Delineation of the clotrimazole/TRAM-34 binding site on the intermediate conductance calcium-activated potassium channel, IKCa1

Selective and potent triarylmethane blockers of the intermediate conductanc e calcium-activated potassium channel, IKCa1, have therapeutic use in sickl e cell disease and secretory diarrhea and as immunosuppressants. Clotrimazo le, a membrane-permeant triarylmethane, blocked IKCa1 with equal affinity w hen applied externally or internally, whereas a membrane-impermeant derivat ive TRAM-30 blocked the channel only when applied to the cytoplasmic side, indicating an internal drug-binding site. Introduction of the S5-P-S6 regio n of the triarylmethane-insensitive small conductance calcium-activated pot assium channel SKCa3 into IKCa1 rendered the channel resistant to triarylme thanes. Replacement of Thr(250) or Val(275) in IKCa1 with the cot-respondin g SKCa3 residues selectively abolished triarylmethane sensitivity without a ffecting the affinity of the channel for tetraethylammonium, charybdotoxin, and nifedipine. Introduction of these two residues into SKCa3 rendered the channel sensitive to triarylmethanes. In a molecular model of IKCa1, Thr(2 50) and Val(275) line a water-filled cavity just below the selectivity filt er. Structure-activity studies suggest that the side chain methyl groups of Thr(250) and Val(275) may lock the triarylmethanes in place via hydrophobi c interactions with the pi -electron clouds of the phenyl rings. The hetero cyclic moiety may project into the selectivity filter and obstruct the ion- conducting pathway from the inside.