Dysregulated Ca2+ signaling, fluid secretion, and mitochondrial function in a mouse model of early Sjögren's disease.

The molecular mechanisms leading to saliva secretion are largely established, but factors that underlie secretory hypofunction, specifically related to the autoimmune disease Sjögren's syndrome (SS) are not fully understood. A major conundrum is the lack of association between the severity of salivary gland immune cell infiltration and glandular hypofunction. SS-like disease was induced by treatment with DMXAA, a small molecule agonist of murine STING. We have previously shown that the extent of salivary secretion is correlated with the magnitude of intracellular Ca2+ signals (Takano et al., 2021). Contrary to our expectations, despite a significant reduction in fluid secretion, neural stimulation resulted in enhanced Ca2+ signals with altered spatiotemporal characteristics in vivo. Muscarinic stimulation resulted in reduced activation of the Ca2+-activated Cl- channel, TMEM16a, although there were no changes in channel abundance or absolute sensitivity to Ca2+. Super-resolution microscopy revealed a disruption in the colocalization of Inositol 1,4,5-trisphosphate receptor Ca2+ release channels with TMEM16a, and channel activation was reduced when intracellular Ca2+ buffering was increased. These data indicate altered local peripheral coupling between the channels. Appropriate Ca2+ signaling is also pivotal for mitochondrial morphology and bioenergetics. Disrupted mitochondrial morphology and reduced oxygen consumption rate were observed in DMXAA-treated animals. In summary, early in SS disease, dysregulated Ca2+ signals lead to decreased fluid secretion and disrupted mitochondrial function contributing to salivary gland hypofunction.

Salinity and prolactin regulate anoctamin 1 in the model teleost, Fundulus heteroclitus.

To maintain internal ion balance in marine environments, teleost fishes leverage seawater (SW)-type ionocytes to actively secrete Na+ and Cl- into the environment. It is well established that SW-type ionocytes use apically expressed cystic fibrosis transmembrane conductance regulator 1 (Cftr1) as a conduit for Cl- to exit the gill. Here, we investigated whether the Ca2+-activated Cl- channel, anoctamin 1 (Ano1), provides an additional path for Cl--secretion in euryhaline mummichogs (Fundulus heteroclitus). Two ano1 gene isoforms, denoted ano1.1a and -1.1b, exhibited higher expression in the gill and opercular epithelium of mummichogs long-term acclimated to SW versus fresh water (FW). Branchial ano1.1b and cftr1 expression was increased in mummichogs sampled 24 h after transfer from FW to SW; ano1.1a and -1.1b were upregulated in the gill and opercular epithelium following transfer from SW to hypersaline SW. Alternatively, the expression of ano1.1a, -1.1b, and cftr1 in the gill and opercular epithelium was markedly decreased after transfer from SW to FW. Given its role in attenuating ion secretion, we probed whether prolactin downregulates ano1 isoforms. In addition to attenuating cftr1 expression, a prolactin injection reduced branchial ano1.1a and -1.1b levels. Given how Ano1 mediates Cl- secretion by mammalian epithelial cells, the salinity- and prolactin-sensitive nature of ano1 expression reported here indicates that Ano1 may constitute a novel Cl--secretion pathway in ionocytes. This study encourages a wider evaluation of this putative Cl--secretion pathway and its regulation by hormones in teleost fishes.NEW & NOTEWORTHY In this study, we provide evidence in a teleost fish that the Ca2+-activated Cl- channel, anoctamin 1 may provide an additional path for Cl- secretion by seawater-type ionocytes. Not only is this the first report of a Cftr-independent Cl--secreting pathway conferring survival in seawater but also the first description of its regulation by the pituitary hormone prolactin.

The effects of Cl- channel inhibitors and pyrethroid insecticides on calcium-activated chloride channels in neurons of Helicoverpa armigera.

