Blockade action of ketanserin and increasing effect of potassium ion on Kv1.3 channels expressed in Xenopus oocytes

Pharmacological Research 56 (2007) 148–154

Blockade action of ketanserin and increasing effect of potassium ion on Kv1.3 channels expressed in Xenopus oocytes Xianpei Wang 1 , Yuhua Liao, Anruo Zou ∗ , Lu Li, Danna Tu Department of Cardiology, Institute of Cardiovascular Diseases, Ion Channelopathy Research Center, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, PR China Accepted 8 May 2007

Abstract The goal of this study was to investigate the pharmacological effects of ketanserin (KT) and elevated extracellular potassium ([K+ ]o) on Kv1.3 potassium channels. Kv1.3 channels were expressed in Xenopus oocytes, and the resulting currents were measured using a two-microelectrode voltage-clamp technique. KT blocked Kv1.3 currents in a concentration-dependent, time-dependent and voltage-independent manner, and accelerated their activation and inactivation. Kv1.3 currents were increased by high [K+ ]o in a concentration-dependent manner. Our results suggest that KT acts directly on the open state of the Kv1.3 channel, whereas augmentation of extracellular [K] enhances current flow through the channel by increasing the channel’s conductance. © 2007 Elsevier Ltd. All rights reserved. Keywords: Ketanserin; Kv1.3 potassium channel; Potassium ion; Immunomodulation; Channel kinetics

1. Introduction Ketanserin (KT) is a selective serotonin (5-HT) 2-receptor antagonist with minor alpha1-receptor blocking properties. It reduces peripheral blood pressure by blocking the activation role of peripheral 5-HT receptors and has been evaluated for the treatment of hypertension, especially the treatment of severe hypertension in pregnancy and certain peripheral vascular diseases [1]. Furthermore, it has been suggested that KT diminishes arteriosclerotic development by its effects on 5HT-induced platelet aggregation and thrombus formation [2]. KT also inhibits the transient outward current (Ito ) encoded by Kv1.4, Kv4.2 and Kv4.3 channel genes, the rapid component of delayed rectifier K+ current (Ikr ) encoded by HERG, the ultra-rapidly activated delayed rectifier K+ current (Ikur ) encoded by Kv1.5 channel genes, and the ATP-sensitive potassium current (IKATP ), encoded by the Kir 5.2 gene. KT also prolongs the action potential duration and QT interval of the electrocardiogram, thereby showing both antiarrhythmic and proarrhythmic

∗

Corresponding author. Tel.: +86 27 85799274; fax: +86 27 85727340. E-mail addresses: xianpei [email protected] (X. Wang), [email protected] (A. Zou). 1 Tel.: +86 27 85799274; fax: +86 27 85727340. 1043-6618/$ – see front matter © 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.phrs.2007.05.002

actions [3–5]. These results suggest that ketanserin could play different pharmacological roles by its non-selective blockade of several types of potassium channels. Accordingly, it is important to investigate the potential actions of KT on other potassium channels. Kv1.3 is a Shaker-related, voltage-gated potassium (Kv) channel expressed in lymphocytes [6], CNS [7], fat and numerous other tissues [8]. In T and B lymphocytes, a concerted interplay between plasma membrane Kv1.3 channels, other K channels, Ca channels and the membrane potential promotes the sustained Ca2+ signaling required for T-cell receptor (TCR)mediated cell activation, gene transcription and proliferation [6]. Pharmacological interference with Kv1.3 inhibits T- and B-cell proliferation in vitro as well as in vivo, thereby defining this ion channel as a potential therapeutic target for immunosuppression in autoimmune diseases and graft rejection [6,9–11]. Levite et al. have suggested that elevated extracellular potassium ([K+ ]o), in the absence of “classical” immunological stimulatory signals, is a sufficient stimulus to activate T-cell ␤1 integrin moieties, and to induce integrin-mediated adhesion and migration [12]. This could be explained by the fact that opening of Kv1.3 channels leads to function, whereas their blockage prevents function [12]. Nevertheless, a direct interaction between K+ and Kv1.3 channels is still unclear. Therefore we investigated the effect of KT on Kv1.3 channels expressed in Xenopus laevis oocytes

