European Journal of Pharmacology 740 (2014) 1–8
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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Neuropharmacology and analgesia
Effects of haloperidol on Kv4.3 potassium channels Hong Joon Lee a, Ki-Wug Sung a,n, Sang June Hahn b,nn a Department of Pharmacology, Cell Death and Disease Research Center, College of Medicine, The Catholic University of Korea, 222 Banpo-daero, Seocho-gu, , Seoul 137-701, Republic of Korea b Department of Physiology, Cell Death and Disease Research Center, College of Medicine, The Catholic University of Korea, 222 Banpo-daero, Seocho-gu, Seoul 137-701, Republic of Korea
art ic l e i nf o
a b s t r a c t
Article history: Received 12 February 2014 Received in revised form 23 June 2014 Accepted 24 June 2014 Available online 3 July 2014
Haloperidol is commonly used in clinical practice to treat acute and chronic psychosis, but it also has been associated with adverse cardiovascular events. We investigated the effects of haloperidol on Kv4.3 currents stably expressed in CHO cells using a whole-cell patch-clamp technique. Haloperidol did not significantly inhibit the peak amplitude of Kv4.3, but accelerated the decay rate of inactivation of Kv4.3 in a concentration-dependent manner. Thus, the effects of haloperidol on Kv4.3 were estimated from the integral of the Kv4.3 currents during the depolarization pulse. The Kv4.3 was decreased by haloperidol in a concentration-dependent manner with an IC50 value of 3.6 μM. Haloperidol accelerated the decay rate of Kv4.3 inactivation and activation kinetics in a concentration-dependent manner, thereby decreasing the time-to-peak. Haloperidol shifted the voltage dependence of the steady-state activation and inactivation of Kv4.3 in a hyperpolarizing direction. Haloperidol also caused an acceleration of the closed-state inactivation of Kv4.3. Haloperidol produced a use-dependent block of Kv4.3, which was accompanied by a slowing of recovery from the inactivation of Kv4.3. These results suggest that haloperidol blocks Kv4.3 by both interacting with the open state of Kv4.3 channels during depolarization and accelerating the closed-state inactivation at subthreshold membrane potentials. & 2014 Elsevier B.V. All rights reserved.
Keywords: Kv4.3 Haloperidol Open-channel block Closed-state inactivation
1. Introduction Haloperidol is structurally classified as a butyrophenone antipsychotic drug, and has long been used to treat schizophrenia (Lopez-Munoz and Alamo, 2009; Miyamoto et al., 2012). With the advent of newer atypical antipsychotics, the clinical use of haloperidol for the long-term treatment of schizophrenia has declined (Meltzer, 2013). However, it continues to be commonly used in treating certain forms of psychotic symptoms, particularly delirium associated with several clinical situations (Meyer-Massetti et al., 2010). Although haloperidol is a known antagonist of dopamine D2 receptors in the central nervous system (Seeman, 2006), it also blocks different ion channels at concentrations that are within the therapeutic range. For example, the acute application of haloperidol blocked numerous types of potassium channels, including delayed-rectifier potassium channels in cortical neurons (Yang et al., 2005), the ATP-sensitive potassium channels in pancreatic β cells (Yang et al., 2004), the G protein-activated inwardly rectifying potassium channels expressed in Xenopus n
Corresponding author. Tel.: þ 82 2 2258 7325; fax: þ 82 2 536 2485. Corresponding author. Tel.: þ 82 2 2258 7275; fax: þ 82 2 532 9575. E-mail addresses: [email protected] (K.-W. Sung), [email protected] (S.J. Hahn). nn
http://dx.doi.org/10.1016/j.ejphar.2014.06.043 0014-2999/& 2014 Elsevier B.V. All rights reserved.
oocytes (Kobayashi et al., 2000), and the human ether-a-go-gorelated gene (HERG) potassium channels expressed in Xenopus oocytes (Suessbrich et al., 1997). These reports suggest that potassium channels possibly are associated with the therapeutic, and/or neurological and non-neurological adverse effects of haloperidol. Kv4.3 is an α-subunit of a Shal-type voltage-gated potassium channel (Kv). It generates rapidly activating and inactivating Kv currents (Ohya et al., 1997), and contributes to the A-type Kv currents in neurons and transient outward Kv currents in cardiac myocytes (Birnbaum et al., 2004; Dixon et al., 1996). Chronic treatment with haloperidol dampened the brain dopamine system by increasing the expression of Kv4.3 mRNA, which underlies the A-type Kv channels in the isolated midbrain dopamine neurons (Hahn et al., 2003). These results also support the possibility that changes in the Kv4.3 channel activities by drugs in certain clinical situations can contribute to the therapeutic action of haloperidol and/or induce serious adverse drug reactions. Accordingly, intravenous haloperidol used to treat delirium has shown an increased risk of torsades de pointes, a life-threatening ventricular arrhythmia via a prolongation of the QT-interval (Hassaballa and Balk, 2003; Meyer-Massetti et al., 2010). However, a limited amount of information is available to explain how haloperidol modulates Kv4.3 channel function to elucidate the therapeutic and/or adverse effects of haloperidol. Therefore, we examined the effects of
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haloperidol on cloned Kv4.3 channels stably expressed in Chinese hamster ovary (CHO) cells using the whole-cell patch-clamp technique.
2. Materials and methods 2.1. Cell culture The CHO cells were transfected with cDNA encoding Kv4.3 channels using the Lipofectamine reagent (Invitrogen, Grand Island, NY, USA), as previously described in detail (Ahn et al., 2005). A stable CHO cell line expressing Kv4.3 channels was maintained in Iscove's modified Dulbecco's medium (Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen), 2 mM glutamine (Invitrogen), 0.1 mM hypoxanthine (Invitrogen), 0.01 mM thymidine (Invitrogen), 0.3 mg/ml G418 (Invitrogen), and 1% of a 100 antibiotic antimycotic mixture (Invitrogen) at 37 1C with 5% CO2-enriched air. The cells treated with trypsin– EDTA solution were plated on glass coverslips (12 mm diameter; Fisher Scientific, Pittsburgh, PA, USA) and placed in 35 mm Petri dishes. Patch-clamp recordings were obtained 12–24 h later. For electrphysiology, the coverslips containing adherent CHO cells were mounted on the glass bottom of the recording chamber (RC-13; Warner Instrument, Hamden, CT, USA) and were continuously perfused with an extracellular bath solution. 2.2. Solutions and drugs The extracellular bath solution contained (in mM) 140 NaCl, 5 KCl, 1 CaCl2, 1 MgCl2, 10 HEPES, and 10 glucose and was adjusted to pH 7.3 using NaOH. The intracellular 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 osmolarity of the solutions was measured with a vapor pressure osmometer (Vapro 5520, Logan, UT, USA) at 300–310 mOsm. Haloperidol was dissolved in dimethyl sulfoxide (DMSO) as a stock solution of 100 mM, and the stock solution was then diluted with the bath solution to obtain the desired concentration. The concentration of DMSO in the final dilution was less than 0.1%, and this concentration had no effect on the Kv4.3 currents.
