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Available online at www.sciencedirect.com

www.elsevier.com/locate/brainres

Research Report

Effects of donepezil on hERG potassium channels Yun Ju Chaea,1, Hong Joon Leeb,1, Ji Hyun Jeonc, In-Beom Kimc, Jin-Sung Choid, Ki-Wug Sungb, Sang June Hahna,n a

Department of Physiology, College of Medicine, The Catholic University of Korea, Seoul 137-701, 222 Banpo-daero, Seocho-gu, Korea b Department of Pharmacology, College of Medicine, The Catholic University of Korea, Seoul 137-701, Korea c Department of Anatomy, College of Medicine, The Catholic University of Korea, Seoul 137-701, Korea d College of Pharmacy, Integrated Research Institute of Pharmaceutical, The Catholic University of Korea, 43-1 Yeokgok 2-dong, Wonmi-gu, Bucheon, Gyeonggi-do, Korea

art i cle i nfo

ab st rac t

Article history:

Donepezil is a potent, selective inhibitor of acetylcholinesterase, which is used for the

Accepted 27 November 2014

treatment of Alzheimer’s disease. Whole-cell patch-clamp technique and Western blot analyses were used to study the effects of donepezil on the human ether-a-go-go-related

Keywords:

gene (hERG) channel. Donepezil inhibited the tail current of the hERG in a concentration-

hERG

dependent manner with an IC50 of 1.3 μM. The metabolites of donepezil, 6-ODD and 5-ODD,

Donepezil

inhibited the hERG currents in a similar concentration-dependent manner; the IC50 values

Alzheimer's disease

were 1.0 and 1.5 μM, respectively. A fast drug perfusion system demonstrated that

Channel trafficking

donepezil interacted with both the open and inactivated states of the hERG. A fast application of donepezil during the tail currents inhibited the open state of the hERG in a concentration-dependent manner with an IC50 of 2.7 μM. Kinetic analysis of donepezil in an open state of the hERG yielded blocking and unblocking rate constants of 0.54 mM  1s  1 and 1.82 s  1, respectively. The block of the hERG by donepezil was voltage-dependent with a steep increase across the voltage range of channel activation. Donepezil caused a reduction in the hERG channel protein trafficking to the plasma membrane at low concentration, but decreased the channel protein expression at higher concentrations. These results suggest that donepezil inhibited the hERG at a supratherapeutic concentration, and that it did so by preferentially binding to the activated (open and/or inactivated) states of the channels and by inhibiting the trafficking and expression of the hERG channel protein in the plasma membrane. & 2014 Published by Elsevier B.V.

n

Corresponding author. Fax: þ82 2 532 9575. E-mail address: [email protected] (S.J. Hahn). 1 Yun Ju Chae and Hong Joon Lee equally contributed as first author.

http://dx.doi.org/10.1016/j.brainres.2014.11.057 0006-8993/& 2014 Published by Elsevier B.V.

Please cite this article as: Chae, Y.J., et al., Effects of donepezil on hERG potassium channels. Brain Research (2014), http://dx. doi.org/10.1016/j.brainres.2014.11.057

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1.

