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CARDIOVASCULAR

Open Channel Block of A-Type, K_v4.3, and Delayed Rectifier K⁺ Channels, K_v1.3 and K_v3.1, by Sibutramine

Departments of Physiology (S.E.K., H.S.A., H.-J.J., M.-J.K., D.-J.R., S.-H.Y., Y.-H.J., M.-S.K., S.J.H.) and Pharmacology (K.-W.S.), Medical Research Center, College of Medicine, The Catholic University of Korea, Seoul, Korea; and Department of Pharmacology, Chonbuk National University, Jeonju, Korea (B.H.C.)

Received November 27, 2006; accepted February 13, 2007.

	Abstract

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The effects of sibutramine on voltage-gated K⁺ channel (K_v)4.3, K_v1.3, and K_v3.1, stably expressed in Chinese hamster ovary cells, were investigated using the whole-cell patch-clamp technique. Sibutramine did not significantly decrease the peak K_v4.3 currents, but it accelerated the rate of decay of current inactivation in a concentration-dependent manner. This phenomenon was effectively characterized by integrating the total current over the duration of a depolarizing pulse to +40 mV. The IC₅₀ value for the sibutramine block of K_v4.3 was 17.3 µM. Under control conditions, the inactivation of K_v4.3 currents could be fit to a biexponential function, and the time constants for the fast and slow components were significantly decreased after the application of sibutramine. The association (k₊₁) and dissociation (k_–1) rate constants for the sibutramine block of K_v 4.3 were 1.51 µM^–1s^–1 and 27.35 s^–1, respectively. The theoretical K_D value, derived from k_–1/k₊₁, yielded a value of 18.11 µM. The block of K_v4.3 by sibutramine displayed a weak voltage dependence, increasing at more positive potentials, and it was use-dependent at 2 Hz. Sibutramine did not affect the time course for the deactivating tail currents. Neither steady-state activation and inactivation nor the recovery from inactivation was affected by sibutramine. Sibutramine caused the concentration-dependent block of the K_v1.3 and K_v3.1 currents with an IC₅₀ value of 3.7 and 32.7 µM, respectively. In addition, sibutramine reduced the tail current amplitude and slowed the deactivation of the tail currents of K_v1.3 and K_v3.1, resulting in a crossover phenomenon. These results indicate that sibutramine acts on K_v4.3, K_v1.3, and K_v3.1 as an open channel blocker.

The activity of K_v channels is critical in maintaining the resting membrane potential, regulating action potential duration and frequency, and determining pacemaker activity in a variety of excitable cells (Rudy, 1988). K_v4.3, one of the major transient outward K_v currents, is mainly expressed in a variety of tissues, including neurons, cardiac myocytes, and smooth muscle cells (Ohya et al., 1997; Birnbaum et al., 2004). This channel can contribute to distinct functional roles due to differences in the membrane potential of these cells. For example, in many neurons, K_v4.3 does not activate at the resting membrane potential, but, in vascular smooth muscle cells, K_v4.3 is open in the window current range, and it is involved in the maintenance of membrane potential and the regulation of excitability (Amberg et al., 2003; Sergeant et al., 2005). Several studies have shown that some serotoninnorepinephrine reuptake inhibitors are effective blockers of K_v channels. Dexfenfluramine inhibits delayed rectifier K⁺ channels in rat lingual taste cells (Hu et al., 1998) and rat vascular smooth muscle cells (Weir et al., 1996). Aminorex, phentermine, dexfenfluramine, sibutramine, and fluoxetine also cause a concentration-dependent inhibition of the hK_v1.5 current stably expressed in human embryonic kidney cells (Perchenet et al., 2001). However, some of these drugs have been withdrawn from the market, due to primary pulmonary hypertension as a side effect. Although the mechanism by which these drugs cause pulmonary hypertension is not known, the inhibitory effect of the drug on K_v channels has been proposed as one of the important mechanisms responsible for causing vasoconstriction and for initiating pulmonary hypertension (Weir et al., 1996). From a consideration of drugs that are involved in the modulation of K_v channels, the vasoconstriction of mesenteric arterial smooth muscle cells due to the block of these currents reduces mesenteric blood flow and diminishes the transport of absorbed nutrients (McDaniel et al., 2001). These potential mechanisms for inhibiting nutrient absorption can be considered to be a favorable anorectic effect. Therefore, to understand the therapeutic action of sibutramine and its side effects, the present study was designed to evaluate the effects of sibutramine on A-type, K_v4.3, and the delayed rectifier K⁺ channels K_v1.3 and K_v3.1, which may play important roles in regulating the membrane potential of vascular smooth muscle cells, and to compare block of K_v4.3, and K_v1.3 and K_v3.1, by sibutramine.