the normalized integral of Kv4.3 currents to determine the concentration–response curves for haloperidol: ƒ ¼ 1=½1 þ ðIC50 =½DÞn
ð1Þ
where ƒ is the fractional block of the integral of Kv4.3 currents at a given haloperidol concentration [D], IC50 is the concentration of haloperidol for a 50% block, and n is the Hill coefficient. To evaluate steady-state activation, the amplitude of tail currents were measured at 60 mV after the application of 8 ms depolarizing pulses at potentials between 80 and þ80 mV in 10 mV increments every 10 s from a holding potential of 80 mV, and were then normalized to the maximal tail current amplitude and plotted against the test potential. The Boltzmann equation was fitted to the steady-state activation curves: y ¼ 1=f1 þexp½ ðV V 1=2 Þkg
ð2Þ
where V1/2 is the voltage at which the conductance was halfmaximal, V is the test potential and k represents the slope factor of the activation curve. The voltage dependence of steady-state inactivation for Kv4.3 in the absence and presence of haloperidol was obtained using a conventional two-pulse protocol: holding the membrane potential at 80 mV and then stepping from 110 to þ10 mV for 1 s in increments of 10 mV at 10-s intervals followed by a 500-ms depolarizing pulse to þ40 mV. The Boltzmann equation was fitted to the steady-state inactivation curves: ðI–I c Þ=ðI max –I c Þ ¼ 1=½1 þ expðV–V 1=2 Þ=k
ð3Þ
where Imax represents the current measured at the most hyperpolarized preconditioning pulse, and Ic represents a non-inactivating current at the most depolarized preconditioning pulse. V, V1/2, and k are the test potential, the point where channels are halfinactivated, and the slope factor, respectively. The noninactivating residual current was removed by subtracting it from the actual value. 2.5. Statistics The data are summarized as the mean 7S.E.M. A paired Student's t-test was used for the statistical analysis of paired data. A one-way analysis of variance with Bonferroni's test was applied for the comparison of multiple groups. A P o0.05 was considered statistically significant.
2.3. Electrophysiology Kv4.3 currents were recorded using a Multiclamp 700B microelectrode amplifier and pClamp 10.1 software (Molecular Devices, Sunnyvale, CA, USA) in a whole-cell configuration of the patch-clamp technique at room temperature (22–24 1C). Glass micropipettes were pulled from glass capillaries (PG10165-4; World Precision Instruments, Sarasota, FL, USA) using a programmable horizontal microelectrode puller (P-97; Sutter Instrument, Novato, CA, USA). The tip resistances of the patch pipettes were between 2–4 MΩ when filled with the internal solution. The liquid junction potentials between pipette and bath solutions were 3–5 mV, and were zeroed online before the pipette touched the cell. The current signal was filtered at 4 kHz and sampled at 10 kHz. In the whole-cell configuration, the mean series resistance was 5.970.3 MΩ (n¼ 33). Maximum amplitude of Kv4.3 currents at 40 mV was averaged 5.270.2 nA (n¼33). The series resistances were usually compensated by up to 80% if Kv4.3 currents exceeded 1 nA, yielding a maximum voltage error of about 6.1 mV after compensation. 2.4. Data analysis Data analysis was performed using Origin 8.6 software (Origin Lab Corp., Northampton, MA, USA). The Hill equation was fitted to
3. Results 3.1. Concentration-dependent block of Kv4.3 by haloperidol Fig. 1A illustrates the representative Kv4.3 currents evoked by depolarizing pulses ranging from 70 to þ 80 mV in steps of 10 mV every 10 s from a holding potential of 80 mV in the absence and presence of haloperidol. Under control conditions, Kv4.3 currents were activated to a peak and then were rapidly inactivated, as reported previously (Ohya et al., 1997; Sung and Hahn, 2013). Haloperidol did not significantly decrease Kv4.3 currents in the voltage range where Kv4.3 currents were activated. The effects of haloperidol on the current–voltage (I–V) relationships for the peak amplitude of Kv4.3 currents are shown. To obtain concentration–response data, Kv4.3 currents were evoked by a 500 ms depolarizing pulses to þ40 mV from a holding potential of 80 mV in the absence and presence of haloperidol (Fig. 1B). Haloperidol did not affect the peak amplitude of Kv4.3 currents at concentration up to 10 μM, but accelerated the decay rate of Kv4.3 inactivation in a concentration-dependent manner. Thus, the concentration-dependent block of Kv4.3 currents by haloperidol was quantified from the integral of the Kv4.3 current
H.J. Lee et al. / European Journal of Pharmacology 740 (2014) 1–8
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Fig. 1. Concentration-dependent block of Kv4.3 by haloperidol. (A) The representative whole-cell Kv4.3 current traces recorded from CHO cells in the absence and presence of haloperidol. Current–voltage relationship for Kv4.3 block by haloperidol. The data were taken from the peak amplitude of Kv4.3 currents under control conditions and after the addition of haloperidol. (B) The representative Kv4.3 currents were elicited by a 500-ms depolarizing pulse to þ 40 mV from a holding potential of 80 mV at 10 s intervals in the absence and presence of 0.3, 1, 3, 10, 30 and 100 μM haloperidol. The integral of Kv4.3 current during depolarizing pulse was normalized to the current recorded under control conditions. The Hill equation was fitted to the normalized data. The inset shows the first 50 ms of the Kv4.3 current recordings on an expanded time scale. Data are expressed as the mean 7S.E.M.
during the 500-ms depolarizing pulse. The integrals of Kv4.3 currents at 0.3, 1, 3, 10, 30, and 100 μM of haloperidol were normalized to the control and then plotted against the concentration of haloperidol. The IC50 value for the blocking effects of haloperidol on the integral of Kv4.3 was 3.6 70.2 μM with a Hill coefficient of 0.5 7 0.02 (n ¼11). Under control conditions, a biexponential function was fitted to the inactivation of Kv4.3 at þ 40 mV with a fast time constant of 19.1 71.7 ms and a slow time constant of 146.6 716.4 ms (n¼ 11) (Fig. 2A). The fast component of Kv4.3 inactivation was predominant at þ40 mV under control conditions: the fast and slow components of inactivation were 87.874.5% and 12.2 70.3% (n ¼11) of total inactivation, respectively. After application of haloperidol, a biexponential function was also best fitted to the apparent inactivation of Kv4.3. At the concentrations of 0.3, 1, 3, 10, 30 and 100 mM, haloperidol decreased the fast time constant to 16.7 71.7, 14.8 71.4, 11.971.0, 7.5 70.5, 4.0 70.2 and 2.7 70.03 ms (F6, 70 ¼29.70, Po 0.01) and the slow time constant to 104.6 711.5, 95.4 7 9.5, 89.5 79.2, 83.8 77.1, 83.17 5.7 and 82.6 76.6 ms, respectively (F6, 70 ¼5.23, P ¼0.05). However, the relative contribution of the fast component to the total inactivation of Kv4.3 was not significantly changed 85.3–89.9%) by haloperidol. The activation kinetics of the Kv4.3 current was also
accelerated by haloperidol in a concentration-dependent manner (Fig. 2B). Under control conditions, the activation time constant was 0.36 70.03 ms (n ¼11). The time constants of activation were decreased to 0.28 70.02, 0.26 70.02, 0.2470.01, 0.23 70.01, 0.22 70.01, and 0.207 0.01 (n ¼ 11) for 0.3, 1, 3, 10, 30, and 100 μM of haloperidol, respectively. In the presence of haloperidol (3 μM), the time-to-peak was reduced at all voltages tested (Fig. 2C). Taken together, both inactivation and activation kinetics of Kv4.3 currents were accelerated by the treatment of haloperidol. 3.2. Effects of haloperidol on the steady-state activation and inactivation of Kv4.3 The voltage dependence of the steady-state activation of Kv4.3 currents was investigated using a two-pulse protocol in the absence and presence of haloperidol (Fig. 3A). The normalized tail current amplitudes were plotted against depolarizing pulse potentials, and a Boltzmann equation was fitted to the data points to estimate the voltage dependence of the steady-state activation of Kv4.3 currents (Fig. 3B). Under control conditions, the halfactivation potential (V1/2) and the slope factor (k) were 27.8 71.2 and 7.270.6 mV (n ¼6), respectively. In the presence
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Fig. 2. Effects of haloperidol on the kinetics of inactivation and activation, and time-to-peak of Kv4.3. (A) A biexponential function was fitted to the inactivation of Kv4.3 at þ 40 mV with the slow (τs) and fast (τf) time constants of the inactivation. (B) The dominant time constant (τa) of activation was determined by fitting a single exponential function to the latter 50% of activation (Snyders et al., 1993). The fitted lines obtained by exponential functions are superimposed over the Kv4.3 current traces. The time constants estimated from exponential fits to the decay and activation phases of Kv4.3 currents are shown. (C) The kinetics of Kv4.3 current activation was also analyzed by measuring the time-to-peak, which reflects the transition from the channel close to the channel open. Time-to-peak as a function of test potential in the absence and presence of haloperidol. nP o0.05, significant difference from the control. Data are expressed as the mean 7 S.E.M.
