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Characterization and molecular basis for the block of Kv1.3 channels induced by carvedilol in HEK293 cells
European Journal of Pharmacology 834 (2018) 206–212
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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Molecular and cellular pharmacology
Characterization and molecular basis for the block of Kv1.3 channels induced by carvedilol in HEK293 cells Jin-Feng Yanga, Neng Chenga, Sheng Rena, Xiang-Ming Liub, Xian-Tao Lia, a b
T
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College of Life Science, South-Central University for Nationalities, Wuhan 430074, China GongQing Institute of Science and Technology, Gongqing City 332020, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Carvedilol Kv1.3 channels Hyperpolarizing shift Closed- and open-channel block, molecular determinants
Carvedilol is a non-selective β-adrenoreceptor antagonist and exhibits a wide range of biological activities. The voltage-gated K+ (Kv) channel is one of the target ion channels of this compound. The rapidly activating Kv1.3 channel is expressed in several different tissues and plays an important role in the regulation of physiological functions, including cell proliferation and apoptosis. However, little is known about the possible action of carvedilol on Kv1.3 currents. Using the whole-cell configuration of the patch-clamp technique, we have revealed that exposure to carvedilol produced a concentration-dependent blocking of Kv1.3 channels heterologously expressed in HEK293 cells, with an IC50 value of 9.7 μM. This chemical decelerated the deactivation tail current of Kv1.3 currents, resulting in a tail crossover phenomenon. In addition, carvedilol generated a markedly hyperpolarizing shift (20 mV) of the inactivation curve, but failed to affect the activation curve. Mutagenesis experiments of Kv1.3 channels identified G427 and H451, two related sites of TEA block, as important residues for carvedilol-mediated blocking. The present results suggest that carvedilol acts directly on Kv1.3 currents by inducing closed- and open-channel block and helps to elucidate the mechanisms of action of this compound on Kv channels.
1. Introduction Functional Kv1.3 is composed of a homotetramer of pore forming α subunits, each of which comprises six transmembrane helices, arranged around a central K+-selective pore (Wulff et al., 2009). Expression of Kv1.3 channels is detected in distinct cells, including T and B lymphocytes (Wulff et al., 2004), osteoclasts (Arkett et al., 1994), neurons (Tubert et al., 2016), and smooth muscle cells (Perez-Garcia et al., 2018). Accumulated evidence indicates that Kv1.3 channels exhibit multiple regulatory actions in setting the resting membrane potential, apoptosis, cell volume and proliferation (Cahalan and Chandy, 2009; Deutsch and Chen, 1993; Gulbins et al., 2010; Liu et al., 2002). As a predominant Kv channel in T lymphocyte, Kv1.3 channels could be potently blocked by immunosuppressants, and accordingly its inhibition leads to a reduction of the proliferation in effector memory T (TEM) cells (Wulff et al., 2001). Thus, Kv1.3 channels are taken as a substantial therapeutic target for the treatment of autoimmune diseases, such as multiple sclerosis (Rus et al., 2005) and rheumatoid arthritis (Beeton et al., 2006). This channel is also detected in insulin-sensitive tissues such as adipose tissue, liver and skeletal muscle and therefore is considered as a potential drug target in diabetic therapy (Choi and
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Hahn, 2010). In addition, Kv1.3 channels have been considered a potentially new molecular target in both the diagnostics and therapy of some cancer diseases (Teisseyre et al., 2015). The inhibition of Kv1.3 channel activity has a beneficial action on above diseases. Hence, extensive works have been conducted to explore inhibitors of Kv1.3 channels as a treatment of related diseases. Concurrently, it is gradually revealed that Kv1.3 channels are also suppressed by chemicals that are originally assumed with other specific effects, such as verapamil (Kuras and Grissmer, 2009) and clofazimine (Faouzi et al., 2015). Nevertheless, only limited data are available for the molecular explanation of the interaction between drugs and Kv1.3 channels. To avoid unwanted side actions and reveal new pharmacological effects, it is worth exploring more about the mechanism of effects of drugs on Kv1.3 channels. Carvedilol is an α- and β-adrenoceptor antagonist that has been in clinical practice in patients with hypertension (McTavish et al., 1993) or myocardial infarction (Ruffolo and Feuerstein, 1997). Besides blocking adrenergic receptors, this drug also has multiple biological actions, such as scavenging free radicals (Yue et al., 1992), as well as protecting against apoptosis, inflammation and mitochondrial damage (Abreu et al., 2000; Savitz et al., 2000). A growing body of evidence
Corresponding author. E-mail address: [email protected] (X.-T. Li).
https://doi.org/10.1016/j.ejphar.2018.07.025 Received 25 May 2018; Received in revised form 4 July 2018; Accepted 13 July 2018 0014-2999/ © 2018 Elsevier B.V. All rights reserved.
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Fig. 1. Blocking of Kv1.3 currents in HEK293 cells by carvedilol. (A) Representative Kv1.3 current traces were shown under control conditions and treatment with 10 and 50 μM carvedilol. Currents in HEK293 cells were routinely evoked in response to 1 s voltage pulses from a holding potential of − 80 mV, to test potentials of + 60 mV. (B) Plot of current-voltage (I - V) relationships of Kv1.3 currents before and after exposure to 10 μM and 50 μM carvedilol (n = 10). (C) The relative currents in the presence of different concentrations of carvedilol have been obtained, and the solid line was the concentration-response relations of carvedilol action on Kv1.3 currents, which was generated by fitting data points of relative currents using Hill equation (n = 6). (D) Normalized block shown as relative current (Icarvedilol/I control) with 10 μM and 50 μM carvedilol was plotted against the varying voltages (n = 6).
experiments.
suggests that Kv channel is one of its targets in distinct tissues. For example, several components of cardiac outward K+ currents, including the slowly and rapidly activating delayed rectifier currents (IKr and IKs) as well as the ultrarapid activating delayed currents (IKur) (Deng et al., 2007), are suppressed by carvedilol. Treatment with carvedilol also inhibits the transient outward K+ currents (Ito), which play important roles in the phase 1 repolarization of cardiac action potential (Liu et al., 1993). Nevertheless, there is not any report for the interaction between carvedilol and Kv1.3 channels. In the current study, we examine the inhibitory effect of carvedilol on Kv1.3 channels and explore the molecular basis of this action.
