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The Ca2+-activated chloride channel ANO1/TMEM16A: An emerging therapeutic target for epithelium-originated diseases?

Anoctamin 1 (ANO1) or TMEM16A gene encodes a member of Ca2+ activated Cl– channels (CaCCs) that are critical for physiological functions, such as epithelial secretion, smooth muscle contraction and sensory signal transduction. The attraction and interest in ANO1/TMEM16A arise from a decade long investigations that abnormal expression or dysfunction of ANO1 is involved in many pathological phenotypes and diseases, including asthma, neuropathic pain, hypertension and cancer. However, the lack of specific modulators of ANO1 has impeded the efforts to validate ANO1 as a therapeutic target. This review focuses on the recent progress made in understanding of the pathophysiological functions of CaCC ANO1 and the current modulators used as pharmacological tools, hopefully illustrating a broad spectrum of ANO1 channelopathy and a path forward for this target validation.


Introduction
Ca 2þ -activated chloride channels (CaCCs) are a heterogeneous group of Cl e channels that can be synergistically activated by intracellular calcium and voltage. The CaCCs are found in almost all species ranging from invertebrates to mammals, and the ubiquitous expression of CaCCs indicates a variety of functions important for physiology, including regulation of epithelial Cl e secretion, excitability of neuronal and cardiac cells, smooth muscle contraction and nociception 1e3 .
A subtype of CaCCs was first described in Xenopus oocytes nearly 40 years ago and its molecular identity was debated until in 2008 when three independent laboratories reported that anoctamin-1 (ANO1) or transmembrane protein 16A (TMEM16A) underlies the molecular basis of a subgroup of CaCCs 4e6 . The Ano1/Tmem16a gene encodes a 986-amino-acid protein that belongs to anoctamin family consisting of 10 members (ANO1eANO10) in mammals. ANO1, as a CaCC, is primarily expressed in epithelial cells, smooth muscle cells and sensory neurons. ANO2, also as a CaCC, is expressed in the olfactory sensory neurons 7 , photoreceptor synaptic terminals 8 , hippocampal pyramidal neurons 9 , thalamocortical neurons 10 , and inferior olive neurons 11 in the brain. Other ANOs family members including ANO6 and ANO7 were onetime considered to be CaCCs 12 , but evidence shows that ANOs3e7 neither generate Ca 2þ -activated Cl e currents nor traffic into membrane, indicating that they are endoplasmic reticulum proteins 13 . More studies reveal that ANOs3e7 and ANO9 are linked to the Ca 2þ -dependent membrane phospholipid scramblases that are responsible for translocation of phospholipids 14e16 . It is generally accepted that ANO1 and ANO2 are two members of the CaCC subfamily, whereas ANO3eANO10 have been debated for their functions as CaCCs or Ca 2þ -dependent membrane phospholipid scramblases, or other physiological proteins 17,18 . In addition, an integral membrane protein bestrophin-1 (BEST1) encoded by the BEST1 gene also functions as a CaCC. BEST1 is predominantly expressed in retinal pigment epithelium 19 , non-neuronal tissue, peripheral and central neurons 20,21 .
ANO1 was initially thought to have eight transmembrane domains, possessing multiple protein isoforms generated by alternative splicing of four segments (a, b, c, and d) located at the C-terminus and the first intracellular loop 5 . ANO1 splice variant lacking segment b, for instance, increases the calcium sensitivity, whereas deletion of the segment c decreases apparent Ca 2þ sensitivity and increases voltage-dependent activity of ANO1 channel 22,23 . The X-ray structure of a Ca 2þ -activated lipid scramblase ANO1 from fungus Nectria haematococca (nhANO1) shows a homodimer with each subunit containing a tentransmembrane a-helices 24 . Several critical amino acid residues for Ca 2þ sensitivity and ion selectivity are subsequently found 25,26 . Recently, a high-resolution cryo-EM structure reveals an overall structure of mouse ANO1 similar to nhANO1 27,28 , exhibiting two Ca 2þ binding sites within the inner vestibule of the pore, and Ca 2þ binding triggering conformational changes of a-helix rendering the pore conductive ( Fig. 1 26e29 ). The mechanisms underlying ANO1 activation and modulation are beginning to emerge. It has been shown that calmodulin, protons, cell volume and thermal stimuli can regulate the channel activation 30,31 , and phosphatidylinositol 4,5-bisphosphate (PIP 2 ) regulates the activation and desensitization of ANO1 32,33 .
ANO1 channel appears to be preferentially activated by local rise of intracellular Ca 2þ release from the endoplasmic reticulum (ER) Ca 2þ stores, which may represent a general mechanism of ANO1 activation 34 . ANO1 expression is regulated by multiple signaling cascade pathways such as mitogen-activated protein kinase (MAPK), nuclear factor kB (NF-kB) and transforming growth factor-b (TGF-b) in pathological functions 35 . Several recent reviews have substantially covered the molecular basis, structure and pathophysiological functions of ANO1 17,35e37 . In this review we will mainly focus on the aspect of channelopathies and pharmacological validation of ANO1 as an emerging therapeutic target.

