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

Abstract

A synthetic Cl- channel-forming peptide, C-K4-M2GlyR, applied to the apical membrane of human epithelial cell monolayers induces transepithelial Cl- and fluid secretion. The sequence of the core peptide, M2GlyR, corresponds to the second membrane-spanning region of the glycine receptor, a domain thought to line the pore of the ligand-gated Cl- channel. Using a pharmacological approach, we show that the flux of Cl- through the artificial Cl- channel can be regulated by modulating basolateral K+ efflux through Ca2+-dependent K+ channels. Application of C-K4-M2GlyR to the apical surface of monolayers composed of human colonic cells of the T84 cell line generated a sustained increase in short-circuit current (I(SC)) and caused net fluid secretion. The current was inhibited by the application of clotrimazole, a non-specific 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 induced by C-K4-M2GlyR. The effect of the combination of C-K4-M2GlyR and 1-EBIO 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 also increased the magnitude of fluid secretion induced by the peptide. The cooperative action of C-K4-M2GlyR and 1-EBIO on I(SC) was attenuated by Cl- transport inhibitors, by removing Cl- from the bathing solution and by basolateral 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 coordinate the apical Cl- conductance and the basolateral K+ conductance, thereby providing a pharmacological approach to modulating Cl- and fluid secretion by human epithelia deficient in cystic fibrosis transmembrane conductance regulator Cl- channels. Copyright (C) 2000 Elsevier Science B.V.