Introduction

Donepezil is a selective inhibitor of acetylcholinesterase, and is used for the symptomatic treatment of Alzheimer's disease (Dooley and Lamb, 2000; Wilkinson, 1999). This drug is effective and well tolerated with less adverse effects than other inhibitors of acetylcholinesterase, such as physostigmine and tacrine (Sugimoto et al., 2000). However, several case reports have stated that donepezil induces adverse side effects of cardiac rhythm, such as prolonged QT interval and Torsade de Pointes arrhythmias (Takaya et al., 2009; Tanaka et al., 2009). One of the mechanisms of QT prolongation and Torsade de Pointes includes the blockage of the human ethera-go-go-related gene (hERG) current, which underlies the rapidly activating delayed rectifier voltage-gated Kþ (Kv) currents in human cardiomyocytes (Thomas et al., 2002; Witchel et al., 2002). In this context, several acetylcholinesterase inhibitors block various Kv channels in different preparations. For example, rivastigmine, tetrahydroaminoacridine and huperzine A are known to have inhibited transient outward Kv currents and delayed rectifier Kv currents in dissociated rat hippocampal neurons. (Li and Hu, 2002; Pan et al., 2003a; Rogawski, 1987). Donepezil has also caused significant inhibition of delayed rectifier Kv currents and fast transient Kv currents in rat dissociated hippocampal neurons (Yu and Hu, 2005) and in molluscan neurons (Solntseva et al., 2007). Although some acetylcholinesterase inhibitors, such as galantamine are known to cause QT interval prolongation by blocking the hERG current (Vigneault et al., 2012), to the best of our knowledge, the effects of donepezil on hERG currents remain unknown. Since hERG channels are highly expressed in both the heart and the brain (Vandenberg et al., 2012), we studied the effect of donepezil on hERG currents using the patch-clamp technique to determine the electrophysiological basis for cardiac and neuronal action. Donepezil is metabolized through the cytochrome P450 enzymes in the liver into several metabolites, including 6-O-desmethyl donepezil (6ODD), a major metabolite with activity that is similar to that of the parent drug, and 5-O-desmethyl donepezil (5-ODD) (Dooley and Lamb, 2000). Therefore, we also studied the action of these metabolites on hERG currents. Because many drugs have been known to inhibit hERG channel protein trafficking to the plasma membrane (Rajamani et al., 2006), long-term modulation (24 h) of the hERG channels by donepezil was also investigated using Western blot analyses.

2.

Results

2.1. Concentration-dependent inhibition of the hERG by donepezil and its metabolites,6-ODD and 5-ODD Fig. 1A shows the effects of donepezil on the hERG currents expressed in HEK cells. The hERG currents were elicited by a 4-s depolarizing step to þ20 mV from a holding potential of 80 mV followed by a repolarization step to  50 mV for 6 s every 15 s in the absence and presence of donepezil. To study the concentration dependence of the hERG current inhibition by donepezil, the peak tail currents of the hERG at 50 mV

Fig. 1 – Concentration-response relationships for the inhibition of the hERG by donepezil. (A) Whole-cell hERG currents were elicited by a 4-s depolarizing pulse to þ20 mV from a holding potential of 80 mV and repolarization to 50 mV for 6 s to measure the tail currents every 15 s in the absence and presence of donepezil. The dotted line marks zero current. (B) Normalized inhibitions of the hERG tail current are plotted as a function of donepezil concentrations. The experimental data were fitted with a Hill equation. Data are expressed as the means7S.E.

were normalized to the respective control values and plotted against a drug concentration (Fig. 1B). Donepezil inhibited the tail current of the hERG in a concentration-dependent manner, i.e., donepezil at concentrations of 0.3, 1, 3, and 10 μM reduced the tail current by 16.171.1, 40.871.6, 67.071.1 and 85.570.6% (n ¼8), respectively. The IC50 value for the inhibition of tail currents was 1.370.2 μM with a Hill coefficient of 1.170.1 (n ¼8). The metabolites of donepezil, 6-ODD and 5-ODD, inhibited the hERG current in a similar concentration-dependent manner (Fig. 2A and B). The IC50 values for the inhibition of tail currents were 1.070.1 μM with a Hill coefficient of 1.170.1 (n ¼8) for 6-ODD and 1.570.1 μM with a Hill coefficient of 1.170.1 (n¼ 6) for 5-ODD. Therefore, donepezil and its metabolites, 6-ODD and 5-ODD, inhibited the hERG currents with similar potency.

Please cite this article as: Chae, Y.J., et al., Effects of donepezil on hERG potassium channels. Brain Research (2014), http://dx. doi.org/10.1016/j.brainres.2014.11.057

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Fig. 2 – Concentration-response relationships for the inhibition of the hERG by donepezil metabolites, 6-ODD and 5-ODD. Whole-cell hERG currents were elicited by a 4-s depolarizing pulse to þ20 mV from a holding potential of  80 mV and repolarization to 50 mV for 6 s to measure the tail currents every 15 s under control conditions and presence of 6-ODD (A) and 5-ODD (B). The dotted line marks zero current. Normalized inhibitions of the hERG tail current are plotted as a function of 6-ODD and 5-ODD concentrations. The experimental data were fitted with a Hill equation. Data are expressed as the means7S.E.

