High affinity inhibition of Ca(2+)-dependent K+ channels by cytochrome P-450 inhibitors.

The Ca(2+)-dependent K+ channel of human red cells was inhibited with high affinity by several imidazole antimycotics which are potent inhibitors of cytochrome P-450. IC50 values were (in microM): clotrimazole, 0.05; tioconazole, 0.3; miconazole, 1.5; econazole, 1.8. Inhibition of the channel was also found with other drugs with known cytochrome P-450 inhibitory effect. However, no inhibition was obtained with carbon monoxide (CO). This suggests that, given the high selectivity of the above inhibitors for the heme moiety, a different but closely related to cytochrome P-450 kind of hemoprotein may be involved in the regulation of the red cell Ca(2+)-dependent K+ channel. Clotrimazole also inhibited two other charybdotoxin-sensitive Ca(2+)-dependent K+ channels, those of rat thymocytes (IC50 = 0.1-0.2 microM) and of Ehrlich ascites tumor cells (IC50 = 0.5 microM). Imidazole antimycotics inhibit also receptor-operated Ca2+ channels (Montero, M., Alvarez, J. and García-Sancho, J. (1991) Biochem. J. 277, 73-79). This suggests that both Ca2+ and Ca(2+)-dependent K+ channels might have a similar regulatory mechanism involving a cytochrome.

Imidazole antimycotics are a relatively new series of drugs with proved effectivity against a wide range of fungal pathogens. Some of them have found clinical use for the treatment of topical and systemic mycoses. Their fungistatic action is due to inhibition of sterol 14a-demethylase, a microsomal cytochrome P-450-dependent enzyme (1,2). Additionally, these drugs have also been shown to be potent inhibitors of many mammalian cytochrome P-450-mediated reactions (3-7). Structure-activity studies suggest that inhibition by these N1-substituted imidazoles results primarily from coordination of the NB of the imidazole with the fifth or sixth ligand of the heme iron of cytochrome P-450 (2, [6][7][8]. We have reported previously that imidazole antimycotics are potent inhibitors of the plasma membrane Ca2+ channels that are activated by emptying the intracellular Ca2+ stores in rat thymocytes (9). From these results we have proposed that a cytochrome P-450, sited at the Ca2+ stores, would mediate the activation of the plasma membrane Ca2+ channels when the stores are emptied (9). The same mechanism was found to be responsible for receptor-activated Ca2+ influx in human neutrophils (10) and platelets (11).
* This investigation was supported by Grant PB89-0359 from the Spanish Direccibn General de Investigacibn Cientifica y Tbcnica. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. We have also reported before that the Ca2+-dependent K+ channels of human red cells (12)(13)(14), rat thymocytes (15), and Ehrlich ascites tumor cells (16) are regulated by the redox state of a membrane component with an apparent standard redox potential of about 50 mV (13). Regulation of a different K+ channel by an hemoprotein has also been proposed recently in the carotid body chemoreceptor cells (17).
In this paper we find a potent inhibition of Ca2+-dependent K+ channels by imidazole antimycotics and other cytochrome P-450 inhibitors. These findings further support the involvement of a cytochrome in the regulation of Ca2+-dependent K+ channels and evidence striking similarities with the regulation of receptor-operated Ca2+ channels.

MATERIALS AND METHODS
Ca2+-dependent K' channels were activated in all the cases by increasing the cytoplasmic free calcium concentration ([Ca2+],)' by incubation of the cells with Ca" and a divalent cation ionophore. Either A23187 or ionomycin were used, and their concentrations were adjusted to give maximal activation of the channels.
