Calcium-dependent chloride channels are required for normal electrolyte and fluid secretion, olfactory perception, and neuronal and smooth muscle excitability. The molecular identity of these membrane proteins is still unclear. Treatment of bronchial epithelial cells with interleukin-4 (IL-4) causes increased calcium-dependent chloride channel activity, presumably by regulating expression of the corresponding genes. We performed a global gene expression analysis to identify membrane proteins that are regulated by IL-4. Transfection of epithelial cells with specific small interfering RNA against each of these proteins shows that TMEM16A, a member of a family of putative plasma membrane proteins with unknown function, is associated with calcium-dependent chloride current, as measured with halide-sensitive fluorescent proteins, short-circuit current, and patch-clamp techniques. Our results indicate that TMEM16A is an intrinsic constituent of the calcium-dependent chloride channel. Identification of a previously unknown family of membrane proteins associated with chloride channel function will improve our understanding of chloride transport physiopathology and allow for the development of pharmacological tools useful for basic research and drug development.

Key Points: Chloride channels are important for proper hydration of the airway surface. TMEM16A protein is an important component of calcium‐activated chloride channels. Interleukin‐4, a cytokine that induces mucous cell metaplasia, also upregulates calcium‐dependent chloride secretion in human bronchial epithelial cells. In bronchial epithelial cells treated with interleukin‐4, we found that TMEM16A protein becomes highly expressed in goblet but not in ciliated cells. Upregulation of TMEM16A by interleukin‐4 may be important for secretion and proper expansion of mucins. Abstract  The TMEM16A protein has a potential role as a Ca²⁺‐activated Cl⁻ channel (CaCC) in airway epithelia where it may be important in the homeostasis of the airway surface fluid. We investigated the function and expression of TMEM16A in primary human bronchial epithelial cells and in a bronchial cell line (CFBE41o–). Under resting conditions, TMEM16A protein expression was relatively low. However, TMEM16A silencing with short‐interfering RNAs caused a marked inhibition of CaCC activity, thus demonstrating that a low TMEM16A expression is sufficient to support Ca²⁺‐dependent Cl⁻ transport. Following treatment for 24–72 h with interleukin‐4 (IL‐4), a cytokine that induces mucous cell metaplasia, TMEM16A protein expression was strongly increased in approximately 50% of primary bronchial epithelial cells, with a specific localization in the apical membrane. IL‐4 treatment also increased the percentage of cells expressing MUC5AC, a marker of goblet cells. Interestingly, MUC5AC was detected specifically in cells expressing TMEM16A. In particular, MUC5AC was found in 15 and 60% of TMEM16A‐positive cells when epithelia were treated with IL‐4 for 24 or 72 h, respectively. In contrast, ciliated cells showed expression of the cystic fibrosis transmembrane conductance regulator Cl⁻ channel but not of TMEM16A. Our results indicate that TMEM16A protein is responsible for CaCC activity in airway epithelial cells, particularly in cells treated with IL‐4, and that TMEM16A upregulation by IL‐4 appears as an early event of goblet cell differentiation. These findings suggest that TMEM16A expression is particularly required under conditions of mucus hypersecretion to ensure adequate secretion of electrolytes and water.

Cystic fibrosis (CF) is caused by mutations in the CFTR chloride channel. Deletion of phenylalanine 508 (F508del), the most frequent CF mutation, impairs the maturation and gating of the CFTR protein. Such defects may be corrected in vitro by pharmacological modulators named as correctors and potentiators, respectively. We have evaluated a panel of correctors and potentiators derived from various sources to assess potency, efficacy, and mechanism of action. For this purpose, we have used functional and biochemical assays on two different cell expression systems, Fischer rat thyroid (FRT) and A549 cells. The order of potency and efficacy of potentiators was similar in the two cell types considered, with phenylglycine PG-01 and isoxazole UCCF-152 being the most potent and least potent, respectively. Most potentiators were also effective on two mutations, G551D and G1349D, that cause a purely gating defect. In contrast, corrector effect was strongly affected by cell background, with the extreme case of many compounds working in one cell type only. Our findings are in favor of a direct action of potentiators on CFTR, possibly at a common binding site. In contrast, most correctors seem to work indirectly with various mechanisms of action. Combinations of correctors acting at different levels may lead to additive F508del-CFTR rescue.

SCN⁻ (thiocyanate) is an important physiological anion involved in innate defense of mucosal surfaces. SCN⁻ is oxidized by H₂O₂, a reaction catalyzed by lactoperoxidase, to produce OSCN⁻ (hypothiocyanite), a molecule with antimicrobial activity. Given the importance of the availability of SCN⁻ in the airway surface fluid, we studied transepithelial SCN⁻ transport in the human bronchial epithelium. We found evidence for at least three mechanisms for basolateral to apical SCN⁻ flux. cAMP and Ca²⁺ regulatory pathways controlled SCN⁻ transport through cystic fibrosis transmembrane conductance regulator and Ca²⁺-activated Cl⁻ channels, respectively, the latter mechanism being significantly increased by treatment with IL-4. Stimulation with IL-4 also induced the strong up-regulation of an electroneutral SCN⁻/Cl⁻ exchange. Global gene expression analysis with microarrays and functional studies indicated pendrin (SLC26A4) as the protein responsible for this SCN⁻ transport. Measurements of H₂O₂ production at the apical surface of bronchial cells indicated that the extent of SCN⁻ transport is important to modulate the conversion of this oxidant molecule by the lactoperoxidase system. Our studies indicate that the human bronchial epithelium expresses various SCN⁻ transport mechanisms under resting and stimulated conditions. Defects in SCN⁻ transport in the airways may be responsible for susceptibility to infections and/or decreased ability to scavenge oxidants.

The Ca²⁺‐activated Cl⁻ channels (CaCCs) are involved in a variety of physiological functions, such as transepithelial anion transport, smooth muscle contraction and olfaction. Recently, the question of the molecular identity of CaCCs has apparently been resolved with the identification of TMEM16A protein (also known as anoctamin‐1). Expression of TMEM16A is associated with the appearance of Ca²⁺‐ and voltage‐dependent Cl⁻ currents with properties similar to those of native CaCCs. The putative structure of TMEM16A consists of eight transmembrane domains, with both the amino‐ and the carboxy‐terminus protruding in the cytosol. TMEM16A is also characterized by the existence of different protein variants generated by alternative splicing. A close paralogue of TMEM16A, TMEM16B (anoctamin‐2), is also associated with CaCC activity, although with different properties. The TMEM16B‐dependent channels require higher intracellular Ca²⁺ concentrations and have faster activation and deactivation kinetics. Expression of other anoctamins is devoid of detectable channel activity. These proteins, such as TMEM16F (anoctamin‐6), may have different functions.