2.2. Mutagenesis and transfection The vectors of pIRES2-EGFP containing the sequence of the human Kv1.3 channels were gifts from Dr. Zhi-Jian Cao (Wuhan University, Wuhan). Mutations to Kv1.3 channels were conducted with the QuikChange Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA). Predicted mutations were clarified by sequence analysis. HEK293 cells transiently expressing Kv1.3 channels were produced as described previously (Chen et al., 2012). Briefly, plasmids with Kv1.3 channels were transfected into HEK293 cells using Lipofectamine 2000 (Life Technologies, Bethesda, MD). At 4 h after treatment, the transfection reagent was replaced with regular culture medium. The next day, cells were allowed to settle on the bottom of recording chamber mounted on an inverted microscope (IX-73, Olympus, Osaka, Japan) for electrical measurements.
2. Materials and methods 2.1. Cell culture The human embryonic kidney cell line, HEK293, was maintained in modified Dulbecco's medium (MEM) with high glucose containing 10% fetal bovine serum in a humidified, 5% CO2 incubator at 37 °C. The cultures were passed every 2–3 days after brief trypsin treatment. Cells were plated onto glass coverslips 1 day prior to use for patch-clamp
2.3. Patch-clamp recording The whole-cell recording mode of the patch-clamp technique was 207
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(Fig. 2A). Under control conditions, the activation time constant (τact) obtained after exponential fitting was 1.9 ± 0.3 ms at −10 mV, and superfusion of 50 μM carvedilol significantly increased the value of τact to 7.1 ± 0.8 ms (n = 6, P < 0.05). This effect of 50 μM carvedilol on τact was obviously voltage-dependent and released at higher potentials (Fig. 2A). The conductance-voltage (G-V) curves of activation were fitted using the Boltzmann equation and shown in Fig. 2B. The value of the half maximal conductance voltage (V1/2) was −16.4 ± 3.5 mV (n = 5) in the absence of carvedilol and exposure to 10 and 50 μM did not affect the G-V curves, with V1/2 values of −15.5 ± 4.0 and −14.6 ± 3.6 mV, respectively (n = 5). Further measurement was performed to study the action of carvedilol on deactivation kinetics of Kv1.3 channels. Cells expressing Kv1.3 channels were repolarized to various voltages ranging from −100 and −40 mV in 10 mV increments following a depolarization pulse to the potential of + 40 mV at a holding potential of −80 mV. Tail currents in the absence and presence of carvedilol were fitted by using exponential function to obtain the deactivation time constant (τdeact). Obviously, the values of τdeact increased in a voltage-dependent way from −80 to −40 mV despite the presence of carvedilol (Fig. 2D). Application of 50 but not 10 μM carvedilol resulted in a potent enhance of τdeact at potentials more positive than −90 mV, showing a deceleration for Kv1.3 current deactivation. The value of τdeact at −40 mV was 25.6 ± 0.9 ms in control and increased to 45.1 ± 6.6 ms after exposure to 50 μM carvedilol (n = 6, P < 0.05), thereby a classical “crossover” deactivation tail currents on repolarization was evident (Fig. 2C). Consistent with the property of open channel block (Armstrong, 1971), carvedilol dissociates from the state of channel deactivation rather slowly compared with that in control, and accordingly channels could only close after the compound dissociated from its binding site.
undertaken throughout all experiments. Cells were superfused with the bath solution at room temperature containing (in mM): 75 Na-gluconate, 70 NaCl, 5 KCl, 5 HEPES and 5 glucose, with pH adjusted to 7.4 using NaOH. Patch pipets were fabricated from borosilicate glass and had resistances between 2.0 and 3.0 MΩ when filled with an artificial internal solution, containing (in mM): 150 KCl, 5 HEPES, 5 EGTA, 5 Glucose, 5 Na2ATP, with pH adjusted to 7.3 using KOH. An Axopatch 200B amplifier was employed to record Kv1.3 currents. The pCLAMP10 software (Molecular Devices, Sunnyvale, CA) was used to produce voltage clamp protocols, acquire data, and analyze current signals. Data were low-pass filtered at 5 kHz and sampled at 10 kHz. 2.4. Data analysis Data are presented as means ± S.E.M. Concentration-dependent effects were quantified by fitting the Hill equation: y = I / (1 + (IC50/x) h ), where IC50 is the drug concentration that produces half maximal inhibition, and h is the slope of the curve. Both conductance-voltage (GV) curves of activation and inactivation curves were fitted using Boltzmann equation: y = 1 ⁄ [1 + exp (V1⁄2 –V) ⁄ k], where V1⁄2 is the half-maximal potential, V is the conditioning potential, and k represents the slope factor. The time constant was obtained after fitting related current traces using the exponential equation: f (t) = A exp(- t ⁄τ), where τ is the time constant, and A is the corresponding current amplitude. Statistical significance was analyzed using a Student's t-test or one-way analysis of variance (ANOVA). A value of P < 0.05 was accepted statistically significant. 3. Results 3.1. Carvedilol directly inhibited Kv1.3 currents in a voltage-independent way
3.3. Effects of carvedilol on the inactivation Whole-cell recording technique was routinely conducted to examine effects of carvedilol on Kv1.3 channels heterologously expressed in HEK293 cells. Macroscopic Kv1.3 currents were activated by 1 s depolarizing voltage steps to potentials from −80 mV to + 60 mV at a holding potential of −80 mV applied every 45 s. Typical examples of current traces illustrated in Fig. 1A obviously implied that exposure to both 10 and 50 μM carvedilol led to a marked reduction in amplitudes of Kv1.3 currents compared with the control. Current-voltage (I - V) relationships before and after treatment with 10 and 50 μM carvedilol were assembled from raw data, and showed that Kv1.3 currents were blocked at all potentials in which Kv1.3 channels were activated (Fig. 1B, n = 10). Carvedilol-induced block on Kv1.3 currents is concentration-dependent, and quantified by concentration-response curves obtained after fitting with a Hill equation, with IC50 value of 9.7 ± 0.8 μM and a Hill coefficient of 1.4 ± 0.2 (Fig. 1C, n = 6). Previous studies indicated that carvedilol could exert an inhibitory action on different Kv channels. We also tested the action of carvedilol on rapidly activating delayed rectifier Kv1.5 channels that contribute to repolarization of action potentials of human atrial myocytes (Fedida et al., 1993). The concentration response curve for block of Kv1.5 currents by carvedilol is shown in Supplementary Figure, with IC50 value of 3.9 ± 0.4 μM and a Hill coefficient of 1.6 ± 0.2 (n = 5). To assess the voltage-dependent block of carvedilol, the relative current (Icarvedilol / Icontrol) was plotted as a function of membrane potential. As shown in Fig. 1D, the decrease of Kv1.3 currents was not voltage-dependent through the voltage over a range from −10 to + 40 mV.