TMEM16A, a member of the Transmembrane protein 16 family, serves as the molecular basis for calcium activated chloride channels (CaCCs). We use RT-PCR to demonstrate the expression of TMEM16A in the neurons of Helicoverpa armigera, and record the CaCCs current of acute isolated neurons of H. armigera for the first time using patch clamp technology. In order to screen effective inhibitors of calcium-activated chloride channels, the inhibitory effects of four chloride channel inhibitors, CaCCinh-A01, NPPB, DIDS, and SITS, on CaCCs were compared. The inhibitory effects of the four inhibitors on the outward current of CaCCs were CaCCinh-A01 (10 µM, 56.31 %), NPPB (200 µM, 43.69 %), SITS (1 mM, 12.41 %) and DIDS (1 mM, 13.29 %). Among these inhibitors, CaCCinh-A01 demonstrated the highest efficacy as a blocker. To further explore whether calcium channel proteins can serve as potential targets of pyrethroids, we compared the effects of (type I) tefluthrin and (type II) deltamethrin on CaCCs. 10 µM and 100 µM tefluthrin can stimulate a large tail current in CaCCs, prolonging their deactivation time by 10.44 ms and 31.49 ms, and the V0.5 shifted in the hyperpolarization by 2-8 mV. Then, deltamethrin had no obvious effect on the deactivation and activation of CaCCs. Therefore, CaCCs of H. armigera can be used as a potential target of pyrethroids, but type I and type II pyrethroids have different effects on CaCCs.

Effect of Synthetic Vitreous Fiber Exposure on TMEM16A Channels in a Xenopus laevis Oocyte Model.

Many years ago, asbestos fibers were banned and replaced by synthetic vitreous fibers because of their carcinogenicity. However, the toxicity of the latter fibers is still under debate, especially when it concerns the early fiber interactions with biological cell membranes. Here, we aimed to investigate the effects of a synthetic vitreous fiber named FAV173 on the Xenopus laevis oocyte membrane, the cell model we have already used to characterize the effect of crocidolite asbestos fiber exposure. Using an electrophysiological approach, we found that, similarly to crocidolite asbestos, FAV173 was able to stimulate a chloride outward current evoked by step membrane depolarizations, that was blocked by the potent and specific TMEM16A channel antagonist Ani9. Exposure to FAV173 fibers also altered the oocyte cell membrane microvilli morphology similarly to crocidolite fibers, most likely as a consequence of the TMEM16A protein interaction with actin. However, FAV173 only partially mimicked the crocidolite fibers effects, even at higher fiber suspension concentrations. As expected, the crocidolite fibers' effect was more similar to that induced by the co-treatment with (Fe3+ + H2O2), since the iron content of asbestos fibers is known to trigger reactive oxygen species (ROS) production. Taken together, our findings suggest that FAV173 may be less harmful that crocidolite but not ineffective in altering cell membrane properties.

TMEM16A regulates satellite cell-mediated skeletal muscle regeneration by ensuring a moderate level of caspase 3 activity.

It has been documented that caspase 3 activity is necessary for skeletal muscle regeneration, but how its activity is regulated is largely unknown. Our previous report shows that intracellular TMEM16A, a calcium activated chloride channel, significantly regulates caspase 3 activity in myoblasts during skeletal muscle development. By using a mouse line with satellite cell (SC)-specific deletion of TMEM16A, we examined the role of TMEM16A in regulating caspase 3 activity in SC (or SC-derived myoblast) as well as skeletal muscle regeneration. The mutant animals displayed apparently impaired regeneration capacity in adult muscle along with enhanced ER stress and elevated caspase 3 activity in Tmem16a-/- SC derived myoblasts. Blockade of either excessive ER stress or caspase 3 activity by small molecules significantly restored the inhibited myogenic differentiation of Tmem16a-/- SCs, indicating that excessive caspase 3 activity resulted from TMEM16A deletion contributes to the impaired muscle regeneration and the upstream regulator of caspase 3 was ER stress. Our results revealed an essential role of TMEM16A in satellite cell-mediated skeletal muscle regeneration by ensuring a moderate level of caspase 3 activity.

Interstitial cells of the sip syncytium regulate basal membrane potential in murine gastric corpus.