	Materials and Methods

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Cell Cultures. The K_v4.3, K_v1.3, and K_v3.1 cDNA were stably transfected into CHO cells (American Type Culture Collection, Manassas, VA) using the Lipofectamine reagent (Invitrogen, Grand Island, NY) as described previously (Hahn et al., 1996; Choi et al., 1999; Ahn et al., 2006). CHO cells were maintained in Iscove's modified Dulbecco's medium (Invitrogen) supplemented with 10% fetal bovine serum, 0.1 mM hypoxanthine, and 0.01 mM thymidine in a humidified 5% CO₂ incubator at 37°C. The transfected cells were exchanged with fresh Iscove's modified Dulbecco's medium containing 0.3 mg/ml Geneticin (G-418; Invitrogen), and they were passed at 2- to 3-day intervals using a brief trypsin-EDTA treatment. The cells were dissociated and seeded onto glass coverslips (diameter 12 mm; Fisher Scientific Co., Pittsburgh, PA) in a 35-mm dish 1 day before use. For electrophysiological experiments, coverslips with attached cells were transferred to a recording chamber (RC-13; Warner Instruments, Hamden, CT). The chamber was superfused at a rate of 0.5 ml/min with normal external solution at room temperature (22–24°C).

View larger version (10K): [in this window] [in a new window]   Fig. 1. Concentration dependence for sibutramine block of Kv4.3 whole-cell currents expressed in CHO cells. Kv4.3 currents were obtained by applying 500-ms depolarizing pulses from a holding potential of –80 to +40 mV at 10-s intervals in the absence and presence of 1, 3, 10, and 30 µM sibutramine. The dotted line represents the peak level of the Kv4.3 current in the control. Concentration-response curve for the block of Kv4.3 currents by sibutramine. The integral of a depolarizing pulse was normalized to the current recorded under control conditions. The normalized currents were plotted against sibutramine concentrations and fit to the Hill equation. Data are expressed as means ± S.E.

Solutions and Drugs. The external bath solution contained 140 mM NaCl, 5 mM KCl, 1 mM CaCl₂, 1 mM MgCl₂, and 10 mM HEPES, and pH was adjusted to 7.3 with NaOH. The internal pipette solution contained 140 mM KCl, 1 mM CaCl₂, 1 mM MgCl₂, 10 mM HEPES, and 10 mM EGTA, and pH was adjusted to pH 7.3 with KOH. The measured osmolarity of the solutions was 300 to 340 mOsm. Sibutramine (Hanmi Pharmaceutical, Seoul, Korea) was dissolved in the bath solution to give a 30 mM stock solution.