We tested whether the steady-state inactivation curves of Kv1.3 channels were shifted by application of 10 and 50 μM carvedilol. Steady-state inactivation was determined by measuring the peak current at + 40 mV, following the 60 s conditioning prepulses from a holding potential of −80 mV to potentials between −80 mV and + 40 mV in increments of 20 mV. The current amplitude during each test pulse was normalized to maximal amplitude (I ⁄ Imax) and plotted against voltages of conditioning prepulses. These data were well fitted by the Boltzmann equation and shown in Fig. 3A. The values of V1/2 and k at control were −19.8 ± 0.4 mV and 9.8 ± 0.4 mV (n = 6), respectively. Exposure to 50 but not 10 μM carvedilol induced an approximately 20 mV hyperpolarizing shift in the inactivation curve, with values of V1/2 and k being −39.3 ± 0.8 mV and 9.0 ± 0.6 mV (n = 6), respectively. 3.4. Molecular determinants of Kv1.3 channel block by carvedilol The residue at the position 449 in the Shaker channel is located at the external mouth of the pore and participates in the modulation of TEA sensitivity and P/C type inactivation rate in Kv channels (LopezBarneo et al., 1993; Spencer et al., 1993; Zsiros et al., 2009). The amino acid equivalent to Shaker 449 in Kv channels (H451 in hKv1.3) has been identified as a common binding site for several drugs (Barana et al., 2010; Li et al., 2015; Lin et al., 2001). Analysis of a sequence alignment suggested that another residue equivalent to site responsible for TEA interaction in Kv1.5 channels (Abid et al., 2015) was a glycine at Kv1.3 position 427. Therefore, mutagenesis of above residues of TEA block (H451 and G427) were conducted and effects of carvedilol on these mutations were assessed to determine the molecular binding sites. Detectable currents were not evident for two of five mutations (H451D and G427D), and significant elimination of carvedilol's action on three other mutations (H451Y, H451V and G427K), but to a different degree,
3.2. Actions of carvedilol on kinetics of activation and deactivation Kv1.3 channels were activated by depolarizing pulses to potentials ranging from −80 to + 40 mV with increment of 10 mV at a holding potential of −80 mV. Application of 50 but not 10 μM carvedilol mediated a deceleration of activation process for Kv1.3 channels 208
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Fig. 2. Actions of carvedilol on kinetics of activation and deactivation. Plot of the activation time constants as a function of depolarizing pulses from − 10 to + 40 mV in the absence and presence of 10 and 50 μM carvedilol (n = 6). (B) Normalized conductance vs. test voltage (G-V) relationships for control as well as 10 and 50 μM carvedilol (n = 5). (C) Representative tail currents of deactivation were shown without and with 50 μM carvedilol. Crossover of these tail currents was illustrated at the bottom panel. (D) Voltage dependence of τdeact under control conditions and during application of 10 and 50 μM carvedilol (n = 6).
(n = 5, P > 0.05), with V1/2 values of 17.6 ± 3.0 mV and −16.4 ± 3.5 mV, respectively. The G-V curves of H451Y with and without carvedilol were shown in Fig. 4A. After addition of 50 but not 10 μM carvedilol, however, the V1/2 value of H451Y was weakly but significantly shifted right to 12.5 ± 5.8 mV (n = 5, P < 0.05), indicating that mutant channel activated at more positive potentials. The mutation of H451Y also changed the effects of carvedilol on deactivation kinetics of channels. Compared with wild type channels, exposure to 50 μM carvedilol potently increased the τdeact value of H451Y to 74.2 ± 2.9 ms at the potential of −40 mV (Fig. 4B; n = 5, P < 0.05). For steady-state inactivation of H451Y, the V1/2 was – 8.6 ± 14.2 mV and shifted left to – 26.1 ± 18.6 mV (Fig. 4C; n = 5, P < 0.05). These data suggest that the alteration of mutation's response to carvedilol may possibly contribute to the attenuation of this drug's action.
was obvious. Previous studies revealed that the residue, near the external TEA binding site and selectivity filter, was also involved in the modulation of inactivation and TEA block (Cha and Bezanilla, 1997; Kiss et al., 1999). In this study, mutation of valine 453 that is located in the outer vestibule near the TEA binding site (H451) fail to alter the carvedilol's action (n = 6). The summary of action of carvedilol on wild type and mutant Kv1.3 currents was shown as a bar graph in Fig. 3B. Exposure to 10 μM carvedilol resulted in the inhibition of 10.4 ± 4.8, 26.4 ± 2.1 and 33.4 ± 3.0% for H451Y, H451V and G427K, respectively (n = 6). Compared with H451V and G427K, the H451Y mutation released the carvedilol's action to a largest degree and following experiments were performed on this mutation to explore the possible mechanism of interaction.
3.5. Effects of carvedilol on activation, deactivation and inactivation of H451Y mutation
4. Discussion
In the absence of carvedilol, no obvious alteration of activation was detectable between H451Y mutation and wild type Kv1.3 channels
Although previous studies found that carvedilol, a non-selective βadrenoreceptor antagonist, can inhibited different Kv currents in native 209
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Fig. 3. Actions of carvedilol on inactivation curves and mutant Kv1.3 channels. (A) The inactivation of Kv1.3 channels was examined during depolarizing voltage steps to + 40 mV after 60 s conditioning prepulses to potentials ranging from –80 and + 40 mV. The peak currents during each test pulse were normalized to maximal currents (I/Imax) and plotted against conditioning voltages. The solid lines were inactivation curves, which were generated by fitting data points using a Boltzmann equation (n = 6). (B) The bar graph is the summary of carvedilol block on wild type and mutant Kv1.3 channels (n = 6). Compared with wild type currents, the potency of carvedilol block was substantially released in the G427Y, H451V and H451Y Kv1.3 currents. *P < 0.05 compared with the wild type group.
