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Comparison of the effects of antiarrhythmic drugs flecainide and verapamil on fKv1.4ΔN channel currents in Xenopus oocytes.

AIM
To study the effects of Na(+) channel blocker flecainide and L-type Ca(2+) channel antagonist verapamil on the voltage-gated fKv1.4ΔN channel, an N-terminal-deleted mutant of the ferret Kv1.4 K(+) channel.


METHODS
fKv1.4ΔN channels were stably expressed in Xenopus oocytes. The K(+) currents were recorded using a two-electrode voltage-clamp technique. The drugs were administered through superfusion.


RESULTS
fKv1.4ΔN currents displayed slow inactivation, with a half-inactivation potential of -41.74 mV and a slow recovery from inactivation (τ=1.90 s at -90 mV). Flecainide and verapamil blocked the currents with IC(50) values of 512.29 ± 56.92 and 260.71 ± 18.50 μmol/L, respectively. The blocking action of the drugs showed opposite voltage-dependence: it was enhanced with depolarization for flecainide, and was attenuated with depolarization for verapamil. Both the drugs exerted state-dependent blockade on fKv1.4ΔN currents, but verapamil showed a stronger use-dependent blockage compared with flecainide. Flecainide accelerated the C-type inactivation rate without affecting the recovery kinetics and the steady-state activation. Verapamil also accelerated the inactivation kinetics of the currents, but unlike flecainide, it affected both the recovery and the steady-state activation, causing slower recovery of fKv1.4ΔN channel and a depolarizing shift of the steady-state activation curve.


CONCLUSION
The results demonstrate that widely used antiarrhythmic drugs flecainide and verapamil substantially inhibit fKv1.4ΔN channels expressed in Xenopus oocytes by binding to the open state of the channels. Therefore, caution should be taken when these drugs are administered in combination with K(+) channel blockers to treat arrhythmia.


Introduction
Kv1.4 voltage-gated potassium channels are expressed in the subendocardium of human and ferret heart and in the rat ventricular septum [1][2][3] . This channel conducts transient outward potassium currents (I to ). Kv1.4 expression in the mammalian endocardium is upregulated during hypertrophy and heart failure [4][5][6][7] , and this channel plays an important role in the repolarization of cardiac myocytes. The inactivation of Kv1.4 occurs through two distinct mechanisms: fast N-type inactivation and slow C-type inactivation [8] . The slow C-type inactivation involves the conformational changes of the external side of the pore [9,10] . In Xenopus oocytes, C-type inactivation controls the recovery of Kv1.4 channels [11] . Because the recovery rate of Kv1.4 determines the availability of the channels for repolarization of the subsequent action potential, it is important to investigate the effects of cardiac medicines on C-type inactivation of Kv1.4 channels.
From a basic biophysical perspective, the slow inactivation of the Kv1.4 channels resembles the inactivation of sodium and calcium channels [12] . We assumed, therefore, that some calcium and sodium channel antagonists could affect Kv1.4 inactivation. In our previous study, we investigated the effects of the L-type calcium channel antagonist diltiazem and the sodium channel antagonist propafenone on Kv1.4 channels. We discovered that both diltiazem and propafenone accelerated Kv1.4 channel inactivation, providing evidence that C-type inactivation maybe a common mechanism underlying channel inactivation for sodium and calcium channels [13,14] .
The sodium channel antagonist flecainide and the L-type calcium channel antagonist verapamil have been used widely for the treatment of atrial fibrillation, supraventricular arrhythmias, and long QT syndrome [15][16][17][18] . In addition to sodium channels and L-type calcium channels, these antagonists also block several voltage-gated potassium channels. Flecainide may inhibit the wild-type Kv4.2 channel (Kv4.2WT) currents in a concentration-dependent manner, and it has been shown to induce a voltage-dependent block of hERG channels [19,20] . In addition, verapamil has been reported to produce a potent use-and frequency-dependent block of hERG channels [21] and to inhibit the hKv1.5 channel in low micromolar concentrations [22] . Flecainide and verapamil have been shown recently to inhibit the conductance of Kv1.4 channels expressed in Xenopus oocytes [13,23] . The detailed characteristics of this effect, which may lead to a better understanding of their antiarrhythmic mechanism, have not been studied.
In the present study, we studied the inhibitory effects of flecainide and verapamil on Kv1.4∆N, an N-terminal deletion construct of Kv1.4 that lacks rapid N-type inactivation but exhibits robust C-type inactivation [24] , to provide a more detailed understanding of their mechanisms of action.

