nystatin-a1 and clofilium

To study K+ channels in the basolateral membrane of chloride-secreting epithelia, rat tracheal epithelial monolayers were cultured on permeable filters and mounted into an Ussing chamber system. The mucosal membrane was permeabilized with nystatin (180 microg/ml) in the symmetrical high K+ (145 mm) Ringer solution. During measurement of the macroscopic K+ conductance properties of the basolateral membrane under a transepithelial voltage clamp, we detected at least two types of K+ currents: one is an inwardly rectifying K+ current and the other is a slowly activating outwardly rectifying K+ current. The inwardly rectifying K+ current is inhibited by Ba2+. The slowly activating K+ current was potentiated by cAMP and inhibited by clofilium, phorbol 12-myristate 13-acetate (PMA) and lowering temperature. This is consistent with the biophysical characteristics of ISK channel. RT-PCR analysis revealed the presence of ISK cDNA in the rat trachea epithelia. Although 0.1 mM Ba2+ only had minimal affect on short-circuit current (Isc) induced by cAMP in intact epithelia, 0.1 mM clofilium strongly inhibited it. These results indicate that ISK might be important for maintaining cAMP-induced chloride secretion in the rat trachea epithelia.

Topics: Animals; Barium; Cell Membrane; Cell Polarity; Cells, Cultured; Chloride Channels; Chlorides; DNA, Complementary; Epithelium; Ion Channel Gating; Male; Membrane Potentials; Nystatin; Patch-Clamp Techniques; Potassium Channel Blockers; Potassium Channels; Potassium Channels, Voltage-Gated; Quaternary Ammonium Compounds; Rats; Rats, Wistar; Temperature; Tetradecanoylphorbol Acetate; Trachea