Smooth muscle cells (SMCs), Interstitial cells of Cajal (ICC) and Platelet-derived growth factor receptor α positive (PDGFRα+) cells form an integrated, electrical syncytium within the gastrointestinal (GI) muscular tissues known as the SIP syncytium. Immunohistochemical analysis of gastric corpus muscles showed that c-KIT+/ANO1+ ICC-IM and PDGFRα+ cells were closely apposed to one another in the same anatomical niches. We used intracellular microelectrode recording from corpus muscle bundles to characterize the roles of intramuscular ICC and PDGFRα+ cells in conditioning membrane potentials of gastric muscles. In muscle bundles, that have a relatively higher input impedance than larger muscle strips or sheets, we recorded an ongoing discharge of stochastic fluctuations in membrane potential, previously called unitary potentials or spontaneous transient depolarizations (STDs) and spontaneous transient hyperpolarizations (STHs). We reasoned that STDs should be blocked by antagonists of ANO1, the signature conductance of ICC. Activation of ANO1 has been shown to generate spontaneous transient inward currents (STICs), which are the basis for STDs. Ani9 reduced membrane noise and caused hyperpolarization, but this agent did not block the fluctuations in membrane potential quantitatively. Apamin, an antagonist of small conductance Ca2+-activated K+ channels (SK3), the signature conductance in PDGFRα+ cells, further reduced membrane noise and caused depolarization. Reversing the order of channel antagonists reversed the sequence of depolarization and hyperpolarization. These experiments show that the ongoing discharge of STDs and STHs by ICC and PDGFRα+ cells, respectively, exerts conditioning effects on membrane potentials in the SIP syncytium that would effectively regulate the excitability of SMCs.

Prostaglandin E2 depolarises sensory axons in vitro in an ANO1 and Nav1.8 dependent manner.

Prostaglandin E2 (PGE2) is a major contributor to inflammatory pain hyperalgesia, however, the extent to which it modulates the activity of nociceptive axons is incompletely understood. We developed and characterized a microfluidic cell culture model to investigate sensitisation of the axons of dorsal root ganglia neurons. We show that application of PGE2 to fluidically isolated axons leads to sensitisation of their responses to depolarising stimuli. Interestingly the application of PGE2 to the DRG axons elicited a direct and persistent spiking activity propagated to the soma. Both the persistent activity and the membrane depolarisation in the axons are abolished by the EP4 receptor inhibitor and a blocker of cAMP synthesis. Further investigated into the mechanisms of the spiking activity showed that the PGE2 evoked depolarisation was inhibited by Nav1.8 sodium channel blockers but was refractory to the application of TTX or zatebradine. Interestingly, the depolarisation of axons was blocked by blocking ANO1 channels with T16Ainh-A01. We further show that PGE2-elicited axonal responses are altered by the changes in chloride gradient within the axons following treatment with bumetanide a Na-K-2Cl cotransporter NKCC1 inhibitor, but not by VU01240551 an inhibitor of potassium-chloride transporter KCC2. Our data demonstrate a novel role for PGE2/EP4/cAMP pathway which culminates in a sustained depolarisation of sensory axons mediated by a chloride current through ANO1 channels. Therefore, using a microfluidic culture model, we provide evidence for a potential dual function of PGE2 in inflammatory pain: it sensitises depolarisation-evoked responses in nociceptive axons and directly triggers action potentials by activating ANO1 and Nav1.8 channels.

Anoctamin 1, a multi-modal player in pain and itch.

Anoctamin 1 (ANO1/TMEM16A) encodes a Ca2+-activated Cl- channel. Among ANO1's many physiological functions, it plays a significant role in mediating nociception and itch. ANO1 is activated by intracellular Ca2+ and depolarization. Additionally, ANO1 is activated by heat above 44 °C, suggesting heat as another activation stimulus. ANO1 is highly expressed in nociceptors, indicating a role in nociception. Conditional Ano1 ablation in dorsal root ganglion (DRG) neurons results in a reduction in acute thermal pain, as well as thermal and mechanical allodynia or hyperalgesia evoked by inflammation or nerve injury. Pharmacological interventions also lead to a reduction in nocifensive behaviors. ANO1 is functionally linked to the bradykinin receptor and TRPV1. Bradykinin stimulates ANO1 via IP3-mediated Ca2+ release from intracellular stores, whereas TRPV1 stimulates ANO1 via a combination of Ca2+ influx and release. Nerve injury causes upregulation of ANO1 expression in DRG neurons, which is blocked by ANO1 antagonists. Due to its role in nociception, strong and specific ANO1 antagonists have been developed. ANO1 is also expressed in pruritoceptors, mediating Mas-related G protein-coupled receptors (Mrgprs)-dependent itch. The activation of ANO1 leads to chloride efflux and depolarization due to high intracellular chloride concentrations, causing pain and itch. Thus, ANO1 could be a potential target for the development of new drugs treating pain and itch.