2.2. Inhibition of the inactivated and open state of the hERG by donepezil A fast drug perfusion system was used to investigate whether donepezil would inhibit the open and/or inactivated state of the hERG channels (Chae et al., 2014; Ganapathi et al., 2009). The hERG currents were elicited by a 5-s depolarizing prepulse to þ60 mV that inactivated the hERG channels followed by 5 s of repolarization to 40 mV, which induced a recovery from inactivation and an open state of the channels (Fig. 3). Donepezil was applied during the depolarizing and repolarization pulses. Donepezil inhibited the hERG currents by 35.672.7% at the end of the depolarizing pulse to þ60 mV and inhibited the currents by 39.871.5% (n¼ 8) after repolarization to 40 mV. Thus, donepezil inhibited the hERG currents by preferentially binding to both the inactivated and open states of the channels.

2.3. The kinetics of an open channel block of the hERG by donepezil To further investigate the interaction of donepezil with the open state of a hERG channel, hERG currents were elicited by

Fig. 3 – Interaction of donepezil with both the inactivated and open state of the hERG channels. The hERG current was elicited from a holding potential of 80 mV by 5-s depolarizing pulse to þ60 mV, followed by a repolarizing pulse to 40 mV for 5 s to record tail currents. Donepezil was rapidly applied during the depolarizing pulse and continued into the repolarizing pulse as indicated by the bar. The currents were measured during the drug application (∙). Currents were corrected for leak using standard online P/7 leak subtraction. The dotted line marks zero current.

Please cite this article as: Chae, Y.J., et al., Effects of donepezil on hERG potassium channels. Brain Research (2014), http://dx. doi.org/10.1016/j.brainres.2014.11.057

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Fig. 4 – Open channel block of the hERG currents by fast application of donepezil. (A) the hERG current was elicited from a holding potential of 80 mV by 4-s depolarizing pulse to þ60 mV, followed by a repolarizing pulse to 40 mV for 10 s to record tail currents. After a delay of 1 s, donepezil was rapidly applied and removed during the repolarizing pulse. The bar indicates the time for the application of donepezil. (B) Concentration-response relationship for an open channel block of the hERG current by donepezil. The normalized current at the end of the drug application (∙) was used to construct the concentrationresponse relationships for a donepezil block of the hERG current. The experimental data were fitted with a Hill equation. Data are expressed as the means7S.E.

a 1 s depolarizing prepulse to þ60 mV that was followed by 10 s of repolarization to 40 mV, which induced recovery from inactivation and the open state of the channels (Fig. 4A). Donepezil was rapidly applied and removed during the tail currents of the hERG. Fast application of donepezil during the repolarization pulse led to a rapid and reversible block of the hERG current in a concentration-dependent manner. A nonlinear least-squares fit of the Hill equation to the concentrationresponse data yielded an IC50 value of 2.770.3 μM with a Hill coefficient of 0.870.01 (n¼ 5) (Fig. 4B). The time courses for the development of the open-channel block by donepezil were fitted to a biexponential function that yielded the fast and slow time constants (Fig. 5A). The fast time constants were used as an approximation of the time course for drug-channel interaction kinetics. The onset time constants (τon) of the donepezilinduced block were concentration-dependent.

Fig. 5 – Kinetics of donepezil interaction with the open state of the hERG channels. (A) Under control conditions, the hERG current were well fitted with a single exponential function. After the administration of donepezil, the decaying phases of the hERG current were well fitted to a biexponential function with a fast and slow time constant. The unblocking time constants (τoff) were described by monoexponential function. (B) The drug-induced fast time constant (τon) were inverted and plotted against the concentration of donepezil. The data were fitted with a linear regression: 1/τon ¼ kþ1 [D]þ k-1, where τon was the drug-induced time constant and [D] denoted the drug concentration. The τoff was independent of donepezil concentration. 1/τoff ¼ k-1. Apparent blocking (kþ1, ■) and unblocking (k-1, □) rate constants were obtained from the slope and the intercept values, respectively. Data are expressed as the means7S.E.

