title: A synthetic channel-forming peptide induces Cl(-) secretion: modulation by Ca(2+)-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 Ca(2+)-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 Ca(2+)-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.
authors.affiliation: Department of Molecular and Integrative Physiology, University of Kansas Medical Center, 3901 Rainbow Blvd., Kansas City, KS 66160-7401, USA. dwallace@kumc.edu
authors.forename: D P
authors.initials: DP
authors.lastname: Wallace
authors.forename: J M
authors.initials: JM
authors.lastname: Tomich
authors.forename: J W
authors.initials: JW
authors.lastname: Eppler
authors.forename: T
authors.initials: T
authors.lastname: Iwamoto
authors.forename: J J
authors.initials: JJ
authors.lastname: Grantham
authors.forename: L P
authors.initials: LP
authors.lastname: Sullivan
mesh_terms: D000595:Amino Acid Sequence; D001562:Benzimidazoles / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002120:Calcium Channel Agonists / Q000494:pharmacology; D002460:Cell Line; D018118:Chloride Channels / Q000378:metabolism*; D002712:Chlorides / Q000037:antagonists & inhibitors / Q000378:metabolism; D003022:Clotrimazole / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D003550:Cystic Fibrosis / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004347:Drug Interactions; D004553:Electric Conductivity; D004847:Epithelial Cells / Q000187:drug effects* / Q000378:metabolism; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins; D008969:Molecular Sequence Data; D010455:Peptides / Q000494:pharmacology*; D026902:Potassium Channel Blockers; D015221:Potassium Channels / Q000378:metabolism
chemical_list: D001562:Benzimidazoles; D002120:Calcium Channel Agonists; D018118:Chloride Channels; D002712:Chlorides; D036341:Intercellular Signaling Peptides and Proteins; C111087:K4-M2GlyR protein, synthetic; D010455:Peptides; D026902:Potassium Channel Blockers; D015221:Potassium Channels; D000242:Cyclic AMP; D003022:Clotrimazole; C102635:1-ethyl-2-benzimidazolinone; D002118:Calcium