Tmem16A Encodes the Ca2+-activated Cl− Channel in Mouse Submandibular Salivary Gland Acinar Cells

Romanenko, Victor G.; Catalán, Marcelo A.; Brown, David A.; Putzier, Ilva; Hartzell, H. Criss; Marmorstein, Alan D.; González-Begné, Mireya; Rock, Jason R.; Harfe, Brian D.; Melvin, James E.

doi:10.1074/jbc.m109.068544

Cited by 182 publications

(219 citation statements)

References 54 publications

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“…3b,d) and in the non-ciliated cells of the respiratory epithelium ( Fig. 3b-d), consistent with a role in salt secretion, as in other epithelia 18,27,28 .…”

Section: Disruption Of Ano2 and Ano2 Expression Patternsupporting

confidence: 83%

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Ca2+-activated Cl− currents are dispensable for olfaction

et al. 2011

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Graduate student at the Freie Universität Berlin Olfaction in mammals involves the binding of odorants to a subset of the large number of different G protein-coupled receptors that are present in the cilia of olfactory sensory neurons (OSNs) 1 . These neurons are predominantly found in the MOE, which senses and discriminates myriad volatile compounds. In the mouse, the nose contains additional, apparently more specialized olfactory organs, such the septal organ of Masera and the VNO 2 . Sensory neurons in the VNO are morphologically distinct and use odorant receptors and signal transduction cascades that differ from those found in the MOE.In the canonical OSN signal transduction pathway, odorant binding to the receptor locally increases cytosolic cAMP through activation of the olfactory G protein G olf and adenylate cyclase type III 1,2 . cAMP then opens heteromeric cyclic nucleotide-gated (CNG) cation channels 3 . The resulting influx of Na + and Ca 2+ depolarizes the plasma membrane and raises the cytosolic Ca 2+ concentration ([Ca 2+ ] i ), thereby activating Ca 2+ -activated Cl -channels 1,4-10 . All these components of the signal transduction cascade are localized to sensory cilia, which are embedded in the mucus covering the MOE. These cilia provide a large interaction surface for odorants, and the cilia's small diameters facilitate large local increases in cytosolic cAMP and [Ca 2+ ].There has been broad consensus that Ca 2+ -activated Cl -channels powerfully amplify olfactory signal transduction 1,4-10 . These channels are thought to mediate an outward flow of Cl -, which generates a depolarizing current that, in rodents, is five-to tenfold larger than currents through CNG channels 8,11,12 . A prerequisite for Cl -efflux 3 is an inside-out electrochemical Cl --gradient. It is believed that cytosolic chloride concentration of OSNs is raised by the Na + K + 2Cl -co-transporter Nkcc1 9,10,13 and that [Cl -] is low in the mucus surrounding the cilia 14,15 . However, mucosal ion concentrations are difficult to measure in vivo, and Nkcc1 knockout mice display attenuated electro-olfactograms (EOGs) 11,16 , but normal olfactory sensitivity 17 .To investigate the role of Ca 2+ -activated Cl -channels in olfaction, we disrupted Ano2 in mice. Ano2 is a member of the Anoctamin (Tmem16) gene family, which encodes several Ca 2+ -activated Cl -channels [18][19][20] . Agreeing with recent results 13,21-23 , our knockout-controlled immunolabeling showed Ano2 expression in cilia of OSNs in the MOE, in microvilli of VNO sensory neurons and in synapses of photoreceptors. Additionally, we found Ano2 in the olfactory bulb. Ano1 expression overlapped with Ano2 in the VNO and the retina, but not in the MOE. Patch-clamp analysis showed that Ca 2+ -activated Cl -currents were undetectable in Ano2 -/-OSNs.Unexpectedly, however, EOGs of Ano2 -/-mice were reduced by only up to ~40%, and Ano2 -/-mice were able to smell normally. Our work calls for a revision of the current view that Ca 2+ -activated Cl -channels have a crucial role...