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and observed how K+ influenced current flow through the channels. Meanwhile, we explore the potential immunomodulatory therapeutic implications of Kv1.3 channel inhibition by KT and Kv1.3 current activation by K+ . 2. Methods 2.1. In vitro transcription and functional expression in Xenopus laevis oocytes Procedures for in vitro transcription and oocyte injection have been described in detail [13,14]. The human Kv1.3 gene (a kind gift from Dr. Maria L. Garcia, Merck & Co., Inc., USA) was subcloned into a pCI-neo vector. CRNAs were prepared with T7 RNA Polymerase (Roche Applied Science) after linearization of the plasmid with EcoRI, according to manufacturer’s protocols. CRNAs were dissolved in diethyl pyrocarbonate(DEPC)-treated sterile water, stored at −80 ◦ C, and diluted immediately prior to injection. Xenopus frogs were anesthetized by immersion in 0.2% tricaine for 15–30 min. Ovarian lobes were digested with 1.5 mg ml−1 type 1A collagenease (Sigma) in Ca2+ -free ND96 solution for 1 h to remove follicle cells. Stage IV and V oocytes were injected with 46 nl of cRNA and then cultured in ND96 solution supplemented with 100 U/ml penicillin, 100 U/ml streptomycin and 2.5 mM pyruvate at 18 ◦ C. ND96 solution contained (mM): 2 KCl, 96 NaCl, 2.0 MgCl2 , 1.8 CaCl2 , and 5 HEPES; pH was adjusted to 7.5 with NaOH. 2.2. Two-microelectrode voltage clamp of oocytes Unless indicated otherwise, oocytes were bathed in ND96 solution. A standard two-microelectrode voltage-clamp technique was used to record Kv1.3 currents at room temperature (21–23 ◦ C) 2–10 days after injection. Glass microelectrodes were filled with 3 M KCl, and their tips were broken to obtain resistances of 1.0–1.5 M when measured in the bath solution. Oocytes were voltage clamped with a Dagan TEV-200 amplifier. pCLAMP software (version 9.2; Molecular Devices, Union City, CA) and a 1322A interface (Molecular Devices, Union City, CA) were used to generate voltage commands. Currents were digitally sampled at 5 kHz and filtered at 2 kHz. Leak and capacitive currents were not corrected. The oocyte was superfused with ND96 solution at a rate of 1 ml/min, and the membrane potential was held at −80 mV between test pulses, which were applied at a rate of 1–3 min−1 . Currents were measured before and 10 min after drug application to the bath. KT was purchased from Sigma and dissolved in DMSO to make a 10 mM stock solution. 2.3. Data analysis Clampfit9.2 software was employed for data collection and analysis. ORIGN 6.0 software (nonlinear curve fitting) was used to fit the data, calculate time constants and plot histograms. Dose–response curves were fit by the Hill equation:     Icon − IKT IC50 n , = Bmax 1 + Icon D

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where Icon is the control peak current, IKT is the peak current after KT inhibition, Bmax is the maximum block of Kv1.3, IC50 is the concentration of KT for half-maximum block, D is the concentration of KT, and n is the Hill coefficient. The activation curve was approximated by the normalized conductance–voltage relationship. The conductance (G) was calculated from the current (IK ) and associated voltage (V) by G=

IK , V + 80 mV

where −80 mV is the approximate reversal potential. The conductance was normalized to its maximum value under control conditions (Gmax ) and fit by the Boltzmann equation: 1 G =1− , Gmax 1 + exp((V − V1/2 )/k) where V1/2 is the potential at half-maximal conductance, i.e. midpoint potential, and k is a slope factor. The inactivation curve of Kv1.3 was fit by the Boltzmann equation: I Imax