Pharmacological modulation of ANO1 channel
Validating ANO1 as a therapeutic target requires specific modulators that can serve as essential tools for understanding the channel pharmacology. Up to now, many ANO1 modulators have been reported, and unfortunately most of them are lack of potency and efficacy. Therefore, there exists a great need for discovery of more selective and potent modulators, inhibitors in particular, which can be used for ANO1 target validation.

Inhibitors
Some broad-spectrum blockers, such as NFA, FFA, DIDS, NPPB and 9AC, have been used as tools for understanding functions of CaCCs (Table 1 38e84 ). These small molecules can block endogenous CaCCs in Xenopus laevis oocytes 39,43,45,85 and are also known to non-specifically modulate other channels, such as inhibition of volume regulated anion channel (VRAC) and Kv4 by NFA and DIDS and activation of Ca 2þ activated K þ channel by NFA and FFA or potentiation of TRPV1 channel by DIDS 2,38 . CaCC inh -A01 was identified to inhibit CaCC current in 2008 from screen of 50,000 compounds using human intestinal epithelial HT29 cell that highly express endogenous CaCCs without obvious effects on intracellular Ca 2þ , Ca 2þ /calmodulin-dependent protein kinase (CAMK) or cystic fibrosis transmembrane conductance regulator (CFTR) 49 . CaCC inh -A01 as an ANO1 inhibitor has been commonly used in many investigations for the role of ANO1 in interstitial cells of cajal (ICC), cardiac fibroblast and rod bipolar cells of retina 86e88 , and channelopathies, including cancer, hypertension, nociception, diarrhea and high glucose induced renal cyst growth 51,53e55 . CaCC inh -A01 is shown to inhibit cancer cell proliferation through increasing the ubiquitination of ANO1 and facilitating ER-associated, proteasomal degradation of ANO1, while other ANO1 inhibitors, such as T16A inh -A01 and digallic acid, are shown to have no effect on cancer cell proliferation 89e91 . Similarly, CaCC inh -A01 inhibits proliferation of cardiac fibroblast, but not another ANO1 inhibitor T16A inh -A01 87 . CaCC inh -A01 reduces upregulation of ANO1 expression and attenuates brain infarct size and neurological deficits after ischemic stroke whereas T16A inh -A01 shows no effect 56 . That ANO1 protein expression is reduced by CaCC inh -A01 but not T16A inh -A01 may partially explain why the two ANO1 inhibitors show different effects on cell proliferation and ischemic stroke. In addition, intrathecal injection of CaCC inh -A01 reduces tactile allodynia and thermal hyperalgesia and also decreases ANO1 upregulation after spinal nerve injury 54 .
Since the identification of CaCC inh -A01, several ANO1 inhibitors have been screened out using the iodide-sensitive yellow fluorescent protein (YFP)-based high throughput screening (HTS) assay 50,65,67 . T16A inh -A01 was reported to inhibit human ANO1 current expressed in Fisher rat thyroid (FRT) cells with an IC 50 of 1 mmol/L and had no effect on CFTR current 50 . However, our previous study showed that T16A inh -A01 at 10 mmol/L only inhibits mouse ANO1 current expressed in CHO cells about 28% at þ80 mV 38 , and it may because that the efficacy of T16A inh -A01 on ANO1 inhibition dependents on splice variants of ANO1 and intracellular calcium 92 . Nevertheless, T16A inh -A01 has been used as a pharmacological probe to investigate the role of ANO1 in pancreatic ductal adenocarcinoma cells 93 , and also physiological and pathological functions of ANO1 in different tissues, including contraction of vesical smooth muscle, iodide release from thyrocyte, initial waveform modulation in retinal rod bipolar cell and melatonin secretion in pineal glands 52,88,94e96 (Table 1). T16A inh -A01 attenuates the interleukin-13 (IL-13) induced increase of ANO1 expression and secretion of mucin in human nasal polyp epithelial cells from chronic rhinosinusitis patients, cultured human bronchial epithelial cells and goblet cells in the guinea pig asthma model 59,97 , suggesting that T16A inh -A01 may be useful for treatment of hypersecretion in asthma and other inflammatory airway diseases. In addition, several studies show that ANO1 inhibitor T16A inh -A01 exerts therapeutic effect on cancer, neuropathic pain and eosinophilic esophagitis 58,98e100 .
Another small molecule Ani9 was also identified to inhibit hANO1 expressed in FRT cells with an IC 50 of 0.08 mmol/L using YFP-HTS assay ( Table 1). Ani9 belongs to acetamides and shows a relatively high selectivity on ANO1 over ANO2 without inhibitory effects on CFTR, VRAC, epithelial sodium channels (ENaC) and intracellular Ca 2þ signaling at concentration of 30 mmol/L 65 , whereas Ani9 can inhibit inward current and slow the time-dependent activation of ANO6 66 . The Ani9 derivative 5f shows more potent inhibitory effect on ANO1 with an IC 50 of 20 nmol/L without activating on ANO2 68 . Ani9 as a novel potent and selective ANO1 inhibitor has also been used in several investigations for the physiological and pathological functions of ANO1, including hypertension, polyspermy block and cancer 47,57,63 . As one of the 2-acylamino-cycloalkylthiophene-3carboxylic acid arylamides, 10bm shows a potent inhibition on ANO1 current with an IC 50 of 30 nmol/L and exhibits dosedependent inhibition on isometric smooth muscle contraction. The 10bm compound also inhibits ANO2 with an IC 50 of 0.4 mmol/L without effect on CFTR 67 . At present, synthesized ANO1 inhibitors are only used as tools for preclinical studies (Table 1).
Many natural products from diverse plants have been found to inhibit ANO1. Tannic acid presented in the green tea and red wine was identified as a blocker for both ANO1 and ANO2 without effect on CFTR or ENaC, showing an inhibitory effect on arterial smooth muscle contraction and intestinal Cl À secretion 71 . Like the synthesized small molecule ANO1 inhibitors, tannic acid has also been used as a pharmacological tool for investigations of the biophysical feature and physiological functions of ANO1 101e103 . Series of flavonoids were recently identified to inhibit ANO1, exerting anticancer effects 77,78 . Natural products or synthetic analogues of natural products, including idebenone with anticancer activity 81 , plumbagin with antidiarrhea 83 and matrine with anti-lung adenocarcinoma activity 104 have been shown to inhibit ANO1.
Several clinical drugs are recently found to have inhibitory effect on ANO1, including clarithromycin, benzbromarone, niclosamide, nitazoxanide and avermectins. Clarithromycin is a clinical antibiotic that was reported to decrease IL-13 induced ANO1 expression in goblet cells from asthma models 105 . Benzbromarone, a clinical drug for gout treatment, shows antiasthmatic effect through inhibiting IL-13 induced mucin secretion and methacholine induced airway smooth muscle (ASM) contraction via the inhibition of ANO1 70,106 . Niclosamide and nitazoxamide are clinical anthelmintics that as potent ANO1 inhibitors can block ASM depolarization and contraction 70 . Avermectins are a type of macrocyclic lactones that are widely used as pharmaceuticals against roundworms in humans and animals and also for crop protection. Several avermectins, including avermectin B1, ivermectin, doramectin, selamectin and moxidectin, exhibit inhibitory effects on cancer cell proliferation  The Ca 2þ -activated chloride channel ANO1/TMEM16A and migration through inhibition of ANO1 84 . These drugs may have repurposing potential for dysfunctional ANO1 related diseases, such as asthma and hypertension.
It is noticeable that many compounds can act on ANO1 with limited selectivity, which imposes a big challenge for validation of ANO1 as a therapeutic target. The reasons for lack of identifying specific ANO1 modulators can be in multiple folds including chemical designs without guidance of target structure, use of an indirect YFP fluorescence-based HTS assay, and compound screens against cells expressing endogenous xANO1 in oocytes (for MONNA) 61 , hANO1 in HT29 cells (for CaCC inh -A01) 49 , or heterogeneous hANO1 in FRT cells (for T16A inh -A01, Ani9 and 10bm) 50,65,67 . In addition, ANO1 and ANO2 subunits share 62% of sequence 4 , which also presents the selectivity challenge. The recent solved cryo-EM structure of ANO1 may help understand the biophysical features and physiological functions of the channel and also can greatly assist to develop more specific ANO1 modulators 27,28 . A pharmacophore model based on three-dimensional quantitative structureeactivity relationship (3D-QSAR) for predicting best target and compound interactions also appears to be a promising tool for virtual screening and enhancing rational design for novel potent and specific ANO1 modulators 107 .

