Key words

IJMM

International Journal of Molecular Medicine

1107-3756 1791-244X

D.A. Spandidos

10.3892/ijmm.2024.5405

ijmm-54-04-05405

Review

TMEM16 proteins: Ca²⁺-activated chloride channels and phospholipid scramblases as potential drug targets (Review)

Huang

Zeqi

1* Iqbal

Zoya

2* Zhao

Zhe

1 Chen

Xiaoqiang

1 Mahmmod

Ayesha

3 Liu

Jianquan

1 Li

Wencui

1 Deng

Zhiqin

1Department of Hand and Foot Surgery, Shenzhen Second People's Hospital (The First Hospital Affiliated to Shenzhen University), Shenzhen, Guangdong 518000, P.R. China 2Department of Orthopaedics, Shenzhen Second People's Hospital (The First Hospital Affiliated to Shenzhen University), Shenzhen, Guangdong 518000, P.R. China 3Faculty of Pharmacy, The University of Lahore, Lahore, Punjab 58240, Pakistan

Correspondence to: Professor Wencui Li or Dr Zhiqin Deng, Department of Hand and Foot Surgery, Shenzhen Second People's Hospital (The First Hospital Affiliated to Shenzhen University), 3002 Sungang West Road, Shenzhen, Guangdong 518000, P.R. China, E-mail: liwencui@email.szu.edu.cn, E-mail: dengzhiqin1988@szu.edu.cn *

Contributed equally

10 2024

29 07 2024

54 4

26 03 2024 06 06 2024

2024

This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

TMEM16 proteins, which function as Ca²⁺-activated Cl⁻ channels are involved in regulating a wide variety of cellular pathways and functions. The modulators of Cl⁻ channels can be used for the molecule-based treatment of respiratory diseases, cystic fibrosis, tumors, cancer, osteoporosis and coronavirus disease 2019. The TMEM16 proteins link Ca²⁺ signaling, cellular electrical activity and lipid transport. Thus, deciphering these complex regulatory mechanisms may enable a more comprehensive understanding of the physiological functions of the TMEM16 proteins and assist in ascertaining the applicability of these proteins as potential pharmacological targets for the treatment of a range of diseases. The present review examined the structures, functions and characteristics of the different types of TMEM16 proteins, their association with the pathogenesis of various diseases and the applicability of TMEM16 modulator-based treatment methods.

Key words Ca²⁺-activated Cl⁻ channels TMEM16 gene therapy cystic fibrosis coronavirus disease 19 cancer

National Natural Science Foundation of China

81972085

82172465

China University Industry-University-Research Innovation Fund

2021JH037

The Natural Science Foundation of Guangdong Province

2021A1515010706

2023A1515010102

Guangdong Provincial Key Clinical Discipline-Orthopedics

2000005

The Sanming Project of Shenzhen Health and Family Planning Commission

SZSM202311008

Shenzhen Science and Technology Planning

GJHZ20210705142007023

The Shenzhen Key Medical Discipline Construction Fund

SZXK025

This study was supported by The National Natural Science Foundation of China (grant no. 81972085, 82172465), China University Industry-University-Research Innovation Fund (grant no. 2021JH037), The Natural Science Foundation of Guangdong Province (grant nos. 2021A1515010706 and 2023A1515010102), Guangdong Provincial Key Clinical Discipline-Orthopedics (grant no. 2000005), The Sanming Project of Shenzhen Health and Family Planning Commission (grant no. SZSM202311008), Shenzhen Science and Technology Planning (grant no. GJHZ20210705142007023) and The Shenzhen Key Medical Discipline Construction Fund (grant no. SZXK025).

1. Introduction

The Cl⁻ channels present in mammalian cells can be divided into the following five categories: Cystic fibrosis transmembrane conductance regulator (CFTR), Ca²⁺-activated Cl⁻ channels (CaCCs), ligand-gated Cl⁻ channels, volume-regulated Cl⁻ channels and voltage-gated Cl⁻ channels (1). The TMEM16 proteins are Cl⁻ channels that can reveal one or more regulatory mechanism, ensuring the normal functioning of molecules or compounds in an organism through their protein typing (2). At present, 10 subtypes of TMEM16 proteins have been identified, which play a role in various functions of the human body (3). Certain members, such as TMEM16A and B, serve as CaCCs and as phospholipid scramblases, thereby demonstrating the dual functions of channels and scramblases, with others performing other cellular functions (4). Transfection of other TMEM16 proteins (such as 16C, 16F, 16G, 16H, 16J and 16K) into null cells does not result in increased anion transport or membrane current, indicating that these proteins have other cellular functions (4). For instance, research has shown that TMEM16J (anostatin-9) exhibits activity as a cAMP/PKA activated channel (5). The TMEM16 proteins are activated by the Gq protein-coupled receptor, mutations of which lead to cellular dysfunction (6). The TMEM16 proteins link Ca²⁺ signals with cellular electrical activity and lipid transport and play a critical role in the manifestation and proliferation of various human diseases, including cystic fibrosis (CF), jaw dysplasia, nephrolithiasis, myotonia congenita and cancer (7). Therefore, fully understanding the structures and functions of the TMEM16 proteins, unraveling the mechanisms underlying their involvement in complex molecular transport and ascertaining their potential as targets for the design of drugs for the treatment of human diseases are major avenues of pursued research.

2. Materials and methods

The present narrative review followed the Assessment of Narrative Review Articles flowchart (Fig. 1) (8). The main purpose of the present review was to summarize the evidence of potential therapeutic targets in TMEM16 protein research and to understand the basic characteristic structures, gene mutations and treatment strategies for related diseases. The English terms ['TMEM16' (Mesh)] OR ['TMEM16' (Mesh)] AND ['Structure' (Mesh)] OR ['Treatment' (Mesh)] were searched on the PubMed (https://pubmed.ncbi.nlm.nih.gov), Google Scholar (https://scholar.google.com) and Cochrane databases (https://www.cochranelibrary.com). The screening results included literature published in the past 15 years. Articles that met the following criteria were included in the present review: i) The data reported in the study was from animals; ii) the structure of TMEM16 protein could be found in the corresponding data in the Protein Data Bank (PDB, https://www.rcsb.org); iii) cases of clinical diseases were not individual case studies; iv) clearly stated the specific methods and evidence was supported by referenced citations; and v) the articles were not practical guidelines, guidelines, meta-analyses, systematic reviews, narrative reviews, case series and case reports. Articles that did not describe the methods and those that were not strictly related to the research objectives were excluded. The search strategy identified 221 articles, of which 192 were excluded after evaluating the title and abstract. Then, based on the importance of the journal, including comparison of research designs and methods, evaluation of journal papers, journal impact factors, academic reputation of scholars and academic status of institutions, three independent reviewers studied the titles and abstracts of the articles. To prioritize analysis of the various subtypes of TMEM16 protein and their corresponding cellular functions, and to analyze the disease and treatment targets based on the collected literature, 29 articles were selected for quality evaluation. The abstracts and images of all selected articles were reviewed and the data and content from the complete article were ultimately used to write the present review.

3. Structural characteristics of TMEM16 proteins

The TMEM16 superfamily comprises bifunctional Cl⁻ channels and phospholipid scramblases. These two isoforms possess a common homodimeric structure with the transmembrane domains (TM) 3-7 helix forming a hydrophilic groove, the multiple conformations of which allow the passage of ions and lipids (Fig. 2) (9,10). The dimer formed by the TMEM16 proteins is surrounded by TM3-7, with each subunit of the dimer possessing an ion-conducting pore (9). The Ca²⁺-dependent activation of TMEM16, which involves the direct binding of Ca²⁺, induces the formation of three consecutive Ca²⁺-binding sites between TM6 and 8 in each subunit (11). The TMEM16F structure, as with TMEM16A, has a large extracellular domain formed by the extracellular loops of TM1-2, 3-4, 5-6, 7-8 and 9-10, and is stabilized by four disulfide bonds. Disruption of TMEM16F leads to ion channel dysfunction (6,7). Nevertheless, the Ca²⁺-binding regions of TMEM16A and F can adopt a closed permeation pathway in which the pores are too narrow to allow the passage of ions (9). This closed conformation is likely due to a Ca²⁺-dependent run-down (desensitization) (10). The proteins belonging to the TMEM16 family are also known as anoctamins, 10 of which are 800-1,000 amino acids long and are suffixed with the letters A-K, excluding I (9). The structure of each family member comprises 10 transmembrane domains, with the -NH₂ and -COOH termini inserted within the cytoplasmic matrix (9). The -NH₂ terminus consists of a dimeric domain involved in homotypic interactions with TMEM16A proteins (10). Two main models have explained the structural characteristics of the TMEM16A proteins (12). The first model suggests the formation of a repetitive reentrant loop between TM5 and 6, which is important for forming the ion channels (12). A previous study also revealed that the EEEEEEAVK sequence in the first intracellular loop has an essential role in the Ca²⁺-dependent regulation of the voltage mechanism (13). The second model proposes a transmembrane domain that directly crosses the cell membrane into the cytoplasmic matrix to form a loop between TM6 and 7, which is important for Ca²⁺ as this loop forms the sixth transmembrane domain that binds to Ca²⁺, regulating channel activity (13). The main Ca²⁺ binding site of the model is located in the third intracellular loop, between two Glu residues (702 and 705) that mediate the activation of the Ca²⁺ channel. The fourth main intracellular loop may also harbor Ca²⁺ binding sites. The messenger protein, such as calmodulin, possesses three sites for Ca²⁺ binding: Calmodulin-binding motifs 1 and 2, which separately have calmodulin-binding activity (14), and the regulatory calmodulin binding site, which provides two different modes of interaction with calmodulin, one in the submicromolar and the other in the micromolar range of Ca²⁺ concentrations (1). In fact, calmodulin interacts with fragment (b) on the TMEM16A protein, corresponding to the selectively spliced exon 6b, and modulates channel activity (1).

Calmodulin can interact with compounds such as 1-EBIO, DCEBIO and riluzole, that induce the opening of Ca²⁺-activated K⁺ channels with low and medium conductivities and can also activate TMEM16A (15). The TMEM16 proteins link Ca²⁺ signals with cellular electrical activity and lipid transport and play a critical role in CF (16). The aforementioned compounds within amino acid supplement tablets can activate the efflux of Ca²⁺ from cells in CF (15). Moreover, calmodulin can interact with and regulate the activity of Ca²⁺ channels (15). Although several structural analyses of the TMEM16 proteins, including fungal and mouse orthologues, have been completed, the binding sites for lipid scramblase activity are yet to be identified. The TMEM16 proteins that function as phospholipid scramblases have an essential role in almost all human physiological processes (2,17). Therefore, dysregulated activity of TMEM16 as a phospholipid scramblase may lead to unfavorable consequences. In summary, although these structural and functional studies provide important insights into the voltage-dependent activation mechanisms of TMEM16A as a CaCC, further studies are needed to comprehensively understand the dual functionalities of TMEM16 proteins as ion channels and phospholipid scramblases.