A plotting for the reciprocal of the fast time constants (τon) vs. each concentration of donepezil was fitted to a linear equation and yielded the apparent blocking (kþ1) and unblocking (k-1) rate constants of 0.5470.04 mM  1s  1 and 1.8870.11s  1 (n ¼5), respectively (Fig. 5B). The rates of current recovery (τoff) following drug washout were also described by monoexponential function, and were independent of donepezil concentration (Fig. 5B). The unblocking rate constant (k1 1) was calculated from a 1/τoff curve, and was 1.8270.10 s (n ¼5), which was similar to the estimate of 1.8870.11 s  1 (n ¼5) that was obtained from the Y-axis intercept of the 1/τon curve. The resultant KD (k-1/kþ1) was found to be 3.370.2 mM (n ¼5), which was in good agreement with the IC50 value of

Please cite this article as: Chae, Y.J., et al., Effects of donepezil on hERG potassium channels. Brain Research (2014), http://dx. doi.org/10.1016/j.brainres.2014.11.057

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2.7 mM for donepezil that was calculated from the concentrationresponse curve.

2.4.

Voltage-dependent inhibition of the hERG by donepezil

Fig. 6A shows the representative hERG currents elicited after applying 4 s depolarizing pulses from a holding potential of  80 mV to þ50 mV in 10-mV steps every 15 s in the absence and presence of donepezil. The tail currents of the hERG were recorded at  50 mV for 6 s. Fig. 6B shows the average

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current-voltage relationships for the hERG currents measured at the end of depolarizing pulses and for the peak tail current in the absence and presence of donepezil. Donepezil significantly inhibited the hERG currents at all voltages between 40 and þ60 mV. To investigate the effects of donepezil on the voltage dependency of the activation curve, the peak tail currents of the hERG were normalized and plotted against the membrane potential before and after the application of donepezil. The plot of the normalized tail currents was fit to a Boltzmann function. The half-activation potential (V1/2)

Fig. 6 – Effect of donepezil on current-voltage relationships. (A) Whole-cell hERG currents were evoked by depolarizing pulses from  50 mV to þ50 mV for 4 s in steps of 10 mV every 15 s from a holding potential of 80 mV and repolarization to  50 mV for 4 s in the absence and presence of donepezil. (B) Current-voltage relationships of the steady-state and peak tail currents of the hERG under control conditions and after the application of donepezil. The peak amplitudes of tail currents in the presence of donepezil were normalized to those at each voltage in the absence of donepezil. Data were fit to a Boltzmann function. (C) The voltage-dependent inhibition of the hERG currents by donepezil was expressed as a relative current (IDonepezil/IControl). n Significant difference from data obtained at 30 mV (Po0.01). The dotted line represents the activation curve of the hERG currents under control conditions. Data are expressed as the means7S.E. Please cite this article as: Chae, Y.J., et al., Effects of donepezil on hERG potassium channels. Brain Research (2014), http://dx. doi.org/10.1016/j.brainres.2014.11.057

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of the activation curve was significantly shifted in a hyperpolarizing direction from -24.771.1 mV under control conditions to -29.471.6 mV after donepezil (n ¼6, Po0.05). However, there was no significant change in the slope factor in the presence of donepezil (control: k ¼ 7.270.4 mV; donepezil: k¼ 7.170.5 mV, n¼ 6). A fractional inhibition (IDonepezil/ IControl) was calculated for each membrane potential with the normal activation curve to evaluate the voltage dependency of the inhibition by donepezil (Fig. 6C). The inhibition of the hERG current by donepezil increased from -23.278.7% at 30 mV to -43.673.3% (n¼ 6) at 10 mV. Thus, the inhibition was steeply increased in the voltage range coinciding with the activation of the hERG currents (Po0.01), which suggested an interaction with the channel in the open state (Paul et al., 2002). However, there was no voltage-dependent inhibition of the hERG currents between 0 and þ60 mV where the channels were fully activated.

