Selective phenylalkylamine block of I(Kr) over other K(+) currents in guinea-pig ventricular myocytes

Abstract

Previous studies on verapamil and D600 have established that the Ca(2+)-channel blockers also inhibit delayed-rectifier K(+) currents in cardiac tissues and myocytes. However, estimated IC(50) values range over two to three orders of concentration, and it is unclear whether this reflects a high selectivity by one or both of the phenylalkylamines for particular K(+) channels. The purpose of the present study was to determine the concentration-dependent actions of verapamil and D600 on three defined cardiac K(+) currents. Guinea-pig ventricular myocytes in the conventional whole-cell configuration were bathed with normal Tyrode's or K(+)-free solution, and pulsed from -80 mV for measurement of the effects of 0.01 microM to 3 mM verapamil and D600 on the inwardly-rectifying K(+) current (I:(Kl)) and the two delayed-rectifier K(+) currents, rapidly-activating I:(Kr) and slowly-activating I:(Ks). The phenylalkylamines inhibited both inward- and outward-directed I:(Kl). The IC(50) values for outward I:(Kl) were approximately 220 microM. Verapamil and D600 were approximately equipotent inhibitors of the delayed-rectifier K(+) currents. They inhibited I:(Kr) with IC(50) near 3 microM, and I:(Ks) with IC(50) > or =280 microM. These results are discussed in relation to previous findings on K(+) currents and to the clinical actions of the drugs.

Figure 1

Effects of verapamil and D600 on membrane currents in guinea-pig ventricular myocytes. The myocytes were bathed in normal Tyrode's solution, held at −80 mV, depolarized to prepulse −40 mV for 200 ms, and then depolarized to more positive potentials for 500 ms at 0.1 Hz; tail currents (I_K,tail) were recorded on repolarizations to −40 mV. (a) Near-complete inhibition of inward I_Ca,L and partial inhibition of I_K,tail after 10-min exposures of myocytes to 10 μM (top) and 100 μM (bottom) verapamil. (b) Effect of 10 μM D600 on membrane currents in a myocyte pretreated with 1 μM nisoldipine to suppress I_Ca,L. The dashed lines on the records here and in other figures indicate zero-current levels.

Figure 2

I_K,tail–V relationships from myocytes treated with verapamil, D600, and E4031. The myocytes were bathed in normal Tyrode's solution, and tail currents on repolarizations to −40 mV after 500-ms pulses to potentials V were recorded before and 7–10 min after addition of drug. (a) Top: tail currents obtained from a myocyte that was sequentially exposed to 1, 5 and 20 μM verapamil, and then washed for 10 min with drug-free solution. The dotted line indicates steady current at −40 mV, and the calibration bars indicate 200 pA and 200 mS. Left hand plot: I_K,tail–V relationships obtained from measurements of the I_K,tail amplitudes. Right hand plot: verapamil-sensitive current obtained by subtracting drug data from control data. (b) Effects of 3 μM D600. Left: mean I_K,tail–V relationships from five experiments. Right: D600-sensitive current. The latter data are significantly different than zero (P<0.01) at all potentials ⩾−10 mV (one-way ANOVA and Dunnetts Multiple Comparison test). (c) Effects of the selective I_Kr inhibitor E4031 (3 μM) for comparison with those of the phenylalkylamines. Left: mean I_K,tail–V relationships from five experiments. Right: E4031-sensitive current. The latter data are significantly different than zero (P<0.01) at all potentials ⩾−10 mV.

Figure 3

Inhibition of I_Kr by verapamil and D600. Myocytes bathed in Tyrode's solution were held at −80 mV and depolarized for 200 ms from prepulse −40 mV to 0 mV at 0.1 Hz; I_Kr,tail was measured on the repolarizations to −40 mV. (a) Time course of changes in I_Kr,tail amplitude before, during and after exposure of a myocyte to 1 and 10 μM verapamil. (b) Effects of 10 μM D600 in a myocyte pretreated with 1 μM nisoldipine. Left: time course of changes in I_Kr,tail. Right: superimposition of currents elicited by the 200-ms pulses to 0 mV at the times marked on the time plot. Note the inhibition and recovery of outward current at 200 ms. (c) Dependence of I_Kr inhibition on drug concentration. The Hill equation fitting the verapamil data has an IC₅₀ of 2.6±0.2 μM and a Hill coefficient of 0.95 (solid curve); the parameters describing the D600 data are 2.7±0.2 μM and 0.96 (dashed curve), respectively. Myocytes were exposed to one or two concentrations of a drug for 6–10 min, and the numbers of observations ranged from 4 to 20.

Figure 4

Phenylalkylamine-induced inhibition of I_Ks in myocytes bathed with normal Tyrode's solution. The myocytes were held at −80 mV and depolarized from prepulse −40 mV to more positive potentials V for 500 ms at 0.1 Hz for measurement of I_K,tail on repolarizations to −40 mV. (a) The effect of 300 μM verapamil (n=5) on the I_K-tail–V relationship. (b) The effect of 1000 μM on the I_K,tail–V relationship. Also depicted are the measurements taken for calculation of the effect of drug on the amplitude of I_Ks. (c) Dependence of I_Ks inhibition on drug concentration. The Hill equation fitting the verapamil data has an IC₅₀ of 1080±240 μM and a coefficient of 0.87. Myocytes were exposed to one or two concentrations of a drug for 6–10 min, and the numbers of observations are shown in parentheses.

Figure 5

Time course and reversibility of phenylalkylamine-induced inhibition of I_Ks in myocytes bathed with K⁺-, Ca²⁺-free Cd²⁺ solution. (a) Inhibition by 300 μM D600. The myocytes were held at −30 mV and depolarized to +50 mV for 500 ms at 0.1 Hz. Left: changes in the amplitude of I_Ks,tail. Right: example records from this experiment; the records on the 500-ms pulses are (top to bottom) control, wash, and D600. (b) Inhibition by 200 and 500 μM verapamil. The records on the 500-ms pulses are (top to bottom) wash, control, 200 and 500 μM verapamil.

Figure 6

Phenylalkylamine concentration-dependent inhibition of I_Ks in myocytes bathed in K⁺-, Ca²⁺-free Cd²⁺ solution. The myocytes were held at −30 mV and depolarized to more positive potentials for 2-s at 0.1 Hz. (a,b) Records and I_K,tail–V relationships from representative experiments. The calibration bars beside the records indicate 200 pA and 200 ms. (c) Dependence of inhibition on drug concentration. The amplitude of I_Ks,tail elicited after 2-s depolarizations to +70 mV was measured before and 5 to 8 min after addition of drug. The Hill equation fitting the verapamil data has an IC₅₀ of 280±26 μM and a coefficient of 0.96. Myocytes were exposed to one or two concentrations of a drug for 6–10 min, and the numbers of observations are shown in parentheses.

Figure 7

Inhibition of I_K1 by phenylalkylamines. Myocytes were bathed in Tyrode's solution±0.2 mM Cd²⁺, and held at −80 mV. (a) Data from a myocyte treated with 300 μM verapamil for 9 min, Left: records obtained when the myocyte was pulsed from prepulse −40 mV to more positive and negative potentials. Right: end-of-pulse current amplitudes measured in this experiment. (b) Time courses of inhibition of outward I_K1 in representative experiments. (c) Dependence of inhibition of outward I_K1 at −40 mV on phenylalkylamine concentration. The Hill equation that describes the verapamil data has an IC₅₀ of 220±14 μM and a coefficient of 0.96. The numbers of myocytes are shown in parentheses.