=

1 , 1 + exp((V − V1/2 )/k)

where Imax is the maximum steady-state current, V the conditioning voltage, V1/2 the voltage at which half-maximal inactivation is obtained, and k is a slope factor. The activation of Kv1.3 current was well described by a monoexponential equation:    t I = I0 + A 1 − exp − , τact where τ act is the activation time constant. Inactivation of Kv1.3 current was also well fitted by a monoexponential equation:   t − t0 , I = I0 + A exp − τina where τ ina is the inactivation time constant. Data are presented as mean ± S.E.M. Student’s t-tests for paired and unpaired data were used to compare control conditions with intervention factors. A value of P < 0.05 was considered to be statistically significant. 3. Results 3.1. Kv1.3 channel currents are blocked by KT To investigate the effect of KT on cloned Kv1.3 channels we performed two-microelectrode voltage-clamp experiments on Xenopus oocytes heterologously expressing the Kv1.3 channel. After control measurements were obtained (Fig. 1A), the oocytes were superfused with KT (20 ␮M) for 10 min before measuring the Kv1.3 currents (Fig. 1B). Test pulses from −70 to +60 mV, in 10 mV increments (2-s duration) were applied from a holding potential of −80 mV and at a frequency of 0.1 Hz to measure current activation and to avoid the cumulative inactivation of the Kv1.3 channels [15,16]. Kv1.3 currents had an activation

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Fig. 1. Inhibition of Kv1.3 channel currents (IKv1.3) by ketanserin. (A) Control currents elicited during superfusion of Xenopus oocytes with ND96 solution. (B) Inhibition of IKv1.3 by 20 ␮M ketanserin. (C) Peak current as a function of the test pulse potential. Holding potential was −80 mV; test pulses ranged from −70 to +60 mV (2-s duration) in 10-mV increments.

threshold of about −40 mV. To differentiate Kv1.3 currents from other Kv1 currents, we used a voltage step from −100 to +50 mV, the same voltage protocol employed by Heinemann et al. for that purpose [17]. In four oocytes, we confirmed that the kinetics of the current was similar to that seen previously for Kv1.3 but not other Kv1 channels (see Fig. 1 of Ref. [17]). We also sequenced the gene and confirmed that Kv1.3 channels, and not some other Kv channels, were expressed. The peak current–voltage (I–V) relationships for control conditions, 20 and 50 ␮M KT are illustrated in Fig. 1C. After oocytes were exposed to 20 ␮M KT (Fig. 1B), the mean peak current at +50 mV was reduced by 54.1 ± 3.5% (n = 5; Fig. 1C). In control experiments, ritanserin, another 5-HT2A/2C receptor antagonist, had no effect on Kv1.3 channel currents, even at a high concentration (100 ␮M, n = 6). 3.2. Inhibition of Kv1.3 current by KT The concentration dependence of KT-induced inhibition was measured on peak Kv1.3 currents. From a holding potential of −80 mV, test pulses to +50 mV (2-s duration) and return pulses to −30 mV (1-s duration) were applied to elicit large, slowly decaying outward currents. Superfusion of oocytes with ND96 solution containing 10 and 20 ␮M KT, significantly inhibited the current (Figs. 1C and 2A). KT at concentrations of 1, 2, 10, 20, 50,100 and 300 ␮M reduced the peak currents by 12.4 ± 1.8, 18.8 ± 2.1, 43.1 ± 3.2, 54.1 ± 3.5, 66.7 ± 2.8, 78.9 ± 3.9 and 87.6 ± 3.5%, respectively (n = 5). The normalized dose–response curve was fit with the Hill equation and gave an IC50 of 14.7 ± 1.5 ␮M; the Hill coefficient was 0.721 (Fig. 2B). If KT’s concentration was above 300 ␮M, the drug could not be dissolved into the solution.

