Characterization of basolateral K⁺ channels underlying anion secretion in the human airway cell line Calu‐3

Abstract

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 (Isc). The non‐specific K+ channel inhibitor Ba2+ inhibited the basal Isc 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 Isc 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 Isc. 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.

In Situ Measurement of Airway Surface Liquid [K+] Using a Ratioable K+-sensitive Fluorescent Dye

“…KCNQ2, KCNQ4, and KCNN1 mRNA were not expressed in cultures of freshly isolated HBE cells. In contrast, Calu-3 cells did not express KCNE1 mRNA (24). We confirmed KCNE1 expression in HBE cells by RT-PCR using two different sets of KCNE1-specific primers and cDNAs from three human subjects (data not shown).…”

“…Prior data suggest that some KCNQ channels, including KCNQ1, KCNE2, KCNE3, KCNN4, and ATP1AL1, a nongastric H ϩ , K ϩ -ATPase, are expressed in human airway epithelia (13,24,27). RT-PCR was done to test for expression of transcripts encoding KCNQ1-5, KCNE1-4, KCNN1-4, and ATP1AL1.…”

An Inwardly Rectifying Potassium Channel in Apical Membrane of Calu-3 Cells

“…Basal and stimulated secretion in Calu-3 cells requires basolateral K ϩ channels, and two types have been identified (6,43). Most basal I sc and essentially all I sc stimulated by increased [cAMP] i is blocked by the ammonium-derived compound clofilium, but not by clotrimazole.…”

“…The Calu-3 cell line (1) is widely used as a model for human airway gland serous cells (2)(3)(4)(5)(6). When grown to confluence, Calu-3 cells form polarized monolayers and express CFTR 1 apically at high levels (1).…”

A Potassium Channel-MiRP Complex Controls Neurosensory Function in Caenorhabditis elegans

“…The last member of the family, MiRP4 was identified later (6). Although MiRPs were initially identified as cardiac proteins, they were soon found to be expressed and function in other tissues and to cause acquired and congenital disease (2,(7)(8)(9)(10)(11). For instance, MiRP2 is expressed in skeletal muscle where it associates with Kv3.…”