4. Characteristics of the different TMEM16 subtypes

In clinical practice, prognostic markers can predict the poor clinical outcomes of treatment methods in patients with cancer. However, the ambiguity in the molecular functions of these markers makes the accurate prediction of the progression of tumors difficult. TMEM16 protein is not a prognostic marker for tumors, but it is closely related to the occurrence and development of tumors (18). TMEM16 proteins are distributed throughout the human body, with different types distributed in different tissues or organs, and are associated with various diseases (Table I). TMEM16A and B function as both CaCCs and phospholipid scramblases that promote the bidirectional mobility of membrane lipids (6). Additionally, TMEM16A and B control the release of Ca²⁺ stored in the cytoplasmic membrane, enhance intracellular Ca²⁺ signaling, amplify Ca²⁺ signaling activated by G protein-coupled receptors and regulate ion channel trafficking (6). TMEM16A is mainly involved in trans-epithelial Cl⁻ transport (1,4,19) and smooth muscle tone regulation (20-22), and is widely expressed throughout the body, serving as a receptor to sense injury stimuli and cell proliferation (particularly when upregulated in cancer). In addition, an induction of the production of angiotensin II stimulates the contraction of cerebral vessels via the TMEM16A-mediated Ras homolog family member A/Rho-associated protein kinase signaling pathway (23). Furthermore, the P38/JNK signaling pathway is also activated by TMEM16A expression, thereby increasing the apoptosis rate of podocytes in mice with diabetic nephropathy, which can exacerbate the injury caused to the kidneys (24). TMEM16A regulates the proliferation of the epithelial cells lining the bile duct via the ATP-stimulated-Ca²⁺-protein kinase C signaling pathway to induce the synthesis and secretion of bile (25). TMEM16B regulates sensory processes such as smell and vision and can control the excitability of neuronal and glial cells (26-28). TMEM16B mutations can cause multiple sclerosis and schizophrenia (4,29-31). TMEM16C is typically expressed in the central and peripheral nervous systems of humans, mice and rats, and interacts with Na⁺-activated K⁺ channels to improve the susceptibility of Na⁺ and the activity of K⁺ channels (32-34). TMEM16C has a role in certain other cellular functions including the regulation of pain and heat processing (34). Previous studies have revealed that genetic mutations in TMEM16C can cause craniocervical dystonia in humans (35-37). TMEM16D also functions as a non-selective ion channel and a phospholipid scramblase (38), is mainly expressed in the brain and endocrine glands, and it can control the mean arterial pressure and secrete aldosterone (39). A mutation in the gene encoding TMEM16D can lead to neurological diseases, such as Alzheimer's disease (40). TMEM16E acts as a non-selective ion channel and scramblase, and is mainly expressed in skeletal muscle, participating in the repair and maintenance of intracellular calcium stability of skeletal muscle, and activates the janus kinase (JAK)/STAT3 signaling pathway for cell migration and invasion (12). TMEM16E causes gnathodiaphyseal dysplasia (GDD) in cases of missense mutations (41) and muscular dystrophy (MD) in cases of functional mutations (42-44). TMEM16F also acts as a non-selective ion channel and scramblase activated by very high concentrations of Ca²⁺ and promotes the translocation of phospholipid and phosphatidylserine (PS) from the inner leaflet of the plasma membrane to the outer leaflet (45,46). Mutations in the gene encoding TMEM16F are associated with the development of Scott syndrome, a hemorrhagic disease caused by phospholipid-related disorders in the membranes of platelets (7,47,48). Moreover, TMEM16F mediates the proliferation of myoblasts; it plays an essential role in C2C12 myoblast proliferation, likely via regulating the ERK/AKT signaling pathway (49). The roles of TMEM16G and H have not yet been fully elucidated (50). The levels of TMEM16G are upregulated in cancer, particularly prostate cancer, and interact with the other upregulated proteins such as intracellular vesicle proteins (51). Hence, TMEM16G may be a potential biomarker for diagnosis and a target for prostate cancer immunotherapy (51). TMEM16G is also involved in the perturbation of the lipid bilayer in cell lines with a deletion in TMEM16F (7,50). TMEM16H forms junctions between the endoplasmic reticulum (ER) and the cell membrane in intracellular signaling and is involved in the transport of bile salts and the manifestation of intrahepatic cholestasis of pregnancy (52,53). The intrinsic process of TMEM16H may involve an interaction between proteins such as the matrix-interacting molecule 1, and receptors such as the inositol 1,4,5-trisphosphate receptor, that induce the release of Ca²⁺ from the cells (52). TMEM16J is a non-selective cation channel with scramblase activity (54,55), is activated by cAMP-dependent protein kinase A (5) and is associated with the development of certain types of cancer, such as gastric cancer, pancreatic cancer and esophageal squamous cell carcinoma (56-58). TMEM16K is mainly localized to the membranes of intracellular compartments, is the most studied phospholipid scramblase and demonstrates non-specific ion channel activity that is optimally regulated by Ca²⁺ and short-chain lipids (59). TMEM16K is involved in spindle assembly (60) and affects macrophage volume regulation (61). TMEM16K deficiency leads to spinocerebella ataxia autosomal receiving type 10 (59). In addition, TMEM16K also forms contact sites with endosomes and is associated with Ca²⁺ signaling, cell volume regulation and apoptosis (59-61).

5. Expression levels of TMEM16 proteins and their applicability as therapeutic targets in different diseases Association between TMEM16 and CF CF

CF is a genetic disease that affects multiple organs. It is caused by abnormal CFTR transport through the epithelial layer and is characterized by a loss of function in various systems. This disease, caused by mutations in a single gene encoding CFTR, can shorten the lifespan of humans (62,63). Patients with CF present with symptoms that indicate the effects on a wide range of organs in the body (62). These include obstruction of the ducts of the mucinous glands and changes in membrane composition in lung epithelium (62). However, certain non-malignant types of CF are virtually asymptomatic and are diagnosed only in adulthood; they affect only a single organ, as there is no systemic involvement (63). The more severe types of CF cause afflictions that affect multiple organs, including male infertility, severe respiratory dysfunction (including bronchiectasis, emphysema and pulmonary edema) and pancreatic and intestinal complications (64). The progressive deterioration of lung function and multiple organ failure are the main causes of mortality in patients with CF (65). Furthermore, multiple therapies have been clinically approved (NCT04058366) for treating the individual conditions associated with CF (66-68). However, novel modulators, multiple experimental approaches and advanced cellular models in patients with CF should be developed to accurately and reliably predict the drugs in clinical settings.

Regulatory mechanism of CFTR

CFTR, a transmembrane conductance regulator protein with 1,480 amino acids, is a Cl⁻ channel driven by cAMP (64) and is located in the apical membrane of secretory epithelial cells. CFTR can transport both Cl⁻ and HCO₃⁻ into epithelial cells. The channel is an ATP-binding transporter comprising five domains: Two transmembrane domains that form the channel pore, a regulatory domain (R) and two nucleotide-binding domains (NBD1/NBD2) (69). High levels of Cl⁻ are necessary for the important physiological actions of CFTR in the epithelial cells of the airways, including moistening the mucosal surface and removing the mucosal cilia (63,69). Patients with CF lack CFTR, which causes mucus aggregation and tracheal blockage and leads to susceptibility to chronic bacterial infections (63). These patients are therefore at risk of respiratory diseases. In addition, these airway epithelial cells also express TMEM16A, which functions as a second Cl⁻ channel; the cytosolic Ca²⁺ concentrations control its activity, and several inhibitors or agonists have been identified (69) (Fig. 3).

In total, ~90% of patients with CF harbor a mutation referred to as F508del, which leads to the degradation of proteases and retention of the ER. A minimal increase in the occurrence of F508del in the CFTR gene was observed in the plasma membrane of apical cells (66,70,71). In total, ~50% of patients with CF are homozygous for F508del, which not only have a processing defect but also significantly reduces the stability and flexibility of the cell surface in channel gating, if the mutation affects CFTR localization to the plasma membrane (66). In such patients, administering lumacaftor as a monotherapy may reduce the levels of Cl⁻ in the sweat by up to 8 mmol/l, in a dose-dependent manner. However, lumacaftor failed to improve abnormal lung function in phase II trials (71-73). In an in vitro preclinical trial (NCT01225211), the addition of high concentrations of ivacaftor as an enhancer was twice as effective as lumacaftor alone, achieving ~25% of the normal CFTR activity (71). Thus, adding enhancers significantly improved lung dysfunction (predicted forced expiratory volume value of 1%) by 3-4% and reduced lung functionality deterioration rates (71,73,74). In particular, the combined use of two corrective agents and one enhancer [elexacaftor-tezacaftor-ivacaftor (Trikafta) combination, NCT03525444] was significantly effective in treating the most commonly manifested defects in the membrane transport and gating caused by CFTR mutations (69,70,75). The addition of tezacaftor, a corrective agent, for treating patients homozygous for F508del markedly improved diminished respiratory function (70). Administration of Kalydeco^®, a pharmacokinetic enhancer, can be used to restore the damage caused to gated membrane transport by CFTR channels due to missense mutations in CFTR (66,74,76).

TMEM16A as a therapeutic target for the treatment of CF

The known modulators of TMEM16A, including CACC_inh-A01, F_act, E_act, MONNA, TM_inh-23, T16_inh-A01, ETX001, Ani9 and phenyl quinoxalinone (CFTRact-J027) are either inhibitors or activators (Fig. 4) (77). Phenyl quinoxalinone is used to treat conditions such as constipation, dry eyes, cholestatic liver and inflammatory lung diseases; it is a highly effective modulator of CF and can thus change its course and progression (77). This type of CFTR-modulating drugs are potential candidates for use in novel therapeutic methods of CF (77). They can enhance the expression of the mutated gene in the G551D-CFTR mutant to the original functional level and even restore it to the pre-mutation state (66). Notably, TMEM16A may demonstrate functions in addition to the secretion of electrolytes and mucus and further engage in the proliferation of basal cells and the repair mechanisms of epithelial cells (78,79).

Before the discovery of TMEM16A, evidence suggested the occurrence of a second Cl⁻-channel expressed in the epithelial cells of the airways of patients with and without CF (80). This second channel, referred to as a CaCC, is regulated by the cellular concentrations of the Ca solute. Stimulation of the apical membranes of the epithelial cells of the airways with the purinergic agonist ATP in vitro and in vivo elicited a large but transient Cl⁻ secretory response (16,81,82). The putative physiological role of CaCCs in the epithelial cells of the airways can involve mechanical stimuli, such as those caused by normal tidal breathing or coughing, that can induce the release of ATP, thereby promoting the secretion of Cl⁻ by the mucosal layer of the airways through the binding of ATP to the purinergic and CaCC-associated receptors and finally triggering the influx of Ca²⁺ (21,83). The secretion of mucus in the airways involves TMEM16A (84).

By contrast, TMEM16A plays a key role in the movement of the tracheal cilia and reducing the discharge of mucus (85). TMEM16A simultaneously guides chlorine gas and bicarbonate through the airway epithelium and is expressed in the surface epithelium and submucosal glands, removing mucosal cilia by enhancing anion influx (85). In addition, the inhibition of TMEM16A by pharmacological compounds reduced the production of fluids on the surfaces of the airways (84). For instance, niclosamide reduced mucus production in the airways of sensitized mice (16) and it was reported to also affect intracellular calcium homeostasis by inhibiting the SERCA calcium pump (86). Therefore, the reported effects, such as the inhibition of mucus and cytokine release, bronchodilation and antibacterial activity, make niclosamide a potential drug suitable for the treatment of inflammatory diseases of the airways such as CF, asthma and chronic obstructive pulmonary disease (86). A recently identified TMEM16A potentiator, ETX001, triggers the secretion of fluids and accelerates the mucus clearance process without causing bronchoconstriction (77,87). The structure, the detailed mechanism of action, the location of the binding site and the selectivity profile of ETX001 have not yet been reported, although ETX001 may not interfere with Ca²⁺ signaling (77). Moreover, the functional efficiency of CFTR in epithelial cells can be improved by blocking microRNA (miR)-based RNA silencing and post-transcriptional regulation to increase the expression of TMEM16A (88).

Although TMEM16A may represent a potential target for the pharmacotherapy of CF, the widespread expression of TMEM16A is a serious concern since systemic administration may produce a broad range of side effects. Thus, any treatment targeting TMEM16A requires selective treatment regimens using specific drugs.

