk of schizophrenia. Nat. Med. 15, 509–518. Ke, Y., Hunter, M.J., Ng, C.A., Perry, M.D., Vandenberg, J.I., 2014. Role of the cytoplasmic N-terminal Cap and Per-Arnt-Sim (PAS) domain in trafficking and stabilization of Kv11.1 channels. J. Biol. Chem. 289, 13782–13791. Kiehn, J., Lacerda, A.E., Brown, A.M., 1999. Pathways of HERG inactivation. Am. J. Physiol. 277, H199–H210. Lee, H.J., Choi, J.S., Choi, B.H., Hahn, S.J., 2017. Inhibition of cloned hERG potassium channels by risperidone and paliperidone. Naunyn-Schmiedeberg’s Arch. Pharmacol. 390, 633–642. Pacher, P., Kecskemeti, V., 2004. Cardiovascular side effects of new antidepressants and antipsychotics: new drugs, old concerns? Curr. Pharmaceut. Des. 10, 2463–2475.
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Full length article
Effects of cariprazine on hERG 1A and hERG 1A/3.1 potassium channels a
b
c
Hong Joon Lee , Bok Hee Choi , Jin-Sung Choi , Sang June Hahn a b c
T
a,∗
Department of Physiology, College of Medicine, The Catholic University of Korea, Seoul, 06591, South Korea Department of Pharmacology, Institute for Medical Science, Chonbuk National University Medical School, Jeonju, Jeonbuk, 54097, South Korea College of Pharmacy, Integrated Research Institute of Pharmaceutical, The Catholic University of Korea, Gyeonggi-do, 14662, South Korea
A R T I C LE I N FO
A B S T R A C T
Keywords: Cariprazine hERG 1A/3.1 heterotetramer Open channel block
Cariprazine is a novel atypical antipsychotic drug that is widely used for the treatment of schizophrenia and bipolar mania/mixed disorder. We used the whole-cell patch-clamp technique to investigate the effects of cariprazine on hERG channels that are stably expressed in HEK cells. Cariprazine inhibited the hERG 1A and hERG 1A/3.1 tail currents at −50 mV in a concentration-dependent manner with IC50 values of 4.1 and 12.2 μM, respectively. The block of hERG 1A currents by cariprazine was voltage-dependent, and increased over a range of voltage for channel activation. Cariprazine shifted the steady-state inactivation curve of the hERG 1A currents in a hyperpolarizing direction and produced a use-dependent block. A fast application of cariprazine inhibited the hERG 1A currents elicited by a 5 s depolarizing pulse to +60 mV to fully inactivate the hERG 1A currents. During a repolarizing pulse wherein the hERG 1A current was deactivated slowly, cariprazine rapidly and reversibly blocked the open state of the hERG 1A current. However, cariprazine did not affect hERG 1A and hERG 1A/3.1 channel trafficking to the cell membrane. Our results indicated that cariprazine concentration-dependently inhibited hERG 1A and hERG 1A/3.1 currents by preferentially interacting with the open states of the hERG 1A channel, but not by the disruption of hERG 1A and hERG 1A/3.1 channel protein trafficking. Our study examined cariprazine's mechanism of action provides a biophysical profile that is necessary to assess the potential therapeutic effects of this drug.
