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

Bestrophin (BEST1–4 in humans) channels are ligand gated chloride (Cl−) channels that are activated by calcium (Ca2+). Mutations in BEST1 cause retinal degenerative diseases. Partly because these channels have no sequence or structural similarity to other ion channels, the molecular mechanisms underlying gating are unknown. Here, we present a series of cryo-electron microscopy (cryo-EM) structures of chicken BEST1, determined at 3.1 Å resolution or better, that represent the principal gating states of the channel. Unlike other channels, opening of the pore is due to the repositioning of tethered pore-lining helices within a surrounding protein shell that dramatically widens a “neck” of the pore through a concertina of amino acid rearrangements within the protein core. The neck serves as both the activation and the inactivation gate. The binding of Ca2+ to a cytosolic domain instigates pore opening and the structures reveal that, unlike voltage-gated Na+ and K+ channels, similar molecular rearrangements are responsible for inactivation and deactivation. A single aperture within the 95 Å-long opened pore separates the cytosol from the extracellular milieu and controls anion permeability. The studies define the basis for Ca2+-activated Cl− channel function and reveal a new molecular paradigm for gating in ligand-gated ion channels.

The calcium-activated chloride channel anoctamin-2 (Ano2) is thought to amplify transduction currents in olfactory receptor neurons (ORNs), a hypothesis supported by previous studies in dissociated neurons from Ano2−/− mice. Paradoxically, despite a reduction in transduction currents in Ano2−/− ORNs, their spike output for odor stimuli may be higher. We examined the role of Ano2 in ORNs in their native environment in freely breathing mice by imaging activity in ORN axons as they arrive in the olfactory bulb glomeruli. Odor-evoked responses in ORN axons of Ano2−/− animals were consistently larger for a variety of odorants and concentrations. In an open arena, Ano2−/− animals took longer to approach a localized odor source than Ano2+/+ animals, revealing clear olfactory behavioral deficits. Our studies provide the first in vivo evidence toward an alternative or additional role for Ano2 in the olfactory transduction cascade, where it may serve as a feedback mechanism to clamp ORN spike output.

Mutations in the human TMEM16E (ANO5) gene are associated both with the bone disease gnathodiaphyseal dysplasia (GDD; OMIM: 166260) and muscle dystrophies (OMIM: 611307, 613319). However, the physiological function of TMEM16E has remained unclear. We show here that human TMEM16E, when overexpressed in mammalian cell lines, displayed partial plasma membrane localization and gave rise to phospholipid scrambling (PLS) as well as non-selective ionic currents with slow time-dependent activation at highly depolarized membrane potentials. While the activity of wild-type TMEM16E depended on elevated cytosolic Ca2+ levels, a mutant form carrying the GDD-causing T513I substitution showed PLS and large time-dependent ion currents even at low cytosolic Ca2+ concentrations. Contrarily, mutation of the homologous position in the Ca2+-activated Cl− channel TMEM16B paralog hardly affected its function. In summary, these data provide the first direct demonstration of Ca2+-dependent PLS activity for TMEM16E and suggest a gain-of-function phenotype related to a GDD mutation. Electronic supplementary material The online version of this article (doi:10.1007/s00018-017-2704-9) contains supplementary material, which is available to authorized users.

Many animals follow odor trails to find food, nesting sites, or mates, and they require only faint olfactory cues to do so. The performance of a tracking dog, for instance, poses the question on how the animal is able to distinguish a target odor from the complex chemical background around the trail. Current concepts of odor perception suggest that animals memorize each odor as an olfactory object, a percept that enables fast recognition of the odor and the interpretation of its valence. An open question still is how this learning process operates efficiently at the low odor concentrations that typically prevail when animals inspect an odor trail. To understand olfactory processing under these conditions, we studied the role of an amplification mechanism that boosts signal transduction at low stimulus intensities, a process mediated by calcium‐gated anoctamin 2 chloride channels. Genetically altered Ano2 −/− mice, which lack these channels, display an impaired cue‐tracking behavior at low odor concentrations when challenged with an unfamiliar, but not with a familiar, odor. Moreover, recordings from the olfactory epithelium revealed that odor coding lacks sensitivity and temporal resolution in anoctamin 2‐deficient mice. Our results demonstrate that the detection of an unfamiliar, weak odor, as well as its memorization as an olfactory object, require signal amplification in olfactory receptor neurons. This process may contribute to the phenomenal tracking abilities of animals that follow odor trails.

