Calcium-activated Chloride Channel Anoctamin Research Articles

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.

Pacemaking in the lymphatic system.

Lymphatic collecting vessels exhibit spontaneous phasic contractions that are critical for lymph propulsion and tissue fluid homeostasis. This rhythmic activity is driven by action potentials conducted across the lymphatic muscle cell (LMC) layer to produce entrained contractions. The contraction frequency of a lymphatic collecting vessel displays exquisite mechanosensitivity, with a dynamic range from <1 to >20 contractions per minute. A myogenic pacemaker mechanism intrinsic to the LMCs was initially postulated to account for pressure-dependent chronotropy. Further interrogation into the cellular constituents of the lymphatic vessel wall identified non-muscle cell populations that shared some characteristics with interstitial cells of Cajal, which have pacemaker functions in the gastrointestinal and lower urinary tracts, thus raising the possibility of a non-muscle cell pacemaker. However, recent genetic knockout studies in mice support LMCs and a myogenic origin of the pacemaker activity. LMCs exhibit stochastic, but pressure-sensitive, sarcoplasmic reticulum calcium release (puffs and waves) from IP3R1 receptors, which couple to the calcium-activated chloride channel Anoctamin 1, causing depolarisation. The resulting electrical activity integrates across the highly coupled lymphatic muscle electrical syncytia through connexin 45 to modulate diastolic depolarisation. However, multiple other cation channels may also contribute to the ionic pacemaking cycle. Upon reaching threshold, a voltage-gated calcium channel-dependent action potential fires, resulting in a nearly synchronous calcium global calcium flash within the LMC layer to drive an entrained contraction. This review summarizes the key ion channels potentially responsible for the pressure-dependent chronotropy of lymphatic collecting vessels and various mechanisms of IP3R1 regulation that could contribute to frequency tuning.

Anoctamin 1 controls bone resorption by coupling Cl− channel activation with RANKL-RANK signaling transduction

Osteoclast over-activation leads to bone loss and chloride homeostasis is fundamental importance for osteoclast function. The calcium-activated chloride channel Anoctamin 1 (also known as TMEM16A) is an important chloride channel involved in many physiological processes. However, its role in osteoclast remains unresolved. Here, we identified the existence of Anoctamin 1 in osteoclast and show that its expression positively correlates with osteoclast activity. Osteoclast-specific Anoctamin 1 knockout mice exhibit increased bone mass and decreased bone resorption. Mechanistically, Anoctamin 1 deletion increases intracellular Cl− concentration, decreases H+ secretion and reduces bone resorption. Notably, Anoctamin 1 physically interacts with RANK and this interaction is dependent upon Anoctamin 1 channel activity, jointly promoting RANKL-induced downstream signaling pathways. Anoctamin 1 protein levels are substantially increased in osteoporosis patients and this closely correlates with osteoclast activity. Finally, Anoctamin 1 deletion significantly alleviates ovariectomy induced osteoporosis. These results collectively establish Anoctamin 1 as an essential regulator in osteoclast function and suggest a potential therapeutic target for osteoporosis.

Function and Regulation of the Calcium-Activated Chloride Channel Anoctamin 1 (TMEM16A).

Various human tissues express the calcium-activated chloride channel Anoctamin 1 (ANO1), also known as TMEM16A. ANO1 allows the passive chloride flux that controls different physiological functions ranging from muscle contraction, fluid and hormone secretion, gastrointestinal motility, and electrical excitability. Overexpression of ANO1 is associated with pathological conditions such as hypertension and cancer. The molecular cloning of ANO1 has led to a surge in structural, functional, and physiological studies of the channel in several tissues. ANO1 is a homodimer channel harboring two pores - one in each monomer - that work independently. Each pore is activated by voltage-dependent binding of two intracellular calcium ions to a high-affinity-binding site. In addition, the binding of phosphatidylinositol 4,5-bisphosphate to sites scattered throughout the cytosolic side of the protein aids the calcium activation process. Furthermore, many pharmacological studies have established ANO1 as a target of promising compounds that could treat several illnesses. This chapter describes our current understanding of the physiological roles of ANO1 and its regulation under physiological conditions as well as new pharmacological compounds with potential therapeutic applications.