2.5. Effects of donepezil on the hERG channel protein trafficking and protein expression To study whether donepezil affected the hERG channel trafficking, the hERG-HEK cells were incubated for 24 h with donepezil (0.3, 1, 3, and 10 μM), and fluoxetine (30 μM) was used as a positive control (Fig. 7A). Fluoxetine significantly decreased a protein band at 155 kD (the fully glycosylated form of the hERG channel protein) but had no effect at 135 kD (the core glycosylated form), as previously described (Rajamani et al., 2006). At 0.3 μM, donepezil reduced a protein band at 155 kD to 89.473.2% of the control value (n¼3), and had no effect at 135 kD. At concentrations of 1, 3 and 10 μM, however, donepezil decreased the 155 kD protein bands to 68.37 2.7, 66.273.4 and 62.474.2% of the control value (n¼ 3, Po0.05) and 135 kD bands to 71.47 3.8, 66.47 3.4 and 66.17 3.4% (n ¼3, Po0.05), respectively, suggesting a reduction in the hERG channel protein expression. The current densities were measured electrophysiologically to confirm that donepezil reduced the hERG channel surface expression (Fig. 7B). Under control conditions, the peak tail current of hERG was 96.973.9 pA/pF at  50 mV (n¼ 8). After 24 h of incubation with 10 μM donepezil, the peak tail current of hERG was decreased to 35.575.4 pA/pF at  50 mV (n¼ 8, Po0.01), confirming the immunological findings.

3.

Discussion

In the present study, the effects of donepezil on the hERG currents expressed in HEK cells were examined. The main findings can be summarized as follows: 1) donepezil caused a reversible, concentration- and voltage-dependent inhibition of the hERG currents; 2) donepezil inhibited the hERG by preferentially binding to the inactivated state during depolarization and to the open state during repolarization; 3) kinetic analysis gave results consistent with an open-channel block mechanism; and, 4) donepezil also inhibited the hERG channel protein trafficking to the plasma membrane and channel protein expression. These characteristics of the hERG inhibition by donepezil were similar to our previous results showing an activated (open and/or inactivated) channel inhibition

Fig. 7 – (A) Effects of donepezil on the hERG channel protein expression. Western blot analysis of the hERG channel protein under control conditions and after 24 h incubation with increasing concentrations (0.3, 1, 3, and 10 μM) of donepezil and 30 μM fluoxetine. Three independent experiments were conducted. (B) The tail current densities of hERG at  50 mV were measured under control conditions and after 24 h of incubation with donepezil. The pulse protocol is the same as that described in Fig. 6. Data are expressed as the means7S.E. and inhibition of hERG channel protein trafficking by escitalopram (Chae et al., 2014). In line with this observation, the selective serotonin-norepinephrine reuptake inhibitor duloxetine exerted inhibitory effects on the hERG current by both direct channel block of the open and inactivated states and a reduction in the channel surface expression (Fischer et al., 2013). Similarly, ranolazine blocked hERG channels in the activated states with little effect on the closed state, but did not alter the hERG protein trafficking to the plasma membrane (Rajamani et al., 2008). Our fast drug perfusion system demonstrated that donepezil inhibited the hERG currents both at depolarized voltages where most channels are inactivated, and during repolarization when most channels are open (Ganapathi et al., 2009). At higher voltage a large fraction of the hERG channels is in an

Please cite this article as: Chae, Y.J., et al., Effects of donepezil on hERG potassium channels. Brain Research (2014), http://dx. doi.org/10.1016/j.brainres.2014.11.057