1. Introduction Cariprazine, a dopamine D3/D2 receptor partial agonist, is a novel atypical antipsychotic drug that was recently approved in the USA for the treatment of schizophrenia and bipolar mania/mixed disorder (Caccia et al., 2013; Citrome, 2013). Cariprazine caused fewer extrapyramidal side effects and was generally well tolerated with minimal adverse effects (European Medicines Agency, 2017; Scarff, 2016). Many drugs used to treat psychiatric patients, specifically antipsychotic drugs, have been implicated in QT interval prolongation that causes cardiac arrhythmia, such as torsades de pointes (TdP) (Pacher and Kecskemeti, 2004; Sicouri and Antzelevitch, 2008). For example, fluoxetine blocks hERG channels, which is essential to ventricular repolarization (Thomas et al., 2002). Accordingly, this drug can delay ventricular repolarization, thus inducing QT interval prolongation and triggering TdP (Rajamani et al., 2006). However, the most interesting advantage of cariprazine is its low incidence of cardiovascular side effects (Durgam et al., 2017). Cariprazine had no significant effect on either the QT or QTc intervals of the ECG and produced no arrhythmias in experimental animals and human beings (European Medicines Agency,
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2017; Durgam et al., 2017). The cardiotoxicity of these drugs can be mainly attributed to their interactions with the hERG 1A channel, which underlies the rapidly activating delayed rectifier K+ currents in the heart (De Bruin et al., 2005; Sanguinetti and Tristani-Firouzi, 2006). Indeed, cariprazine has shown low potential for inhibiting hERG 1A channel activity that is associated with a risk of QT interval prolongation (European Medicines Agency, 2017). However, previous study has focused only on the concentration-dependent effect that cariprazine exerts on hERG 1A currents. In addition, the state dependence of drug binding often affects the potency of the drug in blocking hERG channels. Thus, it is important to investigate the cellular mechanisms for cariprazine action on hERG 1A channels via two mechanisms: direct block of hERG 1A channels and disruption of hERG 1A channel protein trafficking on the cell membrane (Dennis et al., 2007; Hancox et al., 2008). hERG 3.1 is an alternatively spliced isoform of hERG 1A, and is considered a potential therapeutic target for antipsychotic drugs (Apud et al., 2012; Huffaker et al., 2009). As with other voltage-gated K+ channels, hERG 3.1 assembles as homotetramers or heterotetramers, but is well known for its poor expression and displays impaired trafficking and channel activity (Calcaterra et al., 2016; Ke et al., 2014).
Corresponding author. Department of Physiology College of Medicine The Catholic University of Korea 222 Banpo-daero, Seocho-gu, Seoul, 06591, South Korea. E-mail address: [email protected] (S.J. Hahn).
https://doi.org/10.1016/j.ejphar.2019.04.006 Received 27 August 2018; Received in revised form 1 April 2019; Accepted 2 April 2019 Available online 05 April 2019 0014-2999/ © 2019 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/BY/4.0/).
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polyacrylamide electrophoresis gel. The proteins were transferred onto PVDF membranes and blocked with 5% non-fat dry milk for 1 h at room temperature. The membranes were incubated with anti-HA, anti-hERG, anti-Na+/K+ ATPase (1:1000; Santa Cruz Biotechnology), and anti-βactin (1:5000; Sigma-Aldrich) overnight at 4 °C and then incubated for 1 h at room temperature with HRP-conjugated secondary antibody (Cell Signaling Technology). A chemiluminescent signal was detected by the SuperSignal West Dura Extended Duration Substrate (Thermo Scientific, Rockford, IL, USA) and visualized using a LAS-4000 image analyzer (Fujifilm, Tokyo, Japan). The band intensity was analyzed using Multi Gauge V3.0 software (Fujifilm, Tokyo, Japan).
For that reason, it is very difficult to study the pharmacological effects of the drugs on currents recorded from hERG 3.1 homotetramers. Thus, the present study was also focused on the effect of cariprazine on hERG 1A/3.1 heterotetramers, and its potency was compared with that of hERG 1A channels using a patch-clamp and an immunoblot assay. 2. Materials and methods 2.1. Cell culture The hERG 1A-HEK293 recombinant cell line (CYL3039, Millipore, Billerica, MA, USA) was used for electrophysiological measurements, as described previously (Chae et al., 2014). Cells were maintained in an environment that consisted of 95% humidified air and 5% CO2 at 37 °C in D-MEM/F-12 (Invitrogen, Grand Island, NY, USA) and were supplemented with 10% fetal bovine serum, 1% nonessential amino acids, and 400 μg/ml geneticin, according to the manufacturer's instructions. For cells expressing the heterotetrameric hERG 1A/3.1 channel, hERG 1A stable cells were plated on glass coverslips placed inside a 35-mm dish or in 6-well plates. The cells were transfected 24 h later using a vector carrying the hERG 3.1 cDNA and Fugene® HD (Promega, Madison WI, USA) according to the manufacturer's instructions. The HAtagged hERG 3.1 construct in pIRES2-eGFP was a gift from Prof. Jamie Vandenberg.