The review describes molecular and functional properties of the volume regulated anion channel and Ca(2+)-dependent Cl(-) channels belonging to the anoctamin family with emphasis on physiological importance of these channels in regulation of cell volume, cell migration, cell proliferation, and programmed cell death. Finally, we discuss the role of Cl(-) channels in various diseases.

Transmembrane member 16A (TMEM16A) is involved in many physiological functions, such as epithelial secretion, sensory conduction, nociception, control of neuronal excitability, and regulation of smooth muscle contraction, and may be important in peripheral pain transmission. To explore the role of TMEM16A in the persistent hyperalgesia that results from chronic constriction injury-induced neuropathic pain, a rat model of the condition was established by ligating the left sciatic nerve. A TMEM16A selective antagonist (10 μg T16Ainh-A01) was intrathecally injected at L5-6. For measurement of thermal hyperalgesia, the drug was administered once at 14 days and thermal withdrawal latency was recorded with an analgesia meter. For measurement of other indexes, the drug was administered at 12 days, once every 6 hours, totally five times. The measurements were performed at 14 days. Western blot assay was conducted to analyze TMEM16A expression in the L4-6 dorsal root ganglion. Immunofluorescence staining was used to detect the immunoreactivity of TMEM16A in the L4-6 dorsal root ganglion on the injured side. Patch clamp was used to detect electrophysiological changes in the neurons in the L4-6 dorsal root ganglion. Our results demonstrated that thermal withdrawal latency was shortened in the model rats compared with control rats. Additionally, TMEM16A expression and the number of TMEM16A positive cells in the L4-6 dorsal root ganglion were higher in the model rats, which induced excitation of the neurons in the L4-6 dorsal root ganglion. These findings were inhibited by T16Ainh-A01 and confirm that TMEM16A plays a key role in persistent chronic constriction injury-induced hyperalgesia. Thus, inhibiting TMEM16A might be a novel pharmacological intervention for neuropathic pain. All experimental protocols were approved by the Animal Ethics Committee at the First Affiliated Hospital of Shihezi University School of Medicine, China (approval No. A2017-170-01) on February 27, 2017.

This study compared the contribution of the Ca2+-activated Cl− conductance to the electroolfactogram (EOG) evoked by different odorant classes between the marine Cabinza grunt Isacia conceptionis and rainbow trout Oncorhynchus mykiss. The Ca2+-activated Cl− channel blocker niflumic acid significantly diminished odorant responses in O. mykiss, but had no effect on the EOG in I. conceptionis, supporting the notion that Ca2+-activated Cl− channels may not operate as odorant transduction current amplifiers in this marine teleost.

The gating of Ca²⁺-activated Cl⁻ channels is controlled by a complex interplay among [Ca²⁺](i), membrane potential and permeant anions. Besides Ca²⁺, Ba²⁺ also can activate both TMEM16A and TMEM16B. This study reports the effects of several divalent cations as regulators of TMEM16A channels stably expressed in HEK293T cells. Among the divalent cations that activate TMEM16A, Ca²⁺ is most effective, followed by Sr²⁺ and Ni²⁺, which have similar affinity, while Mg²⁺ is ineffective. Zn²⁺ does not activate TMEM16A but inhibits the Ca²⁺-activated chloride currents. Maximally effective concentrations of Sr²⁺ and Ni²⁺ occluded activation of the TMEM16A current by Ca²⁺, which suggests that Ca²⁺, Sr²⁺ and Ni²⁺ all regulate the channel by the same mechanism.

Secretory diarrhea remains a global health burden and causes major mortality in children. There have been some focuses on antidiarrheal therapies that may reduce fluid losses and intestinal motility in diarrheal diseases. In the present study, we identified shikonin as an inhibitor of TMEM16A chloride channel activity using cell-based fluorescent-quenching assay. The IC50 value of shikonin was 6.5 μM. Short-circuit current measurements demonstrated that shikonin inhibited Eact-induced Cl(-) current in a dose-dependent manner, with IC50 value of 1.5 μM. Short-circuit current measurement showed that shikonin exhibited inhibitory effect against CCh-induced Cl(-) currents in mouse colonic epithelia but did not affect cytoplasmic Ca(2+) concentration as well as the other major enterocyte chloride channel conductance regulator. Characterization study found that shikonin inhibited basolateral K(+) channel activity without affecting Na(+)/K(+)-ATPase activities. In vivo studies revealed that shikonin significantly delayed intestinal motility in mice and reduced stool water content in a neonatal mice model of rotaviral diarrhea without affecting the viral infection process in vivo. Taken together, the results suggested that shikonin inhibited enterocyte calcium-activated chloride channels, the inhibitory effect was partially through inhbition of basolateral K(+) channel activity, and shikonin could be a lead compound in the treatment of rotaviral secretory diarrhea.