cells (Cheng et al., 1999; Deng et al., 2007) and heterologous expression systems (Jeong et al., 2012; Karle et al., 2001), it remained obscure whether there exists interaction between carvedilol and voltage-gated Kv1.3 channels, which are widely expressed in many tissues (Arkett et al., 1994; Tubert et al., 2016; Wulff et al., 2004). In the present study here, collected data demonstrate that application of carvedilol can directly inhibit the macroscopic Kv1.3 currents in a concentration-dependent manner and modulates its kinetic properties in HEK293 cells. Our experiments of mutagenesis also identify two related sites of TEA block as binding sites of carvedilol to Kv1.3 channels. Several lines of evidence indicate that carvedilol preferentially binds to the open state of Kv1.3 channel. First, inhibitory actions of carvedilol were detectable after channel activation. Second, treatment with carvedilol potently slows the deactivation time courses of tail currents and, as a result, crossover phenomena of tail currents was evident in the absence and presence of the drug. This observation shows that carvedilol needs to dissociate from its binding site before channels can close, and accordingly the time course of drug dissociation from deactivating channels is much slower compared with control channels. In accordance with both open- and close-channel block mechanism reported by investigators (Frolov et al., 2010), however, carvedilol also
Fig. 4. The response of H451Y mutations to carvedilol. (A) Normalized conductance vs. test voltage (G-V) relationships for the H451Y mutant channel at various concentrations of carvedilol (n = 5). (B) Effects of 10 and 50 μM carvedilol on τdeact of H451Y mutations. (C) Inactivation curves of H451Y mutations under control conditions and in the presence of 10 and 50 μM carvedilol were obtained by using a two-pulse protocol described above (n = 5).
exerts an inhibitory action on closed state of Kv1.3 channel. For instance, a deceleration of activation time is obviously observed after addition of carvedilol, indicating the slowed activation by the closedchannel blocker (Zhang et al., 1997). Our study reveals that two crucial positions equivalent to the presumed TEA binding site in Shaker (T449), G427 in the S5-P linker and 210
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H451 in the turret region, are responsible for the sensitivity of carvedilol to Kv1.3 channels. These residues identified as binding sites for carvedilol block are highly conserved in other Kv channels, and hence it is reasonablely presumed that inhibitory effects of this compound also occur in HERG (Kawakami et al., 2006) and Kv1.5 channels (Jeong et al., 2012). The value of IC50 for carvedilol-mediated inhibition of Kv1.3 channels is 9.7 μM and is over the range of therapeutic concentrations, from 0.1 to 0.6 μM (Morgan et al., 1990). Similarly, IC50 values of carvedilol-mediated inhibitions for transient outward and slowly activating delayed rectifier K+ currents in rabbit ventricular myocytes are higher than therapeutic plasma levels (Cheng et al., 1999). Note that carvedilol is a highly lipid soluble compound that would accumulate in tissue at concentrations higher than those observed in plasma (Neugebauer et al., 1987; Tomlinson et al., 1992). Thus, it is possible that local levels of carvedilol can be high enough to block Kv1.3 channels in clinical treatment. Additionally, Kv1.3 has been shown to coassemble with Kv1.2 and Kv1.4 in neural tissues (Coleman et al., 1999; Koch et al., 1997), and form heteromultimeric complexs with Kv1.5 in macrophages (Vicente et al., 2006). Hence, it is possible that the assembly of Kv1.3 with other Kv channel subunits could result in the varying sensitivity to carvedilol. Besides taking as an antihypertensive agent because of its β-adrenergic blocking and vasodilating action (Dunn et al., 1997), carvedilol exhibits a wide range of biological activities mentioned in the introduction. Based on the current study, it is reasonablely speculated that blockage of Kv1.3 channels is involved in the functional modulation in response to treatment with carvedilol. In summary, the present study demonstrates that the carvedilol can substantially inhibit Kv1.3 channels expressed in HEK293 cells. Two related residues of TEA block are identified as binding sites of carvedilol to this channel, and both open- and closed-channel block are involved in this process. The blocking actions of carvedilol on Kv1.3 channels may provide an alternative explanation for its effects in different tissues.
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Acknowledgments This work was supported by the grants from Basic research project of SCUN [YCZY12019]. Conflicts of interest The authors state no conflict of interest. Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at doi:10.1016/j.ejphar.2018.07.025. References Abid, S., Ali, S., Baig, M.A., Waheed, A.A., 2015. Is it time to replace propranolol with carvedilol for portal hypertension? World J. Gastrointest. Endosc. 7, 532–539. Abreu, R.M., Santos, D.J., Moreno, A.J., 2000. Effects of carvedilol and its analog BM910228 on mitochondrial function and oxidative stress. J. Pharmacol. Exp. Ther. 295, 1022–1030. Arkett, S.A., Dixon, J., Yang, J.N., Sakai, D.D., Minkin, C., Sims, S.M., 1994. Mammalian osteoclasts express a transient potassium channel with properties of Kv1.3. Recept. Channels 2, 281–293. Armstrong, C.M., 1971. Interaction of tetraethylammonium ion derivatives with the potassium channels of giant axons. J. Gen. Physiol. 58, 413–437. Barana, A., Amoros, I., Caballero, R., Gomez, R., Osuna, L., Lillo, M.P., Blazquez, C., Guzman, M., Delpon, E., Tamargo, J., 2010. Endocannabinoids and cannabinoid analogues block cardiac hKv1.5 channels in a cannabinoid receptor-independent manner. Cardiovasc. Res. 85, 56–67. Beeton, C., Wulff, H., Standifer, N.E., Azam, P., Mullen, K.M., Pennington, M.W., KolskiAndreaco, A., Wei, E., Grino, A., Counts, D.R., Wang, P.H., LeeHealey, C.J., B, S.A., Sankaranarayanan, A., Homerick, D., Roeck, W.W., Tehranzadeh, J., Stanhope, K.L., Zimin, P., Havel, P.J., Griffey, S., Knaus, H.G., Nepom, G.T., Gutman, G.A., Calabresi, P.A., Chandy, K.G., 2006. Kv1.3 channels are a therapeutic target for T cell-mediated
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of Kv1.5 and Kv1.3 contributes to the major voltage-dependent K+ channel in macrophages. J. Biol. Chem. 281, 37675–37685. Wulff, H., Castle, N.A., Pardo, L.A., 2009. Voltage-gated potassium channels as therapeutic targets. Nat. Rev. Drug Discov. 8, 982–1001. Wulff, H., Gutman, G.A., Cahalan, M.D., Chandy, K.G., 2001. Delineation of the clotrimazole/TRAM-34 binding site on the intermediate conductance calcium-activated potassium channel, IKCa1. J. Biol. Chem. 276, 32040–32045. Wulff, H., Knaus, H.G., Pennington, M., Chandy, K.G., 2004. K+ channel expression during B cell differentiation: implications for immunomodulation and autoimmunity. J. Immunol. 173, 776–786. Yue, T.L., Cheng, H.Y., Lysko, P.G., McKenna, P.J., Feuerstein, R., Gu, J.L., Lysko, K.A., Davis, L.L., Feuerstein, G., 1992. Carvedilol, a new vasodilator and beta adrenoceptor antagonist, is an antioxidant and free radical scavenger. J. Pharmacol. Exp. Ther. 263, 92–98. Zhang, X., Anderson, J.W., Fedida, D., 1997. Characterization of nifedipine block of the human heart delayed rectifier, hKv1.5. J. Pharmacol. Exp. Ther. 281, 1247–1256. Zsiros, E., Kis-Toth, K., Hajdu, P., Gaspar, R., Bielanska, J., Felipe, A., Rajnavolgyi, E., Panyi, G., 2009. Developmental switch of the expression of ion channels in human dendritic cells. J. Immunol. 183, 4483–4492.