2.4. Cell surface biotinylation For the cell surface biotinylation experiments, we used a Cell Surface Protein Isolation Kit (Thermo Scientific, Waltham, MA, USA). In brief, hERG 1A and hERG 1A/3.1 cells were treated with cariprazine for 24 h, washed with ice-cold PBS, and incubated with PBS containing 0.5 mg/ml EZ-Link-NHS-SS-biotin (Thermo Scientific) for 1 h at 4 °C. After the biotinylation reaction was quenched by adding the Quenching Solution for 30 min at 4 °C, the cells were then harvested with a gentle scraping and lysed with lysis buffer containing a protease inhibitor mixture (Thermo Scientific). The lysates were centrifuged at 15,000 g for 10 min at 4 C. Biotinylated proteins were isolated by NeutrAvidin agarose beads (Thermo Scientific) for 1 h at room temperature. Beads were then washed five times with PBS plus 0.1% SDS. Biotinylated proteins were eluted in sample buffer and heated at 37 °C for 30 min. The eluted proteins were electrophoresed on 8% SDS polyacrylamide gel and analyzed by Western blotting.
2.2. Electrophysiology hERG currents were measured in a whole-cell configuration of the patch-clamp technique using an Axopatch 700B amplifier (Molecular Devices, Sunnyvale, CA, USA). All experiments were carried out at room temperature (22–24 °C). The recording chamber (RC-13; Warner Instruments, Hamden, CT, USA) was continually perfused with an extracellular bath solution. Patch pipettes were formed from borosilicate glass (PG10165-4, World Precision Instruments, Sarasota, Fl, USA) with a tip resistance of 2–4 MΩ when filled with the internal solution. Series resistance was in the range of 4–5 MΩ and the effective series resistance was usually compensated by 80% if the hERG current exceeded 2 nA. Series resistance was periodically checked and was stable during the recordings of the current. Cells with significant leak currents were rejected and on-line leak subtraction was not used in this study. To accomplish a fast application, cariprazine was applied with a superfusion system using a piezoelectric-driven micromanipulator (P-287.70, Physik Instrumente, Waldbronn, Germany), as described previously (Chae et al., 2014). The internal pipette solution contained (in mM) 140 KCl, 1 CaCl2, 1 MgCl2, 10 HEPES, and 10 EGTA and was adjusted to pH 7.3 using KOH. The external bath solution contained (in mM) 140 NaCl, 5 KCl, 1.3 CaCl2, 1 MgCl2, 20 HEPES, and 10 glucose and was adjusted to pH 7.3 using NaOH. Cariprazine (MedChem Express, Monmouth Junction, NJ, USA) was dissolved in dimethyl sulfoxide (DMSO, Sigma, St. Louis, MO, USA). Stock solutions were diluted to appropriate concentrations with external bath solution, which had no effect of its own on the hERG currents at the highest concentration (30 μM) used (0.05% vol/vol) (Chae et al., 2014).
2.5. Data analysis and statistics Analysis of the data was performed using pClamp 10.0 software (Molecular Devices) and Origin 8.0 software (Microcal Software, Inc., Northampton, MA, USA). The data are expressed as the means ± S.E.M. Statistical analyses were accomplished using a Student's t-test for comparison between two groups and an analysis of variance followed by a Bonferroni test for a comparison of multiple groups. A value of P < 0.05 was considered statistically significant. 3. Results 3.1. Concentration-dependent inhibition of hERG 1A and hERG 1A/3.1 currents by cariprazine Fig. 1A shows the effects of cariprazine on hERG 1A and hERG 1A/ 3.1 currents expressed in HEK cells. The cells were depolarized from a holding potential of −80 mV to +20 mV for 4 s, and the hERG tail currents were elicited by 6 s repolarizing pulses to −50 mV after each voltage step. The hERG 1A/3.1 heterotetramers had gating kinetics that were intermediate between those of the homotetrameric hERG 1A and hERG 3.1currents (Heide et al., 2012). Thus, the hERG 1A/3.1 showed faster deactivation and produced tail currents with smaller amplitude compared with homotetrameric hERG 1A. The peak tail current density of hERG 1A was 105.2 ± 9.4 pA/pF for a voltage step to −50 mV (n = 17). The peak tail current density of hERG 1A/3.1 was decreased to 20.9 ± 1.7 pA/pF for a voltage step to −50 mV (n = 12). To investigate the concentration-dependent inhibition of hERG currents, a range of concentrations (0.3–30 μM) of cariprazine was applied sequentially in the same cell. The peak amplitudes of hERG tail currents at different concentrations of cariprazine were normalized to the fractional block of the control and then plotted against the concentration of cariprazine (Fig. 1B). A nonlinear least-squares fit of the Hill equation to the concentration-response data yielded an IC50 value of 4.1 ± 0.2 μM with a Hill coefficient of 1.1 ± 0.1 for hERG 1A currents (n = 11) and an IC50 value of 12.2 ± 1.1 μM with a Hill coefficient of