Superfusion of oocytes with ND96 solution containing 20 or 50 ␮M KT resulted in both a decrease in current amplitude and an apparent acceleration of current decay (Fig. 2A). The onset of block by KT was analyzed in further detail using the same protocol as in Fig. 2A. After a control period of 20 min, which demonstrated the stability of the experimental conditions, oocytes were exposed to 20 ␮M KT, and a steady-state block to 57% of the initial amplitude was observed after about 10 min. After a 15-min exposure, KT was washed out. Its inhibitory effect was partially reversible within 5 min and soon reached a steady-state (Fig. 2C). At a concentration of 100 ␮M, KT blocked Kv1.3 current within 5–6 min. Nevertheless, complete reversal of the blockade was not observed at this concentration, even 30 min after washout. At more depolarized potentials, the increased Kv1.3 current amplitudes indicated more open channels. To determine whether KT prevented closed or inactivated channels from opening or blocked opened channels, we investigated the extent of Kv1.3 current inhibition by 20 ␮M KT during 2-s voltage steps to +40, +50 and +60 mV. KT inhibition was not significantly different at these three potentials (50.2 ± 2.2, 51.5 ± 1.1, and 52.5 ± 2.5%, respectively) (Fig. 2D, n = 4). 3.3. Effect of KT on steady-state activation, inactivation and kinetic properties of IKv1.3 The steady-state activation values of IKv1.3 were approximated by the normalized conductance–voltage relationship and fit by a Boltzmann equation. The membrane was held at −90 mV, depolarized for 2 s to potentials from −60 to +50 mV in 10-mV increments, and repolarized to −30 mV for 1 s. The voltage steps were presented at a frequency of 0.1 Hz. The activation curves

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Fig. 2. Characterization of Kv1.3 current inhibition by ketanserin. (A) Concentration-dependent inhibition of IKv1.3 by ketanserin. (B) Concentration-response data fit by the Hill equation. The percent block of Kv1.3 peak current by ketanserin was defined as (Icontrol − Iketanserin )/Icontrol and plotted as a function of the logarithm of ketanserin. The IC50 was 14.7 ␮M (n = 5). (C) Time course of ketanserin’s effect on peak IKv1.3. An oocyte was superfused with ND96 solution (control) for 20 min, with 20 ␮M ketanserin, and again with ND96 solution (washout). (D) Voltage-independent block of IKv1.3 at +40, +50 and +60 mV by 20 ␮M ketanserin (P > 0.05, n = 4). Holding potential was −80 mV.

for control, 20 ␮M KT and 50 ␮M KT are illustrated in Fig. 3A. During control conditions, V1/2 was 17.5 ± 2.1 mV and k was 17.7 ± 1.8 mV. KT at 20 and 50 ␮M did not change the activation parameters significantly: for 20 ␮M KT, V1/2 was 23.7 ± 4.7 mV and k was 24.0 ± 3.4 mV; for 50 ␮M KT, V1/2 was 21.6 ± 2.8 mV and k was 19.1 ± 2.1 mV (P > 0.05, n = 5). Steady-state inactivation was generated by holding the membrane at potentials ranging from −100 to +20 mV for 2 s before applying a test pulse at +70 mV. The voltage steps were applied at a frequency of 0.1 Hz. The inactivation curves for control, 20 ␮M KT and 50 ␮M KT are illustrated in Fig. 3B. During control conditions, V1/2 was −55.4 ± 1.4 mV and k was −14.7 ± 1.3 mV. After addition of 20 and 50 ␮M KT, the inactivation curve was shifted significantly to more negative potentials: V1/2 for 20 ␮M was −62.1 ± 6.3 mV and k was −16.4 ± 1.6 mV, V1/2 for 50 ␮M was −67.7 ± 2.2 mV and k was −16.0 ± 1.6 mV (P < 0.05, n = 5). Concentrations as low as 2 ␮M also shifted the inactivation curve to the left; however, those results were not statistically significant (n = 6). The time constant for activation of IKv1.3 was obtained by fitting the activation phase with the monoexponential equation described in Section 2. At a test potential of +60 mV, the activation time constants were 10.8 ± 0.7, 6.5 ± 0.8 and 4.6 ± 1.6 ms under control conditions, 20 and 50 ␮M KT, respectively; at +70 mV, the time constants were 12.7 ± 1.2,

