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. AbstractThe TMEM16A protein has a potential role as a Ca 2+ -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 Ca 2+ -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.

Goblet cell hyperplasia, a feature of asthma and other respiratory diseases, is driven by the Th-2 cytokines IL-4 and IL-13. In human bronchial epithelial cells, we find that IL-4 induces the expression of many genes coding for ion channels and transporters, including TMEM16A, SLC26A4, SLC12A2, and ATP12A. At the functional level, we find that IL-4 enhances calcium- and cAMP-activated chloride/bicarbonate secretion, resulting in high bicarbonate concentration and alkaline pH in the fluid covering the apical surface of epithelia. Importantly, mucin release, elicited by purinergic stimulation, requires the presence of bicarbonate in the basolateral solution and is defective in cells derived from cystic fibrosis patients. In conclusion, our results suggest that Th-2 cytokines induce a profound change in expression and function in multiple ion channels and transporters that results in enhanced bicarbonate transport ability. This change is required as an important mechanism to favor release and clearance of mucus.

Key pointsr TMEM16F is a membrane protein with possible dual function as an ion channel and a phospholipid scramblase.r The properties of ion channels associated with TMEM16F and the link between ion channel and scramblase activity are a matter of debate.r We studied the properties of four isoforms of TMEM16F generated by alternative splicing. r Upregulation of three TMEM16F isoforms or silencing of endogenous TMEM16F increased and decreased, respectively, both scramblase and channel activities.r Introduction of an activating mutation in TMEM16F sequence caused a marked increase in phosphatidylserine scrambling and in ion transport indicating direct involvement of the protein in both functions.Abstract TMEM16F, also known as ANO6, is a membrane protein that has been associated with phospholipid scramblase and ion channel activity. However, the characteristics of TMEM16F-dependent channels, particularly the ion selectivity, are a matter of debate. Furthermore, the direct involvement of TMEM16F in phospholipid scrambling has been questioned. We studied the properties of different TMEM16F variants generated by alternative splicing. Using whole-cell patch-clamp recordings, we found that V1, V2 and V5 variants generated membrane currents activated by very high (micromolar) intracellular Ca 2+ concentrations and positive membrane potentials. These variants showed different degrees of Ca 2+ sensitivity and kinetics of activation but similar ion permeability, characterized by a slight selectivity for Cl − over Na + . A fourth variant (V3) showing a unique carboxy-terminus was devoid of activity, in agreement with its intracellular localization. We also measured scramblase activity using the binding of annexin V to detect phosphatidylserine on the cell surface. V1, V2 and V5 variants were associated with calcium-dependent phosphatidylserine externalization. Interestingly, introduction of an activating mutation, D409G, produced a marked increase in the apparent Ca 2+ sensitivity of TMEM16F-dependent channels. In parallel, this mutation also enhanced the extent of phosphatidylserine externalization that occurred even under resting conditions. These results support the conclusion that TMEM16F proteins are directly involved in dual activity, as a phospholipid scramblase and as an ion channel.

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TMEM16A protein, also known as anoctamin-1, has been recently identified as an essential component of Ca2+-activated Cl− channels. We previously reported the existence of different TMEM16A isoforms generated by alternative splicing. In the present study, we have determined the functional properties of a minimal TMEM16A protein. This isoform, called TMEM16A(0), has a significantly shortened amino-terminus and lacks three alternative segments localized in the intracellular regions of the protein (total length: 840 amino acids). TMEM16A(0) expression is associated with Ca2+-activated Cl− channel activity as measured by three different functional assays based on the halide-sensitive yellow fluorescent protein, short-circuit current recordings, and patch-clamp technique. However, compared to a longer isoform, TMEM16(abc) (total length: 982 amino acids), TMEM16A(0) completely lacks voltage-dependent activation. Furthermore, TMEM16A(0) and TMEM16A(abc) have similar but not identical responses to extracellular anion replacement, thus suggesting a difference in ion selectivity and conductance. Our results indicate that TMEM16A(0) has the basic domains required for anion transport and Ca2+-sensitivity. However, the absence of alternative segments, which are present in more complex isoforms of TMEM16A, modifies the channel gating and ion transport ability.