CFTR and TMEM16A are separate but functionally related Cl- channels

Previous reports point out to a functional relationship of the cystic fibrosis transmembrane conductance regulator (CFTR) and Ca(2+) activated Cl(-) channels (CaCC). Recent findings showing that TMEM16A forms the essential part of CaCC, prompted us to examine whether CFTR controls TMEM16A. Inhibition of endogenous CaCC by activation of endogenous CFTR was found in 16HBE human airway epithelial cells, which also express TMEM16A. In contrast, CFBE airway epithelial cells lack of CFTR expression, but express TMEM16A along with other TMEM16-proteins. These cells produce CaCC that is inhibited by overexpression and activation of CFTR. In HEK293 cells coexpressing TMEM16A and CFTR, whole cell currents activated by IMBX and forskolin were significantly reduced when compared with cells expressing CFTR only, while the halide permeability sequence of CFTR was not changed. Expression of TMEM16A, but not of TMEM16F, H or J, produced robust CaCC, which that were inhibited by CaCCinh-A01 and niflumic acid, but not by CFTRinh-172. TMEM16A-currents were attenuated by additional expression of CFTR, and were completely abrogated when additionally expressed CFTR was activated by IBMX and forskolin. On the other hand, CFTR-currents were attenuated by additional expression of TMEM16A. CFTR and TMEM16A were both membrane localized and could be coimmunoprecipitated. Intracellular Ca(2+) signals elicited by receptor-stimulation was not changed during activation of CFTR, while ionophore-induced rise in [Ca(2+)](i) was attenuated after stimulation of CFTR. The data indicate that both CFTR and TMEM16 proteins are separate molecular entities that show functional and molecular interaction.     

Pharmacological characterization of TMEM16A currents

Recent studies have shown that transmembrane protein 16 A (TMEM16A) is a subunit of calcium-activated chloride channels (CACCs). Pharmacological agents have been used to probe the functional role of CACCs, however their effect on TMEM16A currents has not been systematically investigated. In the present study, we characterized the voltage and concentration-dependent effects of 2 traditional CACC inhibitors (niflumic acid and anthracene-9-carboxcylic acid) and 2 novel CACC / TMEM16A inhibitors (CACC_inhA01 and T16A_inhA01) on TMEM16A currents. The whole cell patch clamp technique was used to record TMEM16A currents from HEK 293 cells that stably expressed human TMEM16A. Niflumic acid, A-9-C, CACC_inhA01 and T16A_inhA01 inhibited TMEM16A currents with IC₅₀ values of 12, 58, 1.7 and 1.5 µM, respectively, however, A-9-C and niflumic acid were less efficacious at negative membrane potentials. A-9-C and niflumic acid reduced the rate of TMEM16A tail current deactivation at negative membrane potentials and A-9-C (1 mM) enhanced peak TMEM16A tail current amplitude. In contrast, the inhibitory effects of CACC_inhA01 and T16A_inhA01 were independent of voltage and they did not prolong the rate of TMEM16A tail current deactivation. The effects of niflumic acid and A-9-C on TMEM16A currents were similar to previous observations on CACCs in vascular smooth muscle, strengthening the hypothesis that they are encoded by TMEM16A. However, CACC_inhA01 and T16A_inhA01 were more potent inhibitors of TMEM16A channels and their effects were not diminished at negative membrane potentials making them attractive candidates to interrogate the functional role of TMEM16A channels in future studies.     