6.3 ± 0.8 and 5.0 ± 1.7 ms, respectively. The three histograms describing the time constants under control conditions, 20 and 50 ␮M KT were significantly different at both test potentials (Fig. 3C, P < 0.05, n = 5), suggesting that KT accelerates Kv1.3 channel activation significantly in a concentration-dependent fashion. Some previous investigations suggested that the time course of Ito decay could be described by a biexponential process or by a monoexponential process, depending on the experimental setting [3]. The decay phase of Kv1.3 current could be well fit by a monoexponential function in a previous study [18] and in our own experiments. The decay phase of control current traces and those recorded in the presence of 20 and 50 ␮M KT were fit by monoexponential functions to obtain the inactivation time constants. Similar to the voltageindependent inhibition of IKv1.3 by KT in Fig. 2D, the time constants of current decay were reduced significantly by 20 and 50 ␮M KT; however, this effect did not vary over the voltage range tested (data not shown). The inactivation time constants at +60 mV were 499.2 ± 94.2, 323.8 ± 15.6 and 224.2 ± 27.4 ms under control condition, 20 and 50 ␮M KT, respectively. Thus, the significant differences among the three groups (Fig. 3D, P < 0.05, n = 6) suggest that KT increases the rate of Kv1.3 channel inactivation in a concentration-dependent fashion.

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Fig. 3. Effect of ketanserin on the gating properties of IKv1.3. (A) Activation data were approximated by normalized conductances and fit by a Boltzmann equation (Section 2). (B) Steady-state inactivation data were obtained from the normalized IKv1.3 currents at +70 mV, which followed 2-s prepulses to potentials between −100 and +20 mV, and were fit by a Boltzmann equation (Section 2). (C) Activation of IKv1.3 at representative test potentials of +60 and +70 mV were fit by a monoexponential equation, and the time constants compared for controls and 20 and 50 ␮M ketanserin (# P < 0.05, ketanserine vs. control, n = 5; *P < 0.05, 50 ␮M vs. 20 ␮M ketanserin, n = 5). (D) Inactivation of IKv1.3 at a representative test potential of +60 mV was fit by a monoexponential equation, and the time constants compared for controls and 20 and 50 ␮M ketanserin (# P < 0.05, ketanserin vs. control, n = 5; *P < 0.05, 50 ␮M vs. 20 ␮M ketanserin, n = 5).

3.4. Concentration-dependent enhancement of Kv1.3 current by elevated extracellular [K+ ] The effect of high extracellular [K+ ] on Kv1.3 channels was investigated in Xenopus oocytes superfused with normal ND96 bath solution. Kv1.3 currents were elicited in the usual way. When the currents reached a steady-state, the control measurements were obtained. Then the oocytes were exposed to ND96 solutions containing different concentrations of KCl for 10 min or until a steady-state current was achieved. Solutions containing 5 mM and 20 mM KCl increased IKv1.3 in a concentrationdependent manner compared with the control, which contained 2 mM KCl. Peak current–voltage relationships (I–V curves) for control conditions and 5 and 20 mM KCl are shown in Fig. 4B. The peak current at +50 mV was increased 62.0 ± 6.5% by 5 mM KCl and 199.1 ± 24.6% by 20 mM KCl (n = 5; Fig. 4B). 4. Discussion 4.1. Ketanserin inhibits Kv1.3 currents expressed in Xenopus oocytes The Kv1.3 channel, a Shaker-like K+ channel that lacks N-type inactivation, is found in immune, neuronal and other

tissues [6–8,16]. The results of the present study show that KT inhibits IKv1.3 in a reversible, time and concentrationdependent, voltage-independent manner. This inhibitory effect was mediated through both a decrease in IKv1.3 amplitude and an apparent acceleration of IKv1.3 decay. Application of 5-HT causes a long-lasting inhibition of Kv1.3 current when 5-HT2 receptors are co-expressed with Kv1.3 channels in Xenopus oocytes [19], and we found no effect of retanserin, another 5-HT2 receptor antagonist, on Kv1.3 currents. Therefore the inhibitory effect of KT, a selective serotonin (5-HT) 2-receptor antagonist, appears to be a direct action on Kv1.3 channels, independent of 5-HT or a1-adrenergic receptors and consistent with the receptor-independent effect of KT on Ito and IKATP [3,5]. The present study demonstrated that KT inhibits Kv1.3 channel current (IC50 was 14.7 ␮M) with a potency slightly higher than the reported half-maximal inhibition concentration of KT for Ito and Isus in rat ventricular myocytes [3] (IC50 was 8.3 ␮M for Ito and 11.2 ␮M for Isus ), for Ito (IC50 was 2.25 ␮M) in rabbit ventricular myocytes [4] and for IKATP (IC50 was 9.36 ␮M) in mouse ventricular myocytes [5]. The discrepancy of IC50 among these three Kv1.x families perhaps is due to the fact that the oocyte membrane is less permeable to drugs than the myocardial membrane. Moreover, the IC50 of KT’s actions on those current was close to the effective concen-