Roles of TMEM16 in diverse tumor types Development and proliferation of tumors mediated by TMEM16 proteins

Tumor growth is critically associated with cell differentiation and proliferation. The regulation of the intracellular Ca²⁺ levels by the TMEM16 proteins may affect tumor development or regulate the exocytosis of the cell membrane by controlling the intracellular concentrations of Cl⁻ (3,11). The activation of CaCCs by cellular Ca²⁺ mainly occurs in the proliferative potential cells and different types of cancer cells (89). The expression levels of TMEM16 proteins in diverse types of cancer, including TMEM16A-mediated gastrointestinal stromal tumor (18), leiomyosarcoma (90), head and neck cancer (91), carcinoma of the lungs (92), pancreatic cancer (93), prostate cancer (94), breast cancer (95), colorectal cancer (96), gastric cancer (97), glioma and glioblastoma (98), esophageal cancer (99) and chondroblastoma (100), are indicated in Table II. TMEM16A participates in cancer proliferation and migration by influencing the MAPK and Ca²⁺/calmodulin-dependent protein kinase (CAMK) signaling pathways and interacts with epidermal growth factor receptor (EGFR) in head and neck squamous cell carcinoma (HNSCC) (91). TMEM16E promotes the development of colorectal (92) and thyroid (101) cancer. TMEM16G promotes the development of prostate (51) and breast (102) cancer, and TMEM16J promotes the development of pancreatic cancer (57). Therefore, ascertaining the links between TMEM16 proteins and pathways or mechanisms in tumor cells is important for inhibiting tumor growth and proliferation and developing therapeutic methods in clinical settings. However, to the best of our knowledge, all commercially available TMEM16 protein detection kits are for research purposes only and not for clinical practice. Due to a lack of availability of antibodies against the human-derived TMEM16 proteins and significant interspecific differences in the sequences of the TMEM16 proteins, cross-reactivity is unlikely to occur. Thus, the development of a TMEM16 detection kit for the diagnosis of cancer requires the production of antibodies against the TMEM16 protein in humans.

In summary, TMEM16 can regulate the biochemical/molecular processes in tumors, and its abnormal expression in malignant tumors provides the possibility of employing it as a clinical biomarker for early diagnosis and a therapeutic target for reducing the occurrence and/or growth of tumors.

Various signaling pathways of TMEM16A in tumors

TMEM16A, in conjunction with EGFR, mediates the growth of tumors through two routes. First, as a CaCC, TMEM16A promotes the expression of cyclin D1 (CCND1) via the CAMK and AKT signaling pathways in succession, thereby leading to tumor proliferation (103). Second, TMEM16A participates in cancer proliferation and migration by influencing the MAPK and CAMK signaling pathways. Subsequently, the MAPK signaling pathway activates the MAPKK signaling pathway, which binds to the Grb2 binding site on EGFR with son of sevenless protein on Ras protein, altering the synthesis and activation of the Raf protein, which subsequently activates CCND1 via the phosphorylation of MEK (104). Furthermore, activation of the MAPKK signaling pathway promotes angiogenesis in vascular endothelial cells (104). As EGFR regulates the expression levels of tumor-associated genes, different signaling pathways mediate the development of several types of cancer (105-107). For instance, the development of glioma is mediated by activation of the NF-κB signaling pathway (108,109) and HNSCC is mediated by the Ras/Raf/MEK/ERK1/2 signaling pathway (110). Another set of signaling pathways, p38 and ERK1/2, are associated with promoting hepatocarcinogenesis (Fig. 5) (111).

The number of chromosomal amplicons, the extent of promoter methylation and miRs regulate the expression levels and functions of TMEM16A. The amplicon in chromosome 11q13 consists of TMEM16A-encoding and apoptosis-related genes, such as FAS-associated death domain protein (112). Upon expression, the amplicon drives the proliferation of cancer cells. Hypermethylation of the TMEM16A gene promoter promotes the metastasis of cancer cells but inhibits their proliferation. By contrast, hypomethylation enhances the proliferation of cancer cells but inhibits their metastasis (113). miR-132 (114), miR-9 (115) and miR-381 (97) directly target the mRNAs of TMEM16A, of which miR-381 downregulates epithelial-mesenchymal transition by suppressing the TGF-signaling pathway, thereby inhibiting germinal center cell proliferation and metastasis (97). In addition to the downregulation of these miRs, the levels of TMEM16A are upregulated by transcription of the genes associated with the IL4/IL13/JAK/STAT3/STAT6 axis, thereby activating histone deacetylase, enhancing the production of steroids such as testosterone, removing cells from their physiological environment, cellular reorganization and promoting mitosis (3,116,117).

Mechanism of action of the drugs targeting TMEM16 proteins

The use of niclosamide, a potent TMEM16A inhibitor that suppresses the expression of NF-κB and the Wnt/β-catenin, IL-6/JAK1/STAT3 and GSK-3 signaling pathways, has been approved for use by the US Food and Drug Administration (83,118-120). Niclosamide not only controls the cell cycle by activating the Let-7d/CDC34 axis (121), but also by blocking the Notch signaling pathway in addition to inhibiting goblet cell metaplasia in asthmatic mice (121-125). Tumorigenesis is also inhibited either via knockdown of the TMEM16A encoding gene or the exogenous administration of low concentrations of TMEM16A (126-128). Thus, the antiproliferative effects of niclosamide are associated with its inhibitory effects on TMEM16A and have been used in clinical trials in patients with prostate and colorectal cancer (86,94,119,129). In summary, the various anticancer effects of niclosamide may be related to inhibiting the multiple cancer-promoting mechanisms of TMEM16A.

Bee venom is a complex mixture of natural products such as peptides, enzymes, bioactive amines and non-peptide components with various pharmacological properties (130,131). Bee venom peptide is a potent activator of TMEM16; it promoted the Cl⁻ currents in cells overexpressing the genes encoding TMEM16A, F, J and K; these proteins can also stimulate phospholipase A2 (PLA2) (7,11,132). Furthermore, reactive oxygen species (ROS) and lipid peroxidation can activate the TMEM16 proteins (133). The enhanced production of ROS and its associated lipid peroxidation causes ferroptosis (134), which is mainly characterized by iron-dependent lipid peroxide damage-induced cell death occurring in the mitochondria. Lipid peroxidation can be induced by erastin inhibition of cysteine import through the transporter system Xc⁻, which leads to the depletion of glutathione and the inactivation of glutathione peroxidase (135). The death of cancer cells can be induced by bee venom peptide, and PLA2-dependent activation of metalloproteinase is essential for this effect (132). Therefore, the bee venom peptide promotes the iron-dependent death of cells, including cancer cells, a process in which TMEM16A and F are activated, in turn leading to the activation of PLA2 (132,136), which then finally induces the death of cancer cells (133,137). A number of studies have unraveled the underlying mechanisms and supported the potential therapeutic applications of TMEM16 protein, but its side effects on the human body still need further study.

Association between TMEM16 proteins and cardiovascular disease

TMEM16F is the most widely expressed TMEM16 protein that functions as both an ion channel and a phospholipid scramblase and plays a significant role in several physiological processes of various cells (46). The PS that arises on the surface of the activated platelets necessitates the involvement of phospholipid scramblases, such as TMEM16F, in the formation of the thrombin and prothrombin complex (138). Hence, TMEM16F-mediated exposure to PS is an important process in platelet aggregation and release to the blood (139). TMEM16F and its closest paralog, TMEM16E, both support coagulation on endothelial cells via PS externalization (138,140). As aforementioned, mutated TMEM16F protein can cause Scott Syndrome, a hemorrhagic disease with symptoms such as defects in blood coagulation, long-term bleeding and thrombosis (141). TMEM16F is a Ca²⁺-activated non-selective channel, which plays an essential role in the exposure of PS and the repair of the plasma membrane after pore formation (141-143). Therefore, TMEM16F may be a target for the innovation of novel drugs that can help treat hemostasis and thrombotic diseases (such as stroke and heart attack) in humans. In addition, endothelial cells play a thrombotic role in hyperuricemia via the TMEM16F-mediated exposure of PS and the release of particulate molecules into the blood (144).

6. Association between coronavirus disease 2019 (COVID-19) and TMEM16 proteins Formation of syncytia in COVID-19

As the name suggests, COVID-19 was first identified in 2019 and has become a serious worldwide health concern due to the large number of fatalities. To reduce the mortality rate in critical patients of COVID-19, targeted therapeutics are continuously being developed based on biological and etiological characteristics. COVID-19 causes severe respiratory conditions, such as pulmonary edema and thrombosis, acute respiratory distress syndrome and other diseases (145-147). The development of syncytia in the lungs of patients is a characteristic feature of infection by coronavirus. The host cell acts on angiotensin-converting enzyme 2 (ACE2) to activate the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein in a two-step hydrolytic process (148). The first step involves breaking the spike between the S1 and S2 subunits of the spike protein before or after binding to the receptor. The second step involves the hydrolysis and exposure of the S2 subunit, which immediately binds to the cell membrane and generates a protease to invade normal lung cells (149). Infected lung cells present with a multinucleated and abnormal morphology; SARS-CoV-2 and Middle East Respiratory Syndrome Coronavirus can fuse with the cells expressing the relevant receptors to form syncytia (150).

Mechanisms underlying the interaction of the SARS-CoV-2 spike protein with TMEM16 proteins

The cells that form syncytia and express the SARS-CoV-2 spike protein on their surface demonstrate increased concentrations and enhanced oscillations in the levels of Ca²⁺ along with high expression of the Ca²⁺-activated TMEM16 proteins in the cytoplasmic membrane, resulting in the relocation of PS and secretion of Cl⁻ (149). The associations between the levels of TMEM16, the levels of Ca²⁺ and the activation of TMEM16 by the SARS-CoV-2 spike protein may enhance the magnitude of Ca²⁺-based signaling spontaneously (150). At least three mechanisms explaining the activation of TMEM16 have been proposed. The first involves the direct cis-binding and activation of spike-protein-expressing cells, the second involves trans-binding and the initiation of protease activity via binding to ACE2 and the third involves indirect activation by inducing the release of Ca²⁺ (150). TMEM16F can significantly reduce calcium oscillations and membrane conductivity in spike expressing cells, used to expose PS on the cell surface (150). However, the overexpression of TMEM16F significantly stimulated the SARS-CoV-2 spike protein-induced formation of syncytia. Therefore, the expression of the spike protein, which is required for the formation of syncytia, can be reduced by the downregulation of TMEM16F. Thus, TMEM16F is identified as the major bifunctional cell membrane-phospholipid scramblase and Ca²⁺ channel these cells (Fig. 6).

TMEM16 participates in COVID-19 related coagulopathy

In patients affected by COVID-19, inflammation-induced damage to endothelial cells may lead to the release of a large amount of plasmin activator, thus producing high concentrations of D-dimers and degradation products of fibrin. A specific cytokine storm composed of high concentrations of proinflammatory cytokines and chemokines occurs in the body (146). These proinflammatory factors include TNF-α, IL-1 and IL-6. TNF-α and IL-1 are the primary mediators driving the inhibition of the endogenous anticoagulant pathway (147). IL-6 can induce the expression of tissue factors on monocytes, subsequently initiating the activation of coagulation and generation of thrombin (150). The PS on the surface of the activated platelets requires the activity of the phospholipid scramblase to participate in the formation of the thrombin and prothrombin complex, and the TMEM16F-mediated exposure of PS is crucial for the aggregation of platelets and release to the blood (151). The activation of TMEM16F increases with the formation of syncytia, enhances the aggregation of platelets and utilization of the coagulation factors, which leads to the production of an abnormal amount of thrombin and fibrin and finally causes disseminated intravascular coagulation (DIC) and microangiopathy (146-151). The involvement of TMEM16F in COVID-19-related disorders in blood coagulation results in a combination of low-grade DIC and local, pulmonary and thrombotic microvascular disease, which may significantly impact the dysfunction of the organs in the most severely affected patients.

Mechanisms underlying a novel coronavirus treatment method using drugs targeting TMEM16

Specific drugs should be developed to combat COVID-19 and to provide strategies for treating similar coronaviruses by analyzing the mechanisms behind the formation of viral syncytia and mining for the drugs acting on the Cl⁻ channels. Niclosamide, a drug that suppresses the formation of syncytium by inhibiting TMEM16F, has been identified as a promising drug for the treatment of severe COVID-19 and has been approved for use by the US Food and Drug Administration (150,152). Niclosamide is highly hydrophobic and therefore has poor solubility in aqueous solution (153). The hydrophobic helix formed by the TM1-6 grooves in both TMEM16A and F and the residues in this helix are essential for drug binding (11). TM6 functions as the main gating element of the channel and is part of the ion-conduction pore formed by TMEM16A and F (2,154). The antagonist simultaneously locks both of these ion-conduction pores by binding to the upper region of the TM6 in a closed configuration. In addition to niclosamide, nitazoxanide and 1PBC bind to the same conserved sites (11,155).

