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. channels observed with iberiotoxin (a Kcnma1/large-conductance, Ca2+-activated K+ channel blocker) and apamin (a Kcnn1C3/small-conductance, Ca2+-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 IC50 of 8.7 2.0 M. 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. oocyte expression system, Kcnn4c, which lacks the predicted second transmembrane-spanning domain, required an accessory protein for its delivery to the plasma membrane, and it exhibited an unusually low sensitivity to inhibition by TRAM-34 (a Kcnn4 channel blocker), with NU 9056 IC50 an IC50 of 7.8 M (1). Hyperpolarization of the membrane potential by the Ca2+-dependent opening of Kcnn4 channels plays a critical role in providing the driving force for agonist (cAMP, Ca2+)-induced anion secretion in human and rat colon (8, 13, 22). Although Kcnn4 channels are localized in both the apical and basolateral membranes, only basolateral Kcnn4 channels have been suggested to be responsible for providing the driving force for agonist-induced anion secretion (8, 22). In a recent study, we showed that, in the absence of serosal Kcnn4 channel activity, the activation of mucosal Kcnn4 channels with 5,6-dichloro-1-ethyl-1,3-dihydro-2H-benzimidazole-2-one (a Kcnn4 channel opener) enhanced the anion secretion in rat distal colon that is mediated by both the cystic fibrosis transmembrane regulator and the Ca2+-activated Cl? channel (17). In this study, we also suggested that mucosal Kcnn4 channels might contribute to stool K+ losses that accompany diarrheal illnesses (17). Although basolateral (serosal) Kcnn4 channels have been well characterized, the biophysical and pharmacological properties of apical (mucosal) Kcnn4 channels have not been characterized (5, 8, 14, 19). A few studies have Mouse monoclonal to KSHV ORF26 identified large-conductance, Ca2+-activated K+ channels (BK) channels in the apical membranes of colonic surface cells (16, 23), but the characterization of NU 9056 IC50 apical ion channels has been generally unsuccessful because the layer of mucus that protects the apical surface of intestinal and colonic epithelial cells also makes it difficult for a patch pipette to access and form a seal on the apical membranes. In the present study, we used cultured IEC-18 cells as a surrogate for intestinal and colonic epithelial cells to enable us to characterize for the first time the Kcnn4 channels in the apical membrane of intestinal epithelial cells. The IEC-18 cell line is a nontransformed, epithelial cell line from intestinal crypts that forms a confluent monolayer when grown on a permeable support (15). The monolayer exhibits a net electrical resistance that is similar to the intact epithelial layer of rat small intestine (15), and it lacks the layer of mucus that interferes with patch seal formation. Our choice of this cell line was also justified by immunofluoresence studies that have localized Kcnn4-like proteins on both the apical and basolateral membranes of surface and crypt cells in rat ileum (6). We report here that we successfully characterized the physiological properties of Kcnn4 channels in the apical membrane of IEC-18 cells. MATERIALS AND METHODS IEC-18 cell culture. IEC-18 cells were grown on transparent, 1 m PET, 6-Well Millicell TransWell Hanging Cell Culture inserts (Millipore, Billerica, MA) in high-glucose Dulbecco’s modified Eagle’s medium (Invitrogen-GIBCO) supplemented with insulin (2 NU 9056 IC50 U/ml), penicillin (100 U/ml), streptomycin (100 g/ml), butyrate (500 M), and 10% FCS and incubated at 37C with 5% CO2. The resistance of monolayers was measured using the Millicell-ERS electrical resistance system (Millipore). A 10-day postconfluent monolayer exhibited a specific resistance of 280 cm2, which indicates the formation of tight junctions. Therefore, 10-day postconfluent IEC-18 cells were used for patch-clamp studies and for the extraction of RNA and proteins that were used in RT-PCR and Western blot analyses, respectively. RT-PCR. RT-PCR analyses were performed as.