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Fig. 4. Concentration-dependent enhancement of peak Kv1.3 current by elevated extracellular [K+ ]. (A) Comparison of control current traces (2 mM KCl) with those recorded during superfusion of oocytes with ND96 solution containing 5 and 20 mM KCl. (B) Corresponding peak current–voltage relationships.

tration (10 ␮M) in animal experiments [20], whereas the clinical plasma concentrations were nearly 1 ␮M [21]. Thus, within the range of concentrations observed experimentally and clinically, KT could block the Kv1.3 channels. However, the effect of KT on Kv1.3 channels expressed in mammalian cell lines should be investigated further. The block of IKv1.3 by KT was characterized by a decrease in peak current and an apparent increase in the rate of activation and inactivation. KT induced a hyperpolarizing (negative) shift in the steady-state inactivation curve but showed no significant effect on steady-state activation. The time needed for the current to reach its peak value after a depolarizing stimulus was prominently shorter in the presence of KT, reflecting a faster entry of Kv1.3 channels to their open (conducting) state or an increase in the transition of the open channels to their inactivated (nonconducting) state [22]. The acceleration of the inactivation rate in the presence of KT coincides with a decrease in peak current compared with the control. Therefore, the shorter activation time constants and the decrease in peak amplitude during exposure to KT might have been caused by the faster entry of activated channels to the inactivated state. Furthermore, the concentration-dependent increase in inactivation suggests that increasing the concentration of KT favors transitions from the open to the inactivated (nonconducting) state of the channels. The inhibitory action of KT was not significantly different at three depolarized potentials. This suggests voltage-independent block and indicates that with more open channels there is more channel inhibition. These time-dependent and voltage-independent inhibitory characteristics of KT stand in opposition with the time-independent and voltage-dependent inhibition of Ito by 4-aminopyridine, which has been reported to bind strongly to closed channels with their inactivation gate open [23]. Thus the block of IKv1.3 by KT and the absence of a significant change in the IKv1.3 activation curve by KT could be attributed to preferential binding of KT to the open state of the channel or “open channel block”. Nevertheless, to explain the negative shift of the inactivation curve, one cannot exclude the possibility that KT could bind to the resting state of the channel and

decrease the channel’s availability at the same negative holding potential. 4.2. High extracellular [K+ ] increased IKv1.3 peak amplitude in concentration mode Pardo et al. reported that the observed [K]-dependent increase in macroscopic RCK4 current is more likely due to an increase in the number of channels available for activation, rather than the changes of single-channel conductance or mean open time of channels [24]. Levite et al. suggested that elevated extracellular [K+ ]o, in the absence of “classical” immunological stimulatory signals, is, itself, a sufficient stimulus to activate T-cell ␤1 integrin moieties, and to induce integrin-mediated adhesion and migration. Thus, Kv1.3 channels could be a target of immunomodulatory therapy. These actions might be explained by high [K+ ]o increasing the Kv1.3 channel current, because an inhibitor of the Kv1.3 channel prevented those functions. However, those investigators thought high [K+ ]o depolarized the membrane, thereby opening Kv1.3 channels [12]. Our experiments show that high [K+ ]o can directly increase the conductance of Kv1.3 channels. Based on evidence from the literature, the enhancement of Kv1.3 channel current by high extracellular K can be explained by one or more of the following factors: (i) slowing the time course of C-type inactivation [25]; (ii) increasing the number of open channels [25]; and (iii) accelerating the recovery from C-type inactivation [16]. 4.3. Clinical implications Kv1.3 channels are expressed in T and B lymphocyte in a distinct pattern that depends on the state of lymphocyte activation and differentiation. The channel phenotype changes during the progression from the resting to the activated cell state and from na¨ıve to effector memory cells, affording promise for specific immunomodulatory actions of Kv1.3 channel blockers. KT is a selective serotonin (5-HT) 2-receptor antagonist and has been evaluated for the treatment of hypertension, especially the treatment of severe hypertension in pregnancy, and certain