Matrine is a novel inhibitor of the TMEM16A chloride channel with antilung adenocarcinoma effects

Calcium-activated chloride channels (CaCCs) are ion channels with key roles in physiological processes. They are abnormally expressed in various cancers, including esophageal squamous cell cancer, head and neck squamous cell carcinoma, colorectal cancer, and gastrointestinal stromal tumors. The CaCC component TMEM16A/ANO1 was recently shown to be overexpressed in lung adenocarcinoma cells and may serve as a tumorigenic protein. In this study, we determined that matrine is a potent TMEM16A inhibitor that exerts anti-lung adenocarcinoma effects. Patch clamp experiments showed that matrine inhibited TMEM16A current in a concentration-dependent manner with an IC ₅₀ of 27.94 ± 4.78 μM. Molecular simulation and site-directed mutation experiments demonstrated that the matrine-sensitive sites of the TMEM16A channel involve the amino acids Y355, F411, and F415. Results of cell viability and wound healing assays showed that matrine significantly inhibited the proliferation and migration of LA795 cells, which exhibit high TMEM16A expression. In contrast, matrine has only weak inhibitory effect on CCD-19Lu and HeLa cells lacking TMEM16A expression. Matrine-induced effects on the proliferation and migration of LA795 cells were abrogated upon shRNA-mediated TMEM16A knockdown in LA795 cells. Finally, in vivo experiments demonstrated that matrine dramatically inhibited the growth of lung adenocarcinoma xenograft tumors in mice but did not affect mouse body weight. Collectively, these data indicate that matrine is an effective and safe TMEM16A inhibitor and that TMEM16A is the target of matrine anti-lung adenocarcinoma activity. These findings provide new insight for the development of novel treatments for lung adenocarcinoma.     

Expression cloning of TMEM16A as a calcium-activated chloride channel subunit

Calcium-activated chloride channels (CaCCs) are major regulators of sensory transduction, epithelial secretion, and smooth muscle contraction. Other crucial roles of CaCCs include action potential generation in Characean algae and prevention of polyspermia in frog egg membrane. None of the known molecular candidates share properties characteristic of most CaCCs in native cells. Using Axolotl oocytes as an expression system, we have identified TMEM16A as the Xenopus oocyte CaCC. The TMEM16 family of "transmembrane proteins with unknown function" is conserved among eukaryotes, with family members linked to tracheomalacia (mouse TMEM16A), gnathodiaphyseal dysplasia (human TMEM16E), aberrant X segregation (a Drosophila TMEM16 family member), and increased sodium tolerance (yeast TMEM16). Moreover, mouse TMEM16A and TMEM16B yield CaCCs in Axolotl oocytes and mammalian HEK293 cells and recapitulate the broad CaCC expression. The identification of this new family of ion channels may help the development of CaCC modulators for treating diseases including hypertension and cystic fibrosis.     

Identification of the Novel TMEM16A Inhibitor Dehydroandrographolide and Its Anticancer Activity on SW620 Cells

TMEM16A, a calcium-activated chloride channel (CaCC), is highly amplified and expressed in human cancers and is involved in the growth and metastasis of some malignancies. Inhibition of TMEM16A represents a novel pharmaceutical approach for the treatment of cancers and metastases. The purpose of this study is to identify a new TMEM16A inhibitor, investigate the effects of this inhibitor on the proliferation and metastasis of TMEM16A-amplified SW620 cells, and to elucidate the underlying molecular mechanism in vitro. We identified a novel small-molecule TMEM16A inhibitor dehydroandrographolide (DP). By using patch clamp electrophysiology, we showed that DP inhibited TMEM16A chloride currents in Fisher rat thyroid (FRT) cells that were transfected stably with human TMEM16A and in TMEM16A-overexpressed SW620 cells but did not alter cystic fibrosis transmembrane conductance regulator (CFTR) chloride currents. Further functional studies showed that DP suppressed the proliferation of SW620 cells in a dose- and time-dependent manner using MTT assays. Moreover, DP significantly inhibited migration and invasion of SW620 cells as detected by wound-healing and transwell assays. Further mechanistic study demonstrated that knockdown of human TMEM16A decreased the inhibitory effect of DP on the proliferation of SW620 cells and that TMEM16A-dependent cells (SW620 and HCT116) were more sensitive to DP than TMEM16A-independent cells (SW480 and HCT8). In addition, we found that treatment of SW620 cells with DP led to a decrease in TMEM16A protein levels but had no effect on TMEM16A mRNA levels. The current work reveals that DP, a novel TMEM16A inhibitor, exerts its anticancer activity on SW620 cells partly through a TMEM16A-dependent mechanism, which may introduce a new targeting approach for an antitumour therapy in TMEM16A-amplified cancers.     

