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  • Inhibitors of the G rdos effect

    2022-05-13

    Inhibitors of the Gárdos effect include the Ca2+-channel blocker nitrendipine with an IC50 of ∼103nM (Ellory et al., 1992, Ellory et al., 1992); its mode of action is likely to be from direct blockage of K+ flux through the Gárdos channel (Ellory et al., 1992, Ellory et al., 1992), and the peptidic high-affinity scorpion venom-derived charbybdotoxin, which also blocks large-conductance Ca2+-activated K+ channels (Brugnara et al., 1993, Brugnara et al., 1993, Wolff et al., 1988). Volatile anaesthetics can inhibit the Gárdos effect in resealed erythrocytes in vitro (Caldwell & Harris, 1985) and IK currents in Xenopus oocytes expressing the channel protein (Namba et al., 2000). Halothane, enflurane, isoflurane and sevoflurane inhibit hIK1, but not the structurally related hSK2, at clinically relevant concentrations. Halothane has also been shown to inhibit the Gárdos channel in intact erythrocytes (Scharff & Foder, 1989). While the mechanism of anaesthesia is still poorly understood, the detailed effects of such agents on blood remains an important consideration. The desire for increased selectivity of channel blockers for IK channels over SK, BK and other types of K+ channels have resulted in the development of new types of non-peptidic inhibitors. Malik-Hall, Ganellin, Galanakis, and Jenkinson (2000) developed a set of non-quaternary and bisquaternary derivatives of dequalinium that inhibit IK channels with various levels of affinity. The mode of action of some types of blockers is related to their hydrophobicity, suggesting an internal block, or at least action at a site within the membrane. Of the known Gárdos channel blockers, one of the best-characterised is the anti-fungal agent clotrimazole. Its fungicidal activity derives from inhibition of cytochrome P-450-dependent reactions; but how does it inhibit the Gárdos channel? An initial report profiled the action of a set of cytochrome P-450 inhibitors on the Gárdos effect (Alvarez, Montero, & Garcia-Sancho, 1992), showing that clotrimazole decreases the magnitude of K+ loss following treatment with A23187. Subsequent treatment of the 6063 with valinomycin revealed that the inhibition is not due to blockage of the anion permeability. Gassing with CO (which competes with O2 for the active site of cytochrome P-450s) gives results that suggest that inhibition is due to a direct interaction with the channel. Analogous experiments proved that clotrimazole has the same effect on erythrocytes of sickle-cell patients (Brugnara et al., 1993, Brugnara et al., 1993). The same study also revealed an increase in the affinity of clotrimazole for its binding site in media of low-ionic strength (29 versus 51nM in normal saline). The ability to displace bound iodinated charybdotoxin (Wolff, Cecchi, Spalvins, & Canessa, 1988) re-affirmed that the inhibitory action is via direct blockage of the channel. Clotrimazole was identified as a potential ameliorator of the symptoms of sickle-cell anaemia, and early clinical trials of patients administered the drug exhibited reduced dehydration of mature erythrocytes obtained from them (Brugnara et al., 1995a, Brugnara et al., 1995b). Inside-out patch-clamping studies show that increasing the concentration of clotrimazole produces a different pattern of channel activity compared to reducing the intracellular calcium concentration, suggesting that it does not interfere with calcium binding to the channel (Dunn, 1998). The observed lack of voltage dependence for inhibition of the Gárdos channel by clotrimazole in this study implies that it does not bind in the channel pore. Recently, a new compound was designed with similar potency but much higher specificity than clotrimazole. “TRAM-34” (1-((2-chlorophenyl)diphenylmethyl)-1H-pyrazole) was one of 83 similar compounds synthesized and tested for their effect on Ca2+-activated K+ currents. It displays high inhibition of IK channels, with a much lower affinity for other types of K+ channels and almost no inhibitory action on mammalian cytochrome P-450. This study offers further insights into the structural moiety for optimal inhibition of the channel, i.e. a tri-phenyl group (with one of those phenyls being ortho-halogenated) connected to a π-electron-rich pyrazole or tetrazole.