Differences in the actions of some blockers of the calcium-activated potassium permeability in mammalian red cells

Abstract

1. The actions of some inhibitors of the Ca2+-activated K+ permeability in mammalian red cells have been compared. 2. Block of the permeability was assessed from the reduction in the net loss of K+ that followed the application of the Ca2+ ionophore A23187 (2 microM) to rabbit red cells suspended at a haematocrit of 1% in a low potassium solution ([K]0 0.12-0.17 mM) at 37 degrees C. Net movement of K+ was measured using a K+-sensitive electrode placed in the suspension. 3. The concentrations (microM +/- s.d.) of the compounds tested causing 50% inhibition of K+ loss were: quinine, 37 +/- 3; cetiedil, 26 +/- 1; the cetiedil congeners UCL 1269, UCL 1274 and UCL 1495, approximately 150, 8.2 +/- 0.1, 0.92 +/- 0.03 respectively; clotrimazole, 1.2 +/- 0.1; nitrendipine, 3.6 +/- 0.5 and charybdotoxin, 0.015 +/- 0.002. 4. The characteristics of the block suggested that compounds could be placed in two groups. For one set (quinine, cetiedil, and the UCL congeners), the concentration-inhibition curves were steeper (Hill coefficient, nH, > or = 2.7) than for the other (clotrimazole, nitrendipine, charybdotoxin) for which nH approximately 1. 5. Compounds in the first set alone became less active on raising the concentration of K+ in the external solution to 5.4 mM. 6. The rate of K+ loss induced by A23187 slowed in the presence of high concentrations of cetiedil and its analogues, suggesting a use-dependent component to the inhibitory action. This was not seen with clotrimazole. 7. The blocking action of the cetiedil analogue UCL 1274 could not be overcome by an increase in external Ca2+ and its potency was unaltered when K+ loss was induced by the application of Pb2+ (10 microM) rather than by A23187. 8. These results, taken with the findings of others, suggest that agents that block the red cell Ca2+-activated K+ permeability can be placed in two groups with different mechanisms of action. The differences can be explained by supposing that clotrimazole and charybdotoxin act at the outer face of the channel whereas cetiedil and its congeners may block within it, either at or near the K+ binding site that determines the flow of K+.

Figure 1

The dependence on preincubation time of the P_K(Ca) blocking action of nitrendipine, cetiedil and two analogues of cetiedil, UCL 1269 and UCL 1274. Each compound was incubated with a suspension of rabbit red blood cells for the periods indicated (abscissa) prior to adding the Ca²⁺-ionophore A23187 to the suspension in order to initiate K⁺ loss through Ca²⁺-activated K⁺ channels. The amount of K⁺ lost was measured using a K⁺-sensitive electrode placed in the suspension (see also Figure 4a). The total K⁺ loss 3 min after applying A23187 in the presence of the test compound was expressed as a percentage of the loss in its absence (ordinate). Each point is the mean of 3–6 observations and the error bars indicate the s.e.mean. For cetiedil, UCL 1269 and UCL 1274 the curves have been drawn by fitting the expression y=y_max (1−e^−kt), where y is percentage inhibition, k is a rate constant and t is time (see also Table 1). The onset of the action of nitrendipine was too rapid to be resolved by present technique and the broken line has been constructed using a value of k of 7 min⁻¹, to indicate a lower limit.

Figure 2

The concentration dependence of the inhibitory actions of clotrimazole, nitrendipine quinine, cetiedil, UCL 1274 and UCL 1495 on A23187-induced K⁺ loss from rabbit red cells bathed in a low K⁺ solution. The preincubation times were 60 min for cetiedil, UCL 1274 and UCL 1495, and 3 min for quinine, clotrimazole and nitrendipine. Each point is the mean of 3–8 observations and the error bars show the s.e.means. The lines have been drawn according to the Hill equation, using the parameter values listed in Table 2.

Figure 3

The effect of increasing the external concentration of K⁺ on the blocking action of (a) cetiedil, (b) UCL 1495, (c) charybdotoxin and nitrendipine, (d) clotrimazole. Each point is the mean of 3–8 observations and the error bars show the s.e.mean. The cells were incubated with the blocking agents for 1 h before the application of A23187 to initiate K⁺ loss, except for nitrendipine and clotrimazole which were applied 3 min beforehand.

Figure 4

(a) Records showing the loss of K⁺ from a suspension of rabbit erythrocytes exposed to A23187 (2 μM) either alone (i) or in the presence of either 50 μM cetiedil (ii) or 3 μM clotrimazole (iii). A23187 was applied at the first of each pair of arrows, followed ∼3 min later (second arrow) by digitonin (100 μM) to cause lysis and so allow the total K⁺ content of the cells to be determined. Cetiedil and clotrimazole were added 3 min prior to A23187. Because the absolute value of the initial and final K⁺ concentration varied a little, the traces have been scaled to the same amplitude and accordingly the ordinate shows K⁺ loss as a percentage of the total K⁺ content, as measured following the addition of digitonin. Typically, the concentration of K⁺ in the suspension rose from ∼0.15 (before A23187) to 0.8 mM (after digitonin). The nonlinearity of the ordinate scale reflects mainly the logarithmic relationship between [K⁺] and the potential measured by the K⁺-sensitive electrode used to detect the net movement of K⁺ between the erythrocytes and the solution in which they were suspended. (b) Averaged results. The values are the means of six observations for the control and three for the test compounds. Vertical bars indicate the s.e.mean.

Figure 5

(a) The time course of changes in the rate coefficient (ordinate: note log scale) for A23187-induced K⁺ loss from erythrocytes in the absence (n=6) or presence of either 50 μM cetiedil (n=3) or 3 μM clotrimazole (n=3). Abscissa: time after the introduction of A23187. (b) The ratios of the rate coefficients at 70 and 10 s after applying A23187. Results are shown for clotrimazole (clot, μM concentrations) and cetiedil (cet, μM concentrations). This ratio becomes smaller as the concentration of cetiedil is increased. ^* denotes significance at the 5% level as indicated by an unpaired t-test.

Figure 6

The concentration dependence of the effect of clotrimazole and cetiedil on the rate coefficients for K⁺ loss measured at 10 s (filled symbols) and 170 s (open symbols) after the application of A23187. The values plotted are the coefficients in the presence of the blocker expressed as a percentage of the corresponding values in its absence. Vertical bars indicate the s.e.mean (n=3). The lines have been drawn according to the Hill equation (see text).

Figure 7

(a) Inhibition by UCL 1274 of K⁺ loss from rabbit erythrocytes exposed to either A23187 (2 μM) or Pb²⁺ (10 μM). Each point is the mean of 3–4 observations and the vertical bars indicate the s.e.mean. (b) The relationship between the concentration of external Ca²⁺ and the release of K⁺ in response to A23187 (2 μM) applied in the absence or presence of UCL 1274 (6 μM). The erythrocytes were suspended in solutions containing buffered Ca²⁺ at the concentrations shown on the abscissa. The lines have been drawn according to the Hill equation (E_max=69±3 and 39±2%, and n_H=1.9±0.3 and 2.6±1.1, in the absence and presence respectively of UCL 1274).