highly expressed on inflammatory infiltrates in multiple sclerosis brain. Proc. Natl. Acad. Sci. USA 102, 11094–11099. Savitz, S.I., Erhardt, J.A., Anthony, J.V., Gupta, G., Li, X., Barone, F.C., Rosenbaum, D.M., 2000. The novel beta-blocker, carvedilol, provides neuroprotection in transient focal stroke. J. Cereb. Blood Flow Metab. 20, 1197–1204. Spencer, R.H., Chandy, K.G., Gutman, G.A., 1993. Immunological identification of the Shaker-related Kv1.3 potassium channel protein in T and B lymphocytes, and detection of related proteins in files and yeast. Biochem. Biophys. Res. Commun. 191, 201–206. Teisseyre, A., Gasiorowska, J., Michalak, K., 2015. Voltage-gated potassium channels Kv1.3–potentially new molecular target in cancer diagnostics and therapy. Adv. Clin. Exp. Med. 24, 517–524. Tomlinson, B., Prichard, B.N., Graham, B.R., Walden, R.J., 1992. Clinical pharmacology of carvedilol. Clin. Investig. 70 (Suppl 1), S27–S36. Tubert, C., Taravini, I.R.E., Flores-Barrera, E., Sanchez, G.M., Prost, M.A., Avale, M.E., Tseng, K.Y., Rela, L., Murer, M.G., 2016. Decrease of a current mediated by Kv1.3 channels causes striatal cholinergic interneuron hyperexcitability in experimental Parkinsonism. Cell Rep. 16, 2749–2762. Vicente, R., Escalada, A., Villalonga, N., Texido, L., Roura-Ferrer, M., Martin-Satue, M., Lopez-Iglesias, C., Soler, C., Solsona, C., Tamkun, M.M., Felipe, A., 2006. Association
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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Molecular and cellular pharmacology
Characterization and molecular basis for the block of Kv1.3 channels induced by carvedilol in HEK293 cells Jin-Feng Yanga, Neng Chenga, Sheng Rena, Xiang-Ming Liub, Xian-Tao Lia, a b
T
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College of Life Science, South-Central University for Nationalities, Wuhan 430074, China GongQing Institute of Science and Technology, Gongqing City 332020, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Carvedilol Kv1.3 channels Hyperpolarizing shift Closed- and open-channel block, molecular determinants
Carvedilol is a non-selective β-adrenoreceptor antagonist and exhibits a wide range of biological activities. The voltage-gated K+ (Kv) channel is one of the target ion channels of this compound. The rapidly activating Kv1.3 channel is expressed in several different tissues and plays an important role in the regulation of physiological functions, including cell proliferation and apoptosis. However, little is known about the possible action of carvedilol on Kv1.3 currents. Using the whole-cell configuration of the patch-clamp technique, we have revealed that exposure to carvedilol produced a concentration-dependent blocking of Kv1.3 channels heterologously expressed in HEK293 cells, with an IC50 value of 9.7 μM. This chemical decelerated the deactivation tail current of Kv1.3 currents, resulting in a tail crossover phenomenon. In addition, carvedilol generated a markedly hyperpolarizing shift (20 mV) of the inactivation curve, but failed to affect the activation curve. Mutagenesis experiments of Kv1.3 channels identified G427 and H451, two related sites of TEA block, as important residues for carvedilol-mediated blocking. The present results suggest that carvedilol acts directly on Kv1.3 currents by inducing closed- and open-channel block and helps to elucidate the mechanisms of action of this compound on Kv channels.
1. Introduction Functional Kv1.3 is composed of a homotetramer of pore forming α subunits, each of which comprises six transmembrane helices, arranged around a central K+-selective pore (Wulff et al., 2009). Expression of Kv1.3 channels is detected in distinct cells, including T and B lymphocytes (Wulff et al., 2004), osteoclasts (Arkett et al., 1994), neurons (Tubert et al., 2016), and smooth muscle cells (Perez-Garcia et al., 2018). Accumulated evidence indicates that Kv1.3 channels exhibit multiple regulatory actions in setting the resting membrane potential, apoptosis, cell volume and proliferation (Cahalan and Chandy, 2009; Deutsch and Chen, 1993; Gulbins et al., 2010; Liu et al., 2002). As a predominant Kv channel in T lymphocyte, Kv1.3 channels could be potently blocked by immunosuppressants, and accordingly its inhibition leads to a reduction of the proliferation in effector memory T (TEM) cells (Wulff et al., 2001). Thus, Kv1.3 channels are taken as a substantial therapeutic target for the treatment of autoimmune diseases, such as multiple sclerosis (Rus et al., 2005) and rheumatoid arthritis (Beeton et al., 2006). This channel is also detected in insulin-sensitive tissues such as adipose tissue, liver and skeletal muscle and therefore is considered as a potential drug target in diabetic therapy (Choi and
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Hahn, 2010). In addition, Kv1.3 channels have been considered a potentially new molecular target in both the diagnostics and therapy of some cancer diseases (Teisseyre et al., 2015). The inhibition of Kv1.3 channel activity has a beneficial action on above diseases. Hence, extensive works have been conducted to explore inhibitors of Kv1.3 channels as a treatment of related diseases. Concurrently, it is gradually revealed that Kv1.3 channels are also suppressed by chemicals that are originally assumed with other specific effects, such as verapamil (Kuras and Grissmer, 2009) and clofazimine (Faouzi et al., 2015). Nevertheless, only limited data are available for the molecular explanation of the interaction between drugs and Kv1.3 channels. To avoid unwanted side actions and reveal new pharmacological effects, it is worth exploring more about the mechanism of effects of drugs on Kv1.3 channels. Carvedilol is an α- and β-adrenoceptor antagonist that has been in clinical practice in patients with hypertension (McTavish et al., 1993) or myocardial infarction (Ruffolo and Feuerstein, 1997). Besides blocking adrenergic receptors, this drug also has multiple biological actions, such as scavenging free radicals (Yue et al., 1992), as well as protecting against apoptosis, inflammation and mitochondrial damage (Abreu et al., 2000; Savitz et al., 2000). A growing body of evidence
Corresponding author. E-mail address: [email protected] (X.-T. Li).