Calcium-activated chloride channels (CaCCs) are one of five families of chloride channels, ubiquitously expressed, and essential for a host of biological actions. CaCCs have key roles in processes as diverse as olfactory transduction and epithelial secretion, and also CaCCs are essential in smooth muscle contraction. The corpus cavernosum is a vascular smooth muscle that must relax to facilitate erections. Parasympathetic activation produces relaxation of the corpus cavernosum through a nitric oxide-dependent pathway, and sympathetic stimulation in both preventing and terminating erections by contracting the corpus cavernosum. Both these pathways affect activity of CaCCs. The past 5 years produced many successes in CaCC research. One key area of success was the identification of the elusive 'molecular candidate' of CaCCs, as the TMEM16A protein (dubbed anoctamin-1) and potentially other members of the anoctamin family of transmembrane proteins. However, enthusiasm has been somewhat tempered because of evidence that this family of proteins may not be responsible for calcium-activated chloride currents in certain epithelial tissues. Several studies identified specific inhibitors of CaCCs as well as specific inhibitors for anoctamin-1. Despite the number of recent achievements in this field there are many details that still need to be elucidated. Of particular value would be more details on the identity of the CaCCs in corpus cavernosum smooth muscle, using new inhibitors to gain insight into the signalling pathway, and the evaluation of whether inhibition of CaCCs provides any specific benefit in different models of ED.

The nature of encoded information in neural circuits is determined by neuronal firing patterns and frequencies. This paper discusses the molecular identity and cellular mechanisms of spike-frequency adaptation in the central nervous system (CNS). Spike-frequency adaptation in thalamocortical (TC) and CA1 hippocampal neurons is mediated by the Ca2＋-activated Cl- channel (CACC) anoctamin-2 (ANO2). Knockdown of ANO2 in these neurons results in increased number of spikes, in conjunction with significantly reduced spike-frequency adaptation. No study has so far demonstrated that CACCs mediate afterhyperpolarization currents, which result in the modulation of neuronal spike patterns in the CNS. Our study therefore proposes a novel role for ANO2 in spike-frequency adaptation and transmission of information in the brain. [BMB Reports 2017; 50(3): 109-110].

Calcium-activated chloride channels (CaCCs) are widely expressed in various tissues and implicated in physiological processes such as sensory transduction, epithelial secretion, and smooth muscle contraction. Transmembrane proteins with unknown function 16 (TMEM16A) has recently been identified as a major component of CaCCs. Detailed molecular analysis of TMEM16A will be needed to understand its structure-function relationships. The role this channel plays in physiological systems remains to be established and is currently a subject of intense investigation.

Currents mediated by calcium-activated chloride channels (CaCCs), observed for the first time in Xenopus oocytes, have been recorded in many cells and tissues ranging from different types of neurons to epithelial and muscle cells. CaCCs play a role in the regulation of excitability in neurons including sensory receptors. In addition, they are crucial mediators of chloride movements in epithelial cells where their activity regulates electrolyte and fluid transport. The roles of CaCCs, particularly in epithelia, are briefly reviewed with emphasis on their function in secretory epithelia. The recent identification by three independent groups, using different strategies, of TMEM16A as the molecular counterpart of the CaCC is discussed. TMEM16A is part of a family that has 10 other members in mice. The discovery of the potential TMEM16 anion channel activity opens the way for the molecular investigation of the role of these anion channels in specific cells and in organ physiology and pathophysiology. The identification of TMEM16A protein as a CaCC chloride channel molecule represents a great triumph of scientific perseverance and ingenuity. The varied approaches used by the three independent research groups also augur well for the solidity of the discovery.

This review examines the function of calcium-activated chloride currents (I(Cl(Ca))) in the retina with an emphasis on their physiological role in photoreceptors. Although found in a variety of neurons and glial cells of the retina, I(Cl(Ca)) has been most prominently studied in cones, where it activates in response to depolarization-evoked Ca(2+) influx. The slow and complex gating kinetics of the chloride current have been considered to reflect the changing submembrane concentration of intracellular calcium. It is likely that the role of I(Cl(Ca)) is to stabilize the membrane potential of cones during synaptic activity and presynaptic Ca channel modulation. Several candidates in the molecular identification of the channel have been put forward but the issue remains unresolved.