Exploring the potential of cryptochlorogenic acid as a dietary adjuvant for multi-target combined lung cancer treatment.

BACKGROUND: Lung cancer is a highly malignant disease with limited treatment options and significant adverse effects. It is urgent to develop novel treatment strategies for lung cancer. In recent years, TMEM16A has been confirmed as a specific drug target for lung cancer. The development of TMEM16A-targeting drugs and combined administration for the treatment of lung cancer has become a research hotspot. METHODS: Fluorescence screening and electrophysiological experiments were conducted to confirm the inhibitory effect of CCA on TMEM16A. Molecular dynamics simulation and site-directed mutagenesis were employed to analyze the binding mode of CCA and TMEM16A. CCK-8, colony formation, wound healing, transwell, and annexin-V experiments were conducted to explore the regulatory effects and mechanisms of CCA on the proliferation, migration, and apoptosis of lung cancer cells. Tumor model mice and pharmacokinetic experiments were used to examine the efficacy and safety of CCA and cisplatin in vivo. RESULTS: This study firstly confirmed that CCA effectively inhibits TMEM16A to exert anticancer effects and analyzed the pharmacological mechanism. CCA bound to S517/N546/E623/E633/Q637 of TMEM16A through hydrogen bonding and electrostatic interactions. It inhibited the proliferation and migration, and induced apoptosis of lung cancer cells by targeting TMEM16A. In addition, the combined administration of CCA and cisplatin exhibited a synergistic effect, enhancing the efficacy of lung cancer treatment while reducing side effects. CONCLUSION: CCA is an effective novel inhibitor of TMEM16A, and it synergizes with cisplatin in anticancer treatment. These findings will provide new research ideas and lead compound for the combination therapy of lung cancer.

Structural and theoretical basis for drug development targeting TMEM16A: Inhibition mechanism of tracheloside analogs.

Ion channels play a crucial role in the electrophysiological activities of organisms. The calcium-activated chloride channel TMEM16A is involved in various physiological processes. Therefore, inhibitors of TMEM16A are used to treat diseases caused by TMEM16A dysfunction. However, the unclear inhibition mechanism hinders the progress of drug development. Based on our previous study, we found that the molecular structures of TMEM16A inhibitors tracheloside, matairesinoside and arctigenin are similar. In this study, we conducted a structure-based virtual screening of tracheloside analogs from the PubChem database. The six tracheloside analogs with the highest affinity to TMEM16A were selected, and their inhibitory effects were detected by fluorescence and electrophysiological experiments. Subsequently, the interaction between the tracheloside analogs and TMEM16A was investigated through molecular docking and site-directed mutagenesis. Based on the above results, the mechanism of inhibition of TMEM16A gated conformation by tracheloside analogs was proposed. These findings provide a structural and theoretical basis for drug development targeting TMEM16A.

Involvement of TRPV4 in temperature-dependent perspiration in mice.

Reports indicate that an interaction between TRPV4 and anoctamin 1 (ANO1) could be widely involved in water efflux of exocrine glands, suggesting that the interaction could play a role in perspiration. In secretory cells of sweat glands present in mouse foot pads, TRPV4 clearly colocalized with cytokeratin 8, ANO1, and aquaporin-5 (AQP5). Mouse sweat glands showed TRPV4-dependent cytosolic Ca2+ increases that were inhibited by menthol. Acetylcholine-stimulated sweating in foot pads was temperature-dependent in wild-type, but not in TRPV4-deficient mice and was inhibited by menthol both in wild-type and TRPM8KO mice. The basal sweating without acetylcholine stimulation was inhibited by an ANO1 inhibitor. Sweating could be important for maintaining friction forces in mouse foot pads, and this possibility is supported by the finding that wild-type mice climbed up a slippery slope more easily than TRPV4-deficient mice. Furthermore, TRPV4 expression was significantly higher in controls and normohidrotic skin from patients with acquired idiopathic generalized anhidrosis (AIGA) compared to anhidrotic skin from patients with AIGA. Collectively, TRPV4 is likely involved in temperature-dependent perspiration via interactions with ANO1, and TRPV4 itself or the TRPV4/ANO 1 complex would be targeted to develop agents that regulate perspiration.