https://doi.org/10.1016/j.ejphar.2018.07.025 Received 25 May 2018; Received in revised form 4 July 2018; Accepted 13 July 2018 0014-2999/ © 2018 Elsevier B.V. All rights reserved.
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Fig. 1. Blocking of Kv1.3 currents in HEK293 cells by carvedilol. (A) Representative Kv1.3 current traces were shown under control conditions and treatment with 10 and 50 μM carvedilol. Currents in HEK293 cells were routinely evoked in response to 1 s voltage pulses from a holding potential of − 80 mV, to test potentials of + 60 mV. (B) Plot of current-voltage (I - V) relationships of Kv1.3 currents before and after exposure to 10 μM and 50 μM carvedilol (n = 10). (C) The relative currents in the presence of different concentrations of carvedilol have been obtained, and the solid line was the concentration-response relations of carvedilol action on Kv1.3 currents, which was generated by fitting data points of relative currents using Hill equation (n = 6). (D) Normalized block shown as relative current (Icarvedilol/I control) with 10 μM and 50 μM carvedilol was plotted against the varying voltages (n = 6).
experiments.
suggests that Kv channel is one of its targets in distinct tissues. For example, several components of cardiac outward K+ currents, including the slowly and rapidly activating delayed rectifier currents (IKr and IKs) as well as the ultrarapid activating delayed currents (IKur) (Deng et al., 2007), are suppressed by carvedilol. Treatment with carvedilol also inhibits the transient outward K+ currents (Ito), which play important roles in the phase 1 repolarization of cardiac action potential (Liu et al., 1993). Nevertheless, there is not any report for the interaction between carvedilol and Kv1.3 channels. In the current study, we examine the inhibitory effect of carvedilol on Kv1.3 channels and explore the molecular basis of this action.
2.2. Mutagenesis and transfection The vectors of pIRES2-EGFP containing the sequence of the human Kv1.3 channels were gifts from Dr. Zhi-Jian Cao (Wuhan University, Wuhan). Mutations to Kv1.3 channels were conducted with the QuikChange Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA). Predicted mutations were clarified by sequence analysis. HEK293 cells transiently expressing Kv1.3 channels were produced as described previously (Chen et al., 2012). Briefly, plasmids with Kv1.3 channels were transfected into HEK293 cells using Lipofectamine 2000 (Life Technologies, Bethesda, MD). At 4 h after treatment, the transfection reagent was replaced with regular culture medium. The next day, cells were allowed to settle on the bottom of recording chamber mounted on an inverted microscope (IX-73, Olympus, Osaka, Japan) for electrical measurements.
2. Materials and methods 2.1. Cell culture The human embryonic kidney cell line, HEK293, was maintained in modified Dulbecco's medium (MEM) with high glucose containing 10% fetal bovine serum in a humidified, 5% CO2 incubator at 37 °C. The cultures were passed every 2–3 days after brief trypsin treatment. Cells were plated onto glass coverslips 1 day prior to use for patch-clamp
2.3. Patch-clamp recording The whole-cell recording mode of the patch-clamp technique was 207
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(Fig. 2A). Under control conditions, the activation time constant (τact) obtained after exponential fitting was 1.9 ± 0.3 ms at −10 mV, and superfusion of 50 μM carvedilol significantly increased the value of τact to 7.1 ± 0.8 ms (n = 6, P < 0.05). This effect of 50 μM carvedilol on τact was obviously voltage-dependent and released at higher potentials (Fig. 2A). The conductance-voltage (G-V) curves of activation were fitted using the Boltzmann equation and shown in Fig. 2B. The value of the half maximal conductance voltage (V1/2) was −16.4 ± 3.5 mV (n = 5) in the absence of carvedilol and exposure to 10 and 50 μM did not affect the G-V curves, with V1/2 values of −15.5 ± 4.0 and −14.6 ± 3.6 mV, respectively (n = 5). Further measurement was performed to study the action of carvedilol on deactivation kinetics of Kv1.3 channels. Cells expressing Kv1.3 channels were repolarized to various voltages ranging from −100 and −40 mV in 10 mV increments following a depolarization pulse to the potential of + 40 mV at a holding potential of −80 mV. Tail currents in the absence and presence of carvedilol were fitted by using exponential function to obtain the deactivation time constant (τdeact). Obviously, the values of τdeact increased in a voltage-dependent way from −80 to −40 mV despite the presence of carvedilol (Fig. 2D). Application of 50 but not 10 μM carvedilol resulted in a potent enhance of τdeact at potentials more positive than −90 mV, showing a deceleration for Kv1.3 current deactivation. The value of τdeact at −40 mV was 25.6 ± 0.9 ms in control and increased to 45.1 ± 6.6 ms after exposure to 50 μM carvedilol (n = 6, P < 0.05), thereby a classical “crossover” deactivation tail currents on repolarization was evident (Fig. 2C). Consistent with the property of open channel block (Armstrong, 1971), carvedilol dissociates from the state of channel deactivation rather slowly compared with that in control, and accordingly channels could only close after the compound dissociated from its binding site.