AIM: In the renal collecting ducts, ATP stimulates a Ca(2+) -activated chloride current. The identity of the channel responsible for the current under physiological conditions is not known and it was hypothesized that TMEM16a is a relevant candidate in the renal collecting duct.METHODS: The cortical collecting duct cell line M-1 was used as a model of the collecting duct. The ATP induced Ca(2+) signalling was imaged in cells loaded with Ca(2+) -sensitive fluorescent probes using confocal laser-scanning fluorescence microscopy. Chloride current was determined by mounting M-1 cell layers in Ussing chamber. The expression of TMEM16a in human kidney was tested by immunohistochemistry.RESULTS: M-1 cells displayed a transient increase in intracellular Ca(2+) concentration in response to 100 nm ATP. This response was completely blocked by addition of 100 μm suramin, indicating that ATP signals through purinergic P2 receptors. Apical addition of 100 nm ATP induced a Cl(-) current, which was blocked by suramin, DPC and the cysteine-modifying compound MTSET. M-1 cells were found to express TMEM16a at the mRNA and protein level. Functionally, it was found that knock-down of TMEM16a expression in M-1 cells inhibited the ATP induced Cl(-) -current. In human and mouse kidney sections, TMEM16a protein expression was localized to the collecting duct, and TMEM16a was found to be excreted in human urinary exosomes.CONCLUSION: TMEM16a is a Ca(2+) -activated Cl(-) channel expressed in the collecting ducts.

The functional properties and physiological roles of the calcium-activated chloride channels (CaCCs) encoded by family members of transmembrane proteins of unknown function 16 (TMEM16) have been rigorously studied. Nevertheless, a poorly understood characteristic of CaCCs, namely the channel rundown, significantly interferes electrophysiological measurements of these channels. In our studies, the rundown problem of CaCCs was circumvented by normalizing the Ca2+-induced current to the maximally-activated current within a short period of time in which the rundown was insignificant. Activation of the TMEM16A-encoded CaCC, also known as ANO1, by divalent cations Ca2+, Sr2+, Ba2+, and Mg2+ were thus characterized. Ca2+, Sr2+, and Ba2+ were found to activate the ANO1 channel with different affinities, but these three divalent cations activate current to the same maximal level. On the other hand, Mg2+ was unable to induce current in ANO1, yet, appeared to compete with Ca2+ to shift the Ca2+ concentration-dependent activation curve. We also studied the permeability of the ANO1 pore for various anions, and discovered that the anion occupancy in the pore was inversely correlated with the apparent affinity of the ANO1 inhibition by niflumic acid (NFA), a known CaCC blocker. In contrast, activating the channel by different divalent cations or the different degree of channel activation did not affect NFA inhibition, suggesting that NFA inhibits the channel by altering the pore function rather than by changing the channel gating. In conclusion, our study characterized functional properties of ANO1 without interference from channel rundown, and revealed properties of the channel that have not yet been documented.

Background and Purpose The molecular identity of calcium-activated chloride channels (CaCCs) in vascular endothelial cells remains unknown. This study sought to identify whether anoctamin-1 (Ano1, also known as TMEM16A) functions as a CaCC and whether hypoxia alters the biophysical properties of Ano1 in mouse cardiac vascular endothelial cells (CVECs). Experimental Approach Western blot, quantitative real-time PCR, confocal imaging analysis and patch-clamp analysis combined with pharmacological approaches were used to determine whether Ano1 was expressed and functioned as CaCC in CVECs. Key Results Ano1 was expressed in CVECs. The biophysical properties of the current generated in the CVECs, including the Ca2+ and voltage dependence, outward rectification, anion selectivity and the pharmacological profile, are similar to those described for CaCCs. The density of ICl(Ca) detected in CVECs was significantly inhibited by T16Ainh-A01, an Ano1 inhibitor, and a pore-targeting, specific anti-Ano1 antibody, and was markedly decreased in Ano1 gene knockdown CVECs. The density of ICl(Ca) was significantly potentiated in CVECs exposed to hypoxia, and this hypoxia-induced increase in the density of ICl(Ca) was inhibited by T16Ainh-A01 or anti-Ano1 antibody. Hypoxia also increased the current density of ICl(Ca) in Ano1 gene knockdown CVECs. Conclusions and Implications Ano1 formed CaCC in CVECs of neonatal mice. Hypoxia enhances Ano1-mediated ICl(Ca) density via increasing its expression, altering the ratio of its splicing variants, sensitivity to membrane voltage and to Ca2+. Ano1 may play a role in the pathophysiological processes during ischaemia in heart, and therefore, Ano1 might be a potential therapeutic target to prevent ischaemic damage.