Activators
During the past almost 40 years since the identification of CaCC in 1982 108 , only a few activators of ANO1 have been reported. Two ANO1 activators Eact and Fact are the first discovered using an HTS approach 109 . Eact and Fact are two different classes of chemicals that activate ANO1 through different mechanisms (Table 2 66,109e117 ). Eact is an activator that induces ANO1 current in the absence of intracellular Ca 2þ with an EC 50 of 3 mmol/L. Several studies indicate that Eact might indirectly activate ANO1 through an intracellular Ca 2þ increase 110,111 . Fact is a potentiator that increases ANO1 current in a Ca 2þ -dependent manner with an EC 50 of 6 mmol/L. INO-4995 is an 1-O-octyl-2-O-butyryl-myoinositol 3,4,5,6-tetrakisphosphate octakis(propionoxymethyl) ester directly activates overexpressed ANO1 current but without effect on endogenous ANO1 currents in Xenopus oocytes, human airways and colonic cells 112 .
A small molecule ANO1 potentiator ETX001 is recently shown to increase ANO1 current with an EC 50 of 116 nmol/L without effect on intracellular Ca 2þ signaling 116 (Table 2). ETX001 enhances fluid secretory response in human bronchial epithelial cells from cystic fibrosis (CF) patients, and increases airway mucus clearance in sheep CF models. Several compounds from traditional Chinese medicines, including resveratrol 113 , ginsenoside Rb1 (GRb1) 114 , GRb2 29 , cinnamaldehyde 118 and chitosan oligosaccharides 115 , were recently reported to activate ANO1 channel although their mechanisms of action remain elusive. Activators of ANO1 can be useful for validating ANO1 as a therapeutic target for treatment of disorders associated with ANO1 hypofunction, including Sjogren's syndrome, cystic fibrosis lung disease, and dry eye syndrome.
It should be mentioned that the relatively wide distribution of ANO1 may impose a liability concern or a challenge in developing organ-or tissue-specific therapy. Nevertheless, development of targeted drug delivery systems such as tissue-specific or selective organ targeting nanoparticles and controlled release of therapeutic agents may circumvent potential ANO1-originated complications 119 . In addition, target-related complications can also be minimized thought means of different drug formulations, þ, activation effect; /, no effect; NR, no report.
such as topical preparations, sublingual, buccal and rectal administrations.