Anoctamin 2-chloride channels reduce simple spike activity and mediate inhibition at elevated calcium concentration in cerebellar Purkinje cells.

Modulation of neuronal excitability is a prominent way of shaping the activity of neuronal networks. Recent studies highlight the role of calcium-activated chloride currents in this context, as they can both increase or decrease excitability. The calcium-activated chloride channel Anoctamin 2 (ANO2 alias TMEM16B) has been described in several regions of the mouse brain, including the olivo-cerebellar system. In inferior olivary neurons, ANO2 was proposed to increase excitability by facilitating the generation of high-threshold calcium spikes. An expression of ANO2 in cerebellar Purkinje cells was suggested, but its role in these neurons remains unclear. In the present study, we confirmed the expression of Ano2 mRNA in Purkinje cells and performed electrophysiological recordings to examine the influence of ANO2-chloride channels on the excitability of Purkinje cells by comparing wildtype mice to mice lacking ANO2. Recordings were performed in acute cerebellar slices of adult mice, which provided the possibility to study the role of ANO2 within the cerebellar cortex. Purkinje cells were uncoupled from climbing fiber input to assess specifically the effect of ANO2 channels on Purkinje cell activity. We identified an attenuating effect of ANO2-mediated chloride currents on the instantaneous simple spike activity both during strong current injections and during current injections close to the simple spike threshold. Moreover, we report a reduction of inhibitory currents from GABAergic interneurons upon depolarization, lasting for several seconds. Together with the role of ANO2-chloride channels in inferior olivary neurons, our data extend the evidence for a role of chloride-dependent modulation in the olivo-cerebellar system that might be important for proper cerebellum-dependent motor coordination and learning.

Identification of anoctamin 1 (ANO1) as a key driver of esophageal epithelial proliferation in eosinophilic esophagitis

The pathology of eosinophilic esophagitis (EoE) is characterized by eosinophil-rich inflammation, basal zone hyperplasia (BZH), and dilated intercellular spaces, and the underlying processes that drive the pathologic manifestations of the disease remain largely unexplored. We sought to investigate the involvement of the calcium-activated chloride channel anoctamin 1 (ANO1) in esophageal proliferation and the histopathologic features of EoE. We examined mRNA and protein expression of ANO1 in esophageal biopsy samples from patients with EoE and in mice with EoE. We performed molecular and cellular analyses and ion transport assays on an invitro esophageal epithelial 3-dimensional model system (EPC2-ALI) and murine models of EoE to define the relationship between expression and function of ANO1 and esophageal epithelial proliferation in patients with EoE. We observed increased ANO1 expression in esophageal biopsy samples from patients with EoE and in mice with EoE. ANO1 was expressed within the esophageal basal zone, and expression correlated positively with disease severity (eosinophils/high-power field) and BZH. Using an invitro esophageal epithelial 3-dimensional model system revealed that ANO1 undergoes chromatin modification and rapid upregulation of expression after IL-13 stimulation, that ANO1 is the primary apical IL-13-induced Cl- transport mechanism within the esophageal epithelium, and that loss of ANO1-dependent Cl- transport abrogated esophageal epithelial proliferation. Mechanistically, ANO1-dependent regulation of basal cell proliferation was associated with modulation of TP63 expression and phosphorylated cyclin-dependent kinase 2 levels. These data identify a functional role for ANO1 in esophageal cell proliferation and BZH in patients with EoE and provide a rationale for pharmacologic intervention of ANO1 function in patients with EoE.