Systems Approaches to Unravel Molecular Function: High-content siRNA Screen Identifies TMEM16A Traffic Regulators as Potential Drug Targets for Cystic Fibrosis

An attractive approach to treat people with Cystic Fibrosis (CF), a life-shortening disease caused by mutant CFTR, is to compensate for the absence of this chloride/bicarbonate channel by activating alternative (non-CFTR) chloride channels. One obvious target for such “mutation-agnostic” therapeutic approach is TMEM16A (anoctamin-1/ANO1), a calcium-activated chloride channel (CaCC) which is also expressed in the airways of people with CF, albeit at low levels. To find novel TMEM16A regulators of both traffic and function, with the main goal of identifying candidate CF drug targets, we performed a fluorescence cell-based high-throughput siRNA microscopy screen for TMEM16A trafficking using a double-tagged construct expressed in human airway cells. About 700 genes were screened (2 siRNAs per gene) of which 262 were identified as candidate TMEM16A modulators (179 siRNAs enhanced and 83 decreased TMEM16A traffic), being G-protein coupled receptors (GPCRs) enriched on the primary hit list. Among the 179 TMEM16A traffic enhancer siRNAs subjected to secondary screening 20 were functionally validated. Further hit validation revealed that siRNAs targeting two GPCRs – ADRA2C and CXCR3 – increased TMEM16A-mediated chloride secretion in human airway cells, while their overexpression strongly diminished calcium-activated chloride currents in the same cell model. The knockdown, and likely also the inhibition, of these two TMEM16A modulators is therefore an attractive potential therapeutic strategy to increase chloride secretion in CF.     

Intestinal TMEM16A control luminal chloride secretion in a NHERF1 dependent manner

TMEM16A (Transmembrane protein 16A or Anoctamin1) is a calcium-activated chloride channel.(CaCC),that exerts critical roles in epithelial secretion. However, its localization, function, and regulation in intestinal chloride (Cl^?) secretion remain obscure. Here, we show that TMEM16A protein abundance correlates with Cl^? secretion in different regions of native intestine activated by the Ca²⁺-elevating muscarinic agonist carbachol (CCH). Basal, as well as both cAMP- and CCH-stimulated Isc, was largely reduced in Ano1 ± mouse intestine. We found CCH was not able to increase Isc in the presence of apical to serosal Cl^? gradient, strongly supporting TMEM16A as primarily a luminal Cl^? channel. Immunostaining demonstrated apical localization of TMEM16A where it colocalized with NHERF1 in mouse colonic tissue. Cellular depletion of NHERF1 in human colonic T84 cells caused a significant reduction of both cAMP- and CCH-stimulated Isc. Immunoprecipitation experiments revealed that NHERF1 forms a complex with TMEM16A through a PDZ-based interaction. We conclude that TMEM16A is a luminal Cl^? channel in the intestine that functionally interacts with CFTR via PDZ-based interaction of NHERF1 for efficient and specific cholinergic stimulation of intestinal Cl^? secretion.     