Pathological functions and related diseases
Since ANO1 was identified as a member of CaCCs in 2008, its investigations have been focused on the distribution and expression in numerous organs and tissues, and its roles in pathological conditions and diseases (Fig. 2 51,98,120,121 ).

Epithelial diseases
Epithelial cells are a group of tightly compressed cells that line the inside of all organs and function as a barrier between the inside and outside of an organ. Chloride ion is important for transepithelial secretion, whereas intracellular Ca 2þ is one of two major signals to modulate Cl e secretion 122,123 , indicating an important role of Ca 2þ activated Cl e channels in regulating epithelial secretion. ANO1 is observed in many epithelial tissues, including but not limited airway, bronchus, salivary glands, pancreatic ductal cells and intestinal epithelium 1,4,124 . Numerous literatures not only identify native ANO1 expressions in multiple epithelia tissues but also demonstrate the pathological functions of ANO1 important for the process of fluid and electrolyte secretion 5,37,125e127 . Here, we review several epithelial diseases involved in ANO1 dysfunction.

Asthma
Asthma is a prevalent inflammatory airway disease characterized by chronic inflammation, remodeling, and excessive constriction of the airway. The mechanism underlying asthma is complex and treatment for asthma is still challenging 128 . Hypersecretion of airway epithelium and hypercontraction of airway smooth muscle are two major contributions to asthma 129 . The Ca 2þ activated Cl e channel ANO1 as an anion channel plays a critical role in airway transepithelial mucus function. Mucus secreted by goblet cells is composed of water and mucus protein, which requires anion channel activity to instill Cl e and HCO 3 e for ensurance of proper salination, hydration and pH of the mucus gel layers 130 . ANO1 is found robustly expressed in epithelial goblet cells and involved in mediating native Ca 2þ dependent Cl e current 131 . ANO1 is also highly permeable to HCO 3 e at higher intracellular Ca 2þ levels, likely contributing to mucus release by providing a secretory pathway for HCO 3 e that is an essential ingredient in mucus 132,133 . Downregulation of ANO1 markedly decreases epithelial mucus secretion 126,127 , suggesting ANO1 inhibition beneficial for asthma. ANO1 expression is upregulated by cytokines IL-4, IL-13 and T helper type 2 that are asthmatic biomarkers, and upregulated ANO1 colocalizes to the apical plasma membrane of goblet cells along with the mucin MUC5AC 48,97,131 . In addition, ANO1 is also found abundantly expressed in the smooth muscle cells and plays a role in airway hyperreactivity 48 . ANO1 expression is upregulated both in epithelial cells and airway smooth muscle cells from asthmatic human patients and rodent models 41,48,127 . These investigations suggest that hyperactivity of ANO1 may underlie the pathogenesis of asthma, and inhibition of ANO1 channel activity may represent a therapeutic strategy for mucus hypersecretion in asthma.
The mechanism for ANO1 involving in asthma is unclear. A recent investigation shows that the GPCR/ANO1/VGCC axis contributes to the bronchial hyperresponsiveness in asthma 134 .
The asthmatic inflammatory mediators, including 5-HT, histamine and thromboxane A2 binding to G-protein coupled receptors (GPCRs), activate IP 3 R to release Ca 2þ from sarcoplasmic reticulum for activation of ANO1 and voltage gated calcium channel (VGCC) that leads to Ca 2þ influx and subsequently depolarize the membrane potential and cause airway contraction both ex vivo ASM and in vivo asthmatic animals 134 . Downregulation of ANO1 by either pharmacological inhibitor T16A inh -A01 or knockdown can inhibit the inflammatory mediator-induced Ca 2þ -activated Cl e current and ASM contractile response 134 . Several reports show the involvement of ANO1 in mucus production or secretion 48,59,127 , but the mechanistic insights into mucus production and secretion in airways and validation of the role ANO1 in asthma are required.
Huang and his colleges 48 adopted a HTS assay and identified benzbromarone, a clinical drug for gout treatment, as an ANO1 blocker. Benzbromarone shows anti-asthmatic effect through inhibiting IL-13-induced mucin secretion and methacholineinduced ASM contraction via inhibition of ANO1 function 70,106 . In addition, several ANO1 inhibitors, such as NFA 41 , tannic acid 73 , T16A inh -A01 59 , and niclosamide 70 have been shown to exert anti-asthma effects. However, the anti-asthma effects observed using those ANO1 inhibitors need to be further confirmed in some degree because of their relatively low potency and poor selectivity.