Furthermore, hexachlorophene, dichlorophenol, gefitinib (156), trifluoperazine (16), serotonin reuptake inhibitors and ivermectin (157), which target TMEM16A, can also inhibit the spike protein-induced formation of syncytia. Thus, screening specific drugs may reveal a common mechanism underlying the spike protein-dependent cell-cell fusion. Since the possibility of the recurrence of another COVID-19 pandemic is high, drug development is an indispensable part of future research.

7. Role of TMEM16 proteins in orthopedics TMEM16A as a marker of osteoporosis

Osteoporosis is a systemic disease resulting in a decrease in the mineral density and quality of the bones due to various causes, thus leading to changes in the microstructure and increased fragility of bones, thereby leading to fractures (158). TMEM16A is directly regulated by the cytosolic concentrations of Ca²⁺ and indirectly by interactions with calmodulin (12). Osteoporosis causes changes in the Ca²⁺ concentrations, which affects the levels and function of TMEM16A and therefore can be used as a marker for the early diagnosis and targeted intervention of osteoporosis. Furthermore, a deletion in the gene encoding TMEM16A caused severe defects in the tracheal cartilage in mice, which could be fatal in certain cases (159). In addition, the TMEM16A blockers, benzbromarone and CaCCinh-A0, 1 can significantly inhibit the differentiation of osteoclasts (159).

TMEM16E expression levels in bones and muscles

TMEM16E is a 913 amino acid protein with a Ca²⁺-activated phospholipid scramblase activity (160). TMEM16E is significantly expressed in muscles and bones, with Ca²⁺ activation involving TM4 and 5 (41). Mutations in the gene encoding TMEM16E in humans are associated with two genetic disorders: GDD, a rare skeletal-system-related syndrome associated with bone deformity and increased fragility (161), and limb-girdle MD (LGMD), a type of progressive MD (162,163). A recent model demonstrated that GDD is characterized by the manifestation of tubular bone sclerosis and mandible bone lesions, including those of a giant jawbone, arched tibia, fragility, sclerosis and cortical thickening of the femoral and tibial epiphysis (162). In the blood culture of this model, the number of osteoblasts increased while the number of osteoclasts decreased (162). The p.Cys360Tyr mutation in TMEM16E inhibits receptor activator of NF-κB ligand-induced p65, Erk, p38 and AKT phosphorylation, implying that multiple signal transduction processes contribute to osteoclast maturation and bone resorption (164). A recent study has assisted in generalizing the correlations between the GDD-associated genotype and the phenotype by extending the observations made to the GDD mutants of TMEM16E such as p.Arg215Gly, p.Cys356Gly/Arg/Tyr, p.Cys360Tyr, p.Ser500Phe and p.Gly518Glu (42). In the proposed model, p.Cys360Tyr causes osteosclerosis with a high bone turnover and can be a potential target for treating GDD (Fig. 7).

Mutations in the TMEM16 gene can lead to different orthopedic genetic-related diseases

TMEM16E is highly associated with TMEM16F; both are dual-function proteins with non-selective ion channel and phospholipid scramblase activities. In addition to participating in the exposure of PS and promoting different physiological processes, TMEM16E is also involved in bone mineralization and skeletal muscle repair (11). Expression of TMEM16E is highest in the cardiac and skeletal muscles and in growth plate chondrocytes and osteoblasts (43). Dysfunctional TMEM16E protein causes bone dysplasia and fragility and suppurative osteomyelitis of the lower jaw; in addition, LGMD-related amino acid substitutions cause a loss of function, while GDD-related substitutions lead to a constitutive lipid scramblase activity without the requirement of elevated cytosolic Ca²⁺ levels (42). The dysregulation of TMEM16E also causes arthritis and MD (162,163). Mutated TMEM16C protein causes muscle-related diseases associated with involuntary muscle spasms caused by cranial-neck dystonia (165). The discovery of the relationship between GDD-related mutations and the functional phenotypes confirmed the speculation that interventions based on genetic patterns may greatly improve the treatment outcomes of genetic diseases in humans, which is of great significance for the health and survival of affected individuals. Therefore, it is crucial to understand the mechanism of action of TMEM16 in genetic-related orthopedic diseases and to decipher the molecular mechanisms underlying the activation of gating and regulatory functions to develop novel targeted drug-based therapies towards the early intervention of diseases in the future.

8. Conclusions and future perspectives

In summary, the TMEM16 proteins have emerged as important pharmacological targets for the treatment of several associated diseases. Since the discovery and identification of TMEM16 as a Cl⁻ channel, its roles in various human diseases have been established. The availability of a large amount of structural information has led to significant progress in understanding the role of TMEM16 in disease progression at the molecular level. Thus, its role in the pathogenesis of human diseases, research involving the therapy of such diseases based on the targeting of TMEM16 and understanding the mechanisms of action of the proposed drugs with an enhanced potential should be addressed further. The studies on the structural characteristics of TMEM16 have led to the revelation of its various functions, ranging from ion transport to the modulation of the dynamics of the plasma membrane phospholipids and the underlying molecular mechanisms. However, further research is needed to fully understand the roles played by the other anoctamins besides TMEM16A and B. This requires efforts based on multiple methods, including gene silencing in wild-type cells, overexpression in hematopoiesis systems and generating conditional gene knockouts in mice.

Based on the various research models, such as the 'modular design' model explaining TMEM16 assembly and the 'clam-shell' and the 'pore-dilation' gating/permeation models elaborating the scramblase and channel activities of TMEM16, it is possible to address: i) The study and analyses of the synergistic effects of the three Ca²⁺-binding sites on the TMEM16 protein under normal physiological environments; ii) a more in-depth analysis of the mutated sites in the gene encoding the TMEM16 protein that is related to the manifestation of genetic diseases in humans to develop novel therapeutic drugs with improved specificity and targeting; and iii) the requirement of the careful designing of such drugs to avoid side effects as TMEM16A, F and other anoctamins are expressed in multiple tissues of the human body. It is thus hoped that future research on the structural properties of TMEM16 proteins can capture the open conformations and assist the designing of potential drugs that target Cl⁻ channels via utilizing the existing models more comprehensively and promoting development and innovation in this field.

Availability of data and materials

Not applicable.

Authors' contributions

ZH, ZI, ZZ, XC, AM, JL, WL and ZD participated in the literature review, ZH and ZD performed the figure design and ZH wrote the manuscript. ZH, ZI and ZD revised the paper. All authors have read and approved the final version of the manuscript. Data authentication is not applicable.

Ethics approval and consent for participation

Not applicable.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Authors' information

Wencui Li ORCID ID: 0000-0003-2787-5360. Zhiqin Deng ORCID ID: 0000-0002-0819-8504.

Abbreviations

CFTR

CF transmembrane conductance regulator

GDD

gnathodiaphyseal dysplasia

cystic fibrosis

CaCCs

Ca²⁺-activated Cl⁻ channels

EGFR

epidermal growth factor receptor

CCND1

cyclin D1

phosphatidylserine

Acknowledgements

Not applicable.

References 1

Vocke

Dauner

Hahn

Ulbrich

Broecker

Keller

Frings

Möhrlen

Calmodulin-dependent activation and inactivation of anoctamin calcium-gated chloride channels

J Gen Physiol1423814042013

10.1085/jgp.201311015

24081981

3787769

Whitlock

Hartzell

Anoctamins/TMEM16 proteins: Chloride channels flirting with lipids and extracellular vesicles

Annu Rev Physiol791191432017

10.1146/annurev-physiol-022516-034031

5556385

Kunzelmann

Ousingsawat

Benedetto

Cabrita

Schreiber

Contribution of Anoctamins to cell survival and cell death

Cancers113822019

10.3390/cancers11030382

30893776

6468699

Scudieri

Sondo

Ferrera

Galietta

LJV

The anoctamin family: TMEM16A and TMEM16B as calcium-activated chloride channels

Exp Physiol971771832011

10.1113/expphysiol.2011.058198

21984732

Kim

Lee

Kim

Jung

Lee

Anoctamin 9/TMEM16J is a cation channel activated by cAMP/PKA signal

Cell Calcium7175852018

10.1016/j.ceca.2017.12.003

29604966

Khelashvili

Falzone

Cheng

Lee

B-C

Accardi

Weinstein

Dynamic modulation of the lipid translocation groove generates a conductive ion channel in Ca2+-bound nhTMEM16

Nat Commun1049722019

10.1038/s41467-019-12865-4

6823365

Agostinelli

Tammaro

Polymodal control of TMEM16x channels and Scramblases

Int J Mol Sci2315802022

10.3390/ijms23031580

35163502

8835819

Baethge

Goldbeck-Wood

Mertens

SANRA-a scale for the quality assessment of narrative review articles

Res Integr Peer Rev452019

10.1186/s41073-019-0064-8

30962953

6434870

Falzone

Malvezzi

Lee

Accardi

Known structures and unknown mechanisms of TMEM16 scramblases and channels

J Gen Physiol1509339472018

10.1085/jgp.201711957

29915161

6028493

Falzone

Rheinberger

Lee

Peyear

Sasset

Raczkowski

Eng

Di Lorenzo

Andersen

Nimigean

Accardi

Structural basis of Ca2+-dependent activation and lipid transport by a TMEM16 scramblase

ELife8e432292019

10.7554/eLife.43229

6355197

Cheng

Feng

Puchades

Figueroa

Chen

Han

Identification of a conserved drug binding pocket in TMEM16 proteins

Res Sq10.21203/rs.3.rs-1296933/v1

Pedemonte

Galietta

LJV

Structure and function of TMEM16 proteins (Anoctamins)

Physiol Rev944194592014

10.1152/physrev.00039.2011

24692353

Duran

Cui

Hartzell

Explaining calcium-dependent gating of Anoctamin-1 chloride channels requires a revised topology

Circ Res1109909992012

10.1161/CIRCRESAHA.112.264440

22394518

3558997

Jung

Nam

Park

Yoon

Lee

Dynamic modulation of ANO1/TMEM16A HCO3⁻ permeability by Ca²⁺/calmodulin

Proc Natl Acad Sci USA1103603652012

10.1073/pnas.1211594110

Tian

Kongsuphol

Hug

Ousingsawat

Witzgall

Schreiber

Kunzelmann

Calmodulin-dependent activation of the epithelial calcium-dependent chloride channel TMEM16A

FASEB J25105810682010

10.1096/fj.10-166884

21115851

Hahn

Salomon

Leitz

Feigenbutz

Korsch

Lisewski

Schrimpf

Millar-Büchner

Mall

Frings

Möhrlen

Expression and function of Anoctamin 1/TMEM16A calcium-activated chloride channels in airways of in vivo mouse models for cystic fibrosis research

Pflugers Arch470133513482018

10.1007/s00424-018-2160-x

29860639

Falzone

Feng

Alvarenga

Pan

Lee

Cheng

Fortea

Scheuring

Accardi

TMEM16 scramblases thin the membrane to enable lipid scrambling

Nat Commun1326042022

10.1038/s41467-022-30300-z

35562175

9095706

Jansen

Steurer

DOG1 expression is in common human tumors: A tissue microarray study on more than 15,000 tissue samples

Am J Clin Pathol156SupplS108S1092021

10.1093/ajcp/aqab191.230

Lam

Dutzler

Calcium-dependent electrostatic control of anion access to the pore of the calcium-activated chloride channel TMEM16A

ELife7e391222018

10.7554/eLife.39122

30311910

6195346

Huang

Xiao

Huang

Harfe

Jan

Calcium-Activated chloride channels (CaCCs) regulate action potential and synaptic response in hippocampal neurons

Neuron741791922012

10.1016/j.neuron.2012.01.033

22500639

3329964

Davis

Forrest

Jepps

Valencik

Wiwchar

Singer

Sones

Greenwood

Leblanc

Expression profile and protein translation of TMEM16A in murine smooth muscle

Am J Physiol Cell Physiol299C948C9592010

10.1152/ajpcell.00018.2010

20686072

2980309

Thomas-Gatewood

Neeb

Bulley

Adebiyi

Bannister

Leo

Jaggar

TMEM16A channels generate Ca²⁺-activated Cl-currents in cerebral artery smooth muscle cells

