title: Characteristics of Kcnn4 channels in the apical membranes of an intestinal epithelial cell line.
abstract: Intermediate-conductance K(+) (Kcnn4) channels in the apical and basolateral membranes of epithelial cells play important roles in agonist-induced fluid secretion in intestine and colon. Basolateral Kcnn4 channels have been well characterized in situ using patch-clamp methods, but the investigation of Kcnn4 channels in apical membranes in situ has been hampered by a layer of mucus that prevents seal formation. In the present study, we used patch-clamp methods to characterize Kcnn4 channels in the apical membrane of IEC-18 cells, a cell line derived from rat small intestine. A monolayer of IEC-18 cells grown on a permeable support is devoid of mucus, and tight junctions enable selective access to the apical membrane. In inside-out patches, Ca(2+)-dependent K(+) channels observed with iberiotoxin (a Kcnma1/large-conductance, Ca(2+)-activated K(+) channel blocker) and apamin (a Kcnn1-3/small-conductance, Ca(2+)-activated K(+) channel blocker) present in the pipette solution exhibited a single-channel conductance of 31 pS with inward rectification. The currents were reversibly blocked by TRAM-34 (a Kcnn4 blocker) with an IC(50) of 8.7 ± 2.0 μM. The channels were not observed when charybdotoxin, a peptide inhibitor of Kcnn4 channels, was added to the pipette solution. TRAM-34 was less potent in inhibiting Kcnn4 channels in patches from apical membranes than in patches from basolateral membranes, which was consistent with a preferential expression of Kcnn4c and Kcnn4b isoforms in apical and basolateral membranes, respectively. The expression of both isoforms in IEC-18 cells was confirmed by RT-PCR and Western blot analyses. This is the first characterization of Kcnn4 channels in the apical membrane of intestinal epithelial cells.
authors.affiliation: Department of Biochemistry, West Virginia Univ. School of Medicine, Morgantown, 26506, USA.
authors.forename: Kanthesh M
authors.initials: KM
authors.lastname: Basalingappa
authors.forename: Vazhaikkurichi M
authors.initials: VM
authors.lastname: Rajendran
authors.forename: William F
authors.initials: WF
authors.lastname: Wonderlin
mesh_terms: D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism*; D051660:Intermediate-Conductance Calcium-Activated Potassium Channels / Q000378:metabolism*; D007413:Intestinal Mucosa / Q000166:cytology / Q000378:metabolism*; D007422:Intestines / Q000166:cytology; D008564:Membrane Potentials / Q000502:physiology; D051381:Rats
chemical_list: D051660:Intermediate-Conductance Calcium-Activated Potassium Channels; C491575:Kcnn4 protein, rat