TRPC1 regulates calcium‐activated chloride channels in salivary gland cells

Calcium‐activated chloride channel (CaCC) plays an important role in modulating epithelial secretion. It has been suggested that in salivary tissues, sustained fluid secretion is dependent on Ca²⁺ influx that activates ion channels such as CaCC to initiate Cl^? efflux. However direct evidence as well as the molecular identity of the Ca²⁺ channel responsible for activating CaCC in salivary tissues is not yet identified. Here we provide evidence that in human salivary cells, an outward rectifying Cl^? current was activated by increasing [Ca²⁺]_i, which was inhibited by the addition of pharmacological agents niflumic acid (NFA), an antagonist of CaCC, or T16Ainh‐A01, a specific TMEM16a inhibitor. Addition of thapsigargin (Tg), that induces store‐depletion and activates TRPC1‐mediated Ca²⁺ entry, potentiated the Cl^? current, which was inhibited by the addition of a non‐specific TRPC channel blocker SKF96365 or removal of external Ca²⁺. Stimulation with Tg also increased plasma membrane expression of TMEM16a protein, which was also dependent on Ca²⁺ entry. Importantly, in salivary cells, TRPC1 silencing, but not that of TRPC3, inhibited CaCC especially upon store depletion. Moreover, primary acinar cells isolated from submandibular gland also showed outward rectifying Cl^? currents upon increasing [Ca²⁺]_i. These Cl^? currents were again potentiated with the addition of Tg, but inhibited in the presence of T16Ainh‐A01. Finally, acinar cells isolated from the submandibular glands of TRPC1 knockout mice showed significant inhibition of the outward Cl^? currents without decreasing TMEM16a expression. Together the data suggests that Ca²⁺ entry via the TRPC1 channels is essential for the activation of CaCC. J. Cell. Physiol. 9999: 2848–2856, 2015. © 2015 Wiley Periodicals, Inc.

     

Fractionation of a herbal antidiarrheal medicine reveals eugenol as an inhibitor of Ca2+-Activated Cl- channel TMEM16A

The Ca(2+)-activated Cl(-) channel TMEM16A is involved in epithelial fluid secretion, smooth muscle contraction and neurosensory signaling. We identified a Thai herbal antidiarrheal formulation that inhibited TMEM16A Cl(-) conductance. C18-reversed-phase HPLC fractionation of the herbal formulation revealed >98% of TMEM16A inhibition activity in one out of approximately 20 distinct peaks. The purified, active compound was identified as eugenol (4-allyl-2-methoxyphenol), the major component of clove oil. Eugenol fully inhibited TMEM16A Cl(-) conductance with single-site IC(50)~150 μM. Eugenol inhibition of TMEM16A in interstitial cells of Cajal produced strong inhibition of intestinal contraction in mouse ileal segments. TMEM16A Cl(-) channel inhibition adds to the list of eugenol molecular targets and may account for some of its biological activities.     

Novel 5-substituted benzyloxy-2-arylbenzofuran-3-carboxylic acids as calcium activated chloride channel inhibitors

Transmembrane protein 16A (TMEM16A) channels are recently discovered membrane proteins that functions as a calcium activated chloride channel (CaCC). CaCCs are major regulators of various physiological processes, such as sensory transduction, epithelial secretion, smooth muscle contraction and oocyte fertilization. Thirty novel 5-substituted benzyloxy-2-arylbenzofuran-3-carboxylic acids (B01-B30) were synthesized and evaluated for their TMEM16A inhibitory activity by using short circuit current measurements in Fischer rat thyroid (FRT) cells expressing human TMEM16A. IC(50) values were calculated using YFP fluorescence plate reader assay. Final compounds, having free carboxylic group displayed significant inhibition. Eight of the novel compounds B02, B13, B21, B23, B25, B27, B28, B29 exhibit excellent CaCCs inhibition with IC(50) value <6 μM, with compound B25 exhibiting the lowest IC(50) value of 2.8 ± 1.3 μM. None of the tested ester analogs of final benzofuran derivatives displayed TMEM16A/CaCCs inhibition.     

Synthesis and evaluation of 5,6-disubstituted thiopyrimidine aryl aminothiazoles as inhibitors of the calcium-activated chloride channel TMEM16A/Ano1

Transmembrane protein 16A (TMEM16A), also called Ano1, is a Ca²⁺ activated Cl^? channel expressed widely in mammalian epithelia, as well as in vascular smooth muscle and some tumors and electrically excitable cells. TMEM16A inhibitors have potential utility for treatment of disorders of epithelial fluid and mucus secretion, hypertension, some cancers and other diseases. 4-Aryl-2-amino thiazole T16A_inh-01 was previously identified by high-throughput screening. Here, a library of 47 compounds were prepared that explored the 5,6-disubstituted pyrimidine scaffold found in T16A_inh-01. TMEM16A inhibition activity was measured using fluorescence plate reader and short-circuit current assays. We found that very little structural variation of T16A_inh-01 was tolerated, with most compounds showing no activity at 10?μM. The most potent compound in the series, 9bo, which substitutes 4-methoxyphenyl in T16A_inh-01 with 2-thiophene, had IC₅₀ ～1?μM for inhibition of TMEM16A chloride conductance.     