Autoantibodies in Pandemrix®-induced narcolepsy: Nine candidate autoantigens fail the conformational autoantibody test

Study objectives: Narcolepsy type 1 (NT1) is a chronic sleep disorder characterized by loss of hypocretin-producing neurons. Increased NT1 incidence was observed in Sweden following mass-vaccination with Pandemrix®. Genetic association to HLA DQB1*06:02 implies an autoimmune origin, but target autoantigen remains unknown. Candidate autoantigens for NT1 have previously been identified in solid-phase immunoassays, while autoantibodies against conformation-dependent epitopes are better detected in radiobinding assays. The aims are to determine autoantibody levels against nine candidate autoantigens representing (1) proteins of the hypocretin transmitter system; Preprohypocretin (ppHypocretin), Hypocretin peptides 1 and 2 (HCRT1 and HCRT2) and Hypocretin receptor 2 (HCRTR2); (2) proteins previously associated with NT1; Tribbles homologue 2 (TRIB2), Pro-opiomelanocortin/alpha-melanocyte-stimulating-hormone (POMC/α-MSH) and Prostaglandin D2 Receptor DP1 (DP1); (3) proteins suggested as autoantigens for multiple sclerosis (another HLA DQB1*06:02-associated neurological disease); ATP-dependent Inwardly Rectifying Potassium Channel Kir4.1 (KIR4.1) and Calcium-activated chloride channel Anoctamin 2 (ANO2).Methods: Serum from post-Pandemrix® NT1 patients (n = 31) and their healthy first-degree relatives (n = 66) were tested for autoantibody levels in radiobinding assays separating autoantibody bound from free labelled antigen with Protein A-Sepharose. 125I-labelled HCRT1 and HCRT2 were commercially available while 35S-methionine-labelled ppHypocretin, HCRTR2, TRIB2, α-MSH/POMC, DP1, KIR4.1 or ANO2 was prepared by in vitro transcription translation of respective cDNA. In-house standards were used to express data in arbitrary Units/ml (U/ml).Results: All radiolabelled autoantigens were detected in a concentration-dependent manner by respective standard sera. Levels of autoantibodies in the NT1 patients did not differ from healthy first-degree relatives in any of the nine candidate autoantigens.Conclusions: None of the nine labelled proteins proposed to be autoantigens were detected in the radiobinding assays for conformation-dependent autoantibodies. The results emphasise the need of further studies to identify autoantigen(s) and clarify the mechanisms in Pandemrix®-induced NT1.

Peripheral serotonin receptor 2B and transient receptor potential channel 4 mediate pruritus to serotonergic antidepressants in mice

Peripheral serotonin receptor 2B and transient receptor potential channel 4 mediate pruritus to serotonergic antidepressants in mice

Live Cell Fluorescence Imaging Reveals Intercellular Calcium Waves and Chloride Channel Activation During Rotavirus Infection

BackgroundRotavirus (RV) causes severe diarrheal disease in children worldwide. During infection, RV increases cytosolic calcium in intestinal epithelial cells through release of endoplasmic reticulum calcium and activation of the store‐operated calcium entry (SOCE) pathway. Increased cytosolic calcium is critical for RV replication and activation of secretory pathways in GI epithelium, yet the molecular details of these processes remain poorly defined. Thus, characterizing RV‐induced calcium signaling at the molecular level is needed to understand RV pathogenesis. We conducted live cell fluorescence imaging in cell lines and human intestinal enteroids (HIEs) as a biologically relevant model of the human small intestine to measure calcium signaling dynamics and chloride channel activation during RV infection.Methods &amp; ResultsWe first engineered African green monkey kidney MA104 cells and HIEs to stably express GFP‐based genetically encoded calcium indicators to measure calcium dynamics throughout RV infection using live cell epifluorescence microscopy. We found that RV significantly increases the number and magnitude of calcium transients, which were attenuated with SOCE blockers. Furthermore, we observed that single RV‐infected cells triggered long‐distance intercellular calcium waves (ICWs) that encompassed surrounding uninfected cells in both MA104 cells and both duodenum and jejunum HIEs. Treatment with the ectoNTPase apyrase or purinergic receptor inhibitors blocked ICWs and reduced RV replication, mucin release, and serotonin secretion. These results indicate that extracellular ATP/ADP signaling elicits ICWs and is an important mediator of calcium dynamics during RV infection. Finally, we created HEK293 cell lines stably expressing halide‐sensitive YFP, a chloride channel biosensor, and either the calcium‐activated chloride channel anoctamin 1 (Ano1) or cystic fibrosis transmembrane conductance regulator (CFTR). We found that RV activated both mouse Ano1 and CFTR, which were attenuated with channel‐specific blockers, and suggests that multiple chloride channels can be activated during RV infection.ConclusionsOverall, our results support that RV causes dynamic calcium signaling during infection that is mediated by SOCE and purinergic signaling. Both pathways are potent stimulators of chloride secretion, thus they are potential therapeutic targets for developing life‐saving anti‐diarrheal drugs. Furthermore, we are the first to show that viruses can exploit purinergic signaling to generate ICWs that encompass uninfected cells and thereby potentially amplify the pathophysiological signaling important for diarrhea.Support or Funding InformationBCM Seed Funding, NIH F30 DK112563‐01This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

