A hKv1.4 stable cell line was generated in HEK293 cells by cloning human Kv1.4 cDNA (GenBank number: NM_002233) into pCDH-CMV-MCS-EF1α-Puro vector (System Biosciences, Palo Alto, CA, USA) to obtain hKv1.4-expressing plasmid pCDH-hKv1.4. HEK293 cells were transfected with pCDH-hKv1.4 vector using Lipofectamine 2000 (Life Technologies, Carlsbad, CA, USA) according to the manufacturer's recommendations. Positive clones were selected by the addition of puromycin (Life Technologies, Grand Island, NY, USA) to media. The hKv1.4 stable cell line was established by single colony isolation and characterization of the cellular electrophysiology. The hKv1.4-HEK293 stable cell line was grown in Dulbecco's modified Eagle's medium (Life Technologies, Grand Island, NY, USA) containing 10% fetal bovine serum at 37°C in a 5% CO₂ atmosphere. The cells were plated on sterile glass coverslips placed inside a 35 mm culture dish. One day later, hKv1.4 currents were recorded using patch-clamp technique. A stable CHO cell line expressing Kv4.3 channels was maintained in Iscove’s modified Dulbecco’s medium (Gibco, Life Technologies Co., Grand Island, NY, USA) supplemented with 10% fetal bovine serum (Gibco), 1% HT supplement (Gibco), and 0.3% geneticin (Gibco), as described previously [17]. The cells were treated with trypsin-EDTA solution (Gibco) and seeded on glass coverslips (12 mm diameter) in 35 mm Petri dishes 1 day prior to use in patch-clamp recording.

Kv1.4 and Kv4.3 currents were recorded using the whole-cell patch-clamp technique with a Multiclamp 700B amplifier (Molecular Devices, San Jose, CA, USA) as described previously [18]. Data acquisition was performed with pClamp 10.0 software (Axon Instruments, Inc., Forster City, CA, USA) using a digidata1322A interface (Molecular Devices). The recording chamber (RC-26G; Warner Instruments, Hamden, CT, USA) was continuously perfused with an extracellular bath solution using gravity-fed flow (1 ml/min) at room temperature (22°C–24°C). Patch pipettes (3–4 MΩ) were pulled from borosilicate glass (1B150F-4; World Precision Instruments, Sarasota, Fl, USA) and filled with an internal solution containing (in mM) 140 KCl, 1 CaCl₂, 1 MgCl₂, 10 EGTA, and 10 N‐2-hydroxyethylpiperazine-N_′‐2‐ethanesulfonic acid (HEPES, pH 7.3 with KOH). The external bath solution contained (in mM) 140 NaCl, 5 KCl, 1 CaCl₂, 1 MgCl₂, 10 glucose, and 10 HEPES (pH 7.3 with NaOH). The current signal was sampled at 20 kHz and filtered at 5 kHz. The series resistances were usually compensated up to 80% by amplifier circuits and no leak subtractions were used. Kv1.4 and Kv4.3 currents were evoked by applying 500 ms depolarizing pulses from a holding potential of –80 to +40 mV every 20 sec and 10 sec, respectively.

Aripiprazole and dehydroaripiprazole (MedChem Express, Monmouth Junction, NJ, USA) were dissolved in dimethyl sulfoxide (DMSO) as the stock solution. The stock solution was then further diluted with an external bath solution. The concentration of DMSO in the final dilution was less than 0.1% except for the concentration of 30 μM aripiprazole or dehydroaripiprazole in bath solution (0.3% DMSO). These concentrations of DMSO had no effect on Kv currents (data not shown). All chemicals for bath and internal solution, and DMSO were obtained from Sigma-Aldrich Co. (St. Louis, MO, USA).