of haloperidol, V1/2 was shifted to a hyperpolarized potential ( 35.0 70.9 mV) and k steepened to 5.8 7 0.7 mV (n ¼ 6, Po 0.05). The effect of haloperidol on steady-state inactivation was studied using a two-pulse protocol (Fig. 4A). The normalized peak amplitudes evoked by the test pulse were plotted against conditioning pulse potentials, and a Boltzmann equation was fitted to the data points (Fig. 4B). Under control conditions, the halfinactivation potential (V1/2) and the slope factor (k) were 60.1 70.8 and 4.17 0.1 mV (n ¼8), respectively. The administration of haloperidol resulted in a significant shift in the inactivation curve toward the hyperpolarizing direction (V1/2 ¼ 65.3 7 0.9 mV, n ¼8, Po 0.05) with no significant change in the k (4.57 0.1 mV, n ¼8).
3.3. Effects of haloperidol on the closed-state inactivation of Kv4.3 Because the Kv4.3 channels appeared to be predominantly inactivated in the closed state with no channel opening over the range of subthreshold potentials (Wang et al., 2005), the effects of haloperidol on the kinetics of closed-state inactivation were studied (Fig. 5A). A two-pulse protocol with the prepulse durations of second pulses progressively increasing between 5 ms to 20 s to a potential of 60 mV below the activation threshold was used to quantitate the closed-state inactivation of Kv4.3 currents, and normalized second peak amplitudes were plotted against prepulse durations. Under control conditions, a single exponential function was fitted to the time course for the closed-state inactivation of
H.J. Lee et al. / European Journal of Pharmacology 740 (2014) 1–8
Fig. 3. Effects of haloperidol on the steady-state activation of Kv4.3. (A) The tail current traces of the Kv4.3 channels were obtained at 60 mV after 8-ms depolarizing pulses from 70 to þ 80 mV in the absence or presence of haloperidol. The insets show the tail current traces of Kv4.3 on an expanded time scale. (B) The normalized current was plotted as a function of the test potential, and a Boltzmann equation was fitted to the resultant curve. Data are expressed as the mean 7 S.E.M.
Kv4.3 with the time constant of 2.35 70.42 s (n ¼6). In the presence of haloperidol, the time constant of closed-state inactivation was significantly decreased to 1.27 70.45 s (n ¼6, P o0.05) (Fig. 5B), suggesting that haloperidol significantly accelerated the rate of the closed-state inactivation of Kv4.3 currents.
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Fig. 4. Effects of haloperidol on the steady-state inactivation of Kv4.3. (A) Steadystate inactivation was analyzed using a two-pulse protocol. The currents were evoked by a 1 s prepulse that was varied from 110 to þ10 mV stepped by 10 mV and a 500-ms depolarizing pulse to þ40 mV in the absence and presence of haloperidol. (B) Steady-state inactivation curves of Kv4.3 are shown as a plot of normalized peak currents during the test pulse as a function of the conditioning potential. Data are expressed the mean 7 S.E.M.
control, and 490.4754.1 ms (n¼6, Po0.05) after haloperidol treatment (Fig. 7B). These results suggest that haloperidol significantly attenuated the recovery from inactivation of Kv4.3 currents.
4. Discussion 3.4. Effects of haloperidol on use-dependency and the time course for recovery from inactivation of Kv4.3 Fig. 6A showed the use dependent-block of Kv4.3 currents by haloperidol. Under control conditions, the Kv4.3 currents were slightly reduced by 7.171.2% and 26.572.8% at 1 and 2 Hz, respectively (n¼ 8). In the presence of haloperidol, the Kv4.3 currents were decreased by 21.071.6% at 1 Hz and 44.172.2% at 2 Hz (n¼8, Po0.05) (Fig. 6B). These results suggest that the effect of haloperidol was potentiated by an increase in the stimulation frequency. Fig. 7A shows the effects of haloperidol on the kinetics of the recovery of Kv4.3 currents from inactivation using a two-pulse protocol. The peak amplitudes of the second pulse were normalized to the first peak amplitude and plotted against the inter-pulse intervals. A single exponential function was fitted to the time courses of recovery from inactivation of Kv4.3 with a time constant of 236.4721.8 ms during
The main findings of the present study can be summarized as follows. Haloperidol blocked Kv4.3 currents and accelerated the inactivation and activation kinetics of Kv4.3 in a concentrationdependent manner. These results indicated that haloperidol blocked the open state of Kv4.3 channels (Snyders and Yeola, 1995; Thompson, 1982; Zhang and Steinberg, 1995). The block of Kv4.3 by haloperidol increased as a function of stimulation frequency (use-dependence). This type of block can also be interpreted as the result of an open-channel block (Butterworth and Strichartz, 1990; Snyders et al., 1992). In addition, the block of Kv4.3 increased with depolarizing prepulses, shifting the steadystate inactivation curve to negative potentials and accelerating the closed-state inactivation at subthreshold membrane potentials. These results indicated preferential binding of the drug to an inactivated state of Kv4.3 channels at subthreshold potentials
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Fig. 5. Effects of haloperidol on the closed-state inactivation of Kv4.3. (A) Kv4.3 currents were recorded at þ 40 mV using a double-pulse protocol with a conditioning pulse to 60 mV of variable duration in the absence and presence of haloperidol. (B) The current amplitudes evoked by the second pulse, relative to the amplitude resulting from the initial control pulse, were plotted against the duration of the conditioning pulse. A single exponential function was fitted to the data points. Data are expressed as the mean 7S.E.M.
(Wang et al., 1995). Similarly, haloperidol reduced the amplitude of transient outward Kv current and accelerated the time course of apparent inactivation in rat ventricular myocytes, suggesting an interaction with the channels both in the open and inactivated states (Bebarova et al., 2006). The haloperidol block of HERG was use- and voltage-dependent, suggesting a preferential binding to either the open or inactivated states of channels (Suessbrich et al., 1997). Thus, haloperidol blocked Kv4.3 by both preferentially interacting with the open state and accelerating closed-state inactivation of Kv4.3 channels. Haloperidol, an antipsychotic drug, is commonly used in clinical practice to treat acute and chronic psychosis (LopezMunoz and Alamo, 2009; Miyamoto et al., 2012). Haloperidol has been associated with adverse cardiovascular events, including a prolongation of QT interval in patients receiving either a therapeutic dose or an overdose, suggesting a potential risk for development of cardiac arrhythmia, also known as torsades de pointes (Fayek et al., 2001; Fayer, 1986; Henderson et al., 1991; Hunt and Stern, 1995). Accordingly, numerous studies have demonstrated that haloperidol possesses potent inhibitory properties on different cardiac ion channels in the heart. For instance, haloperidol
Fig. 6. Use-dependent block of Kv4.3 by haloperidol. (A) Ten repetitive 200-ms depolarizing pulses of þ 40 mV from a holding potential of 80 mV were applied at two different frequencies, 1 and 2 Hz under control conditions, and after the application of haloperidol. (B) The peak amplitudes of current 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 mean 7 S.E.M.