Data Analysis. The Origin 7.0 software program (OriginLab Corp., Northampton, MA) was used for the analysis. The concentration-response data were fit to the following Hill equation: y = 1/(1 + ([D]/IC₅₀)ⁿ), where IC₅₀ is the concentration of sibutramine required to produce a 50% block, [D] is the concentration of sibutramine, and n the Hill coefficient. The voltage dependence of the fractional block (f) was fit to the following equation (Woodhull, 1973): f = [D]/([D] + K_D(0) x exp (–zFV/RT)), where K_D(0) represents the apparent affinity at 0 mV, z is the charge valence of sibutramine, is the fractional electrical distance, F is Faraday's constant, R is the gas constant, and T is the absolute temperature. A value of 25.4 mV was used for RT/F at 22°C in the present study. K_v4.3 currents were elicited by applying 500-ms depolarizing pulses from a holding potential of –80 to +40 mV at 10-s intervals. Steady-state activation curves were obtained by normalizing the tail currents measured at –60 mV after the application of 8-ms depolarizing pulses at potentials between –80 and +80 mV in 10-mV increments every 10 s from a holding potential of –80 mV in the absence and presence of sibutramine, and the curves were fit to the Boltzmann equation y = 1/[1 + exp(–(V – V_1/2)/k)], where k represents the slope factor, V is the test potential, and V_1/2 is the potential at which the conductance was half-maximal. The voltage dependence for steady-state inactivation was investigated using a double-pulse voltage protocol; currents were measured by 500-ms depolarizing pulses to +40 mV, whereas 1-s preconditioning pulses were varied from –110 to +10 mV stepped by 10 mV at 10-s intervals in the absence and presence of the drug. The resulting steady-state inactivation data were fit to the Boltzmann equation (I – I_c)/(I_max – I_c) = 1/[1 + exp(V – V_1/2)/k], in which I_max represents the current measured at the most hyperpolarized preconditioning pulse, I_c is a nonzero current that was not inactivated at the most depolarized preconditioning pulse, k is the slope factor, V the test potential, and V_1/2 the potential at which the conductance was half-maximal. We eliminated the nonzero residual current by subtracting it from the actual value. Data are expressed as the mean ± S.E. The one-way analysis of variance, followed by Bonferroni test, was used to evaluate the statistical significance of the observed differences (Wallenstein et al., 1980). Statistical significance was considered at p < 0.05.

View larger version (10K): [in this window] [in a new window]   Fig. 2. Effects of sibutramine on the kinetics of inactivation of Kv4.3 currents. The inactivation time constants of the fast component (fast) and the slow component (slow) were obtained from a biexponential fit. Statistical significance versus control is indicated by *, p < 0.05 (n = 7). Data are expressed as means ± S.E.

	Results

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View larger version (30K): [in this window] [in a new window]   Fig. 3. Time-dependent block of Kv4.3 by sibutramine. A, sibutramine-sensitive currents were first subtracted from the control and then normalized by the control. This procedure was carried out at four different drug concentrations. The time course was fit to a biexponential function that yielded the concentration-dependent time constants. B, inverses of fast time constants were plotted versus sibutramine concentration. The solid line represents the least-squares fit of the data to the following equation: 1/ = k+1[D] + k–1 in which is the drug-induced time constant, k+1 is the association rate constant, and k–1 the dissociation rate constant. Data are expressed as means ± S.E.

View larger version (16K): [in this window] [in a new window]   Fig. 4. Effects of sibutramine on the Kv4.3 current-voltage relationship. A, representative whole-cell Kv4.3 current traces under control conditions and in the presence of 20 µM sibutramine. Whole-cell currents were obtained by applying 500-ms depolarizing pulses from –80 to +60 mV in steps of 10 mV every 10 s from a holding potential of –80 mV. B, current-voltage curves for sibutramine-induced Kv4.3 currents. The data were taken from the integral of the depolarizing pulses shown in Fig. 3A under control conditions and after the addition of 20 µM sibutramine; data were normalized to the control. C, voltage-dependent block of Kv4.3 currents by sibutramine was expressed as a relative current (ISibutramine/IControl). The integral of currents in the presence of sibutramine was normalized to that at each voltage of the control. In the voltage range between –20 and +20 mV for channel opening, the block of Kv4.3 currents by sibutramine increased and was significantly different (n = 8; *, p < 0.05 versus data at –20 mV). The dotted line represents the activation curve of Kv4.3 under control conditions. Data are expressed as means ± S.E.