The Clampfit 10.0 (Molecular devices) and Origin 8.0 software (Origin Lab Corp., Northampton, MA, USA) were used for the data analysis. The concentration-dependent inhibition of Kv currents by aripiprazole was fitted to the Hill equation: f = 1 / [1 + (IC₅₀ / [D])ⁿ], where f denotes the fractional inhibition at drug concentration [D], IC₅₀ is the drug concentration for half-maximum inhibition, and n represents the Hill coefficient. The current activation curves were fitted with the Boltzmann equation : G/G_max = 1 / [1 + exp (–(V–V_1/2) / k)], where V, V_1/2, k, G and G_max represent the test potential, potential at half-maximal current, the slope factor, the conductance, and the maximal conductance, respectively. The conductance was calculated using the formula, G = I / (V–E_K), where I denotes Kv current amplitude and E_K is the calculated equilibrium potential (–85 mV). Voltage dependence of inhibition by aripiprazole was plotted as a function of the membrane potential. The fractional inhibition by aripiprazole ranging between +20 and +60 mV was fitted with the Woodhull equation [19] : f = [D] / {[D] + K_D(0) × exp(–zδFV / RT)}, where K_D(0) denotes the apparent affinity at 0 mV (the reference voltage), z is the charge valence of the drug (+1 for aripiprazole and dehydroaripiprazole), δ represents the fractional electrical distance (i.e., the fraction of the transmembrane electric field sensed by a single charge at the receptor site), F is the Faraday’s constant, R is the gas constant, and T is the absolute temperature. In the present study, 25.4 mV was used as the RT/F value at 22°C.

Data are expressed as mean ± standard error. Statistical comparisons were performed with Student’s t-test and analysis of variance followed by Bonferroni’s test for comparisons of multiple groups using Excel and Origin 8.0 software. A value of p < 0.05 was considered statistically significant.

Kv1.4 current was recorded with the whole-cell patch-clamp technique and its inhibition by aripiprazole was tested. Kv1.4 currents were evoked by 500-ms depolarizing pulses to +40 mV from a holding potential of –80 mV every 20 sec with or without aripiprazole (Fig. 1). Kv1.4 currents were activated and inactivated rapidly in response to membrane depolarization pulses, as described previously [20]. Extracellular application of aripiprazole (0.3 to 30 μM) resulted in moderate reduction of the peak amplitude and acceleration of decay phase of Kv1.4 currents in a concentration-dependent manner (Fig. 1A). The integral of the current during depolarization was measured to quantify the inhibition of Kv1.4 currents. The inhibition measured on the integral of Kv1.4 was fitted by Hill equation and yielded an IC₅₀ value of 4.4 ± 0.4 μM with a Hill coefficient of 2.5 ± 0.2 (n = 8) (Fig. 1B). The concentration-response curve for Kv1.4 was rather steep with a Hill coefficient of 2.5, which suggested more than one binding sites for aripiprazole or a positively cooperative binding mechanism [21]. The time course for the decay of Kv1.4 at +40 mV under control conditions was fitted with a biexponential function with fast and slow time constants of 39.6 ± 3.8 ms and 184.9 ± 25.0 ms, respectively (n = 8) (Fig. 1C). With the application of aripiprazole, the time course for the decay of Kv1.4 was also fitted with a biexponential function. Aripiprazole decreased both the fast and slow time constants (12.3 ± 1.5 ms and 85.3 ± 2.8 ms, respectively, at 10 μM aripiprazole) in a concentration-dependent manner, suggesting that the inactivation kinetics of Kv1.4 were accelerated by aripiprazole. To investigate the effect on the activation kinetics, we measured the time-to-peak values of Kv1.4 currents (Fig. 1D). Under control conditions, the time-to-peak was 5.2 ± 0.2 ms (n = 8). In the presence of aripiprazole, the time-to-peak of Kv1.4 activation (3.7 ± 0.1 ms at 10 μM aripiprazole) was significantly reduced in a concentration-dependent manner. These accelerations in both decay rate and activation time courses of Kv1.4 suggests open-channel block mechanisms [22,23].

The effects of voltage on the aripiprazole-induced inhibition of Kv1.4 were investigated by applying a series of 500-ms depolarizing pulses between –70 and +60 mV from a holding potential of –80 mV with and without 10 μM aripiprazole (Fig. 2A). Aripiprazole inhibited the amplitude of Kv1.4 currents at all membrane potentials. Normalized conductance (G/G_max) at every membrane potential tested was plotted (Fig. 2B). The data were fitted to Boltzmann equation. The activation curve of Kv1.4 yielded the half-activation potential (V_1/2 = –14.0 ± 4.0 mV, n = 8) and the slope factor (k = 22.6 ± 1.4) under control conditions. In the presence of aripiprazole, both half-activation potential (V_1/2 = –17.3 ± 7.0 mV, p = 0.436) and slope factor (k = 24.1 ± 1.1, p = 0.413) of activation curve were unchanged. To analyze the voltage-dependence of inhibition by aripiprazole, the fractional block of the integral (I_aripiprazole/I_control) was plotted as a function of membrane potential (Fig. 2C). The aripiprazole-induced inhibition was steeply increased in a voltage range corresponding to that of Kv1.4 channel activation (F_4,35 = 3.37, p < 0.05). The inhibition was also voltage-dependent at more positive potentials (+20 to +60 mV) where the channels were fully activated. This voltage dependence of aripiprazole-induced inhibition was fitted to a Woodhull’s equation: δ = 0.17 ± 0.05 (n = 8).