Activation and Inhibition of TMEM16A Calcium-Activated Chloride Channels

Calcium-activated chloride channels (CaCC) encoded by family members of transmembrane proteins of unknown function 16 (TMEM16) have recently been intensely studied for functional properties as well as their physiological roles as chloride channels in various tissues. One technical hurdle in studying these channels is the well-known channel rundown that frequently impairs the precision of electrophysiological measurements for the channels. Using experimental protocols that employ fast-solution exchange, we circumvented the problem of channel rundown by normalizing the Ca²⁺-induced current to the maximally-activated current obtained within a time period in which the channel rundown was negligible. We characterized the activation of the TMEM16A-encoded CaCC (also called ANO1) by Ca²⁺, Sr²⁺, and Ba²⁺, and discovered that Mg²⁺ competes with Ca²⁺ in binding to the divalent-cation binding site without activating the channel. We also studied the permeability of the ANO1 pore for various anions and found that the anion occupancy in the pore–as revealed by the permeability ratios of these anions–appeared to be inversely correlated with the apparent affinity of the ANO1 inhibition by niflumic acid (NFA). On the other hand, the NFA inhibition was neither affected by the degree of the channel activation nor influenced by the types of divalent cations used for the channel activation. These results suggest that the NFA inhibition of ANO1 is likely mediated by altering the pore function but not through changing the channel gating. Our study provides a precise characterization of ANO1 and documents factors that can affect divalent cation activation and NFA inhibition of ANO1.     

Functional Swapping between Transmembrane Proteins TMEM16A and TMEM16F

The transmembrane proteins TMEM16A and -16F each carry eight transmembrane regions with cytoplasmic N and C termini. TMEM16A carries out Ca²⁺-dependent Cl⁻ ion transport, and TMEM16F is responsible for Ca²⁺-dependent phospholipid scrambling. Here we established assay systems for the Ca²⁺-dependent Cl⁻ channel activity using 293T cells and for the phospholipid scramblase activity using TMEM16F^−/− immortalized fetal thymocytes. Chemical cross-linking analysis showed that TMEM16A and -16F form homodimers in both 293T cells and immortalized fetal thymocytes. Successive deletion from the N or C terminus of both proteins and the swapping of regions between TMEM16A and -16F indicated that their cytoplasmic N-terminal (147 amino acids for TMEM16A and 95 for 16F) and C-terminal (88 amino acids for TMEM16A and 68 for 16F) regions were essential for their localization at plasma membranes and protein stability, respectively, and could be exchanged. Analyses of TMEM16A and -16F mutants with point mutations in the pore region (located between the fifth and sixth transmembrane regions) indicated that the pore region is essential for both the Cl⁻ channel activity of TMEM16A and the phospholipid scramblase activity of TMEM16F. Some chemicals such as epigallocatechin-3-gallate and digallic acid inhibited the Cl⁻ channel activity of TMEM16A and the scramblase activity of TMEM16F with an opposite preference. These results indicate that TMEM16A and -16F use a similar mechanism for sorting to plasma membrane and protein stabilization, but their functional domains significantly differ.     