In the human erythrocyte, the activity of the channels was estimated either from the net loss of K' from cells incubated in low K+ medium (using the light scattering procedure described below) or from the uptake of 42K, measured under equilibrium exchange conditions (18). For light scattering measurements (19), washed red cells were suspended at 3.3% hematocrit in a solution containing (in mM) NaC1, 150; MgC12, 0.2; CaC12, 0.66; K-HEPES, 10; p H 7.5. Immediately before the experiment 1.2 ml of this cell suspension were mixed with 0.4 ml of a solution containing (in mM): NaSCN, 150; MgC12, 0.2; K-HEPES, 10; pH 7.5. The experiment was then started by the addition of 2 ~1 of a 2 mM solution of the Ca2+ ionophore A23187 in ethanol. The changes in cell volume were followed by recording the changes of the transmittance of the cell suspension at 650 nm. The permeant anion SCN-was included to accelerate the rate of K+ loss, which is otherwise limited by the electrogenic permeability to C1-(19, 20).
For 42K+ exchange measurements, washed red cells were incubated a t 10% hematocrit in a medium containing (in mM): NaC1, 75; KCl, 75; MgC12, 0.2; CaC12, 0.1; K-HEPES, 10; pH 7.5, and tracer amounts of "K. Then A23187 was added to give a final concentration of 10 p~ and, at different times, 0.1-ml samples of the cell suspension were mixed with 1 ml of ice-cold incubation medium containing 1 mM quinine and centrifuged immediately over dibuthylphtalate oil in the microfuge as described before (21). Pellets were extracted with 6% trichloroacetic acid and radioactivity was quantified by scintillation counting.
Rat thymocytes were prepared as described before (22) and suspended at 5% cytocrit in a medium containing (in mM): NaC1, 150; MgC12, 1; CaC12, 1; K-HEPES, 10; glucose, 10; pH 7.5. The activity of the Ca2+-dependent K+ channels was estimated from the uptake of 42K induced by increasing the intracellular Ca2+ concentration with the Ca2+ ionophore A23187. The experiments were started by adding a tracer amount of 42K 30 s after the addition of 10 p~ A23187. Then, a t different times, 0.1-ml samples of the cell suspension were mixed ' The abbreviations used are: [Ca2+Iz, cytoplasmic free Ca2+ concentration; EGTA, [ethylenebis(oxyethylenenitrilo)]tetraacetic acid HEPES, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid. 11789 in 1.5-ml Eppendorf tubes with 1 ml of ice-cold incubation medium containing 1 mM EGTA (instead of CaC12) and 1 mM quinine, centrifuged in the microfuge, and washed once more with the same medium. Radioactivity in the pellets was measured as described above.
NADH-ferricyanide and NADH-cytochrome c reductase activities were measured as described previously (25,26) in membranes prepared from human red cells by hypotonic hemolysis (27).
Ionophore A23187 and ionomycin were obtained from Boehringer Mannheim GmbH and Calbiochem, respectively. "K was produced using a generator provided by Dr. H. Stirner, Technische Universitat, Munchen, Germany. Purified charybdotoxin was kindly provided by Dr. Guillermo Gimenez-Gallego, Centro de Investigaciones Biol6gicas, Madrid, Spain. Tioconazole and the compounds SK&F96365 and SK&F525A were kindly provided by Pfizer Central Research, Sandwich, United Kingdom (U. K.) and Dr. J. E. Merritt, Smith Kline & French Research Ltd., The Frythe, Welwyn, Herts, U. K., respectively. Other chemicals were obtained either from Sigma London, Poole, Dorset, U. K., Aldrich-Chemie, Steinheim, Germany, BioMol Research Laboratories, Plymouth Meeting, PA, or E. Merck, Darmstadt, Germany. . 1 shows the effects of different concentrations of the imidazole antimycotic clotrimazole on the activity of the Ca2+dependent K' channel of human red cells, measured by the light scattering technique (see "Materials and Methods"). The increase of [Ca2+]i produced on addition of the ionophore A23187 induced an immediate loss of KC1 as evidenced by cell shrinkage (decrease of Tsso), which reached a plateau within about 3 min. Clotrimazole produced a concentrationdependent inhibition of the shrinkage with half-maximal effect (Ic60) at about 0.05 PM. Addition of the K+ ionophore valinomycin at the end of every experiment (only shown in the figure for the largest concentration of the inhibitor) produced cell shrinkage, indicating that the inhibition by clotrimazole was not due to a blockade of the anion permeability.