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inactivated state, and there are fewer open states of the channels for the donepezil to block. Since transitions of the hERG channels from the open state into the inactivated state are very fast, open and inactivated channel inhibition cannot be distinguished by comparing the extents of inhibition between depolarization and repolarization. Thus, these results suggest that donepezil inhibited the hERG currents by preferentially binding to the activated (open and/or inactivated) states of the channels. Furthermore, the kinetics of the donepezil interaction with the hERG channels was consistent with an open-channel block mechanism. The blocking rate constant was dependent linearly on donepezil concentration, and the rate of current recovery following drug washout (the unblocking constant) did not significantly change with the concentration of the drug, which is consistent with a simple bimolecular reaction scheme. The IC50 value of 2.7 μM that was estimated for the binding to the open state of the hERG approximated that of the KD of 3.3 μM, which was obtained from kinetic analysis of the blocking and unblocking of donepezil to the open state of the hERG channel. The voltage dependence of a donepezil block of the hERG current was an additional finding of an open channel block. The block of the hERG by donepezil was steeply increased in the voltage range coinciding with the activation of the hERG currents, which suggested an interaction with the channel in the open state of the hERG channels (Paul et al., 2002). In the present study, donepezil interacted with the activated states of the hERG channels with an IC50 of 1.3 μM and with the open state with an IC50 of 2.7 μM. The most likely explanation for this difference in IC50 values is that the hERG channel has a higher affinity for donepezil when it is in the inactivated state compared with the open state. Clinical studies have demonstrated that donepezil is a welltolerated drug that improves cognition and global function in patients with mild to moderate Alzheimer's disease (Rogers et al., 1998; Rogers and Friedhoff, 1998). However, donepezil reportedly causes adverse side effects of cardiac rhythm with QT prolongation including atrioventricular block and Torsade de pointes (Takaya et al., 2009; Tanaka et al., 2009). In the present study, donepezil proved to be a potent blocker of the hERG channel that underlies the rapidly activating component of delayed rectifier currents (Sanguinetti et al., 1995). These currents play an important role in determining the repolarization of the action potential in ventricular myocytes (Snyders, 1999). Abnormal functions in the hERG channel can result in QT prolongation, which can lead to sudden death due to Torsade de pointes arrhythmias (Sanguinetti and TristaniFirouzi, 2006). Therefore, our data provide a molecular mechanism for drug-induced QT prolongation and cardiac arrhythmias under the clinical administration of donepezil. Donepezil is a potent, selective inhibitor of acetylcholinesterase that has resulted in fewer adverse effects compared with other cholinesterase inhibitors, such as physostigmine and tacrine (Dooley and Lamb, 2000; Sugimoto et al., 2000). The hERG gene is highly expressed in the prefrontal cortex and in hippocampal formation, which have been implicated in the cognition and memory processes and in the pathophysiology of Alzheimer's disease (Guasti et al., 2005). In neurons, hERG channels play an important role in controlling neuronal firing

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patterns and cortical network oscillation, and in spikefrequency modulation, and the increase in these currents can counteract Naþ influx, decrease spiking frequency, or even terminate a burst of action potentials (Chiesa et al., 1997; Huffaker et al., 2009). In addition to the inhibition of acetylcholinesterase, the blockade of delayed rectifier K currents by galantamine and tacrine has been linked to the clinical benefits in Alzheimer's disease (Kraliz and Singh, 1997; Pan et al., 2003b). Donepezil also inhibited delayed rectifier Kv currents and fast transient Kv currents in the dissociated hippocampal neurons of rats (Yu and Hu, 2005; Zhong et al., 2002) and in molluscan neurons (Solntseva et al., 2007). Accordingly, the relevance of potassium channels as a potential target for Alzheimer's disease has been reviewed in the literature (Lavretsky and Jarvik, 1992; Wiseman and Jarvik, 1991). The mean donepezil plasma concentrations were 25.970.7 ng/ml and 50.671.9 ng/ml (0.06–0.12 μM) in patients receiving dosages of 5 mg/day and 10 mg/day, respectively (Rogers et al., 1998). In the present study, the concentration at which donepezil inhibited the hERG currents was much higher than the therapeutic plasma concentration in patients being treated for Alzheimer's disease. After multiple-dose administration, however, donepezil accumulates in plasma by 4- to 7-fold in humans (Wilkinson, 1999), and the ratio of the concentration (brain/plasma) of donepezil is 6.1–8.4 in rats (Kosasa et al., 2000). Thus, this concentration was within the blocking range for the hERG channels used in the present study. Donepezil is metabolized to its pharmacologically active metabolites, 6-ODD and 5-ODD, through the cytochrome P450 enzymes in the liver (Dooley and Lamb, 2000). In our study, the potency of 6-ODD and 5-ODD was similar to that of the parent drug in the inhibition of the hERG currents. Furthermore, donepezil inhibited the trafficking of the hERG channel protein to the plasma membrane at low concentrations and affected the hERG channel expression at higher concentrations, resulting in a significant reduction in the density of total hERG currents through the plasma membrane. Therefore, the hERG inhibition by donepezil and its metabolites might be of physiological relevance. In summary, donepezil and its metabolites, 6-ODD and 5ODD produced a concentration-dependent inhibition of the hERG currents. The metabolites of donepezil, 6-ODD and 5ODD, inhibited hERG currents with a potency that was similar to that of the parent drug. Donepezil inhibited the hERG currents by preferentially binding to the activated (open and/ or inactivated) states as well as by inhibiting both the trafficking of the hERG channel protein to the plasma membrane and its expression. Our results might in part account for the ionic mechanism underlying the potential arrhythmogenic action of donepezil.