blocked transient outward Kv currents in rat ventricular myocytes (Bebarova et al., 2006), sodium current in guinea pig ventricular myocytes (Cheng et al., 2007), and HERG currents in human atrial myocytes (Suessbrich et al., 1997). Since one of the mechanisms by which drugs can produce a prolongation of the QT interval is a blockade of cardiac repolarizing ion currents (Kannankeril et al., 2010), a blockade of HERG can explain the lengthening QT interval associated with the use of haloperidol. Kv4.3 channels, shal-type rapidly activating and inactivating Kv channels, encode the transient outward Kv currents in the heart (Dixon et al., 1996). This current is associated with early cardiac repolarizations and affects the activation of the subsequently activating L-type Ca2 þ currents (Niwa and Nerbonne, 2010; Sah et al., 2003). Interactions of the drug with the Kv4.3 channels are associated with alterations in cardiac action potential durations that either prolong or shorten the action potential duration due to secondary changes in subsequently depolarizing currents (Tamargo et al., 2004). Therefore,
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haloperidol to its therapeutic actions is unclear, a considerable amount of evidence demonstrates that some aspects of the clinical effects of haloperidol are mediated by its action on Kv4.3. For example, chronic haloperidol treatment increased the A-type Kv current density and altered the intrinsic pacemaker activity in rat dopaminergic neurons (Hahn et al., 2006). Hahn et al. (2003) also showed that haloperidol increases the number of functional Kv4 A-type currents via the upregulation of Kv4.3 mRNA in dopamine neurons and may serve to decrease excitability in the dopamine system. In general, the inhibition of neuronal A-type Kv currents modulated the refractory period and the duration of action potential, thus altering the firing frequency in neurons (Rudy, 1988). Since Kv4.3 channels are abundant and widely distributed in the CNS (Serodio and Rudy, 1998), the pharmacological block of Kv4.3 channels by haloperidol may influence electrical activity and neurotransmitter release in the dopaminergic neurons, suggesting that some of the antipsychotic and/or side effects of this drug may be attributed to the inhibitory effect on these currents. The therapeutic plasma concentrations of haloperidol are in the range of 10–100 nM (Yang et al., 2005). In the present study, haloperidol blocked Kv4.3 with an IC50 of 3.6 μM, which was much higher than the therapeutic plasma concentrations. Under occasional situations, however, the concentration of haloperidol in tissues can reach micromolar levels. The maximal concentrations of haloperidol in the myocardium were 6 to 10-fold higher than that in plasma after intraperitoneal application (Titier et al., 2004). Moreover, haloperidol concentrations in the human brain tissue were 10–30 times higher than plasma concentrations used in the treatment of schizophrenia (Kornhuber et al., 1999). Thus, the inhibitory effect of haloperidol on Kv4.3 in the present study may be clinically relevant in the range of therapeutic plasma concentrations after chronic administration. In conclusion, haloperidol produced a concentration-, time-, voltage- and use-dependent block of Kv4.3 currents that were stably expressed in CHO cells. These results suggest that haloperidol binds to the open state of Kv4.3 channels during depolarization and accelerates closed-state inactivation at subthreshold membrane potentials. Fig. 7. Effects of haloperidol on recovery from inactivation of Kv4.3. (A) A doublepulse protocol was used to characterize the recovery of Kv4.3 channels from inactivation in the absence and presence of haloperidol. (B) The first pre-pulse of a 500 ms depolarizing pulse of þ 40 mV from a holding potential of 80 mV was followed by a second 100 ms pulse after increasing the inter-pulse intervals between 10 and 10,000 ms at 80 mV. The peak currents elicited during the test pulses were measured and then normalized to the peak currents of the conditioning pulses in the same cells. Normalized data were plotted against the inter-pulse interval. A single exponential function was fitted to the data points. Data are expressed as the mean7 S.E.M.
further study of the action potential duration is required in order to address the possible contribution of the Kv4.3 block by haloperidol to cardiovascular side effects. The clinical pharmacological action of haloperidol is ascribed to dopaminergic receptor inhibition (Seeman and Van Tol, 1994). Atype Kv currents are activated at potentials that are negative to the threshold, thus shaping a spike waveform and regulating the duration of interspike intervals and firing frequency of the neurons in the central nervous system (Rudy, 1988; Zhang and McBain, 1995). The Kv4.3 currents, somatodendritic A-type Kv currents in the CNS, are highly expressed in dopaminergic neurons and play an important role in tuning the pacemaker frequency of the neurons (Liss et al., 2001). Haloperidol inhibits a variety of ionic currents that regulate neuronal activities: delayed rectifier Kv currents in mouse cortical neurons (Yang et al., 2005) and voltage-dependent Na þ currents of mammalian brain neurons (Wakamori et al., 1989). Although the relevance of a Kv4.3 block by
Authorship contributions Participated in research design: KW Sung, SJ Hahn Conducted experiments: HJ Lee Performed data analysis and interpretation: HJ Lee, KW Sung, SJ Hahn Wrote or contributed to the writing of the manuscript: KW Sung, SJ Hahn
Acknowledgments We thank Dr. Imaizumi (Department of Molecular and Cellular Pharmacology, Nagoya City University, Japan) for the Kv4.3 cDNA. This work was supported by a grant from the Catholic Medical Center Research Foundation made in the program year of 2014. References Ahn, H.S., Choi, J.S., Choi, B.H., Kim, M.J., Rhie, D.J., Yoon, S.H., Jo, Y.H., Kim, M.S., Sung, K.W., Hahn, S.J., 2005. Inhibition of the cloned delayed rectifier K þ channels, Kv1.5 and Kv3.1, by riluzole. Neuroscience 133, 1007–1019. Bebarova, M., Matejovic, P., Pasek, M., Novakova, M., 2006. Effect of haloperidol on transient outward potassium current in rat ventricular myocytes. Eur. J. Pharmacol. 550, 15–23. Birnbaum, S.G., Varga, A.W., Yuan, L.L., Anderson, A.E., Sweatt, J.D., Schrader, L.A., 2004. Structure and function of Kv4-family transient potassium channels. Physiol. Rev. 84, 803–833.