Stress, spicy foods and elevated temperatures can all trigger specialized gland cells to move water to the skin ­ in other words, they can make us sweat. This process is one of the most important ways by which our bodies regulate their temperature and avoid life-threatening conditions such as heatstroke. Disorders in which this function is impaired, such as AIGA (acquired idiopathic generalized anhidrosis), pose significant health risks. Finding treatments for sweat-related diseases requires a detailed understanding of the molecular mechanisms behind sweating, which has yet to be achieved. Recent research has highlighted the role of two ion channels, TRPV4 and ANO1, in regulating fluid secretion in glands that produce tears and saliva. These gate-like proteins control how certain ions move in or out of cells, which also influences water movement. Once activated by external stimuli, TRPV4 allows calcium ions to enter the cell, causing ANO1 to open and chloride ions to leave. This results in water also exiting the cell through dedicated channels, before being collected in ducts connected to the outside of the body. TRPV4, which is activated by heat, is also present in human sweat gland cells. This prompted Kashio et al. to examine the role of these channels in sweat production, focusing on mice as well as AIGA patients. Probing TRPV4, ANO1 and AQP5 (a type of water channel) levels using fluorescent antibodies confirmed that these channels are all found in the same sweat gland cells in the foot pads of mice. Further experiments highlighted that TRPV4 mediates sweat production in these animals via ANO1 activation. As rodents do not regulate their body temperature by sweating, Kashio et al. explored the biological benefits of having sweaty paws. Mice lacking TRPV4 had reduced sweating and were less able to climb a slippery slope, suggesting that a layer of sweat helps improve traction. Finally, Kashio et al. compared samples obtained from healthy volunteers with those from AIGA patients and found that TRPV4 levels are lower in individuals affected by the disease. Overall, these findings reveal new insights into the underlying mechanisms of sweating, with TRPV4 a potential therapeutic target for conditions like AIGA. The results also suggest that sweating could be controlled by local changes in temperature detected by heat-sensing channels such as TRPV4. This would depart from our current understanding that sweating is solely controlled by the autonomic nervous system, which regulates involuntary bodily functions such as saliva and tear production.

[Expressions of zinc homeostasis proteins, GPR39 and ANO1 mRNA in the sperm of asthenozoospermia patients and their clinical significance].

OBJECTIVE: To explore the expressions of zinc homeostasis-related proteins, G protein-coupled receptor 39 (GPR39) and ANO1 mRNA in the sperm of patients with asthenozoospermia (AS), and analyze their correlation with sperm motility. METHODS: We collected semen samples from 82 male subjects with PR+NP < 40%, PR < 32% and sperm concentration > 15×106/ml (the AS group, n = 40) or PR+NP ≥ 40%, PR ≥ 32% and sperm concentration > 15×106/ml (the normal control group, n = 42). We analyzed the routine semen parameters and measured the zinc content in the seminal plasma using the computer-assisted sperm analysis system, detected the expressions of zinc transporters (ZIP13, ZIP8 and ZNT10), metallothioneins (MT1G, MT1 and MTF), GPR39, and calcium-dependent chloride channel protein (ANO1) in the sperm by real-time quantitative PCR (RT qPCR), examined free zinc distribution in the sperm by laser confocal microscopy, and determined the expressions of GPR39 and MT1 proteins in the sperm by immunofluorescence staining, followed by Spearman rank correlation analysis of their correlation with semen parameters. RESULTS: There was no statistically significant difference in the zinc concentration in the seminal plasma between the AS and normal control groups (P>0.05). Compared with the controls, the AS patients showed a significantly reduced free zinc level (P<0.05), relative expressions of MT1G, MTF, ZIP13, GPR39 and ANO1 mRNA (P<0.05), and that of the GPR39 protein in the AS group (P<0.05). No statistically significant differences were observed in the relative expression levels of ZIP8, ZNT10 and MT1 mRNA between the two groups (P>0.05). The relative expression levels of GPR39, ANO1, MT1G and MTF mRNA were positively correlated with sperm motility and the percentage of progressively motile sperm (P<0.05). CONCLUSION: The expressions of zinc homeostasis proteins (MT1G, MTF and ZIP13), GPR39 and ANO1 mRNA are downregulated in the sperm of asthenozoospermia patients, and positively correlated with sperm motility.