Diarrhea
Diarrhea is characterized by an increase in the frequency of bowel movements with loose and watery stools and major causes of infectious secretory diarrhea include bacteria, viruses and parasites 135 . Intestinal Cl e channels are important for regulation of excessive mucus secretion induced by bacteria/virus-secreted enterotoxins through increasing apical membrane permeability, suggesting an involvement of Ca 2þ activated Cl e channel ANO1 in pathology of diarrhea. There is an increasing evidence that ANO1 plays an important role in the regulation of intestinal epithelial secretion and the pathogenesis of diarrhea based on the following observations: 1) ANO1 is expressed in intestinal epithelia and ANO1 gene knockdown reduces Ca 2þ activated Cl e secretion induced by muscarinic agonist carbochol 126 ; 2) Ca 2þdependent Cl À secretion increases through enhancement of ANO1 expression or activation of ANO1 current in the distal colon in response to the rotavirus nonstructural glycoprotein NSP4 that causes infantile gastroenteritis 136 ; and 3) ANO1 expression is upregulated in animal models of diarrhea 137 .
The increased activity of gastrointestinal (GI) tract is another important cause for diarrhea. ICC are mesenchymal cells located within the muscle layers in the GI tract and myenteric ICC serve as a pacemaker generating the bioelectrical slow wave potential that leads to contraction of GI smooth muscles in the GI tract 138 . Recent studies have found that ANO1 is also highly expressed in the ICC and mediates the slow wave current in the ICC 4,139,140 . The intracellular Ca 2þ rise from intracellular stores and VGCC in the ICC causes the generation and propagation of pacemaker potential that is also amplified by activation of ANO1 channels 138 , suggesting that inhibition of ANO1 may reduce intestinal mobility for diarrhea. Indeed, pharmacological inhibition or gene silencing of ANO1 blocks slow wave in intestinal smooth muscle 141,142 . It is noted that several ANO1 inhibitors including T16A inh -A01 and CaCC inh -A01 exhibit different potency on blocking gastric and small intestinal slow waves. For instance, CaCC inh -A01 blocks slow waves in the murine stomach at 5 mmol/L and the small intestine at more than 30 mmol/L 143 . The mechanism underlying these different sensitivities is not entirely clear, but several probabilities including other channels contributions, different splice variants and the local Ca 2þ concentrations may contribute to these differences 138 . Nevertheless, both GI epithelium and ICC are synergistically involved in the pathogenesis of diarrhea and downregulation of ANO1 in either type of the two cells may help mitigate diarrhea.
Rotavirus has been known to cause severe diarrhea in infants and young children, whereas red wine extracts with alcohol-free and CaCC inh -A01 can prevent intestinal fluid loss in a neonatal mouse model of rotaviral diarrhea through inhibition of ANO1mediated Ca 2þ -activated Cl e secretion 53 . However, red wine extracts and CaCC inh -A01 show no obvious effect on cholera toxininduced diarrhea or CFTR current in cultured cells or intestinal absorption, suggesting the important role of ANO1 in diarrhea 53 . A recent study demonstrates that glucose and NSP4 synergistically increase the expression of ANO1 and Ca 2þ -activated Cl e secretory in the mouse model of diarrhea 137 . Suppressing ANO1 expression in apical membranes of colonic epithelium decreases Ca 2þ -activated Cl e secretion in chemical dextran sulfate sodiuminduced chronic colitis in mice 144 . There are also observations that inhibition of ANO1 by non-specific ANO1 inhibitors including eugenol 74 , shikonin 145 , plumbagin 83 , resveratrol dimer transε-viniferin (TV) and tetramer g-2-viniferin (RV) 69 can reduce water content in stools, but those observations should be confirmed with specific ANO1 inhibitors.

