M2GlyR is a synthetic 23-amino acid peptide that mimics the second membrane-spanning region of the alpha-subunit of the postsynaptic glycine receptor. This peptide has been shown to form an anion-selective channel in phospholipid bilayers. We have investigated the possibility that the peptide may incorporate into the apical membrane of secretory epithelia and induce the secretion of Cl- and water. We improved the solubility of this peptide by adding four lysine residues to the carboxy terminus, C-K4-M2GlyR, and assayed its channel-forming activity using a subculture of Madin-Darby canine kidney (MDCK) cells. The addition of 100 microM C-K4-M2GlyR to the apical surface of MDCK monolayers significantly increased short-circuit current (Ise), hyperpolarized transepithelial potential difference, and induced fluid secretion. The increase in Ise was inhibited by 100 microM bumetanide and by Cl- channel inhibitors. The effectiveness of the channel blockers followed the sequence niflumic acid > or = 5-nitro-2-(3-phenylpropylamino)benzoate > diphenylamine-2-carboxylate (DPC) > glibenclamide. The effect of the peptide was not inhibited by 4.4'-diisothiocyanostilbene-2-2'-disulfonic acid. Removing Cl from the bathing solutions also inhibited the effect of the peptide. The Cl- efflux pathway induced by C-K4-M2GlyR differs from the native pathway activated by the adenosine 3',5'-cyclic monophosphate (cAMP) agonist, forskolin. First, intracellular cAMP levels were unaffected. Second, the concentration of DPC required to inhibit the effect of the peptide was much lower than that needed to block the forskolin response (100 microM vs. 3 mM). These results support the hypothesis that the synthetic peptide C-K4-M2GlyR can from Cl -selective channels in the apical membrane of secretory epithelial cells and can induce sustained transepithelial secretion of Cl- and fluid.