Anticancer Effect of Hemin through ANO1 Inhibition in Human Prostate Cancer Cells.

Anoctamin1 (ANO1), a calcium-activated chloride channel, is overexpressed in a variety of cancer cells, including prostate cancer, and is involved in cancer cell proliferation, migration, and invasion. Inhibition of ANO1 in these cancer cells exhibits anticancer effects. In this study, we conducted a screening to identify novel ANO1 inhibitors with anticancer effects using PC-3 human prostate carcinoma cells. Screening of 2978 approved and investigational drugs revealed that hemin is a novel ANO1 inhibitor with an IC50 value of 0.45 µM. Notably, hemin had no significant effect on intracellular calcium signaling and cystic fibrosis transmembrane conductance regulator (CFTR), a cyclic AMP (cAMP)-regulated chloride channel, and it showed a weak inhibitory effect on ANO2 at 3 µM, a concentration that completely inhibits ANO1. Interestingly, hemin also significantly decreased ANO1 protein levels and strongly inhibited the cell proliferation and migration of PC-3 cells in an ANO1-dependent manner. Furthermore, it strongly induced caspase-3 activation, PARP degradation, and apoptosis in PC-3 cells. These findings suggest that hemin possesses anticancer properties via ANO1 inhibition and could be considered for development as a novel treatment for prostate cancer.

Modulation of TMEM16B channel activity by the calcium-activated chloride channel regulator 4 (CLCA4) in human cells.

The Ca2+-activated Cl- channel regulator CLCA1 potentiates the activity of the Ca2+-activated Cl- channel (CaCC) TMEM16A by directly engaging the channel at the cell surface, inhibiting its reinternalization and increasing Ca2+-dependent Cl- current (ICaCC) density. We now present evidence of functional pairing between two other CLCA and TMEM16 protein family members, namely CLCA4 and the CaCC TMEM16B. Similar to CLCA1, (i) CLCA4 is a self-cleaving metalloprotease, and the N-terminal portion (N-CLCA4) is secreted; (ii) the von Willebrand factor type A (VWA) domain in N-CLCA4 is sufficient to potentiate ICaCC in HEK293T cells; and (iii) this is mediated by the metal ion-dependent adhesion site motif within VWA. The results indicate that, despite the conserved regulatory mechanism and homology between CLCA1 and CLCA4, CLCA4-dependent ICaCC are carried by TMEM16B, rather than TMEM16A. Our findings show specificity in CLCA/TMEM16 interactions and suggest broad physiological and pathophysiological links between these two protein families.

Interaction between TRP channels and anoctamins.

Anoctamin 1 (ANO1) binds to transient receptor potential (TRP) channels (protein-protein interaction) and then is activated by TRP channels (functional interaction). TRP channels are non-selective cation channels that are expressed throughout the body and play roles in multiple physiological functions. Studies on TRP channels increased after the identification of TRP vanilloid 1 (TRPV1) in 1997. Calcium-activated chloride channel anoctamin 1 (ANO1, also called TMEM16A and DOG1) was identified in 2008. ANO1 plays a major role in TRP channel-mediated functions, as first shown in 2014 with the demonstration of a protein-protein interaction between TRPV4 and ANO1. In cells that co-express TRP channels and ANO1, calcium entering cells through activated TRP channels causes ANO1 activation. Therefore, in many tissues, the physiological functions related to TRP channels are modulated through chloride flux associated with ANO1 activation. In this review, we summarize the latest understanding of TRP-ANO1 interactions, particularly interaction of ANO1 with TRPV4, TRP canonical 6 (TRPC6), TRPV3, TRPV1, and TRPC2 in the salivary glands, blood vessels, skin keratinocytes, primary sensory neurons, and vomeronasal organs, respectively.