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Rudy, B., 1988. Diversity and ubiquity of K channels. Neuroscience 25, 729–749. Sah, R., Ramirez, R.J., Oudit, G.Y., Gidrewicz, D., Trivieri, M.G., Zobel, C., Backx, P.H., 2003. Regulation of cardiac excitation-contraction coupling by action potential repolarization: role of the transient outward potassium current (Ito). J. Physiol. 546, 5–18. Seeman, P., 2006. Targeting the dopamine D2 receptor in schizophrenia. Expert Opin. Ther. Targets 10, 515–531. Seeman, P., Van Tol, H.H., 1994. Dopamine receptor pharmacology. Trends Pharmacol. Sci. 15, 264–270. Serodio, P., Rudy, B., 1998. Differential expression of Kv4 K þ channel subunits mediating subthreshold transient K þ (A-type) currents in rat brain. J. Neurophysiol. 79, 1081–1091. Snyders, D.J., Tamkun, M.M., Bennett, P.B., 1993. A rapidly activating and slowly inactivating potassium channel cloned from human heart. Functional analysis after stable mammalian cell culture expression. J. Gen. Physiol. 101, 513–543. Snyders, D.J., Yeola, S.W., 1995. Determinants of antiarrhythmic drug action. Electrostatic and hydrophobic components of block of the human cardiac hKv1.5 channel. Circ. Res. 77, 575–583. Snyders, J., Knoth, K.M., Roberds, S.L., Tamkun, M.M., 1992. Time-, voltage-, and state-dependent block by quinidine of a cloned human cardiac potassium channel. Mol. Pharmacol. 41, 322–330. Suessbrich, H., Schonherr, R., Heinemann, S.H., Attali, B., Lang, F., Busch, A.E., 1997. The inhibitory effect of the antipsychotic drug haloperidol on HERG potassium channels expressed in Xenopus oocytes. Br. J. Pharmacol. 120, 968–974. Sung, K.W., Hahn, S.J., 2013. Effect of mosapride on Kv4.3 potassium channels expressed in CHO cells. Naunyn Schmiedebergs Arch. Pharmacol. 386, 905–916. Tamargo, J., Caballero, R., Gomez, R., Valenzuela, C., Delpon, E., 2004. Pharmacology of cardiac potassium channels. Cardiovasc. Res. 62, 9–33. Thompson, S., 1982. Aminopyridine block of transient potassium current. J. Gen. Physiol. 80, 1–18. Titier, K., Canal, M., Deridet, E., Abouelfath, A., Gromb, S., Molimard, M., Moore, N., 2004. Determination of myocardium to plasma concentration ratios of five antipsychotic drugs: comparison with their ability to induce arrhythmia and sudden death in clinical practice. Toxicol. Appl. Pharmacol. 199, 52–60. Wakamori, M., Kaneda, M., Oyama, Y., Akaike, N., 1989. Effects of chlordiazepoxide, chlorpromazine, diazepam, diphenylhydantoin, flunitrazepam and haloperidol on the voltage-dependent sodium current of isolated mammalian brain neurons. Brain Res. 494, 374–378. Wang, S., Bondarenko, V.E., Qu, Y.J., Bett, G.C., Morales, M.J., Rasmusson, R.L., Strauss, H.C., 2005. Time- and voltage-dependent components of Kv4.3 inactivation. Biophys. J. 89, 3026–3041. Wang, Z., Fermini, B., Nattel, S., 1995. Effects of flecainide, quinidine, and 4aminopyridine on transient outward and ultrarapid delayed rectifier currents in human atrial myocytes. J. Pharmacol. Exp. Ther. 272, 184–196. Yang, S.B., Major, F., Tietze, L.F., Rupnik, M., 2005. Block of delayed-rectifier potassium channels by reduced haloperidol and related compounds in mouse cortical neurons. J. Pharmacol. Exp. Ther. 315, 352–362. Yang, S.B., Proks, P., Ashcroft, F.M., Rupnik, M., 2004. Inhibition of ATP-sensitive potassium channels by haloperidol. Br. J. Pharmacol. 143, 960–967. Zhang, L., McBain, C.J., 1995. Potassium conductances underlying repolarization and after-hyperpolarization in rat CA1 hippocampal interneurones. J. Physiol. 488, 661–672. Zhang, Z.H., Steinberg, M.I., 1995. LY 97241 accelerates the apparent rate of inactivation of transient outward K þ current: characterization of open channel block. J. Pharmacol. Exp. Ther. 274, 249–257.
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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Neuropharmacology and analgesia
Effects of haloperidol on Kv4.3 potassium channels Hong Joon Lee a, Ki-Wug Sung a,n, Sang June Hahn b,nn a Department of Pharmacology, Cell Death and Disease Research Center, College of Medicine, The Catholic University of Korea, 222 Banpo-daero, Seocho-gu, , Seoul 137-701, Republic of Korea b Department of Physiology, Cell Death and Disease Research Center, College of Medicine, The Catholic University of Korea, 222 Banpo-daero, Seocho-gu, Seoul 137-701, Republic of Korea
art ic l e i nf o
a b s t r a c t
Article history: Received 12 February 2014 Received in revised form 23 June 2014 Accepted 24 June 2014 Available online 3 July 2014
Haloperidol is commonly used in clinical practice to treat acute and chronic psychosis, but it also has been associated with adverse cardiovascular events. We investigated the effects of haloperidol on Kv4.3 currents stably expressed in CHO cells using a whole-cell patch-clamp technique. Haloperidol did not significantly inhibit the peak amplitude of Kv4.3, but accelerated the decay rate of inactivation of Kv4.3 in a concentration-dependent manner. Thus, the effects of haloperidol on Kv4.3 were estimated from the integral of the Kv4.3 currents during the depolarization pulse. The Kv4.3 was decreased by haloperidol in a concentration-dependent manner with an IC50 value of 3.6 μM. Haloperidol accelerated the decay rate of Kv4.3 inactivation and activation kinetics in a concentration-dependent manner, thereby decreasing the time-to-peak. Haloperidol shifted the voltage dependence of the steady-state activation and inactivation of Kv4.3 in a hyperpolarizing direction. Haloperidol also caused an acceleration of the closed-state inactivation of Kv4.3. Haloperidol produced a use-dependent block of Kv4.3, which was accompanied by a slowing of recovery from the inactivation of Kv4.3. These results suggest that haloperidol blocks Kv4.3 by both interacting with the open state of Kv4.3 channels during depolarization and accelerating the closed-state inactivation at subthreshold membrane potentials. & 2014 Elsevier B.V. All rights reserved.
Keywords: Kv4.3 Haloperidol Open-channel block Closed-state inactivation
1. Introduction Haloperidol is structurally classified as a butyrophenone antipsychotic drug, and has long been used to treat schizophrenia (Lopez-Munoz and Alamo, 2009; Miyamoto et al., 2012). With the advent of newer atypical antipsychotics, the clinical use of haloperidol for the long-term treatment of schizophrenia has declined (Meltzer, 2013). However, it continues to be commonly used in treating certain forms of psychotic symptoms, particularly delirium associated with several clinical situations (Meyer-Massetti et al., 2010). Although haloperidol is a known antagonist of dopamine D2 receptors in the central nervous system (Seeman, 2006), it also blocks different ion channels at concentrations that are within the therapeutic range. For example, the acute application of haloperidol blocked numerous types of potassium channels, including delayed-rectifier potassium channels in cortical neurons (Yang et al., 2005), the ATP-sensitive potassium channels in pancreatic β cells (Yang et al., 2004), the G protein-activated inwardly rectifying potassium channels expressed in Xenopus n
Corresponding author. Tel.: þ 82 2 2258 7325; fax: þ 82 2 536 2485. Corresponding author. Tel.: þ 82 2 2258 7275; fax: þ 82 2 532 9575. E-mail addresses: [email protected] (K.-W. Sung), [email protected] (S.J. Hahn). nn
http://dx.doi.org/10.1016/j.ejphar.2014.06.043 0014-2999/& 2014 Elsevier B.V. All rights reserved.
oocytes (Kobayashi et al., 2000), and the human ether-a-go-gorelated gene (HERG) potassium channels expressed in Xenopus oocytes (Suessbrich et al., 1997). These reports suggest that potassium channels possibly are associated with the therapeutic, and/or neurological and non-neurological adverse effects of haloperidol. Kv4.3 is an α-subunit of a Shal-type voltage-gated potassium channel (Kv). It generates rapidly activating and inactivating Kv currents (Ohya et al., 1997), and contributes to the A-type Kv currents in neurons and transient outward Kv currents in cardiac myocytes (Birnbaum et al., 2004; Dixon et al., 1996). Chronic treatment with haloperidol dampened the brain dopamine system by increasing the expression of Kv4.3 mRNA, which underlies the A-type Kv channels in the isolated midbrain dopamine neurons (Hahn et al., 2003). These results also support the possibility that changes in the Kv4.3 channel activities by drugs in certain clinical situations can contribute to the therapeutic action of haloperidol and/or induce serious adverse drug reactions. Accordingly, intravenous haloperidol used to treat delirium has shown an increased risk of torsades de pointes, a life-threatening ventricular arrhythmia via a prolongation of the QT-interval (Hassaballa and Balk, 2003; Meyer-Massetti et al., 2010). However, a limited amount of information is available to explain how haloperidol modulates Kv4.3 channel function to elucidate the therapeutic and/or adverse effects of haloperidol. Therefore, we examined the effects of
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haloperidol on cloned Kv4.3 channels stably expressed in Chinese hamster ovary (CHO) cells using the whole-cell patch-clamp technique.
