Characterization of K + Channels in the Basolateral Membrane of Rat Tracheal Epithelia

Abstract

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.

The Small Conductance K+ Channel, KCNQ1

“…In contrast to other groups, we could neither establish a relevant mRNA expression of KCNE1 with sensitive RT-PCR methods nor could we detect a protein expression with the help of a specific antibody in murine tracheal epithelial cells. As the molecular evidence for an expression of KCNE1 in trachea previously relied on RT-PCR in primary cultured rat tracheal epithelial cells, the apparent discrepancy of these results to our study might be explained by: (a) a species difference between rat and mouse, (b) an altered gene expression due to the 10-day exposure to culture medium (29), or (c) fundamental differences in the experimental protocols used (20). Instead of KCNE1 we found the related ␤-subunit KCNE3 to be expressed in murine tracheal epithelial cells.…”

“…So far there has even been doubt as to whether KCNQ1 plays a functional role in airway epithelium at all (17). In contrast to the ␣-subunit KCNQ1, the ␤-subunit KCNE1 had been associated in several studies with a functional role in airway epithelial cells (20,21). We confirmed the expression of the ␣-subunit KCNQ1 by RT-PCR alone, RT-PCR and subsequent Southern probing, immunofluorescence, and by demonstrating a 293B-sensitive current.…”

“…Pharmacological evidence with the KCNQ1-specific inhibitor, the chromanol 293B, supports a role of KCNQ1 in human nasal epithelium (18,19). Previous reports have suggested a functional role for KCNE1 in rat and mouse airway epithelium (20,21). Here we provide evidence that as in the gut KCNQ1 co-assembles with KCNE3 in murine tracheal epithelial cells to form a basolateral K ϩ channel complex, which functions as the main driving force for epithelial anion secretion and base-line Na ϩ absorption.…”

Prostanoids Secreted by Alveolar Macrophages Enhance Ionic Currents in Swine Tracheal Submucosal Gland Cells

“…To measure the serosal membrane K ϩ channel activity, 180 g/ml nystatin was added to the apical chamber to permeabilize the apical membrane to monovalent ions, such as Cl Ϫ , K ϩ , and Na ϩ (Hwang et al, 1996). A high K ϩ gradient across the serosal membrane was established, and Cl Ϫ current was also minimized by replacing NaCl with potassium gluconate in the apical Ussing solution and sodium gluconate in the serosal solution.…”

Contribution of the IsK (MinK) Potassium Channel Subunit to Regulatory Volume Decrease in Murine Tracheal Epithelial Cells

“…The first hypothesis would involve a principal role for the activation of potassium channels to activate the RVD response, while the second would confer them a more passive role, with the activation of swelling-activated chloride channels being the starting signal. Although we cannot discard the first hypothesis, we favor the second possibility as basolateral I sK currents have been identified in tracheal cells under isosmotic conditions (37). In that respect, I sK current has been associated to the maintenance of transepithelial chloride secretion in the airways (37,38) and colonic cells (36) and potassium secretion in the stria vascularis (39).…”