Am J Physiol Circ Physiol301H1819H18272011

10.1152/ajpheart.00404.2011

Wang

Chen

Jiang

Yin

TMEM16A contributes to angiotensin II-induced cerebral vasoconstriction via the RhoA/ROCK signaling pathway

Mol Med Report13369136992016

10.3892/mmr.2016.4979

Lian

Cheng

TMEM16A exacerbates renal injury by activating P38/JNK signaling pathway to promote podocyte apoptosis in diabetic nephropathy mice

Biochem Biophys Res Commun4872012082017

10.1016/j.bbrc.2017.04.021

28392397

Dutta

Khimji

Liu

Karamysheva

Fujita

Kresge

Rockey

Feranchak

PKCα regulates TMEM16A-mediated Cl-secretion in human biliary cells

Am J Physiol Liver Physiol310G34G422016

Arreola

López-Romero

Pérez-Cornejo

Rodríguez-Menchaca

Phosphatidylinositol 4,5-bisphosphate and cholesterol regulators of the calcium-activated chloride channels TMEM16A and TMEM16B

Adv Exp Med Biol14222793042023

10.1007/978-3-031-21547-6_10

36988885

Lee

Lim

Lee

Cheong

Intracellular loop in the brain isoforms of anoctamin 2 channels regulates calcium-dependent activation

Exp Neurobiol321331462023

10.5607/en22045

37403222

10327929

Pietra

Dibattista

Menini

Reisert

Boccaccio

The Ca²⁺-activated Cl-channel TMEM16B regulates action potential firing and axonal targeting in olfactory sensory neurons

J Gen Physiol1482933112016

10.1085/jgp.201611622

27619419

5037344

Ayoglu

Mitsios

Kockum

Khademi

Zandian

Sjöberg

Forsström

Bredenberg

Lima Bomfim

Holmgren

Anoctamin 2 identified as an autoimmune target in multiple sclerosis

Proc Natl Acad Sci USA113218821932016

10.1073/pnas.1518553113

26862169

4776531

Lee

Kwak

Song

Kwon

Jung

Hong

Chang

Hwang

Shin

The Ca²⁺-activated chloride channel anoctamin-2 mediates spike-frequency adaptation and regulates sensory transmission in thalamocortical neurons

Nat Commun7137912016

10.1038/ncomms13791

Zhang

Xiao

Tien

Jan

Yang

Inferior Olivary TMEM16B mediates cerebellar motor learning

Neuron9511031111.e42017

10.1016/j.neuron.2017.08.010

28858616

5659299

Kim

Lee

Investigation of phosphatidylserine-transporting activity of human TMEM16C isoforms

Membranes (Basel)1210052022

10.3390/membranes12101005

36295764

9611045

Wang

Chen

Huang

Feng

Tien

Braz

Basbaum

Jan

TMEM16C is involved in thermoregulation and protects rodent pups from febrile seizures

Proc Natl Acad Sci USA118e20233421182021

10.1073/pnas.2023342118

33972431

8157992

Huang

Wang

Ostertag

Nuwal

Huang

Jan

Basbaum

Jan

TMEM16C facilitates Na(+)-activated K+ currents in rat sensory neurons and regulates pain processing

Nat Neurosci16128412902013

10.1038/nn.3468

23872594

4034143

Carvalho

Martins

Correia

Costa

Massano

Temudo

Another twist in the tale: Intrafamilial phenotypic heterogeneity in ANO3-related dystonia

Mov Disord Clin Pract87587622021

10.1002/mdc3.13209

34307749

8287195

Stamelou

Charlesworth

Cordivari

Schneider

Kägi

Sheerin

Rubio-Agusti

Batla

Houlden

Wood

Bhatia

The phenotypic spectrum of DYT24 due to ANO3 mutations

Mov Disord299289342014

10.1002/mds.25802

24442708

4150528

Esposito

Trinchillo

Piceci-Sparascio

D'Asdia

Consoli

De Luca

A novel ANO3 variant in two siblings with different phenotypes

Parkinsonism Relat Disord1111054132023

10.1016/j.parkreldis.2023.105413

37116293

Reichhart

Milenkovic

Wetzel

Strauß

Prediction of functional consequences of missense mutations in ANO4 Gene

Int J Mol Sci2227322021

10.3390/ijms22052732

33800471

7962975

Maniero

Scudieri

Haris Shaikh

Zhao

Gurnell

Galietta

LJV

Brown

ANO4 (Anoctamin 4) Is a novel marker of zona glomerulosa that regulates stimulated aldosterone secretion

Hypertension74115211592019

10.1161/HYPERTENSIONAHA.119.13287

31564164

6791498

Sherva

Tripodis

Bennett

Chibnik

Crane

de Jager

Farrer

Saykin

Shulman

Naj

Genome-wide association study of the rate of cognitive decline in Alzheimer's disease

Alzheimers Dement1045522014

10.1016/j.jalz.2013.01.008

Di Zanni

Gradogna

Scholz-Starke

Boccaccio

Gain of function of TMEM16E/ANO5 scrambling activity caused by a mutation associated with gnathodiaphyseal dysplasia

Cell Mol Life Sci75165716702017

10.1007/s00018-017-2704-9

29124309

5897490

Di Zanni

Gradogna

Picco

Scholz-Starke

Boccaccio

TMEM16E/ANO5 mutations related to bone dysplasia or muscular dystrophy cause opposite effects on lipid scrambling

Hum Mutat41115711702020

10.1002/humu.24006

32112655

Whitlock

Cui

Hartzell

Anoctamin 5/TMEM16E facilitates muscle precursor cell fusion

J Gen Physiol150149815092018

10.1085/jgp.201812097

30257928

6219693

Foltz

Cui

Choo

Hartzell

ANO5 ensures trafficking of annexins in wounded myofibers

J Cell Biol220e2020070592021

10.1083/jcb.202007059

33496727

7844426

van Kruchten

Mattheij

Saunders

Feijge

Swieringa

Wolfs

Collins

Heemskerk

Bevers

Both TMEM16F-dependent and TMEM16F-independent pathways contribute to phosphatidylserine exposure in platelet apoptosis and platelet activation

Blood121185018572013

10.1182/blood-2012-09-454314

23303820

Arndt

Alvadia

Straub

Clerico Mosina

Paulino

Dutzler

Structural basis for the activation of the lipid scramblase TMEM16F

Nat Commun1366922022

10.1038/s41467-022-34497-x

36335104

9637102

Fujii

Sakata

Nishimura

Eto

Nagata

TMEM16F is required for phosphatidylserine exposure and microparticle release in activated mouse platelets

Proc Natl Acad Sci USA11212800128052015

10.1073/pnas.1516594112

26417084

4611630

Millington-Burgess

Harper

Gene of the issue: ANO6 and Scott syndrome

Platelets319649672019

10.1080/09537104.2019.1693039

31746257

Gao

Zuo

Yang

Zhao

Chen

Guo

Han

BVES is a novel interactor of ANO5 and regulates myoblast differentiation

Cell Biosci112222021

10.1186/s13578-021-00735-w

34963485

8715634

Guo

Wang

Dong

Gao

Tong

Liu

Zhang

Sun

ANO7: Insights into topology, function, and potential applications as a biomarker and immunotherapy target

Tissue Cell721015462021

10.1016/j.tice.2021.101546

33940566

Kaikkonen

Rantapero

Zhang

Taimen

Laitinen

Kallajoki

Jambulingam

Ettala

Knaapila

Boström

ANO7 is associated with aggressive prostate cancer

Int J Cancer143247924872018

10.1002/ijc.31746

30157291

6589920

Jha

Chung

Vachel

Maleth

Lake

Zhang

Ahuja

Muallem

Anoctamin 8 tethers endoplasmic reticulum and plasma membrane for assembly of Ca²⁺ signaling complexes at the ER/PM compartment

EMBO J38e1014522019

10.15252/embj.2018101452

Liu

Lai

Zeng

Xin

Nie

Liang

Chen

Zheng

Zou

Whole-exome sequencing reveals ANO8 as a genetic risk factor for intrahepatic cholestasis of pregnancy

BMC Pregnancy Childbirth205442020

10.1186/s12884-020-03240-z

32942997

7499841

Schreiber

Ousingsawat

Kunzelmann

Targeting of intracellular TMEM16 proteins to the plasma membrane and activation by purinergic signaling

Int J Mol Sci2140652020

10.3390/ijms21114065

32517157

7312528

Katsurahara

Shiozaki

Kosuga

Kudou

Shoda

Arita

Konishi

Komatsu

Kubota

Fujiwara

ANO9 regulated cell cycle in human esophageal squamous cell carcinoma

Ann Surg Oncol27321832302020

10.1245/s10434-020-08368-y

32227267

Katsurahara

Shiozaki

Kosuga

Shimizu

Kudou

Arita

Konishi

Komatsu

Kubota

Fujiwara

ANO9 regulates PD-L2 expression and binding ability to PD-1 in gastric cancer

Cancer Sci112102610372021

10.1111/cas.14796

33404124

7935785

Jun

Park

Piao

Han

Yun

Zhang

Cha

Shin

Yook

ANO9/TMEM16J promotes tumourigenesis via EGFR and is a novel therapeutic target for pancreatic cancer

Br J Cancer117179818092017

10.1038/bjc.2017.355

29024940

5729472

Schreiber

Talbi

Ousingsawat

Kunzelmann

A TMEM16J variant leads to dysregulated cytosolic calcium which may lead to renal disease

FASEB J37e226832023

10.1096/fj.202200968R

Chrysanthou

Ververis

Christodoulou

ANO10 function in health and disease

Cerebellum224474672022

10.1007/s12311-022-01395-3

35648332

10126014

Wanitchakool

Ousingsawat

Sirianant

Cabrita

Faria

Schreiber

Kunzelmann

Cellular defects by deletion of ANO10 are due to deregulated local calcium signaling

Cell Signal3041492017

10.1016/j.cellsig.2016.11.006

Hammer

Wanitchakool

Sirianant

Papiol

Monnheimer

Faria

Ousingsawat

Schramek

Schmitt

Margos

A coding variant of ANO10, affecting volume regulation of macrophages, is associated with borrelia seropositivity

Mol Med2126372015

10.2119/molmed.2014.00219

25730773

4461583

Gentzsch

Mall

Ion channel modulators in cystic fibrosis

Chest1543833932018

10.1016/j.chest.2018.04.036

29750923

6113631

Shteinberg

Haq

Polineni

Davies

Cystic fibrosis

Lancet397219522112021

10.1016/S0140-6736(20)32542-3

34090606

Lopes-Pacheco

Pedemonte

Veit

Discovery of CFTR modulators for the treatment of cystic fibrosis

Expert Opin Drug Discov168979132021

10.1080/17460441.2021.1912732

33823716

Villamizar

Waters

Scott

Grepo

Jaffe

Morris

Mesenchymal Stem Cell exosome delivered Zinc Finger Protein activation of cystic fibrosis transmembrane conductance regulator

J Extracell Vesicles10e120532021

10.1002/jev2.12053

33532041

7825549

Simon

Csanady

Understanding impact of δF508 and G551D CFTR mutations on CFTR/PKA-c interaction

Biophys J122112a2023

10.1016/j.bpj.2022.11.786

Harrison

Murphy

Plant

Ivacaftor in a G551D homozygote with cystic fibrosis

N Engl J Med369128012822013

10.1056/NEJMc1213681

24066763

Ramsey

Davies

McElvaney

Tullis

Bell

Dřevínek

Griese

McKone

Wainwright

Konstan

A CFTR potentiator in patients with cystic fibrosis and theG551Dmutation

N Engl J Med365166316722011

10.1056/NEJMoa1105185

22047557

3230303

Fiedorczuk

Chen

Mechanism of CFTR correction by type I folding correctors

Cell185158168.e112022

10.1016/j.cell.2021.12.009

34995514

Veit

Roldan

Hancock

Da Fonte

Hussein

Frenkiel

Matouk

Velkov

Lukacs

Allosteric folding correction of F508del and rare CFTR mutants by Elexacaftor-Tezacaftor-Ivacaftor (Trikafta) combination