Cystic fibrosis
CF is an inherited disease of airway obstruction caused by mucus hypersecretion, mucus plugging and bronchoconstriction 146 . The dysfunction of CFTR Cl e channel is considered to be a major cause for CF as defective CFTR Cl e channel mutations are identified in CF patients 147 . It has been shown that small molecules can rescue dysfunctional CFTR mutation through increasing the number or open probability of CFTR Cl e channels. Unfortunately, these CFTR correctors or potentiators only exhibit limited efficacy for CF patients because of their multiple and different mutation sites of CFTR 148 . Therefore, an alternative strategy is CFTR-independent approach for treatment of CF by increasing the activity of ANO1 channel to promote mucus secretion 149 . This hypothesis is supported by observations that: 1) ANO1 is abundantly expressed in the airway goblet cells and upregulated in Figure 2 Distribution of ANO1/TMEM16A in different tissues and its role in diseases. (A) In epithelial cells, ANO1 activation contributes to electrolyte and music secretion. Activation of GPCRs causes an increase in intracellular cAMP and Ca 2þ , further inducing Cl À secretion through the activation of CFTR and ANO1. A crosstalk between CFTR and ANO1-dependent secretions also occurs for regulation of secretory signaling in the airway epithelia. ANO1 high expression or hyperactivity can cause inflammatory diseases such as asthma and diarrhea. (B) Outward flow of Cl À through the activation of ANO1 in smooth muscle cells causes depolarization and smooth muscle contraction. ANO1 high expression or hyperactivity is responsible for asthma diarrhea, and hypertension. (C) In DRG sensory neurons, activation of ANO1 by intracellular Ca 2þ or heat causes Cl À efflux and increases neuronal excitability for induction of neuropathic pain. The functional coupling between TRPV1 and ANO1 is also involved in nociception. (D) In cancer cells, ANO1 upregulation promotes cell proliferation and migration, whereas ANO1 downregulation induces apoptosis through multiple signaling pathways, including EGFR/MAPK signaling pathway 98 , CaMKII/MAPK signaling pathway 51 , TGF-b signaling pathway 120 and NF-kB signaling pathway 121 . Pharmacological activation of ANO1 by activators or potentiators may serve as a therapeutic strategy for treatment of CF, dry mouth and dry eye syndromes, and inhibition of ANO1 by inhibitors may be beneficial for ANO1 related channelopathies including asthma, diarrhea, hypertension, neuropathic pain and cancers. inflammatory conditions 131 ; 2) overexpression of ANO1 in CF human bronchial epithelia suppresses proinflammatory cytokine IL-8 secretion 150 ; 3) specific knockdown of ANO1 in respiratory airways eliminates Ca 2þ -and cAMP-activated Cl e secretion 151 ; 4) Ano1 gene knockout mouse exhibits abnormal trachea morphology, and also mucus obstruction and defective mucociliary clearance that presenting with a CF-like lung phenotyp 125,152 .
Several studies demonstrate that ANO1 has a functional cross talk with CFTR through PSD-95/Dlg/AO-1 proteins 151,153 . Such an interaction between ANO1-mediated Ca 2þ -activated Cl e secretion and CFTR-mediated cAMP-dependent Cl e secretion in airway epithelial cells strongly overlaps through a cAMP sensor protein termed exchange protein directly activated by cAMP and Ca 2þ -sensitive adenylate cyclase type 1 154 . ANO1 expression and activity are deficient in CF patients and upregulation of ANO1 can improve mucus dynamics in CF mice 155 . These lines of evidence support the notion that ANO1 may represent an alternative therapeutic target to circumvent CFTR dysfunction in the airway epithelia of CF patients.
Some CFTR-independent drug candidates have been developed for potential CF therapy. Denufosol, a P2Y2 receptor agonist, promotes airway epithelial chloride secretion through activating CaCCs with P2Y receptors 156 . Unfortunately, denufosol failed in phase III trial because of its short half-life in vivo 147,156,157 . Recently, an ANO1 activator, ET000516-A-2 from pre-clinical study is expected to be evaluated in CF patients 147 . Another novel ANO1 potentiator ETX001 developed by Enterprise Therapeutics (Brighton, UK) was recently reported that it can enhance fluid secretion and improve mucociliary clearance in both primary CF bronchial epithelial cells and sheep model of CF-like airway disease 116 . These two candidates may bring us an exciting prospect for potential development of alternative CF therapy in the clinic.