Fig
The effects of clotrimazole on K' uptake were also studied. These experiments were performed under equilibrium exchange conditions (18), in order to avoid interferences due to possible effects of the drug on the anion permeability or on the membrane potential. Again clotrimazole produced a concentration-dependent inhibition of the uptake of 42K (Fig. 2, left panel). The estimated ICs0 was 0.1 PM, a value somewhat larger than the one obtained in the light scattering experi- Time (min) FIG. 1. Effect of clotrimazole on the Ca2+-dependent K+ channel of human red cells measured by the light scattering technique. Different concentrations of clotrimazole, as stated on the right of each trace in p~, were added to the cell suspension 1 min before the addition of 2.5 p~ A23187 (at t = 0, shown by the arrow). The second arrow indicates the addition of 2 p M valinomycin. ments described above. This difference may be due to the higher hematocrit used in the 42K uptake experiments (10% versus 2.5% in the light scattering experiments).
The right punel of Fig. 2 illustrates, for comparison purposes, the inhibition produced by two concentrations, 20 and 200 nM, of charybdotoxin, a peptidic toxin which inhibits several kinds of Ca2+-dependent K+ channels, including these of red cells (28,29). Inhibition of the Ca2+-dependent K+ channel of human red cells by charybdotoxin has been reported recently to be only partial (73-77%), although a maximal dose of only 20 nM was used (28). In our hands, 200 nM charybdotoxin produced up to a 95% inhibition, confirming previous results with the whole venom of Leiurus quinquiestriutus (29). In additional experiments (not shown), both clotrimazole and charybdotoxin were found to have little effect on the shrinkage induced by the K' ionophore valinomycin (24% inhibition by 2 PM clotrimazole and 17% inhibition by 200 nM charybdotoxin), indicating that they do not interact with this ionophore, nor with the anion permeability. The above results, obtained with two different techniques, confirm that clotrimazole is acting selectively on the Ca2+dependent K+ channels.
A screening of the effects of many other cytochrome P-450 inhibitors on Ca2+-dependent K+ transport by red cells was performed using the light scattering procedure.  (30). This drug is, as the imidazole antimycotics, a N1-substituted imidazole and probably acts through the same mechanism (11). Table I also includes the lipoxygenase inhibitors nordihydroguaiaretic acid, eicosatetraynoic acid, and gossypol, which have also been found to inhibit cytochrome P-450 (11,31) and receptor-operated Ca2+ channels (9-11). Finally, Table I also shows the values obtained with the classical cytochrome P-450 inhibitors metyrapone, compound SKF525A, piperonyl butoxide, and anaphthoflavone (32). It can be observed again that the patterns for inhibition of Ca" channels and Ca2+-dependent K+ channels were different. Table I1 shows the effects of a series of compounds that have been used before as specific inhibitors of particular types of cytochrome P-450 (32). Even though some of the tested compounds were active, the results did not allow the transport activity to relate consistently to any particular cytochrome P-450 subtype.
CO competes with O2 for the active site of cytochrome P-450 and blocks the oxidative activity. The effects of CO on Ca2+-dependent K+ uptake by red cells were studied in experiments with the same design as those of Fig. 2 but gassing with CO 15 min before and during the experiment. Treatment with CO had no significant effect on Ca2+-dependent K+ transport (results not shown). These results should be interpreted with some caution since the high concentration of hemoglobin within the red cell could interfere with the binding of CO to another target. Nevertheless, the simplest interpretation of these results is that the target for inhibition of Ca2+dependent K+ transport may be not a cytochrome P-450 but a closely related hemoprotein not sensitive to CO. In relation to this point, we have also studied the effect of clotrimazole The inhibitors were added, at the concentrations indicated on the right of each trace in PM, 1 min before the addition of 10 +M A23187 at t = 0. 42K uptake in the control reached a stable maximum level (42Km) after about 30 min. Uptake data fitted adequately to an exponential and were linearized by a logarithmic transformation as shown in the figure.