JCI Insight5e1399832020

10.1172/jci.insight.139983

32853178

7526550

Rowe

McColley

Rietschel

Bell

Konstan

Marigowda

Waltz

Boyle

VX09-809-102 Study Group

Lumacaftor/Ivacaftor treatment of patients with cystic fibrosis heterozygous for F508del-CFTR

Ann Am Thorac Soc142132192017

10.1513/AnnalsATS.201609-689OC

5461999

Wainwright

Elborn

Ramsey

Marigowda

Huang

Cipolli

Colombo

Davies

De Boeck

Flume

Lumacaftor-Ivacaftor in patients with cystic fibrosis homozygous for Phe508delCFTR

N Engl J Med3732202312015

10.1056/NEJMoa1409547

25981758

4764353

Clancy

Rowe

Accurso

Aitken

Amin

Ashlock

Ballmann

Boyle

Bronsveld

Campbell

Results of a phase IIa study of VX-809, an investigational CFTR corrector compound, in subjects with cystic fibrosis homozygous for theF508del-CFTRmutation

Thorax6712182011

10.1136/thoraxjnl-2011-200393

Flume

Harris

Paz-Diaz

Ahluwalia

Higgins

Campbell

Berhane

Shih

Sawicki

Long-term tezacaftor/ivacaftor safety and efficacy in people with cystic fibrosis and an F508del-CFTR mutation: 96-week, open-label extension of the EXTEND trial

J Cyst Fibros224644702023

10.1016/j.jcf.2022.12.006

Bruscia

The effects of Elexacaftor/Tezacaftor/Ivacaftor beyond the epithelium: Spurring macrophages to fight infections

Eur Respir J6123002162023

10.1183/13993003.00216-2023

Sawicki

Van Brunt

Booth

Bailey

Millar

Konstan

Flume

Disease burden in people with cystic fibrosis heterozygous for F508del and a minimal function mutation

J Cyst Fibros21961032022

10.1016/j.jcf.2021.07.003

9588169

Galietta

LJV

TMEM16A (ANO1) as a therapeutic target in cystic fibrosis

Curr Opin Pharmacol641022062022

10.1016/j.coph.2022.102206

35364521

Simões

Quaresma

Clarke

Silva

Pankonien

Railean

Kmit

Amaral

TMEM16A chloride channel does not drive mucus production

Life Sci Alliance2e2019004622019

10.26508/lsa.201900462

31732694

6859295

Ruffin

Voland

Marie

Bonora

Blanchard

Blouquit-Laye

Naline

Puyo

Le Rouzic

Guillot

Anoctamin 1 dysregulation alters bronchial epithelial repair in cystic fibrosis

Biochim Biophys Acta1832234023512013

10.1016/j.bbadis.2013.09.012

24080196

Kirk

Wang

A unified view of cystic fibrosis transmembrane conductance regulator (CFTR) gating: Combining the allosterism of a ligand-gated channel with the enzymatic activity of an ATP-binding cassette (ABC) transporter

J Biol Chem28612813128192011

10.1074/jbc.R111.219634

21296873

3075628

Deng

Chen

Lin

Alahdal

Wang

Liu

The homeostasis of cartilage matrix remodeling and the regulation of volume-sensitive ion channel

Aging Dis137878002022

10.14336/AD.2021.1122

35656105

9116913

Talbi

Ousingsawat

Centeio

Schreiber

Kunzelmann

Calmodulin-dependent regulation of overexpressed but not endogenous TMEM16A expressed in airway epithelial cells

Membranes (Basel)117232021

10.3390/membranes11090723

34564540

8471323

Cabrita

Benedetto

Schreiber

Kunzelmann

Niclosamide repurposed for the treatment of inflammatory airway disease

JCI Insight4e1284142019

10.1172/jci.insight.128414

31391337

6693830

Danahay

Fox

Lilley

Charlton

Adley

Christie

Ansari

Ehre

Flen

Tuvim

Potentiating TMEM16A does not stimulate airway mucus secretion or bronchial and pulmonary arterial smooth muscle contraction

FASEB Bioadv24644772020

10.1096/fba.2020-00035

32821878

7429354

Danahay

Lilley

Fox

Charlton

Sabater

Button

McCarthy

Collingwood

Gosling

TMEM16A potentiation: A novel therapeutic approach for the treatment of cystic fibrosis

Am J Respir Crit Care Med2019469542020

10.1164/rccm.201908-1641OC

31898911

7159426

Ousingsawat

Centeio

Cabrita

Talbi

Zimmer

Graf

Göpferich

Schreiber

Kunzelmann

Airway delivery of hydrogel-encapsulated niclosamide for the treatment of inflammatory airway disease

Int J Mol Sci2310852022

10.3390/ijms23031085

35163010

8835663

Centeio

Ousingsawat

Cabrita

Schreiber

Talbi

Benedetto

Doušová

Verbeken

De Boeck

Cohen

Kunzelmann

Mucus release and airway constriction by TMEM16A may worsen pathology in inflammatory lung disease

Int J Mol Sci2278522021

10.3390/ijms22157852

34360618

8346050

Sonneville

Ruffin

Coraux

Rousselet

Le Rouzic

Blouquit-Laye

Corvol

Tabary

MicroRNA-9 downregulates the ANO1 chloride channel and contributes to cystic fibrosis lung pathology

Nat Commun87102017

10.1038/s41467-017-00813-z

28955034

5617894

Kamaleddin

Molecular, biophysical, and pharmacological properties of calcium-activated chloride channels

J Cell Physiol2337877982017

10.1002/jcp.25823

28121009

Sah

McCluggage

DOG1 immunoreactivity in uterine leiomyosarcomas

J Clin Pathol6640432012

10.1136/jclinpath-2012-201150

23038686

Filippou

Pehkonen

Karhemo

Väänänen

Nieminen

Klefström

Grénman

Mäkitie

Joensuu

Monni

ANO1 expression orchestrates p27Kip1/MCL1-Mediated signaling in head and neck squamous cell carcinoma

Cancers (Basel)1311702021

10.3390/cancers13051170

33803266

7967175

Ishaque

Abba

Hauser

Patil

Paramasivam

Huebschmann

Leupold

Balasubramanian

Kleinheinz

Toprak

Whole genome sequencing puts forward hypotheses on metastasis evolution and therapy in colorectal cancer

Nat Commun947822018

10.1038/s41467-018-07041-z

30429477

6235880

Sauter

DRP

Novak

Pedersen

Larsen

Hoffmann

ANO1 (TMEM16A) in pancreatic ductal adenocarcinoma (PDAC)

Pflugers Arch467149515082015

10.1007/s00424-014-1598-8

4464647

Song

Gao

Guan

Chen

Gao

Wang

Inhibition of ANO1/TMEM16A induces apoptosis in human prostate carcinoma cells by activating TNF-α signaling

Cell Death Dis97032018

10.1038/s41419-018-0735-2

Britschgi

Bill

Brinkhaus

Rothwell

Clay

Duss

Rebhan

Raman

Guy

Wetzel

Calcium-activated chloride channel ANO1 promotes breast cancer progression by activating EGFR and CAMK signaling

Proc Natl Acad Sci USA110E1026E10342013

10.1073/pnas.1217072110

23431153

3600458

Sui

Sun

Yang

Zhang

Zhong

Zheng

Fang

Inhibition of TMEM16A expression suppresses growth and invasion in human colorectal cancer cells

PLoS One9e1154432014

10.1371/journal.pone.0115443

25541940

4277312

Cao

Liu

Zhang

Wang

MicroRNA-381 inhibits the metastasis of gastric cancer by targeting TMEM16A expression

J Exp Clin Cancer Res36292017

10.1186/s13046-017-0499-z

28193228

5307754

Lee

Bae

Lee

Kim

Cho

Ryoo

Yoo

Kim

Suppression of 14-3-3γ-mediated surface expression of ANO1 inhibits cancer progression of glioblastoma cells

Sci Rep6264132016

10.1038/srep26413

Shang

Hao

Zhao

Shi

Liu

Jiang

Shi

Yang

ANO1 protein as a potential biomarker for esophageal cancer prognosis and precancerous lesion development prediction

Oncotarget724374243822016

10.18632/oncotarget.8223

27016410

5029708

100

Akpalo

Lange

Zustin

Discovered on gastrointestinal stromal tumour 1 (DOG1): A useful immunohistochemical marker for diagnosing chondroblastoma

Histopathology60109911062012

10.1111/j.1365-2559.2011.04152.x

22335248

101

Chang

Cai

Han

Gao

Feng

Zhang

Zheng

Jin

Zhang

Wei

Anoctamin5 regulates cell migration and invasion in thyroid cancer

Int J Oncol51131113192017

10.3892/ijo.2017.4113

28902351

102

Wang

Vural

Mishra

Cowan

Guda

Exome analysis reveals differentially mutated gene signatures of stage, grade and subtype in breast cancers

PLoS One10e01193832015

10.1371/journal.pone.0119383

25803781

4372331

103

Chen

Gao

Chen

Yang

Xie

Wang

Sun

Wang

The prognostic value and mechanisms of TMEM16A in human cancer

Front Mol Biosci85421562021

10.3389/fmolb.2021.542156

33681289

7930745

104

Liu

Yang

Geng

Huang

Targeting ERK, an Achilles' heel of the MAPK pathway, in cancer therapy

Acta Pharm Sin B85525622018

10.1016/j.apsb.2018.01.008

30109180

6089851

105

Wang

Yao

Luo

Liu

Bai

Chen

Song

Wang

A mutual activation loop between the Ca²⁺-activated chloride channel TMEM16A and EGFR/STAT3 signaling promotes breast cancer tumorigenesis

Cancer Lett45548592019

10.1016/j.canlet.2019.04.027

31042586

106

Bai

Liu

Xiao

The diverse roles of TMEM16A Ca²⁺-activated Cl-channels in inflammation

J Adv Res3353682021

10.1016/j.jare.2021.01.013

34603778

8463915

107

Lin

Deng

Liu

Lin

Chen

Deng

Chloride channel and inflammation-mediated pathogenesis of osteoarthritis

J Inflamm Res159539642022

10.2147/JIR.S350432

35177922

8846625

108

Liu

Ren

Kang

Zhang

Transmembrane protein with unknown function 16A overexpression promotes glioma formation through the nuclear factor-κB signaling pathway

Mol Med Report9106810742014

10.3892/mmr.2014.1888

109

Zhou

Deng

Jiang

Wei

Hong

Chen

Wang

Zhang

Shi

TRIM31 promotes glioma proliferation and invasion through activating NF-κB pathway

Onco Targets Ther12228922972019

10.2147/OTT.S183625

6441556

110

Duvvuri

Shiwarski

Xiao

Bertrand

Huang

Edinger

Rock

Harfe

Henson

Kunzelmann

TMEM 16 A induces MAPK and contributes directly to tumorigenesis and cancer progression

Cancer Res72327032812012

10.1158/0008-5472.CAN-12-0475-T

22564524

3694774

111

Deng

Yang

Chen

Pan

Zhang

Knockdown of TMEM16A suppressed MAPK and inhibited cell proliferation and migration in hepatocellular carcinoma

Onco Targets Ther93253332016

26834491

4716773

112

Ruiz

Martins

Rudin

Schneider

Dietsche

Fischer

Tornillo

Terracciano

Schreiber

Bubendorf

Kunzelmann

Enhanced expression of ANO1 in head and neck squamous cell carcinoma causes cell migration and correlates with poor prognosis

PLoS One7e432652012

10.1371/journal.pone.0043265

22912841

3422276

113

Dixit

Kemp

Kulich

Seethala

Chiosea

Ling

Duvvuri

TMEM16A/ANO1 is differentially expressed in HPV-negative versus HPV-positive head and neck squamous cell carcinoma through promoter methylation

Sci Rep5166572015

10.1038/srep16657

26563938

4643216

114

Mokutani

Uemura

Munakata

Okuzaki

Haraguchi

Takahashi

Nishimura

Hata

Murata

Takemasa

Down-regulation of microRNA-132 is associated with poor prognosis of colorectal cancer