Other epithelial diseases
A recent study shows that ANO1 is overexpressed in mouse pancreatic tissue of acute pancreatitis model 158 . ANO1 promotes the pathogenesis of acute pancreatitis through activating the IP 3 R/ Ca 2þ /NF-kB/IL-6 pathway, and inhibition of ANO1 by T16A inh -A01 reduces the pancreatic damage in acute pancreatitis mice 158 . ANO1 is also likely involved in other epithelial diseases, such as polycystic kidney disease (PKD), diabetic nephropathy and pulmonary fibrosis. The PKD is characterized by multiple bilateral renal cysts that gradually enlarge and can lead to a decline in renal function. ANO1 is found in human kidney and renal epithelial cell lines, and its expression is upregulated in the forskolin induced renal cyst model, autosomal dominant PKD patients and high-fat diet/streptozotocin-induced diabetic nephropathy mice 55,159,160 , indicating a role of ANO1 in kidney disease. ANO1 promotes renal cyst growth via induction of Cl e secretion and proliferation of cyst lining epithelium 159 . Inhibition of ANO1 by pharmacological inhibitors or gene knockdown significantly decreases glucose dependent cyst growth, reduces nephron numbers and also causes albuminuria and tubular damage 55,159,161 . Mechanistically, ANO1 drives the growth of renal cysts through enhancing Ca 2þ release from IP 3 R sensitive Ca 2þ stores 162 and lipid peroxidation also promotes renal cyst growth through activating ANO1 82 . In diabetic nephropathy, ANO1 deletion alleviates renal injury in diabetic mice through increasing nephrin expression, reducing the expression level of apoptosis related factors and also suppressing the activation of P38/JNK signaling pathway 160 .
On the contrary, ANO1 activation aggravates renal injury by activating P38/JNK signaling pathway to promote podocyte apoptosis in diabetic nephropathy mice 160 and exacerbates inflammation via activating the TGF-b-SMAD3 pathway 163 . Downregulation of ANO1 by shRNA can inhibit apoptosis and promote the proliferation of lung fibroblasts in mouse model of idiopathic pulmonary fibrosis 163 . These observations suggest that inhibition of ANO1 may hold therapeutic potential for kidney and other epithelium-originated diseases.
ANO1 upregulation in many kinds of cancers is related to its gene location at chromosome 11q13 that is frequently amplified in many malignant tumors 51,164 and amplification of 11q13 is associated with the increase of ANO1 gene copy numbers 166 . Overexpression of ANO1 promotes proliferation and migration in multiple cancer cell lines 51,169,179 , and ANO1 upregulation is associated with lower overall survival in patients with breast cancer, pancreatic cancer or gastric cancer 51,171,180 (Table 3). ANO1 mRNA is also highly expressed in the blood of GIST patients 184 and patients with epithelial ovarian cancer, and the expression level of ANO1 mRNA decreases after surgical removal of tumors 168 , suggesting that detection of ANO1 gene in blood may serve as a biomarker for early diagnosis of cancer.
Multiple signaling pathways have been shown to be involved in ANO1 modulation in cancer development (Table 3). In breast cancer and HNSCC, downregulation of ANO1 by knockdown or pharmacological inhibition inhibits cancer cell proliferation, induces apoptosis and reduces tumor growth through reducing epidermal growth factor receptor (EGFR) cell signaling pathways 51 . Studies have also shown that ANO1 can affect the progression of intestinal cancer, liver cancer, and pancreatic cancer through the EGFR pathway 169,171,187 , as well as the modulation of glioma by NF-kB signaling 121 . In prostate cancer, ANO1 can regulate TNF-a signaling to contribute to cell growth and apoptosis 57 . In epithelial ovarian cancer, silencing ANO1 can suppress cancer cell proliferation, migration and invasion as well as the growth of xenograft tumors through inactivation of PI3K/ AKT cell signaling pathway 168 . In gastric cancer, ANO1 overexpression can promote tumor invasion and predict poor prognosis through affecting TGF-b signaling function 120,180 , that is also the target for ANO1 regulating cell proliferation, migration and invasion in esophageal squamous cell carcinoma (ESCC) 179 . In glioblastoma, ANO1 expression is regulated by CaMKII-b and suppression of CaMKII-b inhibits ANO1 mediated glioblastoma development 183 , and CaMKII also plays a role in ANO1-mediated The Ca 2þ -activated chloride channel ANO1/TMEM16A  62,175,181 . Small molecules, such as CaCC inh -A01, T16A inh -A01, MONNA and Ani9, as well as natural products including avermectins and flavonoids exhibit anticancer effects through inhibition of ANO1 activity 57,62,78,84 , suggesting that ANO1 may serve as a potential drug target for cancer therapy.

Hypertension
Hypertension or high blood pressure is a common condition in which the long-term force of the blood against the blood vessels is high enough that it may increase the risk of heart diseases and brain stroke. ANO1 is expressed in various smooth muscle cells of arteries and veins 188,189 , suggesting a role of ANO1 in regulation of vasoconstriction. Activation of Ca 2þ -activated Cl e current can lead to Cl e efflux after Ca 2þ influx and membrane depolarization, thus resulting in vasoconstriction and increase of blood pressure. Indeed, ANO1 expression and activity are upregulated in murine pulmonary arterial myocytes induced by chronic hypoxia, which contributes to pulmonary hypertension 60 . Similar results were also observed in monocrotaline-induced pulmonary hypertension rats 188 , spontaneously hypertension rats 63 , idiopathic pulmonary arterial hypertension patients 190,191 , and high-flow-induced pulmonary arterial hypertension (PAH) rats 192 . Conversely, cellspecific knockout of ANO1 reduces blood pressure and attenuates hypertension in mice and spontaneously hypertensive rats 193e195 .
A recent study shows that upregulation of ANO1 depolarizes pulmonary artery smooth muscle cells (PASMC) membrane potential, contributing vasoconstriction and the increased pulmonary vascular resistance in PAH rats 190 . Conversely, pharmacological inhibition or gene silencing of ANO1 reverse the membrane depolarizes of PASMC. For instance, a specific ANO1 inhibitor MONNA hyperpolarizes the rat coronary artery smooth muscle cell membrane potential and increases coronary flow 63 . Vascular smooth muscle cells (VSMC) are the stromal cells of the vascular wall and are responsible for regulating arterial tone, and blood pressure. Overexpression of ANO1 in healthy donor PASMC promotes the cell proliferation and produces an idiopathic pulmonary arterial hypertension (IPAH)-like phenotype. Pharmacological inhibition of ANO1 may reverse vasoconstriction and remodeling of pulmonary arteries in IPAH 190,196 . These investigations suggest the involvement of ANO1 in VSMC contraction and vascular remodeling for hypertension.
Circulating angiotensin II (Ang II) as a major contributor of the renineangiotensin system is upregulated during hypertension, and Ang II is frequently used to establish hypertension models. It has been shown that Ang II significantly enhances ANO1 expression in human umbilical vein endothelial cells and endothelial-specific ANO1 knockout significantly reduces Ang II-induced hypertension through ROS signaling pathway, whereas endothelial-specific transgenic ANO1 shows the opposite effect 195 . These studies suggest that inhibition of ANO1 function may be beneficial for hypertension, and ANO1 inhibitors, including T16A inh -A01, MONNA, and Ani9 can inhibit agonist-induced vesical constriction and cause vasorelaxation 63,94 .

