KCa3.1 K+ Channel Expression and Function in Human Bronchial Epithelial Cells

Abstract

The KCa3.1 K+ channel has been proposed as a novel target for pulmonary diseases such as asthma and pulmonary fibrosis. It is expressed in epithelia but its expression and function in primary human bronchial epithelial cells (HBECs) has not been described. Due to its proposed roles in the regulation of cell proliferation, migration, and epithelial fluid secretion, inhibiting this channel might have either beneficial or adverse effects on HBEC function. The aim of this study was to assess whether primary HBECs express the KCa3.1 channel and its role in HBEC function. Primary HBECs from the airways of healthy and asthmatic subjects, SV-transformed BEAS-2B cells and the neoplastic H292 epithelial cell line were studied. Primary HBECs, BEAS-2B and H292 cells expressed KCa3.1 mRNA and protein, and robust KCa3.1 ion currents. KCa3.1 protein expression was increased in asthmatic compared to healthy airway epithelium in situ, and KCa3.1 currents were larger in asthmatic compared to healthy HBECs cultured in vitro. Selective KCa3.1 blockers (TRAM-34, ICA-17043) had no effect on epithelial cell proliferation, wound closure, ciliary beat frequency, or mucus secretion. However, several features of TGFβ1-dependent epithelial-mesenchymal transition (EMT) were inhibited by KCa3.1 blockade. Treatment with KCa3.1 blockers is likely to be safe with respect to airway epithelial biology, and may potentially inhibit airway remodelling through the inhibition of EMT.

Fig 1. Human bronchial epithelial cells express K_Ca3.1 mRNA and protein, and K_Ca3.1 expression is upregulated in the asthmatic bronchial epithelium.

(A) K_Ca3.1 mRNA (predicted PCR product size: 130 bp) was detected in monolayers of primary HBECs isolated from both patients with asthma (n = 10; denoted with “A”) and non-asthmatic healthy controls (n = 5; denoted with “NA”), alongside the housekeeping gene β-actin (predicted PCR product size: 146 bp). (B) qPCR revealed that K_Ca3.1 mRNA was expressed at similar levels in primary HBECs isolated from patients with asthma (n = 10) and healthy controls (n = 5). (C) An immunoreactive band of the appropriate size (K_Ca3.1: 48 kDa; β-actin: 42 kDa) was detected in lysates of primary HBECs isolated from two patients with asthma and one healthy control. (D) Quantification of K_Ca3.1 immunostaining by threshold analysis revealed that K_Ca3.1 expression was significantly elevated in asthmatic bronchial epithelium (*P = 0.007). (E) K_Ca3.1 immunostaining was significantly increased in severe asthma compared to mild asthma (**P = 0.008) and compared with healthy controls (*P = 0.002). (F) K_Ca3.1 and MUC5AC immunostaining co-localised in bronchial epithelial cells of sequential sections of biopsies from patients with asthma and healthy controls. (G) Quantification of MUC5AC immunostaining by threshold analysis revealed that MUC5AC expression was significantly increased in asthmatic bronchial epithelium (*P = 0.030), and (H) this was driven by a significant difference between the severe asthma and healthy control groups (*P = 0.034). (I) A significant correlation was found between K_Ca3.1 and MUC5AC immunostaining across the different severities of asthma and the healthy control groups (P < 0.001; r_s = 0.608). All data are plotted as median ± interquartile range; horizontal bars represent medians.

Fig 2. Asthmatic HBECs exhibit significantly larger K_Ca3.1 currents than healthy HBECs.

(A) Current-voltage curves demonstrating that baseline whole cell membrane currents recorded from asthmatic and healthy primary HBECs were similar. (B) The 1-EBIO-dependent (1-EBIO minus baseline) currents recorded from asthmatic HBECs (n = 29 cells from 8 donors) were significantly larger than those from healthy controls (n = 16 cells from 5 donors); *P = 0.006 at +40 mV. (C) Raw 1-EBIO-dependent current trace demonstrating characteristic features of K_Ca3.1. The voltage protocol is shown inset. The K_Ca3.1 channel blocker TRAM-34 (200 nM) inhibited currents induced by 1-EBIO in both (D) asthmatic HBECs (n = 19 cells from 8 donors) and (F) healthy HBECs (n = 14 cells from 5 donors). Data are presented as mean ± SEM.

Fig 3. Freshly isolated HBECs, and the H292 and BEAS-2B airway epithelial cell lines exhibit K_Ca3.1 channel activity.