Loss of anoctamin 1 reveals a subtle role for BK channels in lymphatic muscle action potentials.

Ca2+ signalling plays a crucial role in determining lymphatic muscle cell excitability and contractility through its interaction with the Ca2+-activated Cl- channel anoctamin 1 (ANO1). In contrast, the large-conductance (BK) Ca2+-activated K+ channel (KCa) and other KCa channels have prominent vasodilatory actions by hyperpolarizing vascular smooth muscle cells. Here, we assessed the expression and contribution of the KCa family to mouse and rat lymphatic collecting vessel contractile function. The BK channel was the only KCa channel consistently expressed in fluorescence-activated cell sorting-purified mouse lymphatic muscle cell lymphatic muscle cells. We used a pharmacological inhibitor of BK channels, iberiotoxin, and small-conductance Ca2+-activated K+ channels, apamin, to inhibit KCa channels acutely in ex vivo isobaric myography experiments and intracellular membrane potential recordings. In basal conditions, BK channel inhibition had little to no effect on either mouse inguinal-axillary lymphatic vessel (MIALV) or rat mesenteric lymphatic vessel contractions or action potentials (APs). We also tested BK channel inhibition under loss of ANO1 either by genetic ablation (Myh11CreERT2-Ano1 fl/fl, Ano1ismKO) or by pharmacological inhibition with Ani9. In both Ano1ismKO MIALVs and Ani9-pretreated MIALVs, inhibition of BK channels increased contraction amplitude, increased peak AP and broadened the peak of the AP spike. In rat mesenteric lymphatic vessels, BK channel inhibition also abolished the characteristic post-spike notch, which was exaggerated with ANO1 inhibition, and significantly increased the peak potential and broadened the AP spike. We conclude that BK channels are present and functional on mouse and rat lymphatic muscle cells but are otherwise masked by the dominance of ANO1. KEY POINTS: Mouse and rat lymphatic muscle cells express functional BK channels. BK channels make little contribution to either rat or mouse lymphatic collecting vessel contractile function in basal conditions across a physiological pressure range. ANO1 limits the peak membrane potential achieved in the action potential and sets a plateau potential limiting the voltage-dependent activation of BK. BK channels are activated when ANO1 is absent or blocked and slightly impair contractile strength by reducing the peak membrane potential achieved in the action potential spike and accelerating the post-spike repolarization.

Insights into the function and regulation of the calcium-activated chloride channel TMEM16A.

The TMEM16A channel, a member of the TMEM16 protein family comprising chloride (Cl-) channels and lipid scramblases, is activated by the free intracellular Ca2+ increments produced by inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ release after GqPCRs or Ca2+ entry through cationic channels. It is a ubiquitous transmembrane protein that participates in multiple physiological functions essential to mammals' lives. TMEM16A structure contains two identical 10-segment monomers joined at their transmembrane segment 10. Each monomer harbours one independent hourglass-shaped pore gated by Ca2+ ligation to an orthosteric site adjacent to the pore and controlled by two gates. The orthosteric site is created by assembling negatively charged glutamate side chains near the pore´s cytosolic end. When empty, this site generates an electrostatic barrier that controls channel rectification. In addition, an isoleucine-triad forms a hydrophobic gate at the boundary of the cytosolic vestibule and the inner side of the neck. When the cytosolic Ca2+ rises, one or two Ca2+ ions bind to the orthosteric site in a voltage (V)-dependent manner, thus neutralising the electrostatic barrier and triggering an allosteric gating mechanism propagating via transmembrane segment 6 to the hydrophobic gate. These coordinated events lead to pore opening, allowing the Cl- flux to ensure the physiological response. The Ca2+-dependent function of TMEM16A is highly regulated. Anions with higher permeability than Cl- facilitate V dependence by increasing the Ca2+ sensitivity, intracellular protons can replace Ca2+ and induce channel opening, and phosphatidylinositol 4,5-bisphosphate bound to four cytosolic sites likely maintains Ca2+ sensitivity. Additional regulation is afforded by cytosolic proteins, most likely by phosphorylation and protein-protein interaction mechanisms.