2.3. Western blot analyses To measure the cell surface expression of hERG 1A and hERG 1A/ 3.1 channel protein, we performed Western blot analysis as described previously (Lee et al., 2017). Cells were treated with cariprazine for 24 h, washed in cold PBS, and solubilized on ice for 30 min in ice-cold RIPA buffer (Millipore) containing protease/phosphatase inhibitor cocktail (Thermo Scientific, Rockford, IL, USA). Whole cell lysates were pelleted by centrifugation at 15,000 g for 15 min, and the total protein concentrations of the supernatants were analyzed using Bio-Rad Protein Assay dye reagent (Bio-Rad, Hercules, CA, USA). Samples from each cell corresponding to 45 μg of total protein were separated on 8% SDS93
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Fig. 1. Concentration-dependence of the cariprazine inhibition of hERG 1A and hERG 1A/3.1 currents (A). Whole-cell hERG currents were elicited by the voltage protocol shown in the inset. The effects of cariprazine on hERG 1A/3.1 tail currents are shown in the inset. The dotted line marks zero current. (B) The concentrationresponse curves of cariprazine on hERG 1A and hERG 1A/3.1 currents. Data were fitted to the Hill equation. Data are expressed as the means ± S.E.M.
but did not change the slope factor (control: V1/2 = −32.5 ± 1.7 mV, k = 7.2 ± 0.4 mV; cariprazine: V1/2 = −38.4 ± 1.8 mV, k = 6.9 ± 0.8 mV, n = 7, P < 0.05). When quantified as the relative tail current for voltage ranging from −30 to −10 mV (Fig. 2C), the percentage block gradually increased (32.7 ± 4.0% of block at −30 mV, 43.4 ± 3.0% at −20 mV and 48.5 ± 1.9% at −10 mV, n = 7, P < 0.05) and reached a steady state at 0 mV, indicating a voltagedependent block of hERG 1A tail currents by cariprazine. For greater depolarized potentials where the channels are fully activated (between 0 and 60 mV), there was no voltage-dependent block of the hERG 1A currents.
0.7 ± 0.1 for hERG 1A/3.1 currents (n = 10). Thus, cariprazine inhibits both hERG 1A and hERG 1A/3.1 with its effects on hERG 1A being approximately 3-fold more potent. 3.2. Voltage-dependent block of the hERG 1A current by cariprazine Fig. 2A shows the representative whole-cell hERG 1A currents elicited by applying 4 s depolarizing pulses at holding potentials ranging from −80 mV to +60 mV in 10 mV steps in the absence and presence of cariprazine on the same cell. Fig. 2B shows the average normalized current-voltage relationships for the hERG 1A currents measured at the end of depolarizing pulses and the peak hERG 1A tail currents before and after the addition of cariprazine. Cariprazine inhibited the amplitude of hERG 1A currents at all membrane potentials between −50 and + 60 mV. To investigate the effects of cariprazine on the voltage dependency of activation curves, the peak hERG 1A tail currents were normalized and were fitted to a Boltzmann function. Cariprazine shifted the activation curve of hERG 1A currents in a hyperpolarizing direction,
3.3. Effects of cariprazine on the steady-state inactivation of the hERG 1A current Fig. 3A illustrates the effects of cariprazine on the voltage dependence of the steady-state inactivation of hERG 1A currents. The steadystate inactivation curves for hERG 1A under control conditions had a 94
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Fig. 2. Voltage-dependent block of hERG 1A currents. (A) Whole-cell currents were elicited by the voltage protocol shown in the inset under control conditions and in the presence of cariprazine. The dotted line marks zero current. (B) Current-voltage relationships of steady-state and peak hERG 1A tail currents under control conditions and in the presence of cariprazine. The peak amplitudes of hERG 1A tail currents in the presence of cariprazine were normalized to those at each level of voltage under control conditions. Data were fitted to a Boltzmann equation. (C) Voltage dependence of a fractional block, defined as normalized current (ICariprazine/ IControl). * Significant difference from data obtained at −30 mV (P < 0.05). The dotted line represents the activation curve of hERG 1A currents under control conditions. Data are expressed as the means ± S.E.M.
