TMEM16A Channel Function Does Not Influence Goblet Cell Numbers or Mucin Secretion In The Human Airway Epithelium – NACFC – Nashville – October 2019
Henry Danahay1,2, Alexis Flen, Camille Ehre3, Martin Gosling1,2
1University of Sussex, Brighton, UK; 2Enterprise Therapeutics, UK; 3University of North Carolina, Chapel Hill, USA.
A role for the calcium activated chloride channel, TMEM16A, in the regulation of airway goblet cell formation and function has been recently reported (Benedetto et al., 2019; Kondo et al., 2017; Lin et al., 2015; Qin et al., 2016). Much of the published data to support a role for TMEM16A in these processes has been based on either gene silencing or the use of non-specific TMEM16A blockers (e.g. niflumic acid). It is therefore unclear whether these proposed functions are dependent on the ion channel activity of TMEM16A, through an alternative aspect of the proteins function or even a TMEM16A-independent activity of a non-selective pharmacological tool.
Using a potent TMEM16A channel blocker (Ani9) and recently identified TMEM16A potentiator compounds (Enterprise Therapeutics; proprietary), we have evaluated whether channel function can regulate goblet cell number and/or mucin secretion in primary cultures of human bronchial epithelial (HBE) cells.
Primary HBE (3 donor codes) were cultured for 2 weeks at air-liquid interface (ALI) on permeable supports and formed a well-differentiated mucociliary epithelium. On ALI day 15, cells were treated with either: 1) vehicle, 2) IL-13 (10 ng/mL) or 3) the TMEM16A potentiator, ETX001 (1 µM) with each group in either the absence or presence of the TMEM16A blocker, Ani9 (10 µM). ETX001 is a TMEM16A potentiator with EC50 values of 114 and 170 nM for the potentiation of chloride secretion in patch clamp (FRT-TMEM16A) and ion transport (CF-HBE) respectively. Ani9 fully blocks TMEM16A function in both patch clamp and HBE ion transport studies. Cells were cultured under these conditions for 96h before fixation and staining with antibodies directed against MUC5AC (goblet cells) and acetylated α-tubulin (ciliated cells). Goblet and ciliated cell numbers were quantified using an automated image acquisition (Zeiss Axiovert) and analysis system (Image J).
IL-13 induced a significant increase in the density of goblet cells based on the increased staining for MUC5AC, that was unaffected by co-administration of Ani9. Neither ETX001 or Ani9 (alone or in combination) had any effect on goblet cell numbers. Finally, the co-administration of ETX001 with IL-13 also failed to modify goblet cell numbers.
In a separate series of experiments, CF-HBE (±IL-13 pre-treatment; 2 donor codes) were treated with TMEM16A potentiator compounds for 24 hours and mucin secretion was quantified. TMEM16A potentiator compounds were without effect on the secretion of either MUC5B or MUC5B
Together, these data do not support a role for the ion channel function of TMEM16A in either the regulation of goblet cell numbers or the secretion of mucins in primary HBE.
Benedetto et al. (2019) FASEB J. 33(3):4502-4512
Kondo M et al. (2017) Clin Exp Allergy. 47(6):795-804.
Lin J et al. (2015) Exp Cell Res. 334(2):260-9.
Qin et al. (2016) Int Immunopharmacol. 40:106-114