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peripheral vascular diseases [1]. Moreover, KT had been suggested to diminish arteriosclerotic development by its effect on serotonin-induced platelet aggregation and thrombus formation [2]. Our results suggest that KT inhibits Kv1.3 channels in a concentration-dependent manner and is effective in the animal experimental application range, providing a rationale for the potential therapeutic use of KT in immunological disorders. Furthermore, accumulating evidence supports an autoimmune mechanism as one of the prime pathogenic processes involved in the development of atherosclerosis. Autoantibody levels are significantly increased in patients with atherosclerosis, and T lymphocytes specifically responding to these autoantigens have been demonstrated within atherosclerotic plaques [26]. While activation, proliferation and infiltration into the atherosclerotic lesion regions by T and B lymphocytes, secretion of cytokines was involved in the function of Kv1.3 channels. So the effect of KT in relieving arteriosclerotic development might be partially due to its immunomodulatory action through the blockade of Kv1.3 channels. 5. Conclusion Our results suggest that ketanserine, a 5-HT2 receptor antagonist, acts directly on the open state of the Kv1.3 channel, reducing current flow through the channel while accelerating channel activation and inactivation. In contrast, augmentation of extracellular [K] enhances current flow through the channel by increasing the channel’s conductance. Because Kv1.3 channels are expressed in T and B lymphocytes in a distinct pattern that depends on the state of lymphocyte activation and differentiation, we speculate that ketanserine might be useful as an immunomodulator in the therapy of cardiovascular diseases, through its inhibitory effects on the channels. Acknowledgments This work was supported by the National Natural Science Foundation of China (No. 30470711). We thank Dr. Maria L. Garcia (Merck & Co., Inc., USA) for providing the cDNA of the Kv1.3 channel, Dr. Richard D. Nathan for editing the revised manuscript and Heping Guo for technical and equipment support. References [1] van Schie DL, de Jeu RM, Steyn DW, Odendaal HJ, van Geijn HP. The optimal dosage of ketanserin for patients with severe hypertension in pregnancy. Eur J Obstet Gynecol Reprod Biol 2002;102:161–6. [2] Geerling RA, de Bruin RW, Scheringa M, Bonthuis F, IJzermans JN, Marquet RL. Ketanserin reduces graft arteriosclerosis after allogeneic aorta transplantation in rats. J Cardiovasc Pharmacol 1996;27:307–11. [3] Zhang ZH, Boutjdir M, el-Sherif N. Ketanserin inhibits depolarizationactivated outward potassium current in rat ventricular myocytes. Circ Res 1994;75:711–21. [4] Le Grand B, Marty A, Colpaert FC, John GW. Ketanserin inhibits the transient outward current in rabbit ventricular myocytes. J Cardiovasc Pharmacol 1995;25:341–4.