Recent advances in TMEM16A: Structure,function, and disease

TMEM16A (also known as anoctamin 1, ANO1) is the molecular basis of the calcium-activated chloride channels, with ten transmembrane segments. Recently, atomic structures of the transmembrane domains of mouse TMEM16A (mTMEM16A) were determined by single-particle electron cryomicroscopy. This gives us a solid ground to discuss the electrophysiological properties and functions of TMEM16A. TMEM16A is reported to be dually regulated by Ca²⁺ and voltage. In addition, the dysfunction of TMEM16A has been found to be involved in many diseases including cystic fibrosis, various cancers, hypertension, and gastrointestinal motility disorders. TMEM16A is overexpressed in many cancers, including gastrointestinal stromal tumors, gastric cancer, head and neck squamous cell carcinoma (HNSCC), colon cancer, pancreatic ductal adenocarcinoma, and esophageal cancer. Furthermore, overexpression of TMEM16A is related to the occurrence, proliferation, and migration of tumor cells. To date, several studies have shown that many natural compounds and synthetic compounds have regulatory effects on TMEM16A. These small molecule compounds might be novel drugs for the treatment of diseases caused by TMEM16A dysfunction in the future. In addition, recent studies have shown that TMEM16A plays different roles in different diseases through different signal transduction pathways. This review discusses the topology, electrophysiological properties, modulators and functions of TMEM16A in mediates nociception, gastrointestinal dysfunction, hypertension, and cancer and focuses on multiple regulatory mechanisms regarding TMEM16A.     

Developmental expression of the calcium‐activated chloride channels TMEM16A and TMEM16B in the mouse olfactory epithelium

Calcium‐activated chloride channels are involved in several physiological processes including olfactory perception. TMEM16A and TMEM16B, members of the transmembrane protein 16 family (TMEM16), are responsible for calcium‐activated chloride currents in several cells. Both are present in the olfactory epithelium of adult mice, but little is known about their expression during embryonic development. Using immunohistochemistry we studied their expression in the mouse olfactory epithelium at various stages of prenatal development from embryonic day (E) 12.5 to E18.5 as well as in postnatal mice. At E12.5, TMEM16A immunoreactivity was present at the apical surface of the entire olfactory epithelium, but from E16.5 became restricted to a region near the transition zone with the respiratory epithelium, where localized at the apical part of supporting cells and in their microvilli. In contrast, TMEM16B immunoreactivity was present at E14.5 at the apical surface of the entire olfactory epithelium, increased in subsequent days, and localized to the cilia of mature olfactory sensory neurons. These data suggest different functional roles for TMEM16A and TMEM16B in the developing as well as in the postnatal olfactory epithelium. The presence of TMEM16A at the apical part and in microvilli of supporting cells is consistent with a role in the regulation of the chloride ionic composition of the mucus covering the apical surface of the olfactory epithelium, whereas the localization of TMEM16B to the cilia of mature olfactory sensory neurons is consistent with a role in olfactory signal transduction. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 74: 657–675, 2014     

Oleic acid blocks the calcium-activated chloride channel TMEM16A/ANO1

The calcium-activated chloride channel TMEM16A (ANO1) supports the passive movement of chloride ions across membranes and controls critical cell functions. Here we study the block of wild-type and mutant TMEM16A channels expressed in HEK293 cells by oleic acid, a monounsaturated omega-9 fatty acid beneficial for cardiovascular health. We found that oleic acid irreversibly blocks TMEM16A in a dose- and voltage-dependent manner at low intracellular Ca²⁺. We tested whether oleic acid interacted with the TMEM16A pore, varying the permeant anion concentration and mutating pore residues. Lowering the permeating anion concentration in the intracellular side did nothing but the blockade was intensified by increasing the anion concentration in the extracellular side. However, the blockade of the pore mutants E633A and I641A was voltage-independent, and the I641A IC₅₀, a mutant with the inner hydrophobic gate in disarray, increased 16-fold. Furthermore, the uncharged methyl-oleate blocked 20–24% of the wild-type and I641A channels regardless of voltage. Our findings suggest that oleic acid inhibits TMEM16A by an allosteric mechanism after the electric field drives oleic acid's charged moiety inside the pore. Block of TMEM16A might be why oleic acid has a beneficial impact on the cardiovascular system.     