TRPV4 heats up ANO1-dependent exocrine gland fluid secretion.

Several ion channels and transporters regulate fluid secretion in salivary and lacrimal glands. In salivary glands, the major anion channel involved in fluid secretion is the calcium-activated chloride channel anoctamin 1 (ANO1). Several members of the transient receptor potential (TRP) channel superfamily regulate ANO1 activity. Here, we report a functional interaction between thermosensitive TRP vanilloid (TRPV)4 and ANO1 in acinar cells isolated from mouse salivary and lacrimal glands. TRPV4 activation induced chloride currents and shrinkage of acinar cells by increasing intracellular calcium concentrations. The chloride currents evoked by a TRPV4-specific activator (GSK1016790A) were identified as ANO1-mediated currents. Moreover, TRPV4 activation by an inositol 1,4,5-trisphosphate (IP3)-dependent mechanism was found to contribute to the muscarinic pathway of fluid secretion. Muscarinic stimulation of saliva and tear secretion was down-regulated in both TRPV4-deficient mice and in acinar cells treated with a TRPV4-specific antagonist (HC-067047). Furthermore, the temperature dependence of muscarinic salivation was shown to depend mainly on TRPV4. Our results suggest that TRPV4 interacts with IP3 receptors and ANO1 to regulate the muscarinic pathway that mediates salivation and lacrimation.-Derouiche, S., Takayama, Y., Murakami, M., Tominaga, M. TRPV4 heats up ANO1-dependent exocrine gland fluid secretion.

4-isopropylcyclohexanol has potential analgesic effects through the inhibition of anoctamin 1, TRPV1 and TRPA1 channel activities

Interactions between calcium-activated chloride channel anoctamin 1 (ANO1) and transient receptor potential vanilloid 1 (TRPV1) enhance pain sensations in mice, suggesting that ANO1 inhibition could have analgesic effects. Here we show that menthol and the menthol analogue isopropylcyclohexane (iPr-CyH) inhibited ANO1 channels in mice. The iPr-CyH derivative 4-isopropylcyclohexanol (4-iPr-CyH-OH) inhibited mouse ANO1 currents more potently than iPr-CyH. Moreover, 4-iPr-CyH-OH inhibited the activities of TRPV1, TRP ankyrin 1 (TRPA1), TRP melastatin 8 (TRPM8) and TRPV4. Single-channel analysis revealed that 4-iPr-CyH-OH reduced TRPV1 and TRPA1 current open-times without affecting unitary amplitude or closed-time, suggesting that it affected gating rather than blocking the channel pore. The ability of 4-iPr-CyH-OH to inhibit action potential generation and reduce pain-related behaviors induced by capsaicin in mice suggests that 4-iPr-CyH-OH could have analgesic applications. Thus, 4-iPr-CyH-OH is a promising base chemical to develop novel analgesics that target ANO1 and TRP channels.

Effects of new-generation inhibitors of the calcium-activated chloride channel anoctamin 1 on slow waves in the gastrointestinal tract.

