H. Wulff et al., Delineation of the clotrimazole/TRAM-34 binding site on the intermediate conductance calcium-activated potassium channel, IKCa1, J BIOL CHEM, 276(34), 2001, pp. 32040-32045
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.