Materials and methods
Molecular biology fKv1.4ΔN cDNA (GenBank accession No U06156) was a gift from Prof Randall L RAsmussoN (University at Buffalo, suNY, usA). The constructs and sequences have been described previously [11,12,24] . Removal of residues 2-146 from the N-terminal domain of Kv1.4 results in the loss of the fast N-type inactivation but leaves slow C-type inactivation intact. Transcribed fKv1.4ΔN cRNA was prepared in vitro using an mmessAGe mmAchiNe kit (T3 kit, Ambion, usA).
Isolation of oocytes and incubation mature female Xenopus laevis frogs were provided by the Chinese Academy of science, Beijing, china. The frogs were cared for using standards approved by the institutional Animal Care and Use Committee of the Wuhan University of China. mature female frogs were anesthetized by immersion in a tricaine solution (1.5 g/L, sigma) for 30 min, and the oocytes were removed surgically through a lateral incision in the lower abdomen, as described previously [14] . The oocytes were digested by placing the ovarian lobes in a collagenase-containing, Ca 2+ -free oocyte Ringer's (oR 2 ) solution (mmol/L): 82.5 Nacl, 2 Kcl, 1 mgcl 2 , and 5 hePes at ph 7.4, with 1 to 1.5 mg/mL collagenase (Type i, sigma, usA). The oocytes were then shaken gently for approximately 1 h and washed several times with Ca 2+ -free oR 2 solution as described previously [14] . Finally, defolliculated (stage IV) oocytes were selected and placed in ND96 solution (mmol/L): 96 Nacl, 2 Kcl, 1 mgcl 2 , 1.8 CaCl 2 , and 5 hePes at ph 7.4. each oocyte was injected with approximately 25 to 50 nL of fKv1.4ΔN cRNA using a microinjector (WPi, sarasota, FL, usA) and incubated at 18 °c in ND96 solution with 100 iu/mL penicillin for a minimum of 16 h.

Electrophysiology
The two-electrode voltage clamp technique was used for the electrophysiological recordings. microelectrodes were pulled with a two-stage puller (Narishige, Japan) and had tip resistances of 0.5 to 1.0 mΩ when filled with 3 mol/L Kcl. The currents in voltage-clamp mode were recorded at room temperature using a preamplifier cA-1B (Dagan, usA), and they were filtered at 2.5 khz. The recordings were made in 2 mmol/L K + o . Flecainide and verapamil were dissolved in distilled water to give stock solutions of 100 mmol/L. All drugs were purchased from sigma chemical co (st Louis, mo, usA). The drugs were superfused for 10 min before testing to allow equilibration with the oocytes. After this wash-on period, a series of 500-ms depolarizing steps (from -90 to +50 mV, 1 min) were applied to ensure steady-state block before beginning the experimental protocols [12,14] .

Data analysis
The data were digitized and analyzed using pcLAmP 9.0 (Axon, usA). Further analysis was performed using clampfit 9.0 (Axon, usA) in combination with microsoft excel (microsoft, usA) and origin 6.0 software (microcal software, usA). Assuming a 1:1 binding stoichiometry between the drug and the receptor, concentration-response curves were generated using the hill equation: f=K D /(K D +D), where f is the fractional current, K D is the apparent dissociation constant, and D is the drug concentration [12] . Inactivation time constants were obtained using a single or double exponential decay model fitted to the raw current tracings. The peak current activation was fitted to the Boltzmann equation: f=1/{1+exp*[(V-V 1/2 )/ k)]}, where V represents the test potential, V 1/2 is the mid-point of activation, and k is the slope factor. The steady-state inactivation was fitted to the Boltzmann equation: I/I max =(1-α)/ {1+exp*[(V-V 1/2 )/k)]}+α, where I max is the maximal peak current, α is the non-inactivating component of the curves, V represents the test potential, V 1/2 is the mid-point of inactivation, and k is the slope factor. The apparent rate constants for association (k +1 ) and dissociation (k -1 ) were obtained from the equation: 1/τ block =k +1 [D]+k -1 , where D is the drug concentra tion [23,25] . The time course of recovery from inactivation was fitted to a single exponential equation: f=1-A*exp(-τ/t), where t is the duration (in seconds), τ is the time constant, and A is the amplitude of the current. A paired student's t-test was used for statistical analysis of the data. The results are expressed as the mean±sem, and values of P<0.05 were considered significant.