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Fig. 3. Effects of cariprazine on the steady-state inactivation of hERG 1A currents. (A) Representative currents elicited by a double pulse protocol shown in the inset in the absence and presence of cariprazine. (B) The normalized currents are plotted against the membrane potential and fitted with a Boltzmann equation. Data are expressed as the means ± S.E.M.
half-inactivation point (V1/2) of 62.1 ± 3.0 and a slope factor (k) of 21.5 ± 0.6 mV (n = 8). In the presence of cariprazine, V1/2 of the inactivation curves and k values were −68.9 ± 1.8 and 20.5 ± 0.6 mV (n = 8), respectively (Fig. 3B). Thus, cariprazine shifted the steadystate inactivation curve of hERG 1A currents in a hyperpolarizing direction.
Fig. 4. Effects of cariprazine on use-dependent block of hERG 1A currents. (A) hERG 1A currents obtained from depolarizing pulses as shown in the inset at 0.2 and 1 Hz under control conditions and in the presence of cariprazine. (B) The peak amplitudes of hERG 1A currents at each pulse were normalized by the peak amplitude measured at the first pulse, and then plotted against the pulse number. Data are expressed as the means ± S.E.M.
3.4. Effects of cariprazine on the use-dependence of the hERG 1A current 3.7 ± 0.6% at 1 Hz, n = 9) (Fig. 4B). In the presence of cariprazine, the inhibition was significantly increased to 4.4 ± 0.3% at 0.2 Hz; 8.5 ± 1.2% at 1 Hz during the 15 depolarizing pulses, indicating that the inhibition of hERG 1A tail currents by cariprazine was use-dependent.
To assess whether the inhibition of hERG 1A currents by cariprazine shows use dependence, cells were depolarized to +20 mV for 300 ms, followed by a repolarizing pulse to −40 mV for 300 ms to record the hERG tail currents at frequencies of 0.2 and 1 Hz (Stork et al., 2007) in the absence and presence of cariprazine (Fig. 4A). Under control conditions, the currents were slightly decreased over the course of 15 repetitive depolarizing pulses for either frequency (0.9 ± 0.4% at 0.2 Hz; 96
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dependent block of hERG 1A currents by cariprazine. A 5 s depolarizing prepulse to +60 mV served to inactivate the channels, followed by a 5 s repolarizing pulse to −40 mV that induced a recovery from inactivation and an open state of the channels (Fig. 5A). Cariprazine inhibited the hERG 1A currents by 16.6 ± 1.6% at the end of the depolarizing pulse to +60 mV, which increased to 36.0 ± 1.2% (n = 10, P < 0.05) during the repolarization to −40 mV. To further investigate the interaction of cariprazine with the open state of hERG 1A channels, hERG 1A tail currents were elicited by a long repolarizing pulse for 10 s, during which the hERG 1A currents recovered from inactivation rapidly and deactivated slowly, which allowed the outward currents (Fig. 5B). Cariprazine reversibly blocked the hERG 1A tail currents to a similar extent at 38.6% ± 1.5 (n = 11), as shown in Fig. 5A. These results suggest that cariprazine mainly interacts with hERG 1A channels in the open state rather than interacting with the inactivated state of the hERG 1A channel. 3.6. Effects of cariprazine on the hERG channel trafficking and surface expression We performed Western blot analysis to examine that disruption of hERG trafficking to the cell membrane inhibited hERG 1A and hERG 1A/3.1 channel currents. In Western blots, hERG 1A channel protein exists as two bands, a core glycosylated (CG) immature ER-resident form (135 kDa) and a fully glycosylated (FG) mature cell surface form (155 kDa) (Fig. 6A). Cariprazine caused no significant changes to either of the bands, when HEK293 cells stably expressing hERG 1A channel was exposed for 24 h to increasing concentrations of cariprazine. As
Fig. 5. Effects of the fast application of cariprazine on hERG 1A currents. (A) To investigate the interaction of cariprazine with the inactivated state of hERG 1A channels, the currents were elicited by a 5 s depolarizing pulse to +60 mV followed by 5 s of repolarization to −40 mV. (B) To investigate the interaction of cariprazine with the open state of hERG 1A channels, hERG 1A tail currents were elicited by a long repolarizing pulse for 10 s, which induced recovery from inactivation and the open state of channels. Cariprazine was rapidly applied during the pulses as indicated by the stippled bars. The hERG 1A current measured at the end of the drug application (●) was used to estimate the potency of the drug.