undertaken throughout all experiments. Cells were superfused with the bath solution at room temperature containing (in mM): 75 Na-gluconate, 70 NaCl, 5 KCl, 5 HEPES and 5 glucose, with pH adjusted to 7.4 using NaOH. Patch pipets were fabricated from borosilicate glass and had resistances between 2.0 and 3.0 MΩ when filled with an artificial internal solution, containing (in mM): 150 KCl, 5 HEPES, 5 EGTA, 5 Glucose, 5 Na2ATP, with pH adjusted to 7.3 using KOH. An Axopatch 200B amplifier was employed to record Kv1.3 currents. The pCLAMP10 software (Molecular Devices, Sunnyvale, CA) was used to produce voltage clamp protocols, acquire data, and analyze current signals. Data were low-pass filtered at 5 kHz and sampled at 10 kHz. 2.4. Data analysis Data are presented as means ± S.E.M. Concentration-dependent effects were quantified by fitting the Hill equation: y = I / (1 + (IC50/x) h ), where IC50 is the drug concentration that produces half maximal inhibition, and h is the slope of the curve. Both conductance-voltage (GV) curves of activation and inactivation curves were fitted using Boltzmann equation: y = 1 ⁄ [1 + exp (V1⁄2 –V) ⁄ k], where V1⁄2 is the half-maximal potential, V is the conditioning potential, and k represents the slope factor. The time constant was obtained after fitting related current traces using the exponential equation: f (t) = A exp(- t ⁄τ), where τ is the time constant, and A is the corresponding current amplitude. Statistical significance was analyzed using a Student's t-test or one-way analysis of variance (ANOVA). A value of P < 0.05 was accepted statistically significant. 3. Results 3.1. Carvedilol directly inhibited Kv1.3 currents in a voltage-independent way
3.3. Effects of carvedilol on the inactivation Whole-cell recording technique was routinely conducted to examine effects of carvedilol on Kv1.3 channels heterologously expressed in HEK293 cells. Macroscopic Kv1.3 currents were activated by 1 s depolarizing voltage steps to potentials from −80 mV to + 60 mV at a holding potential of −80 mV applied every 45 s. Typical examples of current traces illustrated in Fig. 1A obviously implied that exposure to both 10 and 50 μM carvedilol led to a marked reduction in amplitudes of Kv1.3 currents compared with the control. Current-voltage (I - V) relationships before and after treatment with 10 and 50 μM carvedilol were assembled from raw data, and showed that Kv1.3 currents were blocked at all potentials in which Kv1.3 channels were activated (Fig. 1B, n = 10). Carvedilol-induced block on Kv1.3 currents is concentration-dependent, and quantified by concentration-response curves obtained after fitting with a Hill equation, with IC50 value of 9.7 ± 0.8 μM and a Hill coefficient of 1.4 ± 0.2 (Fig. 1C, n = 6). Previous studies indicated that carvedilol could exert an inhibitory action on different Kv channels. We also tested the action of carvedilol on rapidly activating delayed rectifier Kv1.5 channels that contribute to repolarization of action potentials of human atrial myocytes (Fedida et al., 1993). The concentration response curve for block of Kv1.5 currents by carvedilol is shown in Supplementary Figure, with IC50 value of 3.9 ± 0.4 μM and a Hill coefficient of 1.6 ± 0.2 (n = 5). To assess the voltage-dependent block of carvedilol, the relative current (Icarvedilol / Icontrol) was plotted as a function of membrane potential. As shown in Fig. 1D, the decrease of Kv1.3 currents was not voltage-dependent through the voltage over a range from −10 to + 40 mV.
We tested whether the steady-state inactivation curves of Kv1.3 channels were shifted by application of 10 and 50 μM carvedilol. Steady-state inactivation was determined by measuring the peak current at + 40 mV, following the 60 s conditioning prepulses from a holding potential of −80 mV to potentials between −80 mV and + 40 mV in increments of 20 mV. The current amplitude during each test pulse was normalized to maximal amplitude (I ⁄ Imax) and plotted against voltages of conditioning prepulses. These data were well fitted by the Boltzmann equation and shown in Fig. 3A. The values of V1/2 and k at control were −19.8 ± 0.4 mV and 9.8 ± 0.4 mV (n = 6), respectively. Exposure to 50 but not 10 μM carvedilol induced an approximately 20 mV hyperpolarizing shift in the inactivation curve, with values of V1/2 and k being −39.3 ± 0.8 mV and 9.0 ± 0.6 mV (n = 6), respectively. 3.4. Molecular determinants of Kv1.3 channel block by carvedilol The residue at the position 449 in the Shaker channel is located at the external mouth of the pore and participates in the modulation of TEA sensitivity and P/C type inactivation rate in Kv channels (LopezBarneo et al., 1993; Spencer et al., 1993; Zsiros et al., 2009). The amino acid equivalent to Shaker 449 in Kv channels (H451 in hKv1.3) has been identified as a common binding site for several drugs (Barana et al., 2010; Li et al., 2015; Lin et al., 2001). Analysis of a sequence alignment suggested that another residue equivalent to site responsible for TEA interaction in Kv1.5 channels (Abid et al., 2015) was a glycine at Kv1.3 position 427. Therefore, mutagenesis of above residues of TEA block (H451 and G427) were conducted and effects of carvedilol on these mutations were assessed to determine the molecular binding sites. Detectable currents were not evident for two of five mutations (H451D and G427D), and significant elimination of carvedilol's action on three other mutations (H451Y, H451V and G427K), but to a different degree,
3.2. Actions of carvedilol on kinetics of activation and deactivation Kv1.3 channels were activated by depolarizing pulses to potentials ranging from −80 to + 40 mV with increment of 10 mV at a holding potential of −80 mV. Application of 50 but not 10 μM carvedilol mediated a deceleration of activation process for Kv1.3 channels 208
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Fig. 2. Actions of carvedilol on kinetics of activation and deactivation. Plot of the activation time constants as a function of depolarizing pulses from − 10 to + 40 mV in the absence and presence of 10 and 50 μM carvedilol (n = 6). (B) Normalized conductance vs. test voltage (G-V) relationships for control as well as 10 and 50 μM carvedilol (n = 5). (C) Representative tail currents of deactivation were shown without and with 50 μM carvedilol. Crossover of these tail currents was illustrated at the bottom panel. (D) Voltage dependence of τdeact under control conditions and during application of 10 and 50 μM carvedilol (n = 6).
