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

摘要

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.