title: The Epac1 signaling pathway regulates Cl- secretion via modulation of apical KCNN4c channels in diarrhea.
abstract: The apical membrane of intestinal epithelia expresses intermediate conductance K(+) channel (KCNN4), which provides the driving force for Cl(-) secretion. However, its role in diarrhea and regulation by Epac1 is unknown. Previously we have established that Epac1 upon binding of cAMP activates a PKA-independent mechanism of Cl(-) secretion via stimulation of Rap2-phospholipase Cε-[Ca(2+)]i signaling. Here we report that Epac1 regulates surface expression of KCNN4c channel through its downstream Rap1A-RhoA-Rho-associated kinase (ROCK) signaling pathway for sustained Cl(-) secretion. Depletion of Epac1 protein and apical addition of TRAM-34, a specific KCNN4 inhibitor, significantly abolished cAMP-stimulated Cl(-) secretion and apical K(+) conductance (IK(ap)) in T84WT cells. The current-voltage relationship of basolaterally permeabilized monolayers treated with Epac1 agonist 8-(4-chlorophenylthio)-2'-O- methyladenosine 3',5'-cyclic monophosphate showed the presence of an inwardly rectifying and TRAM-34-sensitive K(+) channel in T84WT cells that was absent in Epac1KDT84 cells. Reconstructed confocal images in Epac1KDT84 cells revealed redistribution of KCNN4c proteins into subapical intracellular compartment, and a biotinylation assay showed ∼83% lower surface expression of KCNN4c proteins compared with T84WT cells. Further investigation revealed that an Epac1 agonist activates Rap1 to facilitate IK(ap). Both RhoA inhibitor (GGTI298) and ROCK inhibitor (H1152) significantly reduced cAMP agonist-stimulated IK(ap), whereas the latter additionally reduced colocalization of KCNN4c with the apical membrane marker wheat germ agglutinin in T84WT cells. In vivo mouse ileal loop experiments showed reduced fluid accumulation by TRAM-34, GGTI298, or H1152 when injected together with cholera toxin into the loop. We conclude that Rap1A-dependent signaling of Epac1 involving RhoA-ROCK is an important regulator of intestinal fluid transport via modulation of apical KCNN4c channels, a finding with potential therapeutic value in diarrheal diseases.
authors.affiliation: Division of Molecular Pathophysiology, National Institute of Cholera and Enteric Diseases, P-33 CIT Road, Scheme-XM, Beliaghata, Kolkata 700010, India.
authors.forename: Irshad Ali
authors.initials: IA
authors.lastname: Sheikh
authors.forename: Hemanta
authors.initials: H
authors.lastname: Koley
authors.forename: Manoj K
authors.initials: MK
authors.lastname: Chakrabarti
authors.forename: Kazi Mirajul
authors.initials: KM
authors.lastname: Hoque
mesh_terms: D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D015153:Blotting, Western; D045744:Cell Line, Tumor; D002712:Chlorides / Q000378:metabolism*; D002772:Cholera Toxin; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000494:pharmacology; D003967:Diarrhea / Q000139:chemically induced / Q000378:metabolism*; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism; D020662:Guanine Nucleotide Exchange Factors / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D051660:Intermediate-Conductance Calcium-Activated Potassium Channels / Q000037:antagonists & inhibitors / Q000378:metabolism*; D007413:Intestinal Mucosa / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D018613:Microscopy, Confocal; D026902:Potassium Channel Blockers / Q000494:pharmacology; D011720:Pyrazoles / Q000494:pharmacology; D034622:RNA Interference; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000187:drug effects; D020668:rap GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism; D020827:rap1 GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism; D054460:rho-Associated Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D020742:rhoA GTP-Binding Protein / Q000037:antagonists & inhibitors / Q000378:metabolism
chemical_list: C459092:2-methyl-1-((4-methyl-5-isoquinolinyl)sulfonyl)homopiperazine; D002712:Chlorides; D020662:Guanine Nucleotide Exchange Factors; D051660:Intermediate-Conductance Calcium-Activated Potassium Channels; C496923:KCNN4 protein, human; D026902:Potassium Channel Blockers; D011720:Pyrazoles; C430803:RAP1A protein, human; C116122:RAPGEF3 protein, human; C411671:TRAM 34; C066737:RHOA protein, human; D005576:Colforsin; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D002772:Cholera Toxin; D000242:Cyclic AMP; D054460:rho-Associated Kinases; C116753:RAP1B protein, human; D020668:rap GTP-Binding Proteins; D020827:rap1 GTP-Binding Proteins; D020742:rhoA GTP-Binding Protein
keywords: Chloride Channels; Chloride Transport; Cholera Toxin; Cl- Secretion; Cyclic AMP (cAMP); Diarrhea; Epac1; KCNN4c; Potassium Transport; Rap1a