Chen H et al. (2013), Comparison of the effects of antiarrhythmic dru...

XB-ART-46301

Acta Pharmacol Sin 2013 Feb 01;342:221-30. doi: 10.1038/aps.2012.157.

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

Chen H , Zhang D , Chao SP , Ren JH , Xu L , Jiang XJ , Wang SM .

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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. 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. 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. 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.

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Species referenced: Xenopus
Genes referenced: kcna4 tff3.7

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	Figure 1. Effects of flecainide and verapamil on the fKv1.4ΔN channel expressed in Xenopus oocytes. Representative curves were shown for 5 s depolarizing steps from −90 mV to voltages between −100 and +50 mV in steps of 10 mV. (A and B) Traces recorded under control conditions. (C and D) Current traces obtained in the presence of 500 μmol/L flecainide (C) or 250 μmol/L verapamil (D). (E and F) Effects of 500 μmol/L flecainide (E) or 250 μmol/L verapamil (F) on the peak current-voltage (I–V) relationships. Currents were normalized to the peak current at +50 mV under control conditions. The Idrug/Icontrol ratio was plotted as a function of the membrane potential. Data are shown as mean±SEM (n=5).
	Figure 2. Concentration-response relationships for the inhibition of the fKv1.4ΔN currents by flecainide and verapamil. (A and B) Representative current traces were elicited in the absence and presence of increasing concentrations of flecainide (A) or verapamil (B). Currents were recorded by depolarizing steps to +50 mV from a holding potential of −90 mV. (C and D) The peak currents were normalized to the maximum peak current under control conditions and plotted against flecainide (C) and verapamil (D) concentrations. The curves were derived by fitting data to the Hill equation. Symbols and error bar are mean±SEM (n=5).
	Figure 3. Use-dependent block of the fKv1.4ΔN currents by flecainide and verapamil. (A and B) Sixty repetitive depolarizing steps from −90 to +50 mV for 500 ms each were applied in the absence and presence of flecainide (A) or verapamil (B). (C and D) Each peak current was normalized to the peak current at the first step under control conditions and then plotted against the number of steps. Data are shown as mean±SEM (n=5).
	Figure 4. Effects of flecainide and verapamil on the activation of the fKv1.4ΔN currents. (A–D) Current traces were obtained by applying 80 ms steps to potentials ranging from −100 to +50 mV and were followed by the tail currents obtained upon repolarization to −40 mV in the absence (A and B) and presence of 500 μmol/L flecainide (C) or 250 μmol/L verapamil (D). (E and F) Peak current activation relationships in the absence and presence of 500 μmol/L flecainide (E) and 250 μmol/L verapamil (F). 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: f=1/{1+exp*[(V–V1/2)/k)]}. Average data are shown as mean±SEM (n=5).
	Figure 5. Changes in the inactivation kinetic properties of the fKv1.4ΔN currents by flecainide and verapamil. (A and B) Steady-state inactivation was studied using a two-step voltage protocol, currents were measured at +50 mV, and the 5 s pre-steps to potential varied from −100 to +50 mV in steps of 10 mV. The curves for steady-state inactivation were fitted with the Boltzmann equation: I/Imax=(1−α)/{1+exp*[(V−V1/2)/k)]}+α. (C and D) Time constant of inhibition as a function of the drug concentration. Time constants (τblock) were estimated from a single or double exponential fits to the tracings shown in Figure 1. The apparent rate constants for association (k+1) and dissociation (k-1) were obtained from the equation: 1/τblock=k+1[d]+k−1 (n=5). Data are shown as mean±SEM (n=5).
	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 (P1) from −90 to +50 mV was followed by a second step (P2) to +50 mV after an interval of between 0.1 and 20 s. (A and B) Current traces were elicited in the absence of flecainide (A) or verapamil (B). (C and D) Current traces obtained in the presence of 500 μmol/L flecainide (C) or 250 μmol/L verapamil (D). (E and F) The ratio of the peak current elicited by the P1 and P2 steps (P2/P1) is plotted as a function of the various interstep intervals. The continuous line represents the fit of the data to the equation: f=1−A*exp(−τ/t). Data were normalized between 0 and 1 presented with intervals on a log scale. Data are shown as mean±SEM (n=5).

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