High-throughput screening of compound libraries using genetically encoded fluorescent biosensors has identified several second-generation. low MW inhibitors of the calcium-activated chloride channel anoctamin 1 (CaCC/Ano1). Here we have (i) examined the effects of these Ano1 inhibitors on gastric and intestinal pacemaker activity; (ii) compared the effects of these inhibitors with those of the more classical CaCC inhibitor, 5-nitro-2-(3-phenylpropylalanine) benzoate (NPPB); (ii) examined the mode of action of these compounds on the waveform of pacemaker activity; and (iii) compared differences in the sensitivity between gastric and intestinal pacemaker activity to the Ano1 inhibitors. Using intracellular microelectrode recordings of gastric and intestinal muscle preparations from C57BL/6 mice, the dose-dependent effects of Ano1 inhibitors were examined on spontaneous electrical slow waves. The efficacy of second-generation Ano1 inhibitors on gastric and intestinal pacemaker activity differed significantly. Antral slow waves were more sensitive to these inhibitors than intestinal slow waves. CaCCinh -A01 and benzbromarone were the most potent at inhibiting slow waves in both muscle preparations and more potent than NPPB. Dichlorophene and hexachlorophene were equally potent at inhibiting slow waves. Surprisingly, slow waves were relatively insensitive to T16Ainh -A01 in both preparations. We have identified several second-generation Ano1 inhibitors, blocking gastric and intestinal pacemaker activity. Different sensitivities to Ano1 inhibitors between stomach and intestine suggest the possibility of different splice variants in these two organs or the involvement of other conductances in the generation and propagation of pacemaker activity in these tissues.

Glucose promotes secretion-dependent renal cyst growth.

Polycystic kidney diseases are characterized by the development of numerous bilateral renal cysts that continuously enlarge resulting in a decline of kidney function due to compression of intact nephrons. Cyst growth is driven by transepithelial chloride secretion which depends on both intracellular cAMP and calcium. Mechanisms that are involved in the regulation of the underlying secretory pathways remain incompletely understood. Here we show that glucose concentration has a strong impact on cyst growth of renal tubular cells within a collagen matrix as well as in embryonic kidneys deficient or competent for Pkd1. Glucose-dependent cyst growth correlates with the transcriptional induction of the calcium-activated chloride channel anoctamin 1 (ANO1) and its increased expression in the apical membrane of cyst-forming cells. Inhibition of ANO1 with the specific inhibitor CaCCinh-AO1 significantly decreases glucose-dependent cyst growth in both models. Ussing chamber analyses revealed increased apical chloride secretion of renal tubular cells upon exposure to high glucose medium which can also be inhibited by the use of CaCCinh-AO1. These data suggest that glycemic control may help to reduce renal cyst growth in patients with polycystic kidney disease. Renal cyst growth depends on glucose concentration in two in vitro cyst models. High glucose leads to upregulation of the calcium-activated chloride channel ANO1. High glucose promotes calcium-activated chloride secretion via ANO1. Glucose-dependent secretion can be inhibited by a specific inhibitor of ANO1.

Channel properties of the splicing isoforms of the olfactory calcium-activated chloride channel Anoctamin 2

Anoctamin (ANO)2 (or TMEM16B) forms a cell membrane Ca(2+)-activated Cl(-) channel that is present in cilia of olfactory receptor neurons, vomeronasal microvilli, and photoreceptor synaptic terminals. Alternative splicing of Ano2 transcripts generates multiple variants with the olfactory variants skipping exon 14 and having alternative splicing of exon 4. In the present study, 5' rapid amplification of cDNA ends analysis was conducted to characterize the 5' end of olfactory Ano2 transcripts, which showed that the most abundant Ano2 transcripts in the olfactory epithelium contain a novel starting exon that encodes a translation initiation site, whereas transcripts of the publically available sequence variant, which has an alternative and longer 5' end, were present in lower abundance. With two alternative starting exons and alternative splicing of exon 4, four olfactory ANO2 isoforms are thus possible. Patch-clamp experiments in transfected HEK293T cells expressing these isoforms showed that N-terminal sequences affect Ca(2+) sensitivity and that the exon 4-encoded sequence is required to form functional channels. Coexpression of the two predominant isoforms, one with and one without the exon 4 sequence, as well as coexpression of the two rarer isoforms showed alterations in channel properties, indicating that different isoforms interact with each other. Furthermore, channel properties observed from the coexpression of the predominant isoforms better recapitulated the native channel properties, suggesting that the native channel may be composed of two or more splicing isoforms acting as subunits that together shape the channel properties.