(n = 5, P > 0.05), with V1/2 values of 17.6 ± 3.0 mV and −16.4 ± 3.5 mV, respectively. The G-V curves of H451Y with and without carvedilol were shown in Fig. 4A. After addition of 50 but not 10 μM carvedilol, however, the V1/2 value of H451Y was weakly but significantly shifted right to 12.5 ± 5.8 mV (n = 5, P < 0.05), indicating that mutant channel activated at more positive potentials. The mutation of H451Y also changed the effects of carvedilol on deactivation kinetics of channels. Compared with wild type channels, exposure to 50 μM carvedilol potently increased the τdeact value of H451Y to 74.2 ± 2.9 ms at the potential of −40 mV (Fig. 4B; n = 5, P < 0.05). For steady-state inactivation of H451Y, the V1/2 was – 8.6 ± 14.2 mV and shifted left to – 26.1 ± 18.6 mV (Fig. 4C; n = 5, P < 0.05). These data suggest that the alteration of mutation's response to carvedilol may possibly contribute to the attenuation of this drug's action.
was obvious. Previous studies revealed that the residue, near the external TEA binding site and selectivity filter, was also involved in the modulation of inactivation and TEA block (Cha and Bezanilla, 1997; Kiss et al., 1999). In this study, mutation of valine 453 that is located in the outer vestibule near the TEA binding site (H451) fail to alter the carvedilol's action (n = 6). The summary of action of carvedilol on wild type and mutant Kv1.3 currents was shown as a bar graph in Fig. 3B. Exposure to 10 μM carvedilol resulted in the inhibition of 10.4 ± 4.8, 26.4 ± 2.1 and 33.4 ± 3.0% for H451Y, H451V and G427K, respectively (n = 6). Compared with H451V and G427K, the H451Y mutation released the carvedilol's action to a largest degree and following experiments were performed on this mutation to explore the possible mechanism of interaction.
3.5. Effects of carvedilol on activation, deactivation and inactivation of H451Y mutation
4. Discussion
In the absence of carvedilol, no obvious alteration of activation was detectable between H451Y mutation and wild type Kv1.3 channels
Although previous studies found that carvedilol, a non-selective βadrenoreceptor antagonist, can inhibited different Kv currents in native 209
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Fig. 3. Actions of carvedilol on inactivation curves and mutant Kv1.3 channels. (A) The inactivation of Kv1.3 channels was examined during depolarizing voltage steps to + 40 mV after 60 s conditioning prepulses to potentials ranging from –80 and + 40 mV. The peak currents during each test pulse were normalized to maximal currents (I/Imax) and plotted against conditioning voltages. The solid lines were inactivation curves, which were generated by fitting data points using a Boltzmann equation (n = 6). (B) The bar graph is the summary of carvedilol block on wild type and mutant Kv1.3 channels (n = 6). Compared with wild type currents, the potency of carvedilol block was substantially released in the G427Y, H451V and H451Y Kv1.3 currents. *P < 0.05 compared with the wild type group.
cells (Cheng et al., 1999; Deng et al., 2007) and heterologous expression systems (Jeong et al., 2012; Karle et al., 2001), it remained obscure whether there exists interaction between carvedilol and voltage-gated Kv1.3 channels, which are widely expressed in many tissues (Arkett et al., 1994; Tubert et al., 2016; Wulff et al., 2004). In the present study here, collected data demonstrate that application of carvedilol can directly inhibit the macroscopic Kv1.3 currents in a concentration-dependent manner and modulates its kinetic properties in HEK293 cells. Our experiments of mutagenesis also identify two related sites of TEA block as binding sites of carvedilol to Kv1.3 channels. Several lines of evidence indicate that carvedilol preferentially binds to the open state of Kv1.3 channel. First, inhibitory actions of carvedilol were detectable after channel activation. Second, treatment with carvedilol potently slows the deactivation time courses of tail currents and, as a result, crossover phenomena of tail currents was evident in the absence and presence of the drug. This observation shows that carvedilol needs to dissociate from its binding site before channels can close, and accordingly the time course of drug dissociation from deactivating channels is much slower compared with control channels. In accordance with both open- and close-channel block mechanism reported by investigators (Frolov et al., 2010), however, carvedilol also
Fig. 4. The response of H451Y mutations to carvedilol. (A) Normalized conductance vs. test voltage (G-V) relationships for the H451Y mutant channel at various concentrations of carvedilol (n = 5). (B) Effects of 10 and 50 μM carvedilol on τdeact of H451Y mutations. (C) Inactivation curves of H451Y mutations under control conditions and in the presence of 10 and 50 μM carvedilol were obtained by using a two-pulse protocol described above (n = 5).
exerts an inhibitory action on closed state of Kv1.3 channel. For instance, a deceleration of activation time is obviously observed after addition of carvedilol, indicating the slowed activation by the closedchannel blocker (Zhang et al., 1997). Our study reveals that two crucial positions equivalent to the presumed TEA binding site in Shaker (T449), G427 in the S5-P linker and 210
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H451 in the turret region, are responsible for the sensitivity of carvedilol to Kv1.3 channels. These residues identified as binding sites for carvedilol block are highly conserved in other Kv channels, and hence it is reasonablely presumed that inhibitory effects of this compound also occur in HERG (Kawakami et al., 2006) and Kv1.5 channels (Jeong et al., 2012). The value of IC50 for carvedilol-mediated inhibition of Kv1.3 channels is 9.7 μM and is over the range of therapeutic concentrations, from 0.1 to 0.6 μM (Morgan et al., 1990). Similarly, IC50 values of carvedilol-mediated inhibitions for transient outward and slowly activating delayed rectifier K+ currents in rabbit ventricular myocytes are higher than therapeutic plasma levels (Cheng et al., 1999). Note that carvedilol is a highly lipid soluble compound that would accumulate in tissue at concentrations higher than those observed in plasma (Neugebauer et al., 1987; Tomlinson et al., 1992). Thus, it is possible that local levels of carvedilol can be high enough to block Kv1.3 channels in clinical treatment. Additionally, Kv1.3 has been shown to coassemble with Kv1.2 and Kv1.4 in neural tissues (Coleman et al., 1999; Koch et al., 1997), and form heteromultimeric complexs with Kv1.5 in macrophages (Vicente et al., 2006). Hence, it is possible that the assembly of Kv1.3 with other Kv channel subunits could result in the varying sensitivity to carvedilol. Besides taking as an antihypertensive agent because of its β-adrenergic blocking and vasodilating action (Dunn et al., 1997), carvedilol exhibits a wide range of biological activities mentioned in the introduction. Based on the current study, it is reasonablely speculated that blockage of Kv1.3 channels is involved in the functional modulation in response to treatment with carvedilol. In summary, the present study demonstrates that the carvedilol can substantially inhibit Kv1.3 channels expressed in HEK293 cells. Two related residues of TEA block are identified as binding sites of carvedilol to this channel, and both open- and closed-channel block are involved in this process. The blocking actions of carvedilol on Kv1.3 channels may provide an alternative explanation for its effects in different tissues.
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