Inhibition of intestinal Cl- secretion by clotrimazole: direct effect on basolateral membrane K+ channels

Abstract

We evaluated the effects of clotrimazole and clofibrate on Ca(2+)- and adenosine 3',5'-cyclic monophosphate (cAMP)-mediated Cl- secretion in the colonic cell line, T84. We used 1-ethyl-2-benzimidazolinone (1-EBIO) to activate the Ca(2+)-dependent K+ channel (KCa) in these cells to induce a sustained Cl- secretory current (Isc). Clotrimazole potently inhibited the KCa-dependent Isc, with an inhibition constant (Ki) of 0.27 +/- 0.02 microM. Clofibrate also inhibited the 1-EBIO-induced Isc albeit with lower affinity (Ki = 6.5 +/- 1.2 microM). Clotrimazole (10 microM) inhibited the Isc response to the Ca(2+)-mediated agonist, carbachol, by 82%. Similarly, both clotrimazole and clofibrate inhibited cAMP-mediated Cl- secretion, with Ki values of 5.2 +/- 1.0 and 6.7 +/- 1.1 microM, respectively. We used nystatin to permeabilize the apical or basolateral membrane to determine the effects of clotrimazole and clofibrate on the basolateral K+ (IK) and apical Cl- (ICl) currents following stimulation by either 1-EBIO or forskolin. Both clotrimazole and clofibrate inhibited the 1-EBIO- and forskolin-induced IK without affecting ICl. We determined the effects of clotrimazole and clofibrate on KCa using 86Rb+ uptake studies into membrane vesicles. Both clotrimazole and clofibrate inhibited the 1-EBIO-induced 86Rb+ uptake, with Ki values of 0.31 +/- 0.08 and 10.8 +/- 5.5 microM, respectively. Similarly, clotrimazole inhibited the Ca(2+)-induced 86Rb+ uptake with a Ki of 0.51 +/- 0.15 microM. Charybdotoxin inhibited both the 1-EBIO- and Ca(2+)-induced 86Rb+ uptakes with similar affinities (Ki values of 0.57 +/- 0.07 and 0.47 +/- 0.08 nM, respectively), suggesting 1-EBIO and Ca2+ activate the same channel (KCa) in this assay. In excised, single-channel recordings both clotrimazole and clofibrate inhibited KCa, demonstrating a direct inhibition of the channel by these compounds. We demonstrate that clotrimazole blocks the intestinal KCa, thereby inhibiting Cl- secretion. These results suggest that clotrimazole may be useful as an antidiarrheal.