The calcium-activated chloride channel Anoctamin 1 contributes to the regulation of renal function

The role of calcium-activated chloride channels for renal function is unknown. By immunohistochemistry we demonstrate dominant expression of the recently identified calcium-activated chloride channels, Anoctamin 1 (Ano1, TMEM16A) in human and mouse proximal tubular epithelial (PTE) cells, with some expression in podocytes and other tubular segments. Ano1-null mice had proteinuria and numerous large reabsorption vesicles in PTE cells. Selective knockout of Ano1 in podocytes (Ano1-/-/Nphs2-Cre) did not impair renal function, whereas tubular knockout in Ano1-/-/Ksp-Cre mice increased urine protein excretion and decreased urine electrolyte concentrations. Purinergic stimulation activated calcium-dependent chloride currents in isolated proximal tubule epithelial cells from wild-type but not from Ano1-/-/Ksp-Cre mice. Ano1 currents were activated by acidic pH, suggesting parallel stimulation of Ano1 chloride secretion with activation of the proton-ATPase. Lack of calcium-dependent chloride secretion in cells from Ano1-/-/Ksp-Cre mice was paralleled by attenuated proton secretion and reduced endosomal acidification, which compromised proximal tubular albumin uptake. Tubular knockout of Ano1 enhanced serum renin and aldosterone concentrations, probably leading to enhanced compensatory distal tubular reabsorption, thus maintaining normal blood pressure levels. Thus, Ano1 has a role in proximal tubular proton secretion and protein reabsorption. The results correspond to regulation of the proton-ATPase by the Ano1-homolog Ist2 in yeast.

Presynaptic Localization and Possible Function of Calcium-Activated Chloride Channel Anoctamin 1 in the Mammalian Retina

Calcium (Ca2+)-activated chloride (Cl−) channels (CaCCs) play a role in the modulation of action potentials and synaptic responses in the somatodendritic regions of central neurons. In the vertebrate retina, large Ca2+-activated Cl− currents (ICl(Ca)) regulate synaptic transmission at photoreceptor terminals; however, the molecular identity of CaCCs that mediate ICl(Ca) remains unclear. The transmembrane protein, TMEM16A, also called anoctamin 1 (ANO1), has been recently validated as a CaCC and is widely expressed in various secretory epithelia and nervous tissues. Despite the fact that tmem16a was first cloned in the retina, there is little information on its cellular localization and function in the mammalian retina. In this study, we found that ANO1 was abundantly expressed as puncta in 2 synaptic layers. More specifically, ANO1 immunoreactivity was observed in the presynaptic terminals of various retinal neurons, including photoreceptors. ICl(Ca) was first detected in dissociated rod bipolar cells expressing ANO1. ICl(Ca) was abolished by treatment with the Ca2+ channel blocker Co2+, the L-type Ca2+ channel blocker nifedipine, and the Cl− channel blockers 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) and niflumic acid (NFA). More specifically, a recently discovered ANO1-selective inhibitor, T16Ainh-A01, and a neutralizing antibody against ANO1 inhibited ICl(Ca) in rod bipolar cells. Under a current-clamping mode, the suppression of ICl(Ca) by using NPPB and T16Ainh-A01 caused a prolonged Ca2+ spike-like depolarization evoked by current injection in dissociated rod bipolar cells. These results suggest that ANO1 confers ICl(Ca) in retinal neurons and acts as an intrinsic regulator of the presynaptic membrane potential during synaptic transmission.

Channel properties of the splicing isoforms of the olfactory calcium-activated chloride channel Anoctamin 2

