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 (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.

Figure 1. Inhibition of short circuit current (I_sc) across Calu-3 monolayers by Ba²⁺

Basal I_sc in Calu-3 monolayers was inhibited by the addition of 5 mm Ba²⁺, a non-specific inhibitor of K⁺ channels, to both the basolateral (A) and apical (B) membranes. Forskolin- and thapsigargin-stimulated I_sc were also inhibited by basolateral application of Ba²⁺ (C and D). The transient inhibition frequently seen following the addition of Ba²⁺ was due to precipitation of Ba²⁺ from solution as previously reported (Moon et al. 1997).

Figure 2. Inhibition of the basal I_sc in Calu-3 cells by clofilium and clotrimazole

Addition of 100 μm clofilium to the basolateral membrane of Calu-3 cells reduced the basal I_sc (A, n = 6). Addition of 30 μm clotrimazole to the basolateral membrane resulted in a much smaller decrease in basal I_sc (B, n = 8). C shows the percentage of basal I_sc inhibited by these agents.

Figure 3. Effects of clofilium and clotrimazole on the cAMP-stimulated increase in I_sc in Calu-3 cells

When Calu-3 monolayers were stimulated with 2 μm forskolin applied to both apical and basolateral sides of the membrane, the subsequent addition of 100 μm clofilium to the basolateral membrane reduced the I_sc (A, n = 8). Addition of 30 μm clotrimazole to the basolateral membrane (B, n = 8) resulted in a much smaller decrease in I_sc. At the end of every experiment 5 mm Ba²⁺ was added to the basolateral membrane, which further inhibited the I_sc. C, reduction in I_sc caused by clofilium and clotrimazole expressed as a percentage of the Ba²⁺-inhibitable I_sc (as described in the text).

Figure 4. Effect of prior treatment with clofilium or clotrimazole on the cAMP-stimulated increase in I_sc in Calu-3 cells

Addition of 2 μm forskolin to Calu-3 monolayers produced an increase in I_sc (A, n = 16). When 100 μm clofilium was applied to the basolateral membrane prior to the forskolin stimulus, the increase in I_sc was virtually abolished (B and D). In contrast, the increase in I_sc recorded following the application of 2 μm forskolin when 30 μm clotrimazole had been pre-applied to the monolayers was not significantly different from the forskolin-alone response (* significantly different from forskolin stimulated as determined by Student's t test; P < 0.05).

Figure 5. Thapsigargin stimulation of I_sc in Calu-3 monolayers

Addition of 300 nm thapsigargin to Calu-3 monolayers produced a large increase in I_sc (A; n = 7). When 100 μm clofilium was applied to the basolateral membrane prior to the thapsigargin stimulus, there was a large increase in I_sc, though it was significantly reduced from the thapsigargin-alone response (B and D). Addition of 30 μm clotrimazole prior to the thapsigargin stimulus also significantly reduced the amplitude of the thapsigargin response (C and D). * Significantly different from thapsigargin-stimulated I_sc as determined by Student's t test (P < 0.05).

Figure 6. Clotrimazole, but not clofilium, inhibits the 1-EBIO stimulation of I_sc in Calu-3 cells

1-EBIO (1 mm) produced a large increase in I_sc (A, n = 3), which was unaffected by the subsequent addition of 100 μm clofilium, but entirely abolished by the addition of 30 μm clotrimazole (B).

Figure 7. Basolateral K⁺ conductances in permeabilized Calu-3 cells

The basolateral K⁺ conductance in Calu-3 cells was isolated by permeabilizing the apical membrane. A small basal I_sc was recorded under these conditions and the addition of 2 μm forskolin induced a small increase in I_sc (A, n = 8) while the addition of 1 mm 1-EBIO induced a much larger increase in I_sc (B, n = 3). This 1-EBIO-stimulated response in the permeabilized monolayers was unaffected by the addition of 100 μm clofilium, but entirely abolished by the addition of 30 μm clotrimazole (C).

Figure 8. PCR analysis on RNA extracted from Calu-3 cells

Transcripts were detected for KCNE2 (lane 2), KCNE3 (lane 3), KCNQ1 (lane 4), KCNN4 (lane 5) and CFTR (lane 7) but not KCNE1 (lane 1) or KCNH2 (lane 6).