Anoctamin pharmacology.

TMEM16 proteins, also known as anoctamins, are a family of ten membrane proteins with various tissue expression and subcellular localization. TMEM16A (anoctamin 1) is a plasma membrane protein that acts as a calcium-activated chloride channel. It is expressed in many types of epithelial cells, smooth muscle cells and some neurons. In airway epithelial cells, TMEM16A expression is particularly enhanced by inflammatory stimuli that also promote goblet cell metaplasia and mucus hypersecretion. Therefore, pharmacological modulation of TMEM16A could be beneficial to improve mucociliary clearance in chronic obstructive respiratory diseases. However, the correct approach to modulate TMEM16A activity (activation or inhibition) is still debated. Pharmacological inhibitors of TMEM16A could also be useful as anti-hypertensive agents given the TMEM16A role in smooth muscle contraction. In contrast to TMEM16A, TMEM16F (anoctamin 6) behaves as a calcium-activated phospholipid scramblase, responsible for the externalization of phosphatidylserine on cell surface. Inhibitors of TMEM16F could be useful as anti-coagulants and anti-viral agents. The role of other anoctamins as therapeutic targets is still unclear since their physiological role is still to be defined.

Promoter hypermethylation as a novel regulator of ANO1 expression and function in prostate cancer bone metastasis.

Despite growing evidence implicating the calcium-activated chloride channel anoctamin1 (ANO1) in cancer metastasis, its direct impact on the metastatic potential of prostate cancer and the possible significance of epigenetic alteration in this process are not fully understood. Here, we show that ANO1 is minimally expressed in LNCap and DU145 prostate cancer cell lines with low metastatic potential but overexpressed in high metastatic PC3 prostate cancer cell line. The treatment of LNCap and DU145 cells with DNMT inhibitor 5-aza-2'-deoxycytidine (5-Aza-CdR) potentiates ANO1 expression, suggesting that DNA methylation is one of the mechanisms controlling ANO1 expression. Consistent with this notion, hypermethylation was detected at the CpG island of ANO1 promoter region in LNCap and DU145 cells, and 5-Aza-CdR treatment resulted in a drastic demethylation at promoter CpG methylation sites. Upon 5-Aza-CdR treatment, metastatic indexes, such as cell motility, invasion, and metastasis-related gene expression, were significantly altered in LNCap and DU145 cells. These 5-Aza-CdR-induced metastatic hallmarks were, however, almost completely ablated by stable knockdown of ANO1. These in vitro discoveries were further supported by our in vivo observation that ANO1 expression in xenograft mouse models enhances the metastatic dissemination of prostate cancer cells into tibial bone and the development of osteolytic lesions. Collectively, our results help elucidate the critical role of ANO1 expression in prostate cancer bone metastases, which is epigenetically modulated by promoter CpG methylation.

Niclosamide potentiates TMEM16A and induces vasoconstriction.

The TMEM16A calcium-activated chloride channel is a promising therapeutic target for various diseases. Niclosamide, an anthelmintic medication, has been considered a TMEM16A inhibitor for treating asthma and chronic obstructive pulmonary disease (COPD) but was recently found to possess broad-spectrum off-target effects. Here, we show that, under physiological Ca2+ (200-500 nM) and voltages, niclosamide acutely potentiates TMEM16A. Our computational and functional characterizations pinpoint a putative niclosamide binding site on the extracellular side of TMEM16A. Mutations in this site attenuate the potentiation. Moreover, niclosamide potentiates endogenous TMEM16A in vascular smooth muscle cells, triggers intracellular calcium increase, and constricts the murine mesenteric artery. Our findings advise caution when considering clinical applications of niclosamide as a TMEM16A inhibitor. The identification of the putative niclosamide binding site provides insights into the mechanism of TMEM16A pharmacological modulation and provides insights into developing specific TMEM16A modulators to treat human diseases.