2. Materials and methods 2.1. Cell culture The CHO cells were transfected with cDNA encoding Kv4.3 channels using the Lipofectamine reagent (Invitrogen, Grand Island, NY, USA), as previously described in detail (Ahn et al., 2005). A stable CHO cell line expressing Kv4.3 channels was maintained in Iscove's modified Dulbecco's medium (Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen), 2 mM glutamine (Invitrogen), 0.1 mM hypoxanthine (Invitrogen), 0.01 mM thymidine (Invitrogen), 0.3 mg/ml G418 (Invitrogen), and 1% of a 100 antibiotic antimycotic mixture (Invitrogen) at 37 1C with 5% CO2-enriched air. The cells treated with trypsin– EDTA solution were plated on glass coverslips (12 mm diameter; Fisher Scientific, Pittsburgh, PA, USA) and placed in 35 mm Petri dishes. Patch-clamp recordings were obtained 12–24 h later. For electrphysiology, the coverslips containing adherent CHO cells were mounted on the glass bottom of the recording chamber (RC-13; Warner Instrument, Hamden, CT, USA) and were continuously perfused with an extracellular bath solution. 2.2. Solutions and drugs The extracellular bath solution contained (in mM) 140 NaCl, 5 KCl, 1 CaCl2, 1 MgCl2, 10 HEPES, and 10 glucose and was adjusted to pH 7.3 using NaOH. The intracellular 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 osmolarity of the solutions was measured with a vapor pressure osmometer (Vapro 5520, Logan, UT, USA) at 300–310 mOsm. Haloperidol was dissolved in dimethyl sulfoxide (DMSO) as a stock solution of 100 mM, and the stock solution was then diluted with the bath solution to obtain the desired concentration. The concentration of DMSO in the final dilution was less than 0.1%, and this concentration had no effect on the Kv4.3 currents.
the normalized integral of Kv4.3 currents to determine the concentration–response curves for haloperidol: ƒ ¼ 1=½1 þ ðIC50 =½DÞn
ð1Þ
where ƒ is the fractional block of the integral of Kv4.3 currents at a given haloperidol concentration [D], IC50 is the concentration of haloperidol for a 50% block, and n is the Hill coefficient. To evaluate steady-state activation, the amplitude of tail currents were measured at 60 mV after the application of 8 ms depolarizing pulses at potentials between 80 and þ80 mV in 10 mV increments every 10 s from a holding potential of 80 mV, and were then normalized to the maximal tail current amplitude and plotted against the test potential. The Boltzmann equation was fitted to the steady-state activation curves: y ¼ 1=f1 þexp½ ðV V 1=2 Þkg
ð2Þ
where V1/2 is the voltage at which the conductance was halfmaximal, V is the test potential and k represents the slope factor of the activation curve. The voltage dependence of steady-state inactivation for Kv4.3 in the absence and presence of haloperidol was obtained using a conventional two-pulse protocol: holding the membrane potential at 80 mV and then stepping from 110 to þ10 mV for 1 s in increments of 10 mV at 10-s intervals followed by a 500-ms depolarizing pulse to þ40 mV. The Boltzmann equation was fitted to the steady-state inactivation curves: ðI–I c Þ=ðI max –I c Þ ¼ 1=½1 þ expðV–V 1=2 Þ=k
ð3Þ
where Imax represents the current measured at the most hyperpolarized preconditioning pulse, and Ic represents a non-inactivating current at the most depolarized preconditioning pulse. V, V1/2, and k are the test potential, the point where channels are halfinactivated, and the slope factor, respectively. The noninactivating residual current was removed by subtracting it from the actual value. 2.5. Statistics The data are summarized as the mean 7S.E.M. A paired Student's t-test was used for the statistical analysis of paired data. A one-way analysis of variance with Bonferroni's test was applied for the comparison of multiple groups. A P o0.05 was considered statistically significant.
2.3. Electrophysiology Kv4.3 currents were recorded using a Multiclamp 700B microelectrode amplifier and pClamp 10.1 software (Molecular Devices, Sunnyvale, CA, USA) in a whole-cell configuration of the patch-clamp technique at room temperature (22–24 1C). Glass micropipettes were pulled from glass capillaries (PG10165-4; World Precision Instruments, Sarasota, FL, USA) using a programmable horizontal microelectrode puller (P-97; Sutter Instrument, Novato, CA, USA). The tip resistances of the patch pipettes were between 2–4 MΩ when filled with the internal solution. The liquid junction potentials between pipette and bath solutions were 3–5 mV, and were zeroed online before the pipette touched the cell. The current signal was filtered at 4 kHz and sampled at 10 kHz. In the whole-cell configuration, the mean series resistance was 5.970.3 MΩ (n¼ 33). Maximum amplitude of Kv4.3 currents at 40 mV was averaged 5.270.2 nA (n¼33). The series resistances were usually compensated by up to 80% if Kv4.3 currents exceeded 1 nA, yielding a maximum voltage error of about 6.1 mV after compensation. 2.4. Data analysis Data analysis was performed using Origin 8.6 software (Origin Lab Corp., Northampton, MA, USA). The Hill equation was fitted to
3. Results 3.1. Concentration-dependent block of Kv4.3 by haloperidol Fig. 1A illustrates the representative Kv4.3 currents evoked by depolarizing pulses ranging from 70 to þ 80 mV in steps of 10 mV every 10 s from a holding potential of 80 mV in the absence and presence of haloperidol. Under control conditions, Kv4.3 currents were activated to a peak and then were rapidly inactivated, as reported previously (Ohya et al., 1997; Sung and Hahn, 2013). Haloperidol did not significantly decrease Kv4.3 currents in the voltage range where Kv4.3 currents were activated. The effects of haloperidol on the current–voltage (I–V) relationships for the peak amplitude of Kv4.3 currents are shown. To obtain concentration–response data, Kv4.3 currents were evoked by a 500 ms depolarizing pulses to þ40 mV from a holding potential of 80 mV in the absence and presence of haloperidol (Fig. 1B). Haloperidol did not affect the peak amplitude of Kv4.3 currents at concentration up to 10 μM, but accelerated the decay rate of Kv4.3 inactivation in a concentration-dependent manner. Thus, the concentration-dependent block of Kv4.3 currents by haloperidol was quantified from the integral of the Kv4.3 current
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Fig. 1. Concentration-dependent block of Kv4.3 by haloperidol. (A) The representative whole-cell Kv4.3 current traces recorded from CHO cells in the absence and presence of haloperidol. Current–voltage relationship for Kv4.3 block by haloperidol. The data were taken from the peak amplitude of Kv4.3 currents under control conditions and after the addition of haloperidol. (B) The representative Kv4.3 currents were elicited by a 500-ms depolarizing pulse to þ 40 mV from a holding potential of 80 mV at 10 s intervals in the absence and presence of 0.3, 1, 3, 10, 30 and 100 μM haloperidol. The integral of Kv4.3 current during depolarizing pulse was normalized to the current recorded under control conditions. The Hill equation was fitted to the normalized data. The inset shows the first 50 ms of the Kv4.3 current recordings on an expanded time scale. Data are expressed as the mean 7S.E.M.