Current-voltage curves demonstrating that 100 μM 1-EBIO induced characteristic K_Ca3.1 channel currents in freshly isolated HBECs isolated from (A) one patient with asthma (n = 3 cells) and (B) one healthy control (n = 3 cells). TRAM-34 (200 nM) blocked 1-EBIO-induced currents to near-baseline levels. Characteristic 1-EBIO-dependent K_Ca3.1 channel activity, sensitive to TRAM-34, was also seen in (C) the H292 cell line (n = 6 cells) and (D) the BEAS-2B cell line (n = 8 cells).

Fig 4. The K_Ca3.1 channel does not regulate epithelial mucus production or secretion.

(A) Recombinant human amphiregulin (rh-AR) upregulated MUC5AC mRNA expression in H292 cells cultured in 6 well plates in a concentration-dependent manner after 24 h (*P = 0.010; **P = 0.028; n = 3 individual experiments). (B) Pre-treatment of H292 cells with the K_Ca3.1 blocker TRAM-34 (200 nM) for 30 min prior to stimulation with 10 ng/ml rh-AR for 24 h did not prevent rh-AR-induced MUC5AC mRNA expression (*P = 0.008, **P = 0.042; n = 6 individual experiments). rh-AR dose-dependently increased the mucin content of (C) H292 ALI culture lysates (*P = 0.029; n = 3 individual experiments) and (D) H292 ALI culture apical washes (*P = 0.009; n = 4 individual experiments) after 24 h, analysed by ELLA. Pre-treatment of H292 ALI cultures with 200 nM TRAM-34 did not prevent rh-AR-induced upregulation of mucin content within (E) H292 lysates (*P = 0.019; n = 6 individual experiments) or (F) apical washes (*P = 0.027; n = 3 individual experiments). Data are expressed as mean ± SEM.

Fig 5. The K_Ca3.1 channel does not regulate airway epithelial ciliary beat frequency.

(A) Ciliary beat frequency of epithelial cells from healthy controls treated with either TRAM-34 or DMSO (n = 3). (B) Ciliary beat frequency of epithelial cells from asthmatic subjects treated with either TRAM-34 or DMSO (n = 3). *P = 0.001; data are expressed as mean ± SEM.

Fig 6. Inhibition of the K_Ca3.1 channel attenuates features of TGFβ1-dependent EMT.

(A) Cell elongation, quantified by a ratio of cell length:cell width of vimentin-stained BEAS-2B cells after 72 h, was increased by TGFβ1 or TGFβ1+DMSO compared to untreated cells (0.1% PBS/BSA; *P < 0.001). Pre-treatment of BEAS-2B cells with 200 nM TRAM-34 (#P < 0.001) or 100 nM ICA-17043 (##P = 0.027) significantly inhibited TGFβ1-induced cell elongation compared to TGFβ1+DMSO control. In contrast, TRAM-7, an inactive analog of TRAM-34, did not inhibit TGFβ1-induced cell elongation (**P = 0.022 compared to TRAM-34). Data are presented as mean ± SEM from 6 individual experiments. (B) BEAS-2B cells treated with 10 ng/ml TGFβ1 or TGFβ1+DMSO for 72 h exhibited a loss of E-cadherin expression in comparison to untreated cells (0.1% PBS/BSA; *P < 0.001, **P = 0.001). Pre-treatment with TRAM-34 (200 nM; #P = 0.013) or ICA-17043 (100 nM; ##P = 0.002) attenuated the TGFβ1-dependent loss of E-cadherin compared to TGFβ1+DMSO. TRAM-7 (200 nM) did not prevent TGFβ1-induced loss of E-cadherin immunostaining (***P = 0.014 compared to TRAM-34). Data are presented as mean ± SEM from 6 individual experiments. (C) BEAS-2B cells treated with 10 ng/ml TGFβ1 or 10 ng/ml TGFβ1+DMSO control for 72 h displayed an increase in collagen-1 expression in comparison to untreated cells (0.1% PBS/BSA) (*P = 0.005; **P = 0.028 respectively). However, pre-treatment with TRAM-34 (200 nM; #P = 0.021) or ICA-17043 (100 nM; ##P = 0.024) significantly inhibited this effect compared to TGFβ1+DMSO. TRAM-7 (200 nM) did not inhibit TGFβ1-induced upregulation of collagen-1 immunostaining (***P = 0.021 compared to ICA-17043). Data are presented as mean ± SEM from 5 individual experiments.