Ann Surg Oncol235996082016

10.1245/s10434-016-5133-3

26868958

5149564

115

Lin

Gregory

MicroRNA biogenesis pathways in cancer

Nat Rev Cancer153213332015

10.1038/nrc3932

25998712

4859809

116

Wang

Zou

Wei

Xiao

Cell-specific mechanisms of TMEM16A Ca²⁺-activated chloride channel in cancer

Mol Cancer161522017

10.1186/s12943-017-0720-x

117

Wanitchakool

Wolf

Koehl

Sirianant

Schreiber

Kulkarni

Duvvuri

Kunzelmann

Role of anoctamins in cancer and apoptosis

Philos Trans R Soc Lond B Biol Sci369201300962014

10.1098/rstb.2013.0096

24493744

3917350

118

Ahn

Yang

Kim

Lee

Cha

Yun

Kang

Lee

Choi

Kim

Yook

Anti-helminthic niclosamide inhibits Ras-driven oncogenic transformation via activation of GSK-3

Oncotarget831856318632017

10.18632/oncotarget.16255

28418865

5458253

119

Miner

Labitzke

Liu

Wang

Henckels

Gaida

Elliott

Chen

Liu

Leith

Drug repurposing: The anthelmintics niclosamide and nitazoxanide are potent TMEM16A antagonists that fully bronchodilate airways

Front Pharmacol10512019

10.3389/fphar.2019.00051

30837866

6382696

120

Jin

Ding

Chen

Sun

Zhou

Pan

Antineoplastic mechanisms of niclosamide in acute myelogenous leukemia stem cells: Inactivation of the NF-κB pathway and generation of reactive oxygen species

Cancer Res70251625272010

10.1158/0008-5472.CAN-09-3950

20215516

121

Han

Wang

Guo

Niclosamide induces cell cycle arrest in G1 phase in head and neck squamous cell carcinoma through Let-7d/CDC34 Axis

Front Pharmacol915442019

10.3389/fphar.2018.01544

30687101

6333743

122

Roberts

Arend

Samant

Buchsbaum

Multi-targeted therapy of cancer by niclosamide: A new application for an old drug

Cancer Lett3498142014

10.1016/j.canlet.2014.04.003

24732808

4166407

123

Arend

Londoño-Joshi

Gangrade

Katre

Kurpad

Samant

Landen

Yang

Correction: Niclosamide and its analogs are potent inhibitors of Wnt/β-catenin, mTOR and STAT3 signaling in ovarian cancer

Oncotarget919459194592018

10.18632/oncotarget.25151

124

Lafkas

Shelton

Chiu

de Leon Boenig

Chen

Stawicki

Siltanen

Reichelt

Zhou

Therapeutic antibodies reveal Notch control of transdifferentiation in the adult lung

Nature5281271312015

10.1038/nature15715

26580007

125

Danahay

Pessotti

Coote

Montgomery

Xia

Wilson

Yang

Wang

Bevan

Thomas

Notch2 is required for inflammatory cytokine-driven goblet cell metaplasia in the lung

Cell Rep102392522015

10.1016/j.celrep.2014.12.017

25558064

126

Seo

Kim

Chang

Kim

Namkung

Kim

Synthesis and biological evaluation of novel Ani9 derivatives as potent and selective ANO1 inhibitors

Eur J Medicinal Chem1602452552018

10.1016/j.ejmech.2018.10.002

127

Burock

Daum

Keilholz

Neumann

Walther

Stein

Phase II trial to investigate the safety and efficacy of orally applied niclosamide in patients with metachronous or sychronous metastases of a colorectal cancer progressing after therapy: The NIKOLO trial

BMC Cancer182972018

10.1186/s12885-018-4197-9

29544454

5856000

128

Schweizer

Haugk

McKiernan

Gulati

Cheng

Maes

Dumpit

Nelson

Montgomery

McCune

Correction: A phase I study of niclosamide in combination with enzalutamide in men with castration-resistant prostate cancer

PLoS One13e02027092018

10.1371/journal.pone.0202709

30110398

6093693

129

Yan

Ding

Han

Gao

Liu

Wang

Involvement of TMEM16A/ANO1 upregulation in the oncogenesis of colorectal cancer

Biochim Biophys Acta Mol Basis Dis18681663702022

10.1016/j.bbadis.2022.166370

35231545

130

Khalil

Elesawy

Ali

Ahmed

Bee venom: From venom to drug

Molecules2649412021

10.3390/molecules26164941

34443529

8400317

131

Badawi

Bee venom components as therapeutic tools against prostate cancer

Toxins (Basel)133372021

10.3390/toxins13050337

34067049

8150751

132

Schreiber

Ousingsawat

Wanitchakool

Sirianant

Benedetto

Reiss

Kunzelmann

Regulation of TMEM16A/ANO1 and TMEM16F/ANO6 ion currents and phospholipid scrambling by Ca²⁺ and plasma membrane lipid

J Physiol5962172292017

10.1113/JP275175

133

Simões

Ousingsawat

Wanitchakool

Fonseca

Cabrita

Benedetto

Schreiber

Kunzelmann

CFTR supports cell death through ROS-dependent activation of TMEM16F (anoctamin 6)

Pflugers Arch4703053142017

10.1007/s00424-017-2065-0

28875346

134

Stockwell

Friedmann Angeli

Bayir

Bush

Conrad

Dixon

Fulda

Gascón

Hatzios

Kagan

Ferroptosis: A regulated cell death nexus linking metabolism, redox biology, and disease

Cell1712732852017

10.1016/j.cell.2017.09.021

28985560

5685180

135

Xue

Yan

Chen

Kang

Klionsky

Kroemer

Chen

Tang

Liu

Copper-dependent autophagic degradation of GPX4 drives ferroptosis

Autophagy19198219962023

10.1080/15548627.2023.2165323

36622894

10283421

136

El-Didamony

Amer

El-Osaily

Formulation, characterization and cellular toxicity assessment of a novel bee-venom microsphere in prostate cancer treatment

Sci Rep12132132022

10.1038/s41598-022-17391-w

35918370

9346107

137

Schreiber

Buchholz

Kraus

Schley

Scholz

Ousingsawat

Kunzelmann

Lipid peroxidation drives renal cyst growth in vitro through activation of TMEM16A

J Am Soc Nephrol302282422019

10.1681/ASN.2018010039

30606785

6362630

138

Schmaier

Anderson

Chen

El-Darzi

Aivasovsky

Kaushik

Sack

Hartzell

Parikh

Flaumenhaft

Schulman

TMEM16E regulates endothelial cell procoagulant activity and thrombosis

J Clin Invest133e1638082023

10.1172/JCI163808

36951953

10231993

139

Fujii

Taniguchi

Nagaya

Ueda

Hashizume

Watanabe

Takeya

Kosaka

Okazaki

A novel mechanism of thrombocytopenia by PS exposure through TMEM16F in sphingomyelin synthase 1 deficiency

Blood Adv5426542772021

10.1182/bloodadvances.2020002922

34478523

8945624

140

Filep

Two to tango: Endothelial cell TMEM16 scramblases drive coagulation and thrombosis

J Clin Invest133e1706432023

10.1172/JCI170643

37259922

10231982

141

Cernysiov

Davidson

Song

Tang

Luo

Qian

Gyurova

Waggoner

Critical role of lipid scramblase TMEM16F in phosphatidylserine exposure and repair of plasma membrane after pore formation

Cell Rep3011291140.e52020

10.1016/j.celrep.2019.12.066

31995754

7104872

142

Taylor

Mahaut-Smith

A major interspecies difference in the ionic selectivity of megakaryocyte Ca²⁺-activated channels sensitive to the TMEM16F inhibitor CaCCinh-A01

Platelets309629662019

10.1080/09537104.2019.1595560

6816474

143

Yang

Kim

David

Palmer

Jin

Tien

Huang

Cheng

Coughlin

Jan

TMEM16F Forms a Ca²⁺-activated cation channel required for lipid scrambling in platelets during blood coagulation

Cell1511111222012

10.1016/j.cell.2012.07.036

23021219

3582364

144

Wang

Yan

Wang

Dong

Shi

Gao

Hyperuricemia enhances procoagulant activity of vascular endothelial cells through TMEM16F regulated phosphatidylserine exposure and microparticle release

FASEB J35e218082021

10.1096/fj.202100426R

34390515

145

Goyal

Choi

Pinheiro

Schenck

Chen

Jabri

Satlin

Campion

JrNahid

Ringel

Clinical characteristics of Covid-19 in new york city

N Engl J Med382237223742020

10.1056/NEJMc2010419

32302078

7182018

146

Levi

Thachil

Iba

Levy

Coagulation abnormalities and thrombosis in patients with COVID-19

Lancet Haematol7e438e4402020

10.1016/S2352-3026(20)30145-9

32407672

7213964

147

Edler

Schröder

Aepfelbacher

Fitzek

Heinemann

Heinrich

Klein

Langenwalder

Lütgehetmann

Meißner

Correction to: Dying with SARS-CoV-2 infection-an autopsy study of the first consecutive 80 cases in Hamburg, Germany

Int J Legal Med13419772020

10.1007/s00414-020-02336-7

32562038

7303576

148

Hoffmann

Kleine-Weber

Schroeder

Krüger

Herrler

Erichsen

Schiergens

Herrler

Nitsche

SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2 and is blocked by a clinically proven protease inhibitor

Cell181271280.e82020

10.1016/j.cell.2020.02.052

32142651

7102627

149

Hoffmann

Hofmann-Winkler

Pöhlmann

Priming time: How Cellular Proteases Arm Coronavirus Spike ProteinsSpringer International Publishing

Cham

71982018

150

Braga

Ali

Secco

Chiavacci

Neves

Goldhill

Penn

Jimenez-Guardeño

Ortega-Prieto

Bussani

Drugs that inhibit TMEM16 proteins block SARS-CoV-2 spike-induced syncytia

Nature59488932021

10.1038/s41586-021-03491-6

33827113

7611055

151

Cappelletto

Allan

Crescente

Schneider

Bussani

Ali

Secco

Vodret

Simeone

Mascaretti

SARS-CoV-2 Spike protein activates TMEM16F-mediated platelet procoagulant activity

Front Cardiovasc Med910132622023

10.3389/fcvm.2022.1013262

36684586

9845929

152

Abdulamir

Gorial

Saadi

Maulood

Hashim

Alnuaimi

Abdulrrazaq

A randomised controlled trial of effectiveness and safety of Niclosamide as add on therapy to the standard of care measures in COVID-19 management

Ann Med Surg (Lond)691027792021

34512959

8416702

153

Chen

Mook

JrPremont

Wang

Niclosamide: Beyond an antihelminthic drug

Cell Signal4189962018

10.1016/j.cellsig.2017.04.001

154

Kalienkova

Clerico Mosina

Paulino

The Groovy TMEM16 family: Molecular mechanisms of lipid scrambling and ion conduction

J Mol Biol4331669412021

10.1016/j.jmb.2021.166941

33741412

155

Lam

AKM

Rutz

Dutzler

Inhibition mechanism of the chloride channel TMEM16A by the pore blocker 1PBC

Nat Commun1327982022

10.1038/s41467-022-30479-1

35589730

9120017

156

Bill

Gutierrez

Kulkarni

Kemp

Bonenfant

Voshol

Duvvuri

Gaither

ANO1/TMEM16A interacts with EGFR and correlates with sensitivity to EGFR-targeting therapy in head and neck cancer

Oncotarget6917391882015

10.18632/oncotarget.3277

25823819

4496210

157

Zhang

Zhai

Zhao

Wang

Inhibition of TMEM16A Ca²⁺-activated Cl-channels by avermectins is essential for their anticancer effects

Pharmacol Res1561047632020

10.1016/j.phrs.2020.104763

158

Fang

Deng

Liu

Chen

Deng

The mechanism of bone remodeling after bone aging

Clin Interv Aging174054152022

10.2147/CIA.S349604

35411139

8994557

159

Genovese

Buccirossi

Guidone

De Cegli

Sarnataro

di Bernardo

Galietta

LJV

Analysis of inhibitors of the anoctamin-1 chloride channel (transmembrane member 16A, TMEM16A) reveals indirect mechanisms involving alterations in calcium signalling

