TMEM16A in Cystic Fibrosis: Activating or Inhibiting?

Abstract

The inflammatory airway disease cystic fibrosis (CF) is characterized by airway obstruction due to mucus hypersecretion, airway plugging, and bronchoconstriction. The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is dysfunctional in CF, leading to defects in epithelial transport. Although CF pathogenesis is still disputed, activation of alternative Cl^- channels is assumed to improve lung function in CF. Two suitable non-CFTR Cl^- channels are present in the airway epithelium, the Ca²⁺ activated channel TMEM16A and SLC26A9. Activation of these channels is thought to be feasible to improve hydration of the airway mucus and to increase mucociliary clearance. Interestingly, both channels are upregulated during inflammatory lung disease. They are assumed to support fluid secretion, necessary to hydrate excess mucus and to maintain mucus clearance. During inflammation, however, TMEM16A is upregulated particularly in mucus producing cells, with only little expression in ciliated cells. Recently it was shown that knockout of TMEM16A in ciliated cells strongly compromises Cl^- conductance and attenuated mucus secretion, but does not lead to a CF-like lung disease and airway plugging. Along this line, activation of TMEM16A by denufosol, a stable purinergic ligand, failed to demonstrate any benefit to CF patients in earlier studies. It rather induced adverse effects such as cough. A number of studies suggest that TMEM16A is essential for mucus secretion and possibly also for mucus production. Evidence is now provided for a crucial role of TMEM16A in fusion of mucus-filled granules with the apical plasma membrane and cellular exocytosis. This is probably due to local Ca²⁺ signals facilitated by TMEM16A. Taken together, TMEM16A supports fluid secretion by ciliated airway epithelial cells, but also maintains excessive mucus secretion during inflammatory airway disease. Because TMEM16A also supports airway smooth muscle contraction, inhibition rather than activation of TMEM16A might be the appropriate treatment for CF lung disease, asthma and COPD. As a number of FDA-approved and well-tolerated drugs have been shown to inhibit TMEM16A, evaluation in clinical trials appears timely.

Keywords: COPD; Ca2+ signaling; TMEM16A; anoctamin 1; asthma; cystic fibrosis; mucus secretion.

Figure 1

Accumulation of mucus in airways of TMEM16A^−/− mice. Mucus staining (PAS) in bronchi of wild type littermates (TMEM16Aflox/flox; TMEM16A^+/+) and mice with an airway epithelial-specific knockout of TMEM16A (FOXJ1-Cre-TMEM16Aflox/flox; TMEM16A^−/−). Strong accumulation of mucus in airway epithelial cells is observed in TMEM16A^−/− mice. Bars indicate 20 μm (Benedetto et al., 2019).

Figure 2

Ion channels contributing to fluid/mucus balance in non-CF and CF airways. (A) In non-CF airways, CFTR is expressed in ciliated epithelial cells and ionocytes (not shown). Ca²⁺ activated Cl⁻ channels are sparsely expressed in both ciliated and secretory club and goblet cells. SLC26A9, TMEM16A and CFTR located in ciliated epithelial cells and probably in ionocytes are in charge of fluid secretion, while TMEM16A expressed in secretory club/goblet cells support mucus secretion. Epithelial Na⁺ channels reabsorb Na⁺ thereby causing fluid absorption. (B) In inflamed CF airways, CFTR is dysfunctional and often dislocated intracellularly together with SLC26A9 and TMEM16A in ciliated epithelial cells. TMEM16A is upregulated in secretory cells strongly contributing to mucus secretion. Na⁺ absorption by ENaC is augmented. Inflammation and transport abnormalities lead to excessive mucus production/secretion, airway plugging, and reduced water secretion, strongly reducing mucociliary clearance.

Figure 3

Molecular and functional relationship of CFTR, SLC26A9 and TMEM16A. Both SLC26A9 and TMEM16A interact with CFTR via their C-terminal PDZ (PSD-95/Discs-large/ZO1) -binding domains with the help of the PDZ-protein NHERF1 (Na⁺/H⁺ exchanger regulatory factor 1).

Figure 4

Predominant expression of TMEM16A in mucus producing cells. Distal airways of a control mouse and an ovalbumin-sensitized (asthmatic) mouse. Expression of TMEM16A (green fluorescence) is hardly detectable in control airways, but is clearly detectable in mucus producing club/goblet cells (G) of asthmatic mice with inflamed airways. Little expression of TMEM16A is found in ciliated epithelial cells (C). Blue, DAPI staining of nuclei. Bars indicate 20 μm (Benedetto et al., 2019).

Figure 5

Impact of TMEM16A on intracellular Ca²⁺, exocytosis, and mucus secretion. (A) TMEM16A is colocalized with purinergic P2Y receptors and CFTR within the apical membrane. Stimulation of purinergic receptors leads to endoplasmic reticulum (ER) Ca²⁺ store release through IP3 receptors (IP3R). Ca²⁺ not only activates TMEM16A (T16A) but also stimulates adenylate cyclase type 1 (ADCY1) to produce cAMP and to activate CFTR via protein kinase A (PKA). TMEM16A binds to IP3 receptors and tethers the ER to the apical membrane, thereby facilitating effective compartmentalized Ca²⁺ signaling. (B) Effective apical Ca²⁺ signaling by TMEM16A leads to activation of the exocytic SNARE machinery insertion and improved expression of CFTR in the apical plasma membrane. (C) Effective apical Ca²⁺ signaling by TMEM16A leads to fusion of mucus containing granules, exocytosis, and release of mucus.

Figure 6

Activation of TMEM16A leads to mucus release and airway contraction. Control mouse airways show very little mucus (alcian blue). In contrast, airways from ovalbumin-sensitized mice show pronounced mucus accumulation. Acute exposure of an ovalbumin-sensitized mouse to the known activator of TMEM16A, Eact (4.8 μg, tracheal instillation), induced a rapid mucus release and airway contraction (Benedetto et al., 2019). Bars indicate 20 μm.

Figure 7

Activation of inhibition of TMEM16A in airways of CF patients. (A) Activators of TMEM16A (green arrow, +) will find only few TMEM16A channels in the apical membrane of ciliated epithelial cells, which could enhance fluid secretion by ciliated epithelial cells. (B) In contrast, activators of TMEM16A will prominently activate TMEM16A strongly upregulated in mucus producing cells and airway smooth muscle cells during airway inflammation. This leads to hypersecretion of mucus along with airway contraction. (C) Inhibitors of TMEM16A will have an only limited (adverse) effect on TMEM16A channels expressed (fluid secreting) ciliated epithelial cells. In contrast, inhibition of TMEM16 channels in mucus producing cells and airway smooth muscle cells will inhibit mucus production/secretion and induce bronchodilation. (D) Inhibition of TMEM16 channels are likely to restore the balance between mucus and fluid in CF airways.