Compartmentalized crosstalk of CFTR and TMEM16A (ANO1) through EPAC1 and ADCY1

Airway epithelial cells express both Ca²⁺ activated TMEM16A/ANO1 and cAMP activated CFTR anion channels. Previous work suggested a significant crosstalk of intracellular Ca²⁺ and cAMP signaling pathways, leading to activation of both chloride channels. We demonstrate that in airway epithelial cells, stimulation of purinergic or muscarinic G-protein coupled receptors (GPCRs) activates TMEM16A and CFTR. Additional expression of G_q/11 and phospholipase C coupled GPCRs strongly enhanced the crosstalk between Ca²⁺- and cAMP-dependent signaling. Knockdown of endogenous GRCRs attenuated crosstalk and functional coupling between TMEM16A and CFTR. The number of receptors did not affect expression or membrane localization of TMEM16A or CFTR, but controlled assembly of the local signalosome. GPCRs translocate Ca²⁺-sensitive adenylate cyclase type 1 (ADCY1) and exchange protein directly activated by cAMP (EPAC1) to particular plasma membrane domains containing GPCRs, CFTR and TMEM16A, thereby producing compartmentalized Ca²⁺ and cAMP signals and significant crosstalk. While biosynthesis and membrane trafficking of CFTR requires a functional Golgi apparatus, maturation and membrane trafficking of TMEM16A may occur independent of the Golgi. Because Ca²⁺ activated TMEM16A currents are only transient, continuous Cl⁻ secretion by airway epithelial cells requires CFTR. The present data also explain why receptor-dependent activation of TMEM16A is more efficient than direct stimulation by Ca²⁺.     

The multifaceted role of TMEM16A in cancer

The calcium-activated chloride channel TMEM16A is intimately linked to cancers. Over decades, TMEM16A over-expression and contribution to prognosis have been widely studied for multiple cancers strengthening the idea that TMEM16A could be a valuable biomarker and a promising therapeutic target. Surprisingly, from the survey of the literature, it appears that TMEM16A has been involved in multiple cancer-related functions and a large number of molecular targets of TMEM16A have been proposed. Thus, TMEM16A appears to be an ion channel with a multifaceted role in cancers.In this review, we summarize the latest development regarding TMEM16A contribution to cancers. We will survey TMEM16A contribution in cancer prognosis, the origins of its over-expression in cancer cells, the multiple biological functions and molecular pathways regulated by TMEM16A. Then, we will consider the question regarding the molecular mechanism of TMEM16A in cancers and the possible basis for the multifaceted role of TMEM16A in cancers.     

Development of the Olfactory Epithelium and Nasal Glands in TMEM16A-/- and TMEM16A+/+ Mice

TMEM16A/ANO1 is a calcium-activated chloride channel expressed in several types of epithelia and involved in various physiological processes, including proliferation and development. During mouse embryonic development, the expression of TMEM16A in the olfactory epithelium is dynamic. TMEM16A is expressed at the apical surface of the entire olfactory epithelium at embryonic day E12.5 while from E16.5 its expression is restricted to a region near the transition zone with the respiratory epithelium. To investigate whether TMEM16A plays a role in the development of the mouse olfactory epithelium, we obtained the first immunohistochemistry study comparing the morphological properties of the olfactory epithelium and nasal glands in TMEM16A^-/- and TMEM16A^+/+ littermate mice. A comparison between the expression of the olfactory marker protein and adenylyl cyclase III shows that genetic ablation of TMEM16A did not seem to affect the maturation of olfactory sensory neurons and their ciliary layer. As TMEM16A is expressed at the apical part of supporting cells and in their microvilli, we used ezrin and cytokeratin 8 as markers of microvilli and cell body of supporting cells, respectively, and found that morphology and development of supporting cells were similar in TMEM16A^-/- and TMEM16A^+/+ littermate mice. The average number of supporting cells, olfactory sensory neurons, horizontal and globose basal cells were not significantly different in the two types of mice. Moreover, we also observed that the morphology of Bowman’s glands, nasal septal glands and lateral nasal glands did not change in the absence of TMEM16A. Our results indicate that the development of mouse olfactory epithelium and nasal glands does not seem to be affected by the genetic ablation of TMEM16A.