Voltage-Dependent Block of K_v4.3. Figure 4A shows the current-voltage relationships in the absence and presence of 20 µM sibutramine in a typical experiment. The K_v4.3 currents began to activate at –50 mV, and the control current-voltage relationship was almost linear for the depolarizations (Fig. 4B). Sibutramine reduced the K_v4.3 currents over the entire voltage range over which this current is activated. When the current block was expressed as a function of the test potential (Fig. 4C), the blocking effects of sibutramine on K_v4.3 were found to be voltage-dependent: the block increased between –20 and +20 mV, corresponding to the voltage range for channel opening (n = 8; p < 0.05). Furthermore, the reduction in the amount of charge crossing the membrane during depolarization increased in a shallow manner at membrane potentials where the maximal conductance was reached (+30 to +60 mV). This voltage dependence was fit to Woodhull's equation (see Materials and Methods) and yielded = 0.16 ± 0.07 (n = 8).

View larger version (15K): [in this window] [in a new window]   Fig. 5. Use-dependent block of Kv4.3 by sibutramine. A, repetitive 20- or 200-ms depolarizing pulses of +40 mV from a holding potential of –80 mV were applied at the 2-Hz frequency under control conditions and after the application of 20 µM sibutramine. The peak amplitudes of the current at each pulse were normalized to that of the current obtained at the first pulse and then plotted versus pulse number (n = 5). B, current in the presence of sibutramine at 10th pulse was normalized to that in the absence of the drug. Data are expressed as means ± S.E.

Effect of Sibutramine on the Time Course of Tail Currents. In the absence of the drug, K_v4.3 currents were completely deactivated at –60 mV with a time constant of 10.0 ± 0.9 ms (n = 6) (Fig. 6). Sibutramine at a concentration of 20 µM reduced the tail current amplitude, but it failed to alter the time course for the deactivation of the tail current. The average value was 13.1 ± 2.4 ms (n = 6), which was not significantly different from the control value.

View larger version (9K): [in this window] [in a new window]   Fig. 6. Effect of sibutramine on the time course of tail currents. Tail currents were induced at the repolarizing pulses to –60 mV after a 20-ms depolarizing pulse of +40 mV from a holding potential of –80 mV in the absence and presence of sibutramine.

View larger version (23K): [in this window] [in a new window]   Fig. 7. Effects of sibutramine on the steady-state activation and inactivation of Kv4.3. A, tail currents were recorded at –60 mV after 8-ms depolarizing pulses between –80 and +80 mV in steps of 10 mV from a holding potential of –80 mV under control conditions and in the presence of 20 µM sibutramine. The steady-state activation curves were drawn by fitting normalized tail currents to the Boltzmann equations (n = 8). B, effects of sibutramine on the steady-state inactivation of Kv4.3. The currents were evoked by 1-s prepulses that were varied from –110 to +10 mV stepped by 10 mV and a 500-ms depolarizing pulse to +40 mV in the absence and presence of sibutramine. Steady-state inactivation curves were shown as a plot of normalized peak currents during the depolarizing pulse as a function of the conditioning potential. The curves represent the best-fit Boltzmann equations (n = 8). Data are expressed as means ± S.E.

Effect of Sibutramine on the Recovery from Inactivation of K_v4.3. Figure 8 shows a typical example of the recovery kinetics of K_v4.3 in the absence and presence of 20 µM sibutramine. The peak currents elicited by the second depolarizing pulse were divided by those evoked by the first prepulse, and the normalized data are plotted against the interpulse interval. Under control conditions, the plotted data fit well to a single exponential function with a time constant of 0.25 ± 0.03 ms (n = 7). In the presence of sibutramine, the time constant for recovery was 0.33 ± 0.04 ms (n = 7), and there was no significant drug effect in altering the recovery from the inactivation of K_v4.3.

View larger version (17K): [in this window] [in a new window]   Fig. 8. Effects of sibutramine on the recovery from inactivation of Kv4.3. A double-pulse protocol was used to characterize the recovery of Kv4.3 channels from inactivation: the first prepulse of a 500-ms depolarizing pulse of +40 mV from a holding potential of –80 mV was followed by a second identical pulse after increasing the interpulse intervals between 5 and 5,000 ms at –80 mV. Typical Kv4.3 current traces for recovery from inactivation were shown under control conditions and in the presence of 20 µM sibutramine. The peak currents elicited by the second depolarizing pulse were divided by those evoked by the first prepulse and the normalized data were plotted against the interpulse interval. The plotted data were fit well to a single exponential function under control conditions and in the presence of 20 µM sibutramine (n = 7). Data are expressed as means ± S.E.