4.

Experimental procedures

The hERG-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 1C in D-MEM/F-12 (Invitrogen, Grand Island, NY, USA), and were supplemented with 10%

Please cite this article as: Chae, Y.J., et al., Effects of donepezil on hERG potassium channels. Brain Research (2014), http://dx. doi.org/10.1016/j.brainres.2014.11.057

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fetal bovine serum, 1% nonessential amino acid, and 400 μg/ml geneticin, according to the manufacturer's instructions. The cells used for electrophysiological recordings were seeded on glass coverslips (12-mm diameter; Fisher Scientific, Pittsburgh, PA, USA) 24 h before use. The whole-cell patch-clamp recordings were performed at room temperature (22–24 1C) using an Axopatch 700B amplifier (Molecular Devices, Sunnyvale, CA, USA). Patch pipettes were formed from borosilicate glass (PG10165-4, World Precision Instruments, Sarasota, FL, USA) with tip resistances of 2 to 4 MΩ when filled with an internal pipette solution. The leak currents measured by a pulse from -80 to  70 mV were relatively small in most experiments (8.971.8 pA, n ¼18), and cells with significant leak currents were rejected. Unless otherwise stated, no leak subtraction was performed during the experiments. For fast drug application, donepezil 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; Choi et al., 2003 ). The rate of solution change was 20.972.5 ms (n ¼14), and this solution exchange time was not a limiting factor to the determination of the blocking time course. 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. Donepezil and its metabolites, 6-ODD and 5-ODD (Santa Cruz Biotechnology, Inc., Dallas, Texas, USA), were dissolved in dimethyl sulfoxide (DMSO, Sigma, St. Louis, MO, USA). The concentration of DMSO in the final dilution was less than 0.1%. The tail current of the hERG was not significantly changed after 5 min of 0.1% DMSO (95.170.7% of the control, n ¼12). Western blot analyses were performed with minor modifications, as described previously (Jeon et al., 2013). Briefly, cells were homogenized in ice-cold RIPA buffer (50 mM Tris buffer, pH 8.0; 150 mM NaCl; 1% NP-40; 0.5% deoxycholate; and 0.1% SDS). Equal amounts (20 mg) of total protein from each group of cells were separated by SDS-PAGE, and the proteins were transferred to a nitrocellulose membrane. Nonspecific binding was blocked with 5% non-fat dry milk in Tris buffer saline with 0.1% Tween-20 for 1 h at room temperature, followed by incubation with the anti-hERG antibody (dilution, 1:500; Alomone labs, Jerusalem, Israel) overnight at 4 1C. The membrane was rinsed three times, and incubated for 2 h at room temperature in a 1:1,000 dilution of the appropriate biotin-conjugated IgG antibody (Vector Laboratories, Burlingame, CA, USA). Afterwards, the membrane was rinsed three times, and incubated for 1 h at room temperature in ABC solution (Vector Laboratories). The blot was washed three times, and the immunoreactive bands were detected using an Enhanced Chemiluminescence Detection Kit (Amersham, Arlington Heights, IL, USA). Band intensities obtained from immunoblot assays were measured by densitometry. 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 results are expressed as the means7S.E. A paired Student's t-test for comparison between two groups and an analysis of variance

for comparisons of multiple groups followed by a Bonferroni's test were used for the statistical analyses. A value of Po0.05 was considered statistically significant.

Conflict of interest none declared

Authorship contributions Participated in research design: JS Choi, K-W Sung, and SJ Hahn Conducted experiments: YJ Chae, JH Jeon, and HJ Lee, Performed data analysis and interpretation: YJ Chae, HJ Lee, JH Jeon, and JS Choi Wrote or contributed to the writing of the manuscript: I-B Kim, and SJ Hahn

Acknowledgments This work was supported by a grant from the Catholic Medical Center Research Foundation made in the program year of 2014.

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Please cite this article as: Chae, Y.J., et al., Effects of donepezil on hERG potassium channels. Brain Research (2014), http://dx. doi.org/10.1016/j.brainres.2014.11.057

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Effects of donepezil on hERG potassium channels.

Donepezil is a potent, selective inhibitor of acetylcholinesterase, which is used for the treatment of Alzheimer's disease. Whole-cell patch-clamp tec...
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