Calcium-activated chloride channel TMEM16A modulates mucin secretion and airway smooth muscle contraction

Abstract

Mucous cell hyperplasia and airway smooth muscle (ASM) hyperresponsiveness are hallmark features of inflammatory airway diseases, including asthma. Here, we show that the recently identified calcium-activated chloride channel (CaCC) TMEM16A is expressed in the adult airway surface epithelium and ASM. The epithelial expression is increased in asthmatics, particularly in secretory cells. Based on this and the proposed functions of CaCC, we hypothesized that TMEM16A inhibitors would negatively regulate both epithelial mucin secretion and ASM contraction. We used a high-throughput screen to identify small-molecule blockers of TMEM16A-CaCC channels. We show that inhibition of TMEM16A-CaCC significantly impairs mucus secretion in primary human airway surface epithelial cells. Furthermore, inhibition of TMEM16A-CaCC significantly reduces mouse and human ASM contraction in response to cholinergic agonists. TMEM16A-CaCC blockers, including those identified here, may positively impact multiple causes of asthma symptoms.

Fig. 1.

TMEM16A is up-regulated in epithelial cells of asthmatic human patients and asthma models. (A–D) The abundance of TMEM16A protein is increased in airway epithelial cells of the mouse asthma model of ovalbumin sensitization and challenge. (A and B) TMEM16A antibody staining in the airway epithelium of control (saline-treated) mice. (C and D) TMEM16A staining in the airway of OVA-treated asthmatic mice. Epithelial cells are marked with E-cadherin (red, overlay images in B and D). (E and F) The abundance and distribution of TMEM16A were visualized using TMEM16A:GFP fusion protein knock-in mice in which a functional and properly localized fusion protein of TMEM16A and GFP is under control of the endogenous TMEM16A regulatory elements. (E) Relatively little TMEM16A (green, anti-GFP) is observed in control airways. (F) When crossed onto a transgenic line that expresses high levels of IL-13 in the respiratory epithelium, the abundance of apical TMEM16A is increased, particularly in mucous cells (red, anti-Muc5AC). Multiciliated epithelial cells are visualized with differential interference contrast (arrowheads). (G–I) Immunostaining for TMEM16A and Muc5AC in healthy human bronchial biopsy tissue. Arrows point to the weak signal for TMEM16A in the Muc5AC-positive cells. HC, healthy control. (J–L) Immunostaining for TMEM16A and Muc5AC in asthmatic human bronchial epithelial cells. Arrows indicate TMEM16A staining in Muc5AC-positive cells. Arrowheads indicate secretory cells in submucosal glands.

Fig. 2.

TMEM16A is strongly expressed in the mucin-secreting cells in asthma models. (A–C) TMEM16A (red) is expressed in the mucin-secreting cells (green, MUC5AC) in the airway epithelium of OVA-challenged asthma mice. Note the expression of TMEM16A in ASM as well. (D–H) Bronchial epithelial cells from FoxJ1-GFP mice were cultured at the ALI and treated with IL-13 (10 ng/mL). (D and E) Antibody staining for TMEM16A (red) is stronger in the rare mucous cells (MUC5AC, blue staining), compared with the abundant multiciliated cells (FoxJ1-GFP, green) in control, nontreated (NT) cultures. (F and G) IL-13 treatment increases the number of mucous cells (blue, MUC5AC), and the abundance of TMEM16A (red) is increased at the apical surfaces of these cells. (H) High-magnification picture with the orthogonal views to show the apical localization of TMEM16A in the Muc5AC-positive cells.

Fig. 3.

High-throughput screening yields three blockers of TMEM16A. (A and B) High-throughput screening yields small molecules that block TMEM16A-CaCC. (A) The assay design. HEK293 cells stably express TMEM16A-CaCC and the genetically encoded fluorescent YFP I⁻ ion biosensor. Upon the addition of agonists of calcium motility ionomycin to cells, the increased Ca²⁺ will activate CaCC and, consequently, the increase of iodine ion flux into the cytoplasm through the open CaCC. The increased I⁻ ion levels in the cytoplasm will quench the fluorescence of YFP, which is monitored in real time by a fluorescence imaging plate reader. The validation set library of 2,000 compounds was screened for molecules that block TMEM16A-CaCC (inhibit the ability of ionomycin to induce quenching of the YFP biosensor). (B and C) Structure and properties of selected hits from the screen: dichlorophen, benzbromarone, and hexachlorophene. (D) Benzbromarone blocks CaCC currents from HEK293 cells expressing mouse TMEM16A. The patch was voltage-clamped from the holding potential of −80 mV with a 500-ms voltage ramp to +80 mV. The pipette solution contained 2 μM free Ca²⁺. Extracellular benzbromarone exhibits concentration-dependent block of CaCC current at +80 mV. The data were fitted to I/I_zero = I_min + (I_max − I_min)/{1 + ([blocker]/K_i)^p}, where K_i = 10.4 μM and P = 0.8; n = 5–13. (E) An inside-out patch from an Axolotl oocyte expressing mouse TMEM16A was exposed to intracellular benzbromarone. The patch was voltage-clamped from the holding potential of −80 mV with a 500-ms voltage ramp to +80 mV. All benzbromarone solutions contained 100 μM free Ca²⁺. Intracellular benzbromarone also exhibits concentration-dependent block of CaCC current at −80 mV. The data were fitted to I/I_Zero = I_min + (I_max − I_min)/{1 + ([blocker]/K_i)^p}, where K_i = 3.6 μM and P = 1.0; n = 4–11.

Fig. 4.

TMEM16A blockers negatively regulate agonist-stimulated mucin secretion in IL-13–treated NHBE cells. (A and B) NHBE cells were grown at the ALI and treated with IL-13. Benzbromarone blocks the ATP-induced CaCC current measured by short circuit current (Isc) in Ussing chamber. Currents (A), normalized to baseline before ATP, are plotted as mean ± SEM (B), n = 4, ***P < 0.001. (C–F) Benzbromarone impairs ATP-induced mucin depletion in IL-13–treated NHBE cells as shown by Muc5AC staining of the mucin stores. (C) Control. (D) 100 μM ATP with 10 μM benzbromarone. (E) 100 μM ATP. (F) Quantification and statistical analysis of Muc5AC staining intensity, ***P < 0.0001. Statistical significance was determined by one-way ANOVA followed by Bonferroni's multiple comparison test.

Fig. 5.

CaCC blockers inhibit MCh-induced ASM contraction. (A) Sections of mouse airways were stained with antibodies against TMEM16A (green) and α-SMA (red). TMEM16A is expressed in the ASM cells. (B and C) Benzbromarone inhibits the MCh-induced contraction of human ASM (B) but not KCl-induced contraction, as shown in C. **P < 0.01, n = 5. (Note that all five bronchial rings are from the same human lung.)