TMEM16A and TMEM16B are calcium-activated chloride channels (CaCCs) with important functions in mammalian physiology. Whether distant relatives of the vertebrate TMEM16 families also form CaCCs is an intriguing open question. Here we report that a TMEM16 family member from Drosophila melanogaster, Subdued (CG16718), is a CaCC. Amino acid substitutions of Subdued alter the ion selectivity and kinetic properties of the CaCC channels heterologously expressed in HEK 293T cells. This Drosophila channel displays characteristics of classic CaCCs, thereby providing evidence for evolutionarily conserved biophysical properties in the TMEM16 family. Additionally, we show that knockout flies lacking subdued gene activity more readily succumb to death caused by ingesting the pathogenic bacteria Serratia marcescens, suggesting that subdued has novel functions in Drosophila host defense. DOI: http://dx.doi.org/10.7554/eLife.00862.001, eLife digest Ions are at the root of most processes that occur in the body, so they must be able to move in and out of cells. However, because they have an electric charge, ions are not usually able to pass through the fatty membrane that encloses all cells. Instead, they must be imported or exported by a variety of dedicated proteins in the cell membrane. These include ion channels – proteins that, under certain conditions, open to form pores – and ion transporters. Calcium-activated chloride channels are ion channels that permeate chloride ions when the concentration of calcium ions inside the cell increases. Two important calcium-activated chloride channels in mammals belong to the TMEM16 family of proteins, which is conserved in many organisms. However, to date all the examples of TMEM16 proteins forming calcium-activated chloride channels have been found in vertebrates. Moreover, it is not known how many members of the TMEM16 family can act as ion channels. Wong et al. have now isolated a protein belonging to the TMEM16 family from fruit flies and, in a series of experiments on human cells, showed that it acts as a calcium-activated chloride channel. Previous work has shown that fruit flies lacking this protein, which is called Subdued, are more susceptible than wild-type flies to a pathogenic bacterium called Serratia marrescens, which implies that the Subdued ion channel might be involved in the immune system. Indeed, Wong et al. found that the mutant flies died more often than wild-type flies after eating these bacteria; the mutant files also had higher levels of the bacteria in their digestive tracts. These results will be of interest to researchers trying to understand how TMEM16 ion channels evolved to be involved in processes as diverse as vision, the secretion of bodily fluids and the immune system. DOI: http://dx.doi.org/10.7554/eLife.00862.002

Background and purposeT16A(inh) -A01 is a recently identified inhibitor of the calcium-activated chloride channel TMEM16A. The aim of this study was to test the efficacy of T16A(inh) -A01 for inhibition of calcium-activated chloride channels in vascular smooth muscle and consequent effects on vascular tone.Experimental approachSingle channel and whole cell patch clamp was performed on single smooth muscle cells from rabbit pulmonary artery and mouse thoracic aorta. Isometric tension studies were performed on mouse thoracic aorta and mesenteric artery as well as human abdominal visceral adipose artery.Key resultsIn rabbit pulmonary artery myocytes T16A(inh) -A01 (1-30 μM) inhibited single calcium (Ca(2+) )-activated chloride (Cl(-) ) channels and whole cell currents activated by 500 nM free Ca(2+) . Similar effects were observed for single Ca(2+) -activated Cl(-) channels in mouse thoracic aorta, and in both cell types, channel activity was abolished by two antisera raised against TMEM16A but not by a bestrophin antibody. The TMEM16A potentiator, F(act) (10 μM), increased single channel and whole cell Ca(2+) -activated Cl(-) currents in rabbit pulmonary arteries. In isometric tension studies, T16A(inh) -A01 relaxed mouse thoracic aorta pre-contracted with methoxamine with an IC(50) of 1.6 μM and suppressed the methoxamine concentration-effect curve. T16A(inh) -A01 did not affect the maximal contraction produced by 60 mM KCl and the relaxant effect of 10 μM T16A(inh) -A01 was not altered by incubation of mouse thoracic aorta in a cocktail of potassium (K(+) ) channel blockers. T16A(inh) -A01 (10 μM) also relaxed human visceral adipose arteries by 88 ± 3%.Conclusions and implicationsT16A(inh) -A01 blocks calcium-activated chloride channels in vascular smooth muscle cells and relaxes murine and human blood vessels.