Fig. 6. Effects of cariprazine on hERG 1A and hERG 1A/3.1 channel trafficking. Western blot analyses of hERG 1A (A) and hERG 1A/3.1 (B) protein under control conditions and after 24 h incubation with increasing concentrations of cariprazine (0.3–30 μM). Cell lysates extracted from HEK 293 cells that expressed hERG 1A and hERG 1A/3.1 channels were probed with anti-hERG and anti-HA. β–Actin was used as a loading control and fluoxetine was used as a positive control. Molecular weight marker is indicated in kDa. Data shown are representative of four independent experiments. FLX, fluoxetine; FG, a fully glycosylated mature cell surface form; CG, a core glycosylated immature ERresident form.
3.5. Inhibition of the inactivated and open state of the hERG 1A current by cariprazine We used a fast drug application system to investigate the state97
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Fig. 7. Effects of cariprazine on the surface membrane expression of hERG 1A and hERG 1A/3.1 channels. The HEK 293 cells expressing hERG 1A (A) and hERG 1A/ 3.1 (B) channels were treated by the indicated concentrations of cariprazine for 24 h. Whole cell lysates and surface membrane proteins were probed with anti-hERG and anti-HA antibody to detect hERG 1A and hERG 1A/3.1 channels, respectively. Na+/K+ ATPase was used as a loading control for membrane fraction. Molecular weight marker is indicated in kDa. Data shown are representative of three independent experiments. FG, a fully glycosylated mature cell surface form; CG, a core glycosylated immature ER-resident form.
reported previously, fluoxetine (FLX) was used as a positive control to inhibit hERG 1A channel trafficking (Rajamani et al., 2006). On the other hand, hERG 1A/3.1 channel exists in only 1 band (a core glycosylated (CG) immature ER-resident form (about 135 kDa)), as described previously (Calcaterra et al., 2016; Ke et al., 2014; Lee et al., 2017) (Fig. 6B). In a similar manner, cariprazine had no effect on hERG 1A stable cells transiently transfected with HA-tagged hERG 3.1 to express heteromeric hERG 1A/3.1 channel protein. To examine whether cariprazine affects the surface membrane expression of hERG 1A and hERG 1A/3.1 channel, we isolated surface membrane protein using a biotinylation method. The mature hERG 1A protein on the plasma membrane was isolated from hERG 1A cells under control conditions or treated with indicated concentrations of cariprazine (0.3–30 μM) for 24 h and was analyzed by Western blot with anti-hERG antibody. Treatment with cariprazine had no effect on whole cell lysate and surface membrane expression of hERG 1A channels (Fig. 7A). The only immature form of hERG 1A/3.1 protein (CG, approximately 135 kDa) was detected in the whole cell proteins and was not changed by treatment of cariprazine compared with control (Fig. 7B). As shown in Fig. 6B, when probed with anti-HA antibody, the mature form of hERG 1A/3.1 protein was not seen by Western blot analysis. However, we detected visible bands (FG, approximately 155 kDa) for the mature hERG 1A/3.1 in the biotinylated surface proteins collected from hERG 1A/3.1 cells. Likewise, the expression levels
of the mature hERG 1A/3.1 protein were not altered in cells treated with cariprazine compared with control cells (Fig. 7B). Although this hERG 1A/3.1 protein is poorly expressed at the plasma membrane, our data provide supportive evidence for recordings of heterotetrameric hERG 1A/3.1 currents. These results suggest that cariprazine do not affect the disruption of hERG 1A and hERG 1A/3.1 channel trafficking and surface membrane expression. 4. Discussion Our data can be summarized as follows: 1) cariprazine inhibited hERG 1A and hERG 1A/3.1 currents in a concentration-dependent manner, 2) a fast application study showed that cariprazine blocked hERG 1A currents mainly in the open state, 3) cariprazine did not affect hERG 1A and hERG 1A/3.1 channel protein trafficking to the cell membrane. In the present study, cariprazine produced voltage-dependent block; the block was steeply increased in the activation range (−30 mV to −10 mV) of the hERG 1A currents. Cariprazine also induced a shift of the steady-state inactivation curve in the hyperpolarizing direction, and produced use-dependent block. These results are consistent with the interaction of cariprazine with activated (open and inactivated states) states of the channels (Paul et al., 2002; Wang et al., 1995). hERG 1A channels are found mainly in one of three states: closed, open and 98