Results
Inhibition of fKv1.4ΔN currents by flecainide and verapamil Figure 1 shows superimposed traces of currents from fKv1.4ΔN channels expressed in Xenopus oocytes under control conditions (A and B) and in the presence of flecainide (c) or verapamil (D). The cells were held at -90 mV, and the fKv1.4ΔN currents were examined with a series of 5-s step stimuli from -100 to +50 mV at 10-mV increments, followed by www.chinaphar.com Chen H et al Acta Pharmacologica Sinica npg a step clamping to +50 mV. Under control conditions, positive depolarization to -30 mV elicited outward currents that declined slowly during the maintained depolarization (slow c-type inactivation). Figures 1c and 1D show the effects of flecainide (500 μmol/L) and verapamil (250 μmol/L). Both drugs not only reduced the current amplitude but also accelerated the inactivation. Flecainide and verapamil reduced the amplitude of the peak current at +50 mV to 44.5%±2.3% and 53.5%±6.2% (n=5) of control, respectively. Flecainide and verapamil showed similar efficacy in inhibiting fKv1.4ΔN currents, and the inhibitory effects were completely abrogated by a 10-min washout of the drug (data not shown).
The effects of 500 μmol/L flecainide and 250 μmol/L verapamil on the peak current-voltage (I-V) relationships for the fKv1.4ΔN channel are shown in Figures 1e and 1F. The I-V relationships were constructed by plotting the normalized currents as a function of the membrane potential. Flecainide and verapamil induced a voltage-dependent inhibition of the fKv1.4ΔN currents. To quantify the voltage dependency of the fKv1.4ΔN block, the relative current (I drug /I control ) was plotted as a function of the membrane potential. The current began to activate at the activation threshold (between -40 and -20 mV), and both drugs decreased the peak current at positive potentials to the level of the activation threshold. The blockage by flecainide and verapamil showed differential voltage-dependence, with a voltage-dependent enhancement for flecainide and a voltage-dependent attenuation for verapamil.
Flecainide and verapamil inhibit fKv1.4ΔN currents in a concen tra tiondependent manner Figure 2 shows representative fKv1.4ΔN currents superim-  Effects of flecainide and verapamil on the activation of fKv1.4ΔN currents Figure 4 shows the representative currents and peak current activation relationships. The activation curves were measured in the absence (A and B) and presence of either 500 μmol/L flecainide (c) or 250 μmol/L verapamil (D). The steady-state activation curves were depicted by plotting the peak tail currents (obtained at -40 mV) against the depolarizing potentials (from -100 to +50 mV). continuous lines represent the fit of the data to a Boltzmann equation. We found that flecainide did not affect the steady-state activation:  .07 seconds at +50 mV (n=5). in the presence of flecainide, the inactivation was best fitted to a bi-exponential function, as the inactivation processes were composed of fast and slow components. The slow component (τ slow ) was regarded as intrinsic c-type inactivation of the fKv1.4ΔN channel. The fast component (τ fast ) was thought to represent the time constant for the drug-induced blockade of the fKv1.4ΔN currents. After application of 500 μmol/L flecainide, τ fast was 0.36±0.04 seconds and τ slow was 0.79±0.05 seconds (at +50 mV, n=5). in the presence of verapamil, the inactivation of fKv1.4ΔN was well fitted to a bi-exponential function, with τ fast =0.01±0.002 seconds and τ slow =0.27±0.04 seconds at +50 mV (n=5). We found that 500 μmol/L flecainide and 250 μmol/L verapamil accelerated the intrinsic C-type inactivation.
We then compared the effects of flecainide and verapamil on the steady inactivation curve for the fKv1.4ΔN channel ( Figure 5). The membrane potential was held at various levels between -100 mV and +50 mV, and the tail currents were obtained upon depolarization to +50 mV. The peak currents were normalized, plotted against the membrane potential, and fitted by a Boltzmann equation. The steadystate inactivation relationships were normalized, as shown in Figures 5A and 5B. Although 500 μmol/L flecainide produced a slight leftward shift in the steady-state inactivation curve, V 1/2 was not significantly changed: V 1/2 control =-42.84±5.72 mV; V 1/2 flecainide =-42.28±4.33 mV (n=5, P>0.05). In the presence of 250 μmol/L verapamil, the steady-state inactivation curve was shifted to the left: V 1/2 control =-41.81±6.20 mV; V 1/2 verapamil =-52.12±6.35 mV (n=5, P<0.05). These results indicate that verapamil, but not flecainide, alters the voltagedependency of the steady-state inactivation of the fKv1.4ΔN channel. Figures 5c and 5D show τ fast at +50 mV plotted against the various drug concentrations (τ fast values were not shown). From this fit, in the presence of flecainide (C), an apparent association (k +1 ) of 0.0022±0.0004 μmol·L -1 ·s -1 and (k -1 ) of 1.20±0.27 s -1 was obtained. The k D value derived on the basis of a first-order reaction between the drug and the channel was 486 μmol/L in the presence of flecainide. The k D value of verapamil (D) was 242 μmol/L (k +1 =0.07±0.02 μmol·L -1 ·s -1 and k -1 =17.16±7.67 s -1 ).
We further compared the effects of flecainide and verapamil on the recovery kinetics of the fKv1.4ΔN channel. The recovery kinetics were examined using a standard gappedstep protocol with a variable inter-stimulus interval as shown at the top of Figure 6. Figures 6A and 6B show the recovery traces of two cells under control conditions, and Figures 6C  and 6D show the recovery traces of those two cells in the presence of flecainide (6c) or verapamil (6D), respectively. The ratio of the current amplitude initiated by the second step

