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

AbstractA 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 (ISC) 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 ISC 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 ISC 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