View larger version (22K): [in this window] [in a new window]   Fig. 9. Concentration dependence for sibutramine block of Kv1.3 (A) and Kv3.1 (B) whole-cell currents expressed in CHO cells. Kv1.3 currents were obtained by 200-ms depolarizing pulses from a holding potential of –80 to +40 mV at 30-s intervals in the absence and presence of 0.03, 0.3, 3, and 30 µM sibutramine. The steady-state currents of depolarizing pulses were normalized to the current recorded under control conditions. The normalized currents were plotted against sibutramine concentrations and fit to the Hill equation. The data yielded an IC50 value of 3.7 ± 0.7 µM and a Hill coefficient of 1.1 ± 0.2 (n = 6). Tail currents were recorded during 200-ms repolarizing pulses of –40 mV after 200-ms depolarizing pulses of +40 mV from a holding potential of –80 mV in the absence and presence of 3 µM sibutramine. Kv3.1 currents were obtained by 250-ms depolarizing pulses from a holding potential of –80 to +40 mV at 10-s intervals in the absence and presence of 3, 10, 30, and 100 µM sibutramine. The steady-state currents of depolarizing pulses were normalized to the current recorded under control conditions. The normalized currents were plotted against sibutramine concentrations and fit to the Hill equation. The data yielded an IC50 value of 32.7 ± 5.0 µM and a Hill coefficient of 1.6 ± 0.2 (n = 5). Tail currents were recorded during 250-ms repolarizing pulses of –40 mV after 250-ms depolarizing pulses of +40 mV from a holding potential of –80 mV in the absence and presence of 30 µM sibutramine. The dotted lines in A and B represent zero current. Data are expressed as means ± S.E.

	Discussion

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The main finding of the present study is that sibutramine has two effects on K_v4.3: 1) an acceleration of the time-dependent decrease in current in a concentration-dependent manner and 2) use-dependent block. Similar blocking effects were also observed for K_v1.3 and K_v3.1 in terms of time- and concentration-dependent block. Furthermore, the deactivation kinetics of K_v1.3 and K_v3.1 upon repolarization was slowed, with a crossover in the tail currents. Thus, the effects of sibutramine on these currents may be explained by the block of these channels in the open states.

In the present study, the action of sibutramine on accelerating the rate of current inactivation of K_v4.3 in a concentration-dependent manner can be explained by an open channel block mechanism. Similar findings have previously been reported as characteristics of the block of K_v1.5 channels by sibutramine (Perchenet et al., 2001), bisindolylmaleimide (Choi et al., 2000), zatebradine (Valenzuela et al., 1996), and terfenadine (Yang et al., 1995). They are all characterized as open channel blockers: the acceleration in the current decay by the drugs is due to the slow binding of the drug to the channels after channel activation rather than the modulation of intrinsic inactivation. Thus, the mechanism of channel block by sibutramine in K_v4.3 and K_v1.5 channels is similar. Additional evidence of an open channel block is the voltage dependence of the sibutramine block. The block increased with channel opening over the voltage range of activation (–20 to +20 mV), suggesting the preferential binding to the open state of the channel. Because sibutramine is predominantly in the cationic form at physiological pH (Perchenet et al., 2001), the interaction of sibutramine and channels would be expected to experience a transmembrane electrical field. Indeed, the block of K_v1.5 by sibutramine exhibits a shallow voltage dependence, which was described by an electrical distance () of 0.17 (Perchenet et al., 2001). At potentials positive to +30 mV where the maximal conductance is reached, the block of K_v4.3 increased with a shallow voltage dependence. Because the voltage-dependent block over this voltage range is generally considered to be an open channel block (Snyders et al., 1992; Valenzuela et al., 1996), this reflects the effects of the electrical field on the interaction between the charged form of sibutramine and the channel. We obtained a fractional of 0.16 for sibutramine, similar to that reported previously for sibutramine for K_v1.5 (Perchenet et al., 2001). This analysis suggests that sibutramine binds to a similar site in the internal mouth of the channel. Although the possibility that sibutramine increases the rate of intrinsic inactivation of K_v4.3 cannot be excluded, all of the actions of sibutramine may be explained by a single mechanism of open channel block.