during the 500-ms depolarizing pulse. The integrals of Kv4.3 currents at 0.3, 1, 3, 10, 30, and 100 μM of haloperidol were normalized to the control and then plotted against the concentration of haloperidol. The IC50 value for the blocking effects of haloperidol on the integral of Kv4.3 was 3.6 70.2 μM with a Hill coefficient of 0.5 7 0.02 (n ¼11). Under control conditions, a biexponential function was fitted to the inactivation of Kv4.3 at þ 40 mV with a fast time constant of 19.1 71.7 ms and a slow time constant of 146.6 716.4 ms (n¼ 11) (Fig. 2A). The fast component of Kv4.3 inactivation was predominant at þ40 mV under control conditions: the fast and slow components of inactivation were 87.874.5% and 12.2 70.3% (n ¼11) of total inactivation, respectively. After application of haloperidol, a biexponential function was also best fitted to the apparent inactivation of Kv4.3. At the concentrations of 0.3, 1, 3, 10, 30 and 100 mM, haloperidol decreased the fast time constant to 16.7 71.7, 14.8 71.4, 11.971.0, 7.5 70.5, 4.0 70.2 and 2.7 70.03 ms (F6, 70 ¼29.70, Po 0.01) and the slow time constant to 104.6 711.5, 95.4 7 9.5, 89.5 79.2, 83.8 77.1, 83.17 5.7 and 82.6 76.6 ms, respectively (F6, 70 ¼5.23, P ¼0.05). However, the relative contribution of the fast component to the total inactivation of Kv4.3 was not significantly changed 85.3–89.9%) by haloperidol. The activation kinetics of the Kv4.3 current was also
accelerated by haloperidol in a concentration-dependent manner (Fig. 2B). Under control conditions, the activation time constant was 0.36 70.03 ms (n ¼11). The time constants of activation were decreased to 0.28 70.02, 0.26 70.02, 0.2470.01, 0.23 70.01, 0.22 70.01, and 0.207 0.01 (n ¼ 11) for 0.3, 1, 3, 10, 30, and 100 μM of haloperidol, respectively. In the presence of haloperidol (3 μM), the time-to-peak was reduced at all voltages tested (Fig. 2C). Taken together, both inactivation and activation kinetics of Kv4.3 currents were accelerated by the treatment of haloperidol. 3.2. Effects of haloperidol on the steady-state activation and inactivation of Kv4.3 The voltage dependence of the steady-state activation of Kv4.3 currents was investigated using a two-pulse protocol in the absence and presence of haloperidol (Fig. 3A). The normalized tail current amplitudes were plotted against depolarizing pulse potentials, and a Boltzmann equation was fitted to the data points to estimate the voltage dependence of the steady-state activation of Kv4.3 currents (Fig. 3B). Under control conditions, the halfactivation potential (V1/2) and the slope factor (k) were 27.8 71.2 and 7.270.6 mV (n ¼6), respectively. In the presence
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Fig. 2. Effects of haloperidol on the kinetics of inactivation and activation, and time-to-peak of Kv4.3. (A) A biexponential function was fitted to the inactivation of Kv4.3 at þ 40 mV with the slow (τs) and fast (τf) time constants of the inactivation. (B) The dominant time constant (τa) of activation was determined by fitting a single exponential function to the latter 50% of activation (Snyders et al., 1993). The fitted lines obtained by exponential functions are superimposed over the Kv4.3 current traces. The time constants estimated from exponential fits to the decay and activation phases of Kv4.3 currents are shown. (C) The kinetics of Kv4.3 current activation was also analyzed by measuring the time-to-peak, which reflects the transition from the channel close to the channel open. Time-to-peak as a function of test potential in the absence and presence of haloperidol. nP o0.05, significant difference from the control. Data are expressed as the mean 7 S.E.M.
of haloperidol, V1/2 was shifted to a hyperpolarized potential ( 35.0 70.9 mV) and k steepened to 5.8 7 0.7 mV (n ¼ 6, Po 0.05). The effect of haloperidol on steady-state inactivation was studied using a two-pulse protocol (Fig. 4A). The normalized peak amplitudes evoked by the test pulse were plotted against conditioning pulse potentials, and a Boltzmann equation was fitted to the data points (Fig. 4B). Under control conditions, the halfinactivation potential (V1/2) and the slope factor (k) were 60.1 70.8 and 4.17 0.1 mV (n ¼8), respectively. The administration of haloperidol resulted in a significant shift in the inactivation curve toward the hyperpolarizing direction (V1/2 ¼ 65.3 7 0.9 mV, n ¼8, Po 0.05) with no significant change in the k (4.57 0.1 mV, n ¼8).
3.3. Effects of haloperidol on the closed-state inactivation of Kv4.3 Because the Kv4.3 channels appeared to be predominantly inactivated in the closed state with no channel opening over the range of subthreshold potentials (Wang et al., 2005), the effects of haloperidol on the kinetics of closed-state inactivation were studied (Fig. 5A). A two-pulse protocol with the prepulse durations of second pulses progressively increasing between 5 ms to 20 s to a potential of 60 mV below the activation threshold was used to quantitate the closed-state inactivation of Kv4.3 currents, and normalized second peak amplitudes were plotted against prepulse durations. Under control conditions, a single exponential function was fitted to the time course for the closed-state inactivation of
H.J. Lee et al. / European Journal of Pharmacology 740 (2014) 1–8
Fig. 3. Effects of haloperidol on the steady-state activation of Kv4.3. (A) The tail current traces of the Kv4.3 channels were obtained at 60 mV after 8-ms depolarizing pulses from 70 to þ 80 mV in the absence or presence of haloperidol. The insets show the tail current traces of Kv4.3 on an expanded time scale. (B) The normalized current was plotted as a function of the test potential, and a Boltzmann equation was fitted to the resultant curve. Data are expressed as the mean 7 S.E.M.
Kv4.3 with the time constant of 2.35 70.42 s (n ¼6). In the presence of haloperidol, the time constant of closed-state inactivation was significantly decreased to 1.27 70.45 s (n ¼6, P o0.05) (Fig. 5B), suggesting that haloperidol significantly accelerated the rate of the closed-state inactivation of Kv4.3 currents.
5
Fig. 4. Effects of haloperidol on the steady-state inactivation of Kv4.3. (A) Steadystate inactivation was analyzed using a two-pulse protocol. The currents were evoked by a 1 s prepulse that was varied from 110 to þ10 mV stepped by 10 mV and a 500-ms depolarizing pulse to þ40 mV in the absence and presence of haloperidol. (B) Steady-state inactivation curves of Kv4.3 are shown as a plot of normalized peak currents during the test pulse as a function of the conditioning potential. Data are expressed the mean 7 S.E.M.
control, and 490.4754.1 ms (n¼6, Po0.05) after haloperidol treatment (Fig. 7B). These results suggest that haloperidol significantly attenuated the recovery from inactivation of Kv4.3 currents.
4. Discussion 3.4. Effects of haloperidol on use-dependency and the time course for recovery from inactivation of Kv4.3 Fig. 6A showed the use dependent-block of Kv4.3 currents by haloperidol. Under control conditions, the Kv4.3 currents were slightly reduced by 7.171.2% and 26.572.8% at 1 and 2 Hz, respectively (n¼ 8). In the presence of haloperidol, the Kv4.3 currents were decreased by 21.071.6% at 1 Hz and 44.172.2% at 2 Hz (n¼8, Po0.05) (Fig. 6B). These results suggest that the effect of haloperidol was potentiated by an increase in the stimulation frequency. Fig. 7A shows the effects of haloperidol on the kinetics of the recovery of Kv4.3 currents from inactivation using a two-pulse protocol. The peak amplitudes of the second pulse were normalized to the first peak amplitude and plotted against the inter-pulse intervals. A single exponential function was fitted to the time courses of recovery from inactivation of Kv4.3 with a time constant of 236.4721.8 ms during
The main findings of the present study can be summarized as follows. Haloperidol blocked Kv4.3 currents and accelerated the inactivation and activation kinetics of Kv4.3 in a concentrationdependent manner. These results indicated that haloperidol blocked the open state of Kv4.3 channels (Snyders and Yeola, 1995; Thompson, 1982; Zhang and Steinberg, 1995). The block of Kv4.3 by haloperidol increased as a function of stimulation frequency (use-dependence). This type of block can also be interpreted as the result of an open-channel block (Butterworth and Strichartz, 1990; Snyders et al., 1992). In addition, the block of Kv4.3 increased with depolarizing prepulses, shifting the steadystate inactivation curve to negative potentials and accelerating the closed-state inactivation at subthreshold membrane potentials. These results indicated preferential binding of the drug to an inactivated state of Kv4.3 channels at subthreshold potentials
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Fig. 5. Effects of haloperidol on the closed-state inactivation of Kv4.3. (A) Kv4.3 currents were recorded at þ 40 mV using a double-pulse protocol with a conditioning pulse to 60 mV of variable duration in the absence and presence of haloperidol. (B) The current amplitudes evoked by the second pulse, relative to the amplitude resulting from the initial control pulse, were plotted against the duration of the conditioning pulse. A single exponential function was fitted to the data points. Data are expressed as the mean 7S.E.M.
