A synthetic channel-forming peptide induces Cl- secretion: modulation by Ca2+-dependent K+ channels

A synthetic Cl- channel-forming peptide, C-K4-M2GlyR, applied to the apical membrane of human epithelial cell monolayers induces transepithelial Cl- a nd fluid secretion. The sequence of the core peptide, M2GlyR, corresponds t o the second membrane-spanning region of the glycine receptor, st domain th ought to line the pore of the ligand-gated Cl- channel. Using a pharmacolog ical approach, we show that the flux of Cl- through the artificial Cl- chan nel can be regulated by modulating basolateral K+ efflux through Ca2+-depen dent K+ channels. Application of C-K4-M2GlyR to the apical surface of monol ayers composed of human colonic cells of the T84 cell line generated a sust ained increase in short-circuit current (Isc) and caused net fluid secretio n. The current was inhibited by the application of clotrimazole, a non-spec ific inhibitor of K+ channels, and charybdotoxin, a potent inhibitor of Ca2 +-dependent K+ channels. Direct activation of these channels with 1-ethyl-2 -benzimidazolinone (1-EBIO) greatly amplified the Cl- secretory current ind uced by C-K4-M2GlyR. The effect of the combination of C-K4-M2GlyR and 1-EBI O on I-SC was significantly greater than the sum of the individual effects of the two compounds and was independent of cAMP. Treatment with 1-EBIO als o increased the magnitude of fluid secretion induced by the peptide. The co operative action of C-K4-M2GIyR and 1-EBIO on Ise was attenuated by Cl- tra nsport inhibitors, by removing Cl- from the bathing solution and by basolat eral treatment with K+ channel blockers. These results indicate that apical membrane insertion of Cl- channel-forming peptides such as C-K4-M2GlyR and direct activation of basolateral K+ channels with benzimidazolones may coo rdinate the apical Cl- conductance and the basolateral K+ conductance, ther eby providing a pharmacological approach to modulating Cl- and fluid secret ion by human epithelia deficient in cystic fibrosis transmembrane conductan ce regulator Cl- channels. (C) 2000 Elsevier Science B.V. All rights reserv ed.