Another characteristic of sibutramine block is its use dependence. The degree of block increased with repetitive depolarizations. However, the peak amplitude of K_v4.3 elicited by the first pulse was not significantly modified, and a decrease in current amplitude was observed with subsequent pulses, indicating that the channels are not blocked before channel activation by depolarization. Furthermore, extrapolation to zero at the start of the pulse suggests that there is no block of K_v4.3 in the resting state. Use-dependent block may be accomplished by the binding of the drug either to the open or to the inactivated states of channels (Courtney, 1975; Butterworth and Strichartz, 1990; Wang et al., 1995; Valenzuela et al., 1996; Delpon et al., 1997). To determine whether the binding of sibutramine to the open state of the channel was influenced by the process of inactivation of K_v4.3, we studied the use-dependent block by using variable pulse durations. Although an enhancement in use dependence was observed with long-duration pulses (200 ms), which allow adequate inactivation to occur compared with short-duration pulses (20 ms), which limit the entry of K_v4.3 channels into the inactivated state, no change in the degree of block between the short- and the long-duration pulses in the presence of the drug was observed. Furthermore, sibutramine block was not affected by depolarizing prepulses over the inactivation voltage range as shown in the steady-state inactivation curves. These results suggest that the use-dependent block is due to channel activation but that it is independent of the channel inactivation of K_v4.3, and the interaction of sibutramine with the binding site was not influenced by the gating kinetics of inactivation.

Use-dependent block is generally associated with a slow rate of recovery from inactivation due to the slower dissociation of the drug from its binding site. However, in the present study, the recovery process in control and with sibutramine was the same: the rate of recovery of the blocked channel is similar to that of recovery from intrinsic inactivation. The lack of change in the time course for recovery from inactivation rules out the possibility that the drug interacts with K_v4.3 channels in the inactivated state. Furthermore, these results are similar to previous findings showing that mibefradil induces significant use-dependent block but paradoxically increases the rate of recovery from inactivation of K_v1.5 (Perchenet and Clement-Chomienne, 2000). According to this study, mibefradil exclusively competes with the inactivation gate in the channel pore. Thus, one possible explanation for the use-dependent block is that there is competition between the modulatory site of inactivation and sibutramine, and the blocked channels cannot inactivate after drug binding. The drug is trapped at its binding site after the open channels are blocked and the use-dependent block develops when the rate of drug binding is equal to the rate of drug untrapping.

Sibutramine slowed the deactivation time course of K_v1.3 and K_v3.1, thus inducing a tail crossover. This phenomenon has also been reported for other open channel blockers of K_v1.5, such as sibutramine, terfenadine, and zatebradine (Yang et al., 1995; Valenzuela et al., 1996; Perchenet et al., 2001), and it provides further evidence for the open channel block mechanism. However, the tail crossover phenomenon that occurred in the release of the drug before channel closing was not observed in sibutramine block of K_v4.3. These results suggest that sibutramine is not able to dissociate from its binding site before the channels close and is trapped in the deactivating channels, as described above. Loratadine, which also acts as an open channel blocker, did not induce a tail crossover of K_v1.5 currents, suggesting that the channel can close with the drug bound (Lacerda et al., 1997). Propafenone did not induce a tail crossover in delayed rectifier K⁺ currents due to being trapped in the deactivating channels during closing or due to slow unblocking kinetics, which produced use-dependent effects of the drug (Delpon et al., 1995). The differences in deactivation kinetics between both channels may be due to the slower dissociation rate constant observed for K_v4.3 than for K_v1.5 (Perchenet et al., 2001). Thus, this characteristic block of K_v4.3 by sibutramine differentiates it from other open channel blockers studied on delayed rectifier K⁺ channels K_v1.3, K_v3.1, and K_v1.5.