[5] Ju JM, Hwang JH, Piao LH, Park HW, Park JS, Shin DH, et al. Ketanserin, a 5-HT2 antagonist, directly inhibits the ATP-sensitive potassium channel in mouse ventricular myocytes. J Cardiovasc Pharmacol 2006;47:96– 102. [6] Panyi G, Vamosi G, Bodnar A, Gaspar R, Damjanovich S. Looking through ion channels: recharged concepts in T-cell signaling. Trends Immunol 2004;25:565–9. [7] Mourre C, Chernova MN, Martin-Eauclaire MF, Bessone R, Jacquet G, Gola M, et al. Distribution in rat brain of binding sites of kaliotoxin, a blocker of Kv1.1 and Kv1.3 alpha-subunits. J Pharmacol Exp Ther 1999;291:943–52. [8] Xu J, Wang P, Li Y, Li G, Kaczmarek LK, Wu Y, et al. The voltage-gated potassium channel Kv1.3 regulates peripheral insulin sensitivity. Proc Natl Acad Sci USA 2004;101:3112–7. [9] George Chandy K, Wulff H, Beeton C, Pennington M, Gutman GA, Cahalan MD. K+ channels as targets for specific immunomodulation. Trends Pharmacol Sci 2004;25:280–9. [10] Wulff H, Knaus HG, Pennington M, Chandy KG. K+ channel expression during B cell differentiation: implications for immunomodulation and autoimmunity. J Immunol 2004;173:776–86. [11] Krasznai Z. Ion channels in T cells: from molecular pharmacology to therapy. Arch Immunol Ther Exp (Warsz) 2005;53:127–35. [12] Levite M, Cahalon L, Peretz A, Hershkoviz R, Sobko A, Ariel A, et al. Extracellular K(+) and opening of voltage-gated potassium channels activate T cell integrin function: physical and functional association between Kv1.3 channels and beta1 integrins. J Exp Med 2000;191: 1167–76. [13] Wagner CA, Friedrich B, Setiawan I, Lang F, Broer S. The use of Xenopus laevis oocytes for the functional characterization of heterologously expressed membrane proteins. Cell Physiol Biochem 2000;10:1–12. [14] Sigel E, Minier F. The Xenopus oocyte: system for the study of functional expression and modulation of proteins. Mol Nutr Food Res 2005;49:228–34. [15] Marom S, Levitan IB. State-dependent inactivation of the Kv3 potassium channel. Biophys J 1994;67:579–89. [16] Levy DI, Deutsch C. A voltage-dependent role for K+ in recovery from C-type inactivation. Biophys J 1996;71:3157–66. [17] Heinemann SH, Rettig J, Graack HR, Pongs O. Functional characterization of Kv channel beta-subunits from rat brain. J Physiol 1996;493(Pt 3):625–33. [18] Hahn SJ, Wang LY, Kaczmarek LK. Inhibition by nystatin of Kv1.3 channels expressed in Chinese hamster ovary cells. Neuropharmacology 1996;35:895–901. [19] Attali B, Honore E, Lesage F, Lazdunski M, Barhanin J. Regulation of a major cloned voltage-gated K+ channel from human T lymphocytes. FEBS Lett 1992;303:229–32. [20] Blomeley C, Bracci E. Excitatory effects of serotonin on rat striatal cholinergic interneurones. J Physiol 2005;569:715–21. [21] Hanff LM, Visser W, Steegers EA, Vulto AG. Population pharmacokinetics of ketanserin in pre-eclamptic patients and its association with antihypertensive response. Fundam Clin Pharmacol 2005;19:585–90. [22] Poulopoulou C, Markakis I, Davaki P, Nikolaou C, Poulopoulos A, Raptis E, et al. Modulation of voltage-gated potassium channels in human T lymphocytes by extracellular glutamate. Mol Pharmacol 2005;67: 856–67. [23] Castle NA, Slawsky MT. Characterization of 4-aminopyridine block of the transient outward K+ current in adult rat ventricular myocytes. J Pharmacol Exp Ther 1993;265:1450–9. [24] Pardo LA, Heinemann SH, Terlau H, Ludewig U, Lorra C, Pongs O, et al. Extracellular K+ specifically modulates a rat brain K+ channel. Proc Natl Acad Sci USA 1992;89:2466–70. [25] Lopez-Barneo J, Hoshi T, Heinemann SH, Aldrich RW. Effects of external cations and mutations in the pore region on C-type inactivation of Shaker potassium channels. Receptors Channels 1993;1:61–71. [26] Mandal K, Jahangiri M, Xu Q. Autoimmune mechanisms of atherosclerosis. Handb Exp Pharmacol 2005:723–43.