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produced smaller amplitude currents than homotetrameric hERG 1A, and had a level of gating kinetics that fell between those of the homotetrameric hERG 1A and hERG 3.1 currents (Heide et al., 2012). Cariprazine blocked hERG 1A/3.1 at concentrations much higher than the therapeutic plasma concentration (IC50 of 12.2 μM). However, the physiochemical properties of cariprazine are as follows (Caccia et al., 2013; Citrome, 2013): a highly lipophilic antipsychotic that is rapidly absorbed and extensively distributed in tissues; a drug that is metabolized very slowly, with an elimination half-life of 2–5 days in schizophrenic patients; a drug that readily crosses the blood-brain barrier after oral administration in rats reaching brain concentrations that are approximately eight times those in plasma (Caccia et al., 2013). Thus, the potential clinical relevance of our findings remains to be confirmed. In conclusion, cariprazine inhibited hERG 1A and hERG 1A/3.1 currents in a concentration-dependent manner. At substantially supratherapeutic concentrations, cariprazine blocked hERG 1A currents by preferentially interacting with the open state of hERG 1A channels. Cariprazine did not affect membrane trafficking of either hERG 1A or hERG 1A/3.1 channels to the cell surface. Our study provides a biophysical profile that is necessary to assess the potential therapeutic effects of this drug.
inactivated (Kiehn et al., 1999). In this study, the fast application system was used to investigate the state-dependent block of hERG 1A currents by cariprazine (Chae et al., 2014; Ganapathi et al., 2009). As shown in Fig. 5, cariprazine more potently blocked hERG 1A currents during repolarization (in open state) than at the end of depolarization (in inactivated state), which suggests a preferential interaction with the open state. Furthermore, cariprazine produced a rapid and reversible block of hERG 1A tail currents during repolarization, wherein the channels recover from inactivation and are in an open state. Therefore, these results indicate that cariprazine blocks hERG 1A currents by preferentially binding to the open states of the channels. Another mechanism for inhibition of hERG current is the disruption of hERG channel protein trafficking to the cell surface (Rajamani et al., 2006). In our Western blot experiments, cariprazine had no effect on either hERG 1A or hERG 1A/3.1 channel trafficking to the cell surface membrane. hERG 1A channels are a key determinant in the repolarization of cardiac action potentials, and the inhibition of hERG 1A current can result in a QT interval prolongation of an ECG that is a surrogate marker for predicting the risk of potential cardiac arrhythmia, as in the case of TdP (Finlayson et al., 2004; Hancox et al., 2008). In patients receiving the maximum recommended human dose of 6 mg/day, maximal plasma concentrations of cariprazine were about 54 nM (European Medicines Agency, 2017). In the present study, the concentration at which cariprazine inhibited hERG 1A currents was 55–75-fold higher than the therapeutic plasma concentration. In addition, cariprazine had no effect on hERG 1A channel trafficking to the cell membrane. In dogs, cariprazine had no significant effect on the QT interval of the ECG, and induced no arrhythmogenesis (European Medicines Agency, 2017). Even at supratherapeutic doses in humans, an ECG showed no signs of QT interval prolongation (Caccia et al., 2013). Thus, in terms of the cardiovascular side effects, cariprazine can be considered a relatively safe drug. Because the hERG gene is widely expressed in many regions of the central nervous system, in addition to its well-defined expression in the heart, hERG channels play critical roles in regulating the resting membrane potentials, membrane excitability and spike frequency adaptation in neuronal cells (Pessia et al., 2008; Sacco et al., 2003). Recently, SNPs within the intron 2 of hERG 1A have been significantly associated with a high incidence of schizophrenic disorders, and hERG 1A mRNA is known to be decreased in individuals with schizophrenia, so that the ratio of hERG 3.1 to hERG 1A is 2.5-fold higher in hippocampal formation (Huffaker et