Discussion
The sodium channel antagonist flecainide has distinct effects on cardiac electrophysiology and arrhythmias. Flecainide produces potent voltage-and frequency-dependent inhibition of cardiac sodium channels, attributes that have generally proved to be beneficial in the treatment of cardiac arrhythmias [26] . The effects of flecainide on other channels have also been reported. For example, flecainide inhibits several cloned potassium channels, including Kv1.1, Kv1.2, Kv1.5, Kv2.1, Kv4.2, and hERG channels [19,20,23] . however, blockade of these channels has generally required relatively high drug concentrations. Limited data are available for the Kv1.4 potassium channel.   [13] , and propafenone and diltiazem share its characteristics [14] . Flecainide was also shown to reduce Kv1.4 potassium currents [23] , but its mechanism of action was not examined. our results provide novel information on the inhibition of fKv1.4ΔN potassium currents by flecainide. our findings agree with previous reports, although the IC 50 for a flecainide block (512.29 μmol/L) is relatively higher than that for blockade of the sodium channel [27] . however, the blockade of channels expressed in Xenopus oocytes by lipophilic drugs often requires much higher concentrations (five-to ten-fold) than those are needed to block the channels expressed in mammalian cells [23] . Therefore, flecainide may have a greater potency toward the fKv1.4ΔN channel than reported in this study. our results reveal that flecainide inhibits fKv1.4ΔN channels expressed in Xenopus oocytes in a concentration-, voltage-, and use-dependent manner. The blockade significantly accelerates channel inactivation, suggesting an open channel block [14] . Verapamil shares these characteristics. our results demon-strate that flecainide and verapamil exhibit similar affinities for fKv1.4ΔN, but the drugs do not share high-affinity heRG channel blocking properties [21] .
Because flecainide and verapamil are different types of antiarrhythmic drugs, we examined the electrophysiological effects of both drugs on fKv1.4ΔN channel inactivation. under control conditions, fKv1.4ΔN channels only show slow c-type inactivation, and the decay of the currents can be fitted with a single-exponential function. The binding of open-channel blockers introduces a blocked state. The blocked channel is rapidly inactivated, enters the blocked-inactivated state, and subsequently proceeds to the C-type inactivated state, leading to a bi-exponential decay of fKv1.4ΔN currents [12] .  shifted the steady-state inactivation curve to the left. This may be related to a mechanism in which binding of the drug to the intracellular site of the channel triggers a conformational change at the external mouth of the pore that facilitates C-type inactivation [12,14] . Flecainide also slightly shifted the steady-state inactivation curve to the left, but the V 1/2 was not significantly changed. These results indicate that the voltagedependency of the steady-state inactivation of the fKv1.4ΔN channel is altered by verapamil but not by flecainide. The lack of an effect of flecainide on the voltage dependency of steady-state inactivation suggests that flecainide is unlikely to interact with the inactivated state of the fKv1.4ΔN channel [28] . The sodium channel antagonist propafenone shares these characteristics [14] . Verapamil not only changed the voltage-dependency of the steady-state inactivation of the fKv1.4ΔN channel but also shifted the fKv1.4ΔN steady-state activation curve to the right with a significant change in V 1/2 . Therefore, it is presumed that verapamil may also interact simultaneously with the open and inactivated states of the fKv1.4ΔN channel [29] . Unlike verapamil, diltiazem only interacts with the fKv1.4ΔN channel in the open state and not in the inactivated state [14] . We suggest that the blockade of fKv1.4ΔN channels by verapamil is a more complex process than blockade by diltiazem and flecainide. The binding of diltiazem and flecainide to the channel affects the C-type inactivation without changing channel activation [14] , while the binding of verapamil may alter channel activation [30] . in this study, we used fKv1.4ΔN, which lacks rapid N-type Figure 6. Effects of flecainide and verapamil on the kinetics of the fKv1.4ΔN channel recovery from steady-state inactivation. The degree of recovery was measured by following a standard variable interval gapped step protocol. An initial 5 s step (P 1 ) from -90 to +50 mV was followed by a second step (P 2 ) to +50 mV after an interval of between 0.1 and 20 s.   [12] . The effects of flecainide and verapamil on the currents of native Kv1.4 have been studied previously [23] . The steady-state activation curves were shifted in the positive direction by verapamil but not by flecainide, similar to their effects on fKv1.4ΔN. The steadystate inactivation curves were not affected by either drug. The decay time course of native Kv1.4 was accelerated by verapamil, but it was decelerated by flecainide and quinidine. however, we found that fKv1.4ΔN decayed faster in the presence of flecainide and verapamil, and the effect of quinidine was similar to that of flecainide [12] . Although the effects of flecainide and verapamil inactivation were different for native Kv1.4 and fKv1.4ΔN, the recovery of native channels from inactivation would have to be taken into account. The slower C-type inactivation is critically important for determining the rate of recovery and is likely to be of primary physiological importance for determining the refractory period of Kv1.4 channels [11] . When a drug blocks fKv1.4ΔN, recovery is still governed by the direct C-type inactivated-to-open transition because dissociation of the drug is most likely much faster than recovery from C-type inactivation [12] . In this study, verapamil showed a greater effect on Kv1.4 than flecainide, slowing recovery of the fKv1.4Δ N channel. Flecainide did not alter channel recovery, consistent with its lack of an effect on C-type inactivation. Using the fast time constants in the range from 10 to 1000 μmol/L, which represent the interaction of the drug with the open state, the constants k +1 and k -1 were obtained. The k D values derived on the basis of a first-order reaction between the drug and channel were 486 μmol/L in the presence of flecainide and 242 μmol/L in the presence of vera pamil [14,28,31,32] . The values were close to the IC 50 values obtained from the concentration-response curve. The similarity of the IC 50 values obtained by the two independent methods supports the openchannel block model used to calculate the rate constant for the fKv1.4ΔN channel [33,34] .
Flecainide exerts effects on cardiac arrhythmias that can be distinguished from other sodium channel antagonist drugs. Although the antiarrhythmic efficacy of flecainide has usually been ascribed to its suppression of sodium channels, our findings suggest that the blockade of fKv1.4ΔN channels may also contribute to the cardiac effects of flecainide. The Kv1.4 channel, which conducts I to , contributes to phase 1 and the early part of phase 2 of the cardiac action potential and plays an important role in the repolarization of cardiac myocytes [4][5][6][7] . Flecainide and verapamil share an ability to inhibit the fKv1.4ΔN currents and increase c-type inactivation.
Whether the inhibition of fKv1.4ΔN currents by flecainide or verapamil would exert a beneficial action on arrhythmias remains to be studied. on the one hand, suppression of fKv1.4ΔN channels prolongs action potential duration and increases the refractory period, and it may thereby terminate reentrant circuits [35] . Blockade of fKv1.4ΔN currents and decreasing the rate of channel recovery could attenuate the shortening of the action potential duration that results from the inhibition of L-type calcium channels by verapamil [13] . on the other hand, Kv1.4 channels are expressed in the subendocardium of the human heart [1][2][3] , and the extra inhibition of Kv1.4 would increase the transmural dispersion of repolarization, which could lead to cardiac arrhythmias.
In summary, the present study provides the first indication that the widely used antiarrhythmic drugs flecainide and verapamil substantially inhibit fKv1.4ΔN potassium channels expressed in the Xenopus oocyte by binding to the open state of the channel. Furthermore, inhibition of Kv1.4 by flecainide and verapamil is completely independent of their inhibition of sodium channels and calcium channels. Therefore, caution should be exercised when these drugs are administered in combination with other potassium channel blockers to patients suffering from atrial fibrillation, supraventricular arrhythmias, and long QT syndrome.