Br J Pharmacol1807757852023

10.1111/bph.15995

160

Shaibani

Khan

Shinawi

Autosomal dominant ANO5-Related disorder associated with myopathy and gnathodiaphyseal dysplasia

Neurol Genet7e6122021

10.1212/NXG.0000000000000612

34291158

8290902

161

Liu

Wang

Miao

Tian

Genetic disruption of Ano5 leads to impaired osteoclastogenesis for gnathodiaphyseal dysplasia

Oral Dis30140314152024

10.1111/odi.14562

162

Chandra

Defour

Mamchoui

Pandey

Mishra

Mouly

Sreetama

Mahad Ahmad

Mahjneh

Morizono

Dysregulated calcium homeostasis prevents plasma membrane repair in Anoctamin 5/TMEM16E-deficient patient muscle cells

Cell Death Discov51182019

10.1038/s41420-019-0197-z

31341644

6639303

163

Thiruvengadam

Sreetama

Charton

Hogarth

Novak

Suel-Petat

Chandra

Allard

Richard

Jaiswal

Anoctamin 5 Knockout mouse model recapitulates LGMD2L muscle pathology and offers insight into in vivo functional deficits

J Neuromuscul Dis8SupplS243S2552021

10.3233/JND-210720

34633328

8673513

164

Wang

Chen

Liu

Miao

Tian

Dong

Introduction of a Cys360Tyr Mutation in ANO5 creates a mouse model for gnathodiaphyseal dysplasia

J Bone Miner Res375155302022

10.1002/jbmr.4481

165

Jiang

Liu

Zhang

Pan

Wang

Wan

Jin

The expanding clinical and genetic spectrum of ANO3 dystonia

Neurosci Lett7461355902021

10.1016/j.neulet.2020.135590

33388357

Figure 1

Flowchart of the studies revised in the narrative review. PDB, Protein Data Bank. Created with BioRender.com.

Figure 2

Structural properties of the TMEM16 proteins. (A) The re-entrant loop plays an important role in ion conductance by TMEM16, thereby playing a role in the formation of the Ca²⁺ binding site and promoting the binding efficiency. (B) The binding sites of Ca²⁺ in mTMEM16A between TM6 and 7 (PDB 5OYB). (C) Structure of the Ca²⁺-bound fungal nhTMEM16 (PDB 4WIS). (D and E) Residues that are implicated in controlling the phospholipid scramblase activities of the mTMEM16F (PDB 6QP6) and nhTMEM16 (PDB 4WIS). PDB, Protein Data Bank; TM, transmembrane; mTMEM16, Mus musculus TMEM16; nhTMEM16, Nectria haematococca TMEM16; CBM1/2, calmodulin binding site 1/2; RCBM, regulatory calmodulin binding site; S_E, E313 (on TM3) and R432 (on TM6); S_C, E352 and K353 (both at the intracellular end of TM4). Created with BioRender.com.

Figure 3

Expression of TMEM16A and CFTR in the apical membrane of epithelial cells. The activities of CFTR and TMEM16A are regulated by cAMP and Ca²⁺, respectively, and mediate the secretion of anions such as Cl⁻ and HCO₃⁻. CFTR mainly regulates the cAMP-dependent Cl⁻ transport across the apical membrane. High cAMP levels can activate PKA, leading to the phosphorylation of a Ser in the R region, which is a prerequisite for the activation of CFTR that is strictly controlled by ATP levels. If the Ser in the R region is phosphorylated, ATP binds to NBD2 and if the phosphate group at the terminal of NBD1 is cleaved, stereo conformational changes occur in the CFTR, the domain dimerizes (NBD1:NBD2), thereby opening the channel pore, which results in the efflux of Cl⁻. CFTR, cystic fibrosis transmembrane conductance regulator; PKA, protein kinase A; TMD1/2, transmembrane domain 1/2; R, regulatory domain; NBD1/2, nucleotide-binding domain 1/2. Created with BioRender.com.

Figure 4

Chemical structures of TMEM16A modulators. The chemical structures of the TMEM16A modulators that act either as synthetic or natural inhibitors or synthetic activators were created with BioRender.com. CaCC_inh-A01, 6-t-butyl-2-(furan-2-carboxamido)-4,5,6,7-tetrahydrobenzo(b) thiophene-3-carboxylic acid; T16A_inh-A01, 2-((5-Ethyl-1,6-dihydro-4-methyl-6-oxo-2-pyrimidinyl)thio)-N-(4-(4-methoxyphenyl)-2-thiazolyl)-acetamide; CFTR_act-J027, 1-benzyl-3-(2-amino, 5-nitroso phenyl) quinoxaline-2(1H)-one; Ani9, 2-(4-chloro-2-methylphenoxy)-N-((2-methoxyphenyl) methylidene amino)-acetamide; MONNA, N-((4′-methoxy)-2′-naphthyl)-5-nitroanthranilic acid; TM_inh-23, 2-bromodifluoroacetylamino-5,6,7,8-tetrahydro-4H-cyclohepta(b) thiophene-3-carboxylic acid o-tolylamide; E_act, 3,4,5-trimethoxy-N-(2-methoxyethyl)-N-(4-phenyl-2-thiazolyl)benzamide; F_act, N-(4bromophenyl)-3-(1H-tetrazol-1-yl) benzamide. Created with BioRender.com.

Figure 5

Synergism between TMEM16A and EGFR in mediating the proliferation of tumors. There is a reciprocal exchange of Ca²⁺ between TMEM16A and EGFR. The two signaling pathways that mainly affect the proliferation of tumor cells are indicated by blue and green lines in the figure. The blue line indicates that upon activation, TMEM16A further activates the CAMK and AKT signaling pathways in succession, to act on CCND1 and cause proliferation of the tumor. The green line indicates the MAPKK signaling pathway, in which phosphorylated MEK activates CCND1 and the ERK and AP-1/STAT-3/NF-κB signaling pathways, both of which finally act on the nuclear genes to cause tumor cell proliferation. EGFR, epidermal growth factor receptor; CCND1, cyclin D1; Grb2, growth factor receptor-bound protein 2; SOS, son of sevenless; CAMK, Ca²+/calmodulin-dependent protein kinase. Created with BioRender.com.

Figure 6

Mechanism of activation of the TMEM16 proteins by the coronavirus disease 2019-related syncytia and spike protein-expressing cells. The spike protein of SARS-CoV-2, by acting on ACE2, cleaves the spike between the S1 and S2 subunits of the spike protein to form a syncytium. The host cell hydrolyzes and exposes the S2 subunit, which associates with the cell membrane to encode a protease. The first route for the S2 subunit to bind to the cell membrane is through cis-binding, with the direct activation of spike protein-expressing cells. The second is trans-binding, which activates TMEM16 by binding to ACE2 to activate the protease. The third is the indirect activation of Ca²⁺ release by inducing an increase in the intracellular Ca²⁺ concentrations via the syncytium and S2 subunit. SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; ACE2, angiotensin-converting enzyme 2; TM6, transmembrane 6. Created with BioRender.com.

Figure 7

Diseases or disorders caused by mutations in the TMEM16E encoding gene. TMEM16E is highly expressed in the muscles and bones. A mutation in the TMEM16E encoding gene can lead to GDD, LGMD and MMD. An imbalance in the levels of TMEM16E proteins can lead to arthritis, muscular dystrophy and suppurative osteomyelitis. TMEM16E also participates in bone mineralization and skeletal muscle repair. In addition, the p.Cys360Tyr mutation in the TMEM16 protein that causes GDD may be a target for potential therapeutic applications. GDD, gnathodiaphyseal dysplasia; LGMD, limb girdle muscular dystrophy; MMD, Miyoshi muscular dystrophy. Created with BioRender.com.

Table I

Functions, expression, physiological role and diseases related to TMEM16 proteins.

Protein	Functions	Expression	Physiological role	Related disease
TMEM16A	CaCC/scramblase	Widely expressed.	Transepithelial ion transport, smooth muscle contraction, activation of nociceptive neurons and cell proliferation and invasion.	GIST, HNSCC, pancreatic ductal adenocarcinoma, gastric cancer, colon cancer and esophageal cancer.
TMEM16B	CaCC/scramblase	Synaptic terminals of light and olfactory receptors.	Regulates olfactory and visual senses and controls excitability of neurons and glial cells.	Multiple sclerosis and schizophrenia.
TMEM16C	Scramblase	Cerebral cortex, cerebellum, hippocampus and caudate body.	Enhancing the K/Na⁺ channel and regulation of pain and heat processing.	Cluster headaches, Huntington's disease, osteoarthritis, eczema and craniocervical muscle tone disorder.
TMEM16D	Non-selective cation channel/scramblase	Endocrine tissue and the brain.	Aldosterone secretion and cell proliferation.	Severe hypoglycemia, Alzheimer's disease and multiple sclerosis.
TMEM16E	Non-selective ion channel/scramblase	Gastrointestinal tract and skeletal muscles.	Cell migration and invasion by affecting the JAK/STAT3 pathway, maintaining cytosolic Ca²⁺ homeostasis and muscle repair.	Mutated in GDD, LGMD, and MMD.
TMEM16F	Non-selective ion channel/scramblase	Endothelial cells.	Programmed cell death, lymphocyte apoptosis and activation of coagulation system.	Scott syndrome and amyotrophic lateral sclerosis.
TMEM16G	Ion channel?/scramblase	Cartilage, spleen, prostate and mainly in the gastrointestinal tract	Interactions with intracellular vesicle proteins.	Prostate cancer, breast cancer, familial non-nodular thyroid cancer and neuroblastoma
TMEM16H	CaCC?	Expression level is relatively low in most organisms.	Connecting the plasma membrane and endoplasmic reticulum, intracellular signal transduction and bile salt transport.	Intrahepatic cholestasis of pregnancy.
TMEM16J	cAMP/PKA-activated cation channel/scramblase	The plasma membrane of olfactory epithelium, the endoplasmic reticulum of renal epithelial cells and the brush like plate membrane of proximal tubular epithelial cells.	Inducing cell proliferation, apoptosis and invasion and upregulating calcium influx.	Chronic kidney disease, pancreatic cancer and colorectal cancer.
TMEM16K	Scramblase	Between two endoplasmic reticulum lobules.	Redistribution of phosphatidylserine, regulation of macrophage immune function, cell proliferation and apoptosis.	Spinocerebellar ataxia and CoQ10 deficiency.

GIST, gastrointestinal stromal tumor; HNSCC, head and neck squamous cell carcinoma; CaCC, Ca²⁺-activated Cl⁻ channel; PKA, protein kinase A; GDD, gnathodiaphyseal dysplasia; LGMD, limb girdle muscular dystrophy; MMD, Miyoshi muscular dystrophy; JAK, janus kinase.

Table II

Cancer or physiological processes mediated by various TMEM16 proteins.

TMEM16	Cancer or process	(Refs.)
TMEM16A	gastrointestinal stromal tumor	(18)
	Leiomyosarcoma	(90)
	Head and neck cancer	(91)
	Lung cancer	(92)
	Pancreatic cancer	(93)
	Prostate cancer	(94)
	Breast cancer	(95)
	Colorectal cancer	(96)
	Gastric cancer	(97)
	Glioma, glioblastoma	(98)
	Esophageal cancer	(99)
	Chondroblastoma	(100)
TMEM16B	Regulate olfactory sensation and vision	(29)
TMEM16B	Control the excitability of neurons and glial cells	(31)
TMEM16C	Craniocervical dystonia	(93)
TMEM16D	Control of mean arterial pressure	(39)
TMEM16E	Colorectal cancer	(92)
TMEM16E	Thyroid cancer	(101)
TMEM16F	Myoblast proliferation	(49)
TMEM16G	Prostate cancer	(51)
TMEM16G	Breast cancer	(96)
TMEM16H	Participate in the formation of the connection between ER and cell membrane	(52,53)
TMEM16J	Gastric cancer	(56)
TMEM16J	Pancreatic cancer	(57)
TMEM16K	Spindle formation	(60)
TMEM16K	Volume regulation	(61)