al., 2009). Since these changes in expression patterns may influence the electrical properties of these neurons, it is likely that inhibition of hERG 1A and/or hERG 3.1 contributes in some way to the therapeutic action or to the neurological side effects of the drugs. Alterations in the biophysical properties of dopaminergic neurons by antipsychotics have been implicated in the therapeutic action of these drugs (Grace et al., 1997; White and Wang, 1983). Indeed, the expression of hERG 3.1 influences the treatment response to antipsychotic drugs, such as an improvement in the ratings of a positive syndrome and in general psychopathology, suggesting that the therapeutic effects of antipsychotics, particularly risperidone, may be in part associated with their actions on hERG 3.1 (Apud et al., 2012; Heide et al., 2016; Huffaker et al., 2009). Although it is unknown whether hERG 1A coassembles with hERG 3.1 in native neuronal tissues, the subunits of the erg subfamily are able to form heteromultimers with their subfamily in native tissues and heterologous cells, which is indicated by in situ hybridization and immunoprecipitation (Ke et al., 2014; Wimmers et al., 2001, 2002). Moreover, co-immunoprecipitation of hERG 1A and hERG 3.1 channels from transfected HEK293 cells had been demonstrated (Ke et al., 2014). Since the hERG 3.1 channel is well known for its poor expression and channel activity due to impaired trafficking (Calcaterra et al., 2016), we used hERG 1A/3.1 heterotetramers to study the pharmacological effects of the drugs on these currents. Although the stoichiometry of hERG 1A/3.1 heterotetramers is unknown, in the present study the hERG 1A/3.1 heterotetramers
Conflicts of interest The authors declare no conflicts of interest. Authorship contributions Participated in research design: BH Choi and SJ Hahn. Conducted experiments: HJ Lee. Performed data analysis and interpretation: HJ Lee, JS Choi. Wrote or contributed to the writing of the manuscript: SJ Hahn. Acknowledgements We thank Professor Jamie Vanderberg (Victor Chang Cardiac Research Institute, Darlinghurst, NSW, Australia) for the hERG 3.1 plasmid. This research was supported by the Basic Science Research Program through the National Research Foundation of Korea funded by the Ministry of Education (2017R1D1A1B03028108). References Apud, J.A., Zhang, F., Decot, H., Bigos, K.L., Weinberger, D.R., 2012. Genetic variation in KCNH2 associated with expression in the brain of a unique hERG isoform modulates treatment response in patients with schizophrenia. Am. J. Psychiatry 169, 725–734. Caccia, S., Invernizzi, R.W., Nobili, A., Pasina, L., 2013. A new generation of antipsychotics: pharmacology and clinical utility of cariprazine in schizophrenia. Therapeut. Clin. Risk Manag. 9, 319–328. Calcaterra, N.E., Hoeppner, D.J., Wei, H., Jaffe, A.E., Maher, B.J., Barrow, J.C., 2016. Schizophrenia-associated hERG channel Kv11.1-3.1 exhibits a unique trafficking deficit that is rescued through proteasome inhibition for high throughput screening. Sci. Rep. 6, 19976. Chae, Y.J., Jeon, J.H., Lee, H.J., Kim, I.B., Choi, J.S., Sung, K.W., Hahn, S.J., 2014. Escitalopram block of hERG potassium channels. Naunyn-Schmiedeberg’s Arch. Pharmacol. 387, 23–32. Citrome, L., 2013. Cariprazine: chemistry, pharmacodynamics, pharmacokinetics, and metabolism, clinical efficacy, safety, and tolerability. Expert Opin. Drug Metabol. Toxicol. 9, 193–206. De Bruin, M.L., Pettersson, M., Meyboom, R.H., Hoes, A.W., Leufkens, H.G., 2005. AntiHERG activity and the risk of drug-induced arrhythmias and sudden death. Eur. Heart J. 26, 590–597. Dennis, A., Wang, L., Wan, X., Ficker, E., 2007. hERG channel trafficking: novel targets in drug-induced long QT syndrome. Biochem. Soc. Trans. 35, 1060–1063. Durgam, S., Greenberg, W.M., Li, D., Lu, K., Laszlovszky, I., Nemeth, G., Migliore, R., Volk, S., 2017. Safety and tolerability of cariprazine in the long-term treatment of schizophrenia: results from a 48-week, single-arm, open-label extension study. Psychopharmacology (Berlin) 234, 199–209. European Medicines Agency, 2017. Assessment Report. Reagila. . http://www.ema. europa.eu/docs/en_GB/document_library/EPAR_-_Public_assessment_report/human/ 002770/WC500234926.pdf. Finlayson, K., Witchel, H.J., McCulloch, J., Sharkey, J., 2004. Acquired QT interval
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