'At 2 mM inhibition was 30%. ' At 100 L(M inhibition was 25%. on two red cell plasma membrane oxydoreductase activities, NADH-ferricyanide reductase and NADH-cytochrome c reductase. However, none of these activities was affected by clotrimazole (results not shown). In order to investigate whether Ca2+-dependent K+ channels of other cell types were also blocked by cytochrome P-450 inhibitors, we studied the effects of clotrimazole on Ca2+dependent K' transport in rat thymocytes and in Ehrlich ascites tumor cells. In thymocytes (Fig. 3), the increase of [Ca"], induced by the Ca2+ ionophore A23187 accelerated about 13-fold the initial rate of 42K uptake (truce labeled Control) with respect to cells not treated with A23187 (trace labeled no A23187).* Clotrimazole produced a concentration-' We have shown before that charybdotoxin does not interfere with A23187-induced Ca" influx (29). In experiments with fluo-3-loaded thymocytes suspended in Ca2+-containing medium we find that 2 +M clotrimazole does not significantly modify the increase of [Caz+], induced by either 10 PM A23187 or 1 PM ionomycin (results not shown).

Effect of seueral drugs with specific activity on several cytochrome P -450 subtypes on the human red cell Ca2+-dependent K' channel
The values preceded by > > mean that no inhibition was detected at this concentration.   Fig. 4 shows the effects of clotrimazole on Caz+-dependent K' transport in Ehrlich ascites tumor cells, measured by the light scattering technique (24, see also "Materials and Methods"). In the Ehrlich cell addition of ionomycin induces a transient increase in [Ca"], which produces a simultaneous activation of Ca2+-dependent K+ and C1-channels, leading to cell shrinkage by KC1 loss (16,33,34). Under these conditions, the C1-permeability appears to be increased more than the K' permeability (33,34) and, therefore, the rate of cell shrinkage is limited by the K+ permeability. The cell shrinkage induced by ionomycin is fast (see curue labeled control), and is followed in less than 1 min by a slower return to the initial volume by activation of a Na'/Cl-cotransport system (33)(34)(35). Clotrimazole produced a concentration-dependent inhibition of the shrinkage induced by ionomycin with an IC50 value of about 0.5 p~. The inhibitory effect of the peptidic toxin charybdotoxin is also shown in the figure for comparison purposes.

DISCUSSION
We describe here the effects of a new family of Ca2+dependent K' channels blockers, the imidazole antimycotics. Among them clotrimazole is the most potent non-peptidic inhibitor of the red cell Ca2'-dependent K+ channel found up to date, with an IC50 1-2 orders of magnitude lower that quinine (ICbo = 5 p~, Ref. 36) and the Di-S-C2 carbocyanine dye (ICEo = 0.7 p~, Ref. 37). Two other kinds of charybdotoxin-sensitive Ca2+-dependent K+ channels tested, those of rat thymocytes and of Ehrlich ascites tumor cells, were also sensitive to clotrimazole. Charybdotoxin is a peptide venom widely known as inhibitor of the "maxi K" (conductance, 150-250 pS) Ca2'-dependent K' channel (38-40), but it also inhibits the low conductance (18-36 pS) Ca2'-dependent K' channels of human red cells (28,29,41), lymphoid cells (29,42,43), human macrophages (44), and Aplysiu neurons (45). Ca2+-dependent K' channels of Ehrlich ascites tumor cells (16) are also sensitive to charybdotoxin (29), but their conductance properties have not been studied. The results presented here show that clotrimazole inhibits the charybdotoxin-sensitive low conductance channels of red cells (Figs. 1 and 2) and thymocytes (Fig. 3) and the charybdotoxin-sensitive channel of the Ehrlich cell (Fig. 4). All these channels are insensitive to the bee venom apamin (29). In addition to imidazole antimycotics, many other cytochrome P-450 inhibitors were able to block Ca2+-dependent K+ channels with reasonable affinity (Tables I and 11). However, we did not find inhibition by CO, suggesting that the mediator involved in the control of the channel may be not a cytochrome P-450 but a closely