Recovery from inactivation of Kv1.4 was studied using a two-pulse protocol (Fig. 3). The cell was depolarized to +40 mV for 500 ms by prepulse and then repolarized to –80 mV holding potential for a variable period ranging from 0.3 sec to 14 sec before applying the same test pulses in the absence and presence of 10 μM aripiprazole (Fig. 3A). The peak amplitudes of the test pulse were normalized to those of prepulse and plotted against the inter-pulse intervals. In the control experiments, the time course of recovery from inactivation of Kv1.4 was fitted to a single exponential function with a time constant of 4.9 ± 0.8 s (n = 7) (Fig. 4B). After application of aripiprazole, the recovery from inactivation was also fitted to a single exponential function with a time constant of 5.5 ± 0.7 sec (n = 7, p = 0.105), suggesting that aripiprazole has no effect on the time course of recovery from inactivation of Kv1.4.

To analyze the use-dependent inhibition by aripiprazole, Kv1.4 currents were evoked by 10 consecutive 20-ms and 200-ms pulses to +40 mV from a holding potential of –80 mV at 0.25 Hz in the absence and presence of aripiprazole (Fig. 4A). Since the peak amplitudes of the Kv1.4 currents were diminished to less than 50% by repetitive pulses at 0.5–2 Hz under control conditions (data not shown), we analyzed the use-dependent inhibition with aripiprazole at 0.25 Hz under two pulse durations (20 and 200 ms). Under control conditions, the Kv1.4 currents decreased by 25.0% ± 2.9% and 36.8% ± 2.6% at 20-ms and 200-ms pulses, respectively, after 10th repetitive pulses (n = 6) (Fig. 4B). In the presence of aripiprazole, the Kv1.4 currents decreased by 42.2% ± 3.5% (p < 0.001) and 53.1% ± 3.2% (p < 0.001) at 20-ms and 200-ms pulses, respectively. However, the calculated percentages of inhibition by aripiprazole were similar between 20 ms (17.2%) and 200 ms pulses (16.3%) (p = 0.267). These results suggest that aripiprazole exhibits use-dependent inhibition with successive pulses on Kv1.4 currents.

Next, we investigated the effect of aripiprazole on another voltage-gated A-type Kv channel, Kv4.3 (Fig. 5). Kv4.3 currents expressed in CHO cells were evoked by the same voltage protocol as in Kv1.4 currents except for the 10-sec interval in voltage step and were measured under control conditions and in the presence of various concentrations of aripiprazole (0.3 to 30 μM). Under control conditions, the Kv4.3 currents were activated to a peak and inactivated rapidly as reported previously [17], which was similar to those of Kv1.4 in Fig. 1A. In the presence of aripiprazole, the peak amplitude of Kv4.3 currents also decreased (Fig. 5B). As shown in Kv1.4 currents, to quantify the inhibition of Kv4.3 currents, the integral of the currents during the entire depolarization pulse was measured. The normalized inhibition of Kv4.3 currents plotted as function of drug concentration was fitted by Hill equation and yielded an IC₅₀ value of 4.9 ± 0.4 μM (n = 5, p = 0.580 compared with Kv1.4) with a Hill coefficient of 2.3 ± 0.2 (p = 0.463 compared with Kv1.4) (Fig. 5B). These results suggest that aripiprazole showed similar inhibitory effects on both A-type Kv channels, Kv1.4 and Kv4.3.

A major active metabolite of aripiprazole, dehydroaripiprazole, has been reported to exhibit similar pharmacological activity compared to the parent compound [6,24]. To analyze the effect of dehydroaripiprazole on Kv1.4 currents, a series of dehydroaripiprazole concentrations were tested (Fig. 6). Bath application of dehydroaripiprazole reduced the integral of the Kv1.4 currents in a concentration-dependent manner (Fig. 6A). The normalized Kv1.4 currents plotted as a function of drug concentration was fitted to Hill equation (Fig. 6B) and yielded an IC₅₀ value of 6.3 ± 0.8 μM (n = 5, p < 0.05 compared with aripiprazole) with a Hill coefficient of 2.0 ± 0.2 (p = 0.185 compared with aripiprazole). These results suggest that dehydroaripiprazole has inhibitory effects comparable to the parent compound, aripiprazole.