thapsigargin and 1-ethyl-2-benzimidazolinone

Electrophysiological studies of H441 human distal airway epithelial cells showed that thapsigargin caused a Ca(2+)-dependent increase in membrane conductance (G(Tot)) and hyperpolarization of membrane potential (V(m)). These effects reflected a rapid rise in cellular K(+) conductance (G(K)) and a slow fall in amiloride-sensitive Na(+) conductance (G(Na)). The increase in G(Tot) was antagonized by Ba(2+), a nonselective K(+) channel blocker, and abolished by clotrimazole, a KCNN4 inhibitor, but unaffected by other selective K(+) channel blockers. Moreover, 1-ethyl-2-benzimidazolinone (1-EBIO), which is known to activate KCNN4, increased G(K) with no effect on G(Na). RT-PCR-based analyses confirmed expression of mRNA encoding KCNN4 and suggested that two related K(+) channels (KCNN1 and KCNMA1) were absent. Subsequent studies showed that 1-EBIO stimulates Na(+) transport in polarized monolayers without affecting intracellular Ca(2+) concentration ([Ca(2+)](i)), suggesting that the activity of KCNN4 might influence the rate of Na(+) absorption by contributing to G(K). Transient expression of KCNN4 cloned from H441 cells conferred a Ca(2+)- and 1-EBIO-sensitive K(+) conductance on Chinese hamster ovary cells, but this channel was inactive when [Ca(2+)](i) was <0.2 microM. Subsequent studies of amiloride-treated H441 cells showed that clotrimazole had no effect on V(m) despite clear depolarizations in response to increased extracellular K(+) concentration ([K(+)](o)). These findings thus indicate that KCNN4 does not contribute to V(m) in unstimulated cells. The present data thus establish that H441 cells express KCNN4 and highlight the importance of G(K) to the control of Na(+) absorption, but, because KCNN4 is quiescent in resting cells, this channel cannot contribute to resting G(K) or influence basal Na(+) absorption.

Topics: Animals; Benzimidazoles; Calcium Channel Agonists; Cell Line; Cell Lineage; CHO Cells; Cloning, Molecular; Cricetinae; Enzyme Inhibitors; Gene Expression; Humans; Intermediate-Conductance Calcium-Activated Potassium Channels; Membrane Potentials; Potassium; Respiratory Mucosa; RNA, Messenger; Sodium; Thapsigargin

Cystic fibrosis (CF) is caused by genetic mutations that lead to dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel. The most common mutation, DeltaF508, causes inefficient trafficking of mutant CFTR protein from the endoplasmic reticulum to the cell membrane. Therapeutic efforts have been aimed at increasing the level of DeltaF508-CFTR protein in the membrane using agents such as sodium butyrate. In this study, we investigated the effects of culturing a human airway epithelial cell line, Calu-3, in the presence of 5 mM sodium butyrate. Within 24 h, butyrate exposure caused a significant decrease in the basal, as well as Ca(2+)-activated, anion secretion by Calu-3 cell monolayers, determined by the change in transepithelial short-circuit current in response to the Ca(2+)-elevating agent thapsigargin. The secretory response to 1-ethyl-2-benzimidazolinone, an activator of the basolateral Ca(2+)-activated K(+) channel KCNN4, was similarly reduced by butyrate treatment. Quantitative PCR revealed that these functional effects were associated with dramatic decreases in mRNA for both KCNN4 and CFTR. Furthermore, the KCNQ1 K(+) channel was upregulated after butyrate treatment. We suggest that prolonged exposure to sodium butyrate downregulates the expression of both KCNN4 and CFTR, leading to a functional loss of Ca(2+)-activated anion secretion. Thus, butyrate may inhibit, rather than stimulate, the anion secretory capacity of human epithelial cells that express wild-type CFTR, particularly in tissues that normally exhibit robust Ca(2+)-activated secretion.

Topics: Benzimidazoles; Butyric Acid; Calcium; Calcium Channel Agonists; Cell Line; Chlorides; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Down-Regulation; Enzyme Inhibitors; Epithelial Cells; Humans; Intermediate-Conductance Calcium-Activated Potassium Channels; Lung; Thapsigargin

Transepithelial anion secretion in many tissues depends upon the activity of basolateral channels. Using monolayers of the Calu-3 cell line, a human submucosal serous cell model mounted in an Ussing chamber apparatus, we investigated the nature of the K+ channels involved in basal, cAMP- and Ca2+-stimulated anion secretion, as reflected by the transepithelial short circuit current (I(sc)). The non-specific K+ channel inhibitor Ba2+ inhibited the basal I(sc) by either 77 or 16 % when applied directly to the basolateral or apical membranes, respectively, indicating that a basolateral K+ conductance is required for maintenance of basal anion secretion. Using the K+ channel blockers clofilium and clotrimazole, we found basal I(sc) to be sensitive to clofilium, with a small clotrimazole-sensitive component. By stimulating the cAMP and Ca2+ pathways, we determined that cAMP-stimulated anion secretion was almost entirely abolished by clofilium, but insensitive to clotrimazole. In contrast, the Ca2+-stimulated response was sensitive to both clofilium and clotrimazole. Thus, pharmacologically distinct basolateral K+ channels are differentially involved in the control of anion secretion under different conditions. Isolation of the basolateral K+ conductance in permeabilized monolayers revealed a small basal and forskolin-stimulated I(sc). Finally, using the reverse transcriptase-polymerase chain reaction, we found that Calu-3 cells express the K+ channel genes KCNN4 and KCNQ1 and the subunits KCNE2 and KCNE3. We conclude that while KCNN4 contributes to Ca2+-activated anion secretion by Calu-3 cells, basal and cAMP-activated secretion are more critically dependent on other K+ channel types, possibly involving one or more class of KCNQ1-containing channel complexes.

Topics: Anions; Barium; Benzimidazoles; Calcium Channel Agonists; Clotrimazole; Cyclic AMP; Electric Conductivity; Enzyme Inhibitors; Humans; Ions; Lung; Potassium Channel Blockers; Potassium Channels; Protein Isoforms; Quaternary Ammonium Compounds; Serous Membrane; Thapsigargin; Tumor Cells, Cultured

Topics: Benzimidazoles; Calcium Channel Agonists; Cell Line; Charybdotoxin; Gene Expression Regulation; Humans; Intermediate-Conductance Calcium-Activated Potassium Channels; Membrane Potentials; Patch-Clamp Techniques; Placenta; Potassium Channels; Potassium Channels, Calcium-Activated; Thapsigargin; Transfection