(Wang et al., 1995). Similarly, haloperidol reduced the amplitude of transient outward Kv current and accelerated the time course of apparent inactivation in rat ventricular myocytes, suggesting an interaction with the channels both in the open and inactivated states (Bebarova et al., 2006). The haloperidol block of HERG was use- and voltage-dependent, suggesting a preferential binding to either the open or inactivated states of channels (Suessbrich et al., 1997). Thus, haloperidol blocked Kv4.3 by both preferentially interacting with the open state and accelerating closed-state inactivation of Kv4.3 channels. Haloperidol, an antipsychotic drug, is commonly used in clinical practice to treat acute and chronic psychosis (LopezMunoz and Alamo, 2009; Miyamoto et al., 2012). Haloperidol has been associated with adverse cardiovascular events, including a prolongation of QT interval in patients receiving either a therapeutic dose or an overdose, suggesting a potential risk for development of cardiac arrhythmia, also known as torsades de pointes (Fayek et al., 2001; Fayer, 1986; Henderson et al., 1991; Hunt and Stern, 1995). Accordingly, numerous studies have demonstrated that haloperidol possesses potent inhibitory properties on different cardiac ion channels in the heart. For instance, haloperidol
Fig. 6. Use-dependent block of Kv4.3 by haloperidol. (A) Ten repetitive 200-ms depolarizing pulses of þ 40 mV from a holding potential of 80 mV were applied at two different frequencies, 1 and 2 Hz under control conditions, and after the application of haloperidol. (B) The peak amplitudes of current 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 mean 7 S.E.M.
blocked transient outward Kv currents in rat ventricular myocytes (Bebarova et al., 2006), sodium current in guinea pig ventricular myocytes (Cheng et al., 2007), and HERG currents in human atrial myocytes (Suessbrich et al., 1997). Since one of the mechanisms by which drugs can produce a prolongation of the QT interval is a blockade of cardiac repolarizing ion currents (Kannankeril et al., 2010), a blockade of HERG can explain the lengthening QT interval associated with the use of haloperidol. Kv4.3 channels, shal-type rapidly activating and inactivating Kv channels, encode the transient outward Kv currents in the heart (Dixon et al., 1996). This current is associated with early cardiac repolarizations and affects the activation of the subsequently activating L-type Ca2 þ currents (Niwa and Nerbonne, 2010; Sah et al., 2003). Interactions of the drug with the Kv4.3 channels are associated with alterations in cardiac action potential durations that either prolong or shorten the action potential duration due to secondary changes in subsequently depolarizing currents (Tamargo et al., 2004). Therefore,
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haloperidol to its therapeutic actions is unclear, a considerable amount of evidence demonstrates that some aspects of the clinical effects of haloperidol are mediated by its action on Kv4.3. For example, chronic haloperidol treatment increased the A-type Kv current density and altered the intrinsic pacemaker activity in rat dopaminergic neurons (Hahn et al., 2006). Hahn et al. (2003) also showed that haloperidol increases the number of functional Kv4 A-type currents via the upregulation of Kv4.3 mRNA in dopamine neurons and may serve to decrease excitability in the dopamine system. In general, the inhibition of neuronal A-type Kv currents modulated the refractory period and the duration of action potential, thus altering the firing frequency in neurons (Rudy, 1988). Since Kv4.3 channels are abundant and widely distributed in the CNS (Serodio and Rudy, 1998), the pharmacological block of Kv4.3 channels by haloperidol may influence electrical activity and neurotransmitter release in the dopaminergic neurons, suggesting that some of the antipsychotic and/or side effects of this drug may be attributed to the inhibitory effect on these currents. The therapeutic plasma concentrations of haloperidol are in the range of 10–100 nM (Yang et al., 2005). In the present study, haloperidol blocked Kv4.3 with an IC50 of 3.6 μM, which was much higher than the therapeutic plasma concentrations. Under occasional situations, however, the concentration of haloperidol in tissues can reach micromolar levels. The maximal concentrations of haloperidol in the myocardium were 6 to 10-fold higher than that in plasma after intraperitoneal application (Titier et al., 2004). Moreover, haloperidol concentrations in the human brain tissue were 10–30 times higher than plasma concentrations used in the treatment of schizophrenia (Kornhuber et al., 1999). Thus, the inhibitory effect of haloperidol on Kv4.3 in the present study may be clinically relevant in the range of therapeutic plasma concentrations after chronic administration. In conclusion, haloperidol produced a concentration-, time-, voltage- and use-dependent block of Kv4.3 currents that were stably expressed in CHO cells. These results suggest that haloperidol binds to the open state of Kv4.3 channels during depolarization and accelerates closed-state inactivation at subthreshold membrane potentials. Fig. 7. Effects of haloperidol on recovery from inactivation of Kv4.3. (A) A doublepulse protocol was used to characterize the recovery of Kv4.3 channels from inactivation in the absence and presence of haloperidol. (B) The first pre-pulse of a 500 ms depolarizing pulse of þ 40 mV from a holding potential of 80 mV was followed by a second 100 ms pulse after increasing the inter-pulse intervals between 10 and 10,000 ms at 80 mV. The peak currents elicited during the test pulses were measured and then normalized to the peak currents of the conditioning pulses in the same cells. Normalized data were plotted against the inter-pulse interval. A single exponential function was fitted to the data points. Data are expressed as the mean7 S.E.M.
further study of the action potential duration is required in order to address the possible contribution of the Kv4.3 block by haloperidol to cardiovascular side effects. The clinical pharmacological action of haloperidol is ascribed to dopaminergic receptor inhibition (Seeman and Van Tol, 1994). Atype Kv currents are activated at potentials that are negative to the threshold, thus shaping a spike waveform and regulating the duration of interspike intervals and firing frequency of the neurons in the central nervous system (Rudy, 1988; Zhang and McBain, 1995). The Kv4.3 currents, somatodendritic A-type Kv currents in the CNS, are highly expressed in dopaminergic neurons and play an important role in tuning the pacemaker frequency of the neurons (Liss et al., 2001). Haloperidol inhibits a variety of ionic currents that regulate neuronal activities: delayed rectifier Kv currents in mouse cortical neurons (Yang et al., 2005) and voltage-dependent Na þ currents of mammalian brain neurons (Wakamori et al., 1989). Although the relevance of a Kv4.3 block by
Authorship contributions Participated in research design: KW Sung, SJ Hahn Conducted experiments: HJ Lee Performed data analysis and interpretation: HJ Lee, KW Sung, SJ Hahn Wrote or contributed to the writing of the manuscript: KW Sung, SJ Hahn
Acknowledgments We thank Dr. Imaizumi (Department of Molecular and Cellular Pharmacology, Nagoya City University, Japan) for the Kv4.3 cDNA. This work was supported by a grant from the Catholic Medical Center Research Foundation made in the program year of 2014. References Ahn, H.S., Choi, J.S., Choi, B.H., Kim, M.J., Rhie, D.J., Yoon, S.H., Jo, Y.H., Kim, M.S., Sung, K.W., Hahn, S.J., 2005. Inhibition of the cloned delayed rectifier K þ channels, Kv1.5 and Kv3.1, by riluzole. Neuroscience 133, 1007–1019. Bebarova, M., Matejovic, P., Pasek, M., Novakova, M., 2006. Effect of haloperidol on transient outward potassium current in rat ventricular myocytes. Eur. J. Pharmacol. 550, 15–23. Birnbaum, S.G., Varga, A.W., Yuan, L.L., Anderson, A.E., Sweatt, J.D., Schrader, L.A., 2004. Structure and function of Kv4-family transient potassium channels. Physiol. Rev. 84, 803–833.
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