Sibutramine is a centrally acting anorectic agent for the treatment of obesity (Buckett et al., 1988). Although the precise mechanism by which sibutramine causes weight loss remains unclear, the therapeutic effects of sibutramine are known to involve the selective inhibition of the reuptake of monoamines in the brain, thus inducing satiety and reducing feeding behavior (Halford et al., 2005). Because some anorexic agents, such as aminorex and fenfluramine that interfere with serotonin function, have been associated with an increased incidence of pulmonary hypertension, these drugs have been withdrawn from the market. Sibutramine is known to be an efficacious and safe drug for the management of obesity, and it has not been shown to induce pulmonary hypertension, although some cardiovascular dysfunctions have been reported in patients receiving sibutramine (Luque and Rey, 1999; McMahon et al., 2000). All of these drugs are known to inhibit K_v currents in vascular smooth muscle cells (Weir et al., 1996) and a dysfunctional K_v in pulmonary smooth muscle cells has been implicated in primary pulmonary hypertension (Yuan et al., 1998). Several types of K_v channels, including K_v1.3, K_v1.5, K_v3.1, and K_v4.3, have been identified in human artery smooth muscle cells, and the functional expression of these channels may play a critical role in the regulation of membrane potential and pulmonary vascular tone (Amberg et al., 2003; Platoshyn et al., 2004). These observations suggest that sibutramine may regulate vascular tone through modulating K_v channel activity in pulmonary vascular smooth muscle cells. Thus, special attention to the possibility that pulmonary hypertension may occur is necessary in predisposed patients. Furthermore, because K_v4.3 and K_v1.5 are expressed at high levels in the heart and they are responsible for repolarization of the cardiac action potential, cardiac arrhythmias associated with the administration of sibutramine may be, at least in part, related to block of these channels in cardiac myocytes. On the other hand, anorexic effects of K_v channel blockers have been reported in mesenteric arterial smooth muscle cells (McDaniel et al., 2001). Since K_v4.3, K_v1.3, K_v3.1, and K_v1.5 channels have been identified in vascular smooth muscle cells (McDaniel et al., 2001; Mandegar et al., 2002; Platoshyn et al., 2004; Sergeant et al., 2005), the inhibition of these channels in mesenteric arterial smooth muscle cells leads to membrane depolarization which, in turn, induces calcium entry and vasoconstriction. Thus, sibutramine can reduce mesenteric blood flow, which transports nutrients to the liver and adipose tissue through its action on K_v4.3, K_v1.3, and K_v3.1 in the present study and K_v1.5 (Perchenet et al., 2001). Thus, we proposed that the block of K_v4.3, K_v1.3, and K_v3.1 by sibutramine involves a mechanism underlying another anorectic and metabolic action of this drug.

In conclusion, sibutramine blocked K_v4.3 binding to open channels in a concentration- and time-dependent manner. The open channel block of K_v4.3 by sibutramine is generally similar to that observed for K_v1.3, K_v3.1, and K_v1.5, as reported in previous studies, whereas its effects on current kinetics may vary depending on the channel being studied. Our results may help explain the mechanism underlying some of the therapeutic action of this drug and the side effects associated with its use.

	Acknowledgements

 
We thank Dr. Leonard K. Kaczmarek (Department of Pharmacology, Yale University School of Medicine, New Haven, CT) for the K_v1.3 and K_v3.1 cDNA, and Dr. Yuji Imaizumi (Department of Molecular and Cellular Pharmacology, Nagoya City University, Nagoya, Japan) for the K_v4.3 cDNA.

	Footnotes

 
This work was supported by Grant R13-2002-005-01002-0 from the Medical Research Center, Korea Science and Engineering Foundation, Republic of Korea.

S.E.K. and H.S.A. contributed equally to this study.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.106.117747.

ABBREVIATIONS: K_v, voltage-gated K⁺ channel; CHO, Chinese hamster ovary.

Address correspondence to: Dr. Sang June Hahn, Department of Physiology, College of Medicine, The Catholic University of Korea, 505 Banpo-dong, Socho-gu, Seoul 137-701, Korea. E-mail: sjhahn{at}catholic.ac.kr

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