related hemoprotein. Although cytochrome P-450 has not been detected previously in human red cells (46,47), it has been reported that oxyhemoglobin can act similarly, exhibiting aniline hydroxylase activity and other similarities with cytochrome P-450 in the presence of NADPH (48, 49). However, the involvement of hemoglobin in the clotrimazole-sensitive activation of human red cells Ca2'-dependent K' channels is not likely because (i) hemoglobin has a high affinity for CO and CO does not inhibit the channels, and (ii) the hemoglobin concentration in our experiments is about 3 orders of magnitude above the ICso of clotrimazole. Other components analogous to the microsomal electron transport system have been found in red cells. Cytochrome P-420, usually regarded as a protease degradation product of cytochrome P-450 (50), and cytochrome b5 are present in the red cell membrane (50, 51). A cytoplasmic cytochrome bs and cytochrome 6 5 reductase with methemoglobin reductase activity have also been described (52, 53). Cytochrome b5 is not sensitive to CO, and a form of cytochrome P-420 that does not bind CO has been described (47,54). Any of these hemoproteins could be related to the inhibitory effects of the imidazole antimycotics and other cytochrome P-450 inhibitors reported in this paper and to the redox modulation of the Ca2+-dependent K channels reported previously (see below).
We have reported before that the Ca2+-dependent K' channels of human red cells (12-14), rat thymocytes (15), and Ehrlich ascites tumor cells (16) are regulated by the redox state of a membrane component with an apparent standard redox potential of about 50 mV (13). This value is compatible with a b-type cytochrome or a flavin (13). Ca2+-dependent K' channel activity is easily lost in excised membrane patches of rat thymocytes. This has been attributed to wash-out of an essential intracellular component (42,43). Loss of Ca2' sensitivity of K' channels had been observed before in insideout vesicles (55-58) and ghosts (59) of red cells and in insideout vesicles (60) of thymocytes. In red cell ghosts the responsiveness to Ca2+ was restored on addition of an electron donor system (59). This suggests that inactivation might occur by modification of the redox state of a membrane component, which regulates the activity of the channel, as a result of the loss of a cytoplasmic component. In a previous study we found that there is no correlation between plasma membrane NADH-cytochrome c and NADH-ferricyanide reductase activities and the Ca2'-dependent K' channel activity in red cells of several animal species (26). We now find that clotrimazole has no effect on these oxydoreductase activities.
We have reported previously that imidazole antimycotics block the plasma membrane Ca2+ channels activated by emptying the intracellular Ca2' stores, either by cell agonists or by other means, in several cell lines. In those cases CO was an efficient inhibitor of the Ca2+ channels, so that the involvement of cytochrome P-450 was suggested (9-11). The pattern for inhibition of the Ca2+ channels by imidazole antimycotics was very different to the one found here for the Ca2+-dependent K+ channels (Table I). While econazole and miconazole were the best inhibitors of the Ca2+ channels, with an ICs0 about 1 order of magnitude lower than clotrimazole, here clotrimazole was more than 1 order of magnitude more effective than econazole and miconazole. This indicates that the interaction of the inhibitor with its target is slightly different for each kind of channel and suggests that a study of closely related structures could lead to find a very specific inhibitor for either Ca2+ or Ca2+-dependent K' channels. On the other hand, in spite of these differences, the fact that both kinds of channels can be inhibited by cytochrome ligands suggests a surprising similarity in their structure or in their mechanism of activation. This similarity is reminiscent of the close structural homology recently found between Ca", K' , and Na' channels (61).