Nociception
Nociception is a perception in response to painful or harmful stimuli, such as heat, cold, mechanical and chemical stimulus in the environment. Dorsal root ganglion (DRG) is a cluster of sensory neurons in the dorsal root of spinal nerves responsible for pain signal transmission 197 . ANO1 is mainly expressed in small diameter DRG neurons that are intimately involved in nociception 4,198 , suggesting a modulatory role of ANO1 in pain sensation. It has been shown that an inflammatory mediator bradykinin, released from damaged tissues or applied exogenously can activate ANO1 through B2 receptors and PLC pathway, subsequently depolarizing membrane potential and markedly stimulating firings in DRG neurons, and pharmacological inhibition of ANO1 attenuates pain behaviors 44 . ANO1 expression is upregulated in the spinal cord and DRG neurons after spinal nerve injury, suggesting the involvement of ANO1 in development of neuropathic pain 54 . Protease activated receptor 2 (PAR2), also known as G-protein coupled receptor 11, has been identified to be involved in the pathogenesis of pain 199 . PAR2 and ANO1 are co-localized in DRG neurons, and their expressions are increased in rat pain model of chronic constriction injury 200 . ANO1 can be activated by temperatures over 44 C, and silencing of ANO1 in DRG neurons significantly reduces nociceptive behavior in thermal pain model 198 , inflammation and nerve-injury induced hyperalgesia or allodynia 54,201 . Thus, downregulation of ANO1 activity may present a potential therapeutic strategy for neuropathic pain. ANO1 as a potential target for pain is evidenced by observations that inhibition of ANO1 by small molecules NPPB, NFA, T16A inh -A01 or CaCC inh -A01 reduces capsaicin-induced inward current and action potential firing, and as well as pain-related behaviors 99,100 . Again, these ANO1 inhibitors are not specific and a key question such as whether ANO1 directly affects the excitability of nociceptors should be addressed.

Others
A recent study shows that ANO1 is expressed in mouse brain endothelial cells where ANO1 expression is upregulated after ischemic stroke induced by the middle cerebral artery occlusion 56 . Targeting ANO1 with inhibitor CaCC inh -A01 or silencing attenuates bloodebrain barrier (BBB) breakdown after ischemic stroke through decreasing intracellular adhesion molecule-1 via NF-kB signaling pathway, suggesting that downregulation of ANO1 protects BBB disruption after ischemia stroke. It is supported by another study that ANO1 inhibitor T16A inh -A01 or siRNA inhibits proliferation and migration of brain capillary endothelial cells that comprise BBB 202 . It would be interesting to see more studies that are designed to validate ANO1 as a therapeutic target for brain stroke.

Summary and perspectives
The CaCC ANO1 channel is expressed in a wide variety of epithelial cells, smooth muscle cells and neurons. Although abnormal expression or dysfunction of ANO1 is involved in the pathology of many diseases, validating ANO1 as a therapeutic target still presents a big challenge. While a significant progress has been made for the distribution, expression, structure and pathophysiological functions of ANO1, there still exists an urgent need for selective modulators of the channel for target validation. Current available ANO1 modulators are also in preclinical stage without any treatments ready for clinical utility, which highlights the much-needed efforts in understanding the channel pharmacology and validation of ANO1 as therapeutic target. The recent structure of ANO1 solved by single-particle cryo-electron microscopy can provide a valuable model for the design of more potent and selective ANO1 modulators that can be used to help validate this emerging target for therapeutic potential of diseases, including cancer, inflammatory epithelial diseases, neuropathic pain and hypertension and cystic fibrosis lung disease.

Acknowledgments
We thank Dr. Xiaowen Tang (School of Pharmacy, Qingdao University Medical College, Qingdao, China) for his help in