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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.

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.

Figure 1 Flowchart of the studies revised in the narrative review. PDB, Protein Data Bank. Created with BioRender.com.

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).

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; SE, E313 (on TM3) and R432 (on TM6); SC, E352 and K353 (both at the intracellular end of TM4). Created with BioRender.com.

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.

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).

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

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.

[i] 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.

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).

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.

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 CACCinh-A01, Fact, Eact, MONNA, TMinh-23, T16inh-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).

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. CaCCinh-A01, 6-t-butyl-2-(furan-2-carboxamido)-4,5,6,7-tetrahydrobenzo(b) thiophene-3-carboxylic acid; T16Ainh-A01, 2-((5-Ethyl-1,6-dihydro-4-methyl-6-oxo-2-pyrimidinyl)thio)-N-(4-(4-methoxyphenyl)-2-thiazolyl)-acetamide; CFTRact-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; TMinh-23, 2-bromodifluoroacetylamino-5,6,7,8-tetrahydro-4H-cyclohepta(b) thiophene-3-carboxylic acid o-tolylamide; Eact, 3,4,5-trimethoxy-N-(2-methoxyethyl)-N-(4-phenyl-2-thiazolyl)benzamide; Fact, N-(4bromophenyl)-3-(1H-tetrazol-1-yl) benzamide. Created with BioRender.com.

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.

Table II Cancer or physiological processes mediated by various TMEM16 proteins.

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)
	Control the excitability of neurons and glial cells	(31)
TMEM16C	Craniocervical dystonia	(93)
TMEM16D	Control of mean arterial pressure	(39)
TMEM16E	Colorectal cancer	(92)
	Thyroid cancer	(101)
TMEM16F	Myoblast proliferation	(49)
TMEM16G	Prostate cancer	(51)
	Breast cancer	(96)
TMEM16H	Participate in the formation of the connection between ER and cell membrane	(52,53)
TMEM16J	Gastric cancer	(56)
	Pancreatic cancer	(57)
TMEM16K	Spindle formation	(60)
	Volume regulation	(61)

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).

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.

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).

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).

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.

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.

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).

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.

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.

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

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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:

Acknowledgements

Not applicable.

Funding

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).

References