Anoctamin (ANO)2 (or TMEM16B) forms a cell membrane Ca2+-activated Cl− channel that is present in cilia of olfactory receptor neurons, vomeronasal microvilli, and photoreceptor synaptic terminals. Alternative splicing of Ano2 transcripts generates multiple variants with the olfactory variants skipping exon 14 and having alternative splicing of exon 4. In the present study, 5′ rapid amplification of cDNA ends analysis was conducted to characterize the 5′ end of olfactory Ano2 transcripts, which showed that the most abundant Ano2 transcripts in the olfactory epithelium contain a novel starting exon that encodes a translation initiation site, whereas transcripts of the publically available sequence variant, which has an alternative and longer 5′ end, were present in lower abundance. With two alternative starting exons and alternative splicing of exon 4, four olfactory ANO2 isoforms are thus possible. Patch-clamp experiments in transfected HEK293T cells expressing these isoforms showed that N-terminal sequences affect Ca2+ sensitivity and that the exon 4–encoded sequence is required to form functional channels. Coexpression of the two predominant isoforms, one with and one without the exon 4 sequence, as well as coexpression of the two rarer isoforms showed alterations in channel properties, indicating that different isoforms interact with each other. Furthermore, channel properties observed from the coexpression of the predominant isoforms better recapitulated the native channel properties, suggesting that the native channel may be composed of two or more splicing isoforms acting as subunits that together shape the channel properties.

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

The calcium-activated chloride channel anoctamin 1 (ANO1) is located within the 11q13 amplicon, one of the most frequently amplified chromosomal regions in human cancer, but its functional role in tumorigenesis has remained unclear. The 11q13 region is amplified in ∼15% of breast cancers. Whether ANO1 is amplified in breast tumors, the extent to which gene amplification contributes to ANO1 overexpression, and whether overexpression of ANO1 is important for tumor maintenance have remained unknown. We have found that ANO1 is amplified and highly expressed in breast cancer cell lines and primary tumors. Amplification of ANO1 correlated with disease grade and poor prognosis. Knockdown of ANO1 in ANO1-amplified breast cancer cell lines and other cancers bearing 11q13 amplification inhibited proliferation, induced apoptosis, and reduced tumor growth in established cancer xenografts. Moreover, ANO1 chloride channel activity was important for cell viability. Mechanistically, ANO1 knockdown or pharmacological inhibition of its chloride-channel activity reduced EGF receptor (EGFR) and calmodulin-dependent protein kinase II (CAMKII) signaling, which subsequently attenuated AKT, v-src sarcoma viral oncogene homolog (SRC), and extracellular signal-regulated kinase (ERK) activation in vitro and in vivo. Our results highlight the involvement of the ANO1 chloride channel in tumor progression and provide insights into oncogenic signaling in human cancers with 11q13 amplification, thereby establishing ANO1 as a promising target for therapy in these highly prevalent tumor types.

A comprehensive strategy to identify stoichiometric membrane protein interactomes

There are numerous experimental approaches to identify the interaction networks of soluble proteins, but strategies for the identification of membrane protein interactomes remain limited. We discuss in detail the logic of an experimental design that led us to identify the interactome of a membrane protein of complex membrane topology, the calcium activated chloride channel Anoctamin 1/Tmem16a (Ano1). We used covalent chemical stabilizers of protein-protein interactions combined with magnetic bead immuno-affinity chromatography, quantitative SILAC mass-spectrometry and in silico network construction. This strategy led us to define a putative Ano1 interactome from which we selected key components for functional testing. We propose a combination of procedures to narrow down candidate proteins interacting with a membrane protein of interest for further functional studies.

The calcium-activated chloride channel anoctamin 1 acts as a heat sensor in nociceptive neurons

Nociceptors are a subset of small primary afferent neurons that respond to noxious chemical, thermal and mechanical stimuli. Ion channels in nociceptors respond differently to noxious stimuli and generate electrical signals in different ways. Anoctamin 1 (ANO1 also known as TMEM16A) is a Ca(2+)-activated chloride channel that is essential for numerous physiological functions. We found that ANO1 was activated by temperatures over 44 °C with steep heat sensitivity. ANO1 was expressed in small sensory neurons and was highly colocalized with nociceptor markers, which suggests that it may be involved in nociception. Application of heat ramps to dorsal root ganglion (DRG) neurons elicited robust ANO1-dependent depolarization. Furthermore, knockdown or deletion of ANO1 in DRG neurons substantially reduced nociceptive behavior in thermal pain models. These results indicate that ANO1 is a heat sensor that detects nociceptive thermal stimuli in sensory neurons and possibly mediates nociception.