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“…3b,d) and in the non-ciliated cells of the respiratory epithelium ( Fig. 3b-d), consistent with a role in salt secretion, as in other epithelia 18,27,28 .…”

Section: Disruption Of Ano2 and Ano2 Expression Patternsupporting

confidence: 83%

“…With appropriate driving forces, apical Cl -channels may change mucosal ion composition, similar to the proposed role of Ano1 in secretory epithelia 18,27,28 .…”

Section: Disruption Of Ano2 Moderately Reduces Eogsmentioning

confidence: 79%

Ca2+-activated Cl− currents are dispensable for olfaction

et al. 2011

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“…Because HEK cells do not naturally express Ano1, it is possible that we have missed some interacting proteins that might be present in cell types that natively express Ano1. However, because the currents that we record in transfected HEK cells are virtually identical to those that we have recorded in native cells, such as salivary gland acinar cells (8), Xenopus oocytes (32), and inner medullary collecting duct (IMCD) cells (33), it seems unlikely that essential binding partners are missing. However, our investigation was limited to nominally "basal" intracellular Ca 2+ conditions.…”

Section: Discussionmentioning

confidence: 98%

“…Ano1 is widely expressed in epithelia including salivary gland, pancreas, gut, mammary gland, and airway. Disruption of the Ano1 gene in mice eliminates Ca 2+ -dependent Cl − secretion in several epithelial tissues including salivary gland (3,(5)(6)(7)(8)(9). Ano1 also performs important functions in nonepithelial tissues including pacemaker activity in the gut and regulation of vascular and airway smooth muscle tone.…”

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confidence: 99%

Anoctamin 1 (Tmem16A) Ca ² ⁺ -activated chloride channel stoichiometrically interacts with an ezrin–radixin–moesin network

Pérez‐Cornejo

Gokhale

Duran

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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The newly discovered Ca 2+ -activated Cl − channel (CaCC), Anoctamin 1 (Ano1 or TMEM16A), has been implicated in vital physiological functions including epithelial fluid secretion, gut motility, and smooth muscle tone. Overexpression of Ano1 in HEK cells or Xenopus oocytes is sufficient to generate Ca 2+ -activated Cl − currents, but the details of channel composition and the regulatory factors that control channel biology are incompletely understood. We used a highly sensitive quantitative SILAC proteomics approach to obtain insights into stoichiometric protein networks associated with the Ano1 channel. These studies provide a comprehensive footprint of putative Ano1 regulatory networks. We find that Ano1 associates with the signaling/scaffolding proteins ezrin, radixin, moesin, and RhoA, which link the plasma membrane to the cytoskeleton with very high stoichiometry. Ano1, ezrin, and moesin/radixin colocalize apically in salivary gland epithelial cells, and overexpression of moesin and Ano1 in HEK cells alters the subcellular localization of both proteins. Moreover, interfering RNA for moesin modifies Ano1 current without affecting its surface expression level. Another network associated with Ano1 includes the SNARE and SM proteins VAMP3, syntaxins 2 and -4, and syntaxin-binding proteins munc18b and munc18c, which are integral to translocation of vesicles to the plasma membrane. A number of other regulatory proteins, including GTPases, Ca 2+ -binding proteins, kinases, and lipid-interacting proteins are enriched in the Ano1 complex. These data provide stoichiometrically prioritized information about mechanisms regulating Ano1 function and trafficking to polarized domains of the plasma membrane.calcium | interactome | cross-linker | apical targeting

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“…When mucin secretion is increased, mucus can accumulate within airway lumens. Although still somewhat controversial (11), studies of TMEM16A-null mutant mice, as well as siRNA knockdown, suggest that TMEM16A mediates purinergic receptor-induced chloride transport across airway epithelia (9,12,43) and various exocrine cells (10,11,43,44). To determine if TMEM16A-CaCC mediates mucin secretion in asthma models, we tested the blockers we identified in our high-throughput screening for their effects on ATP-induced mucin depletion in IL-13-treated NHBE cells.…”

Section: Tmem16a Is Preferentially Expressed In Secretory Cells In Asmentioning

confidence: 99%

Calcium-activated chloride channel TMEM16A modulates mucin secretion and airway smooth muscle contraction

Huang

Zhang

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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Mucous cell hyperplasia and airway smooth muscle (ASM) hyperresponsiveness are hallmark features of inflammatory airway diseases, including asthma. Here, we show that the recently identified calciumactivated chloride channel (CaCC) TMEM16A is expressed in the adult airway surface epithelium and ASM. The epithelial expression is increased in asthmatics, particularly in secretory cells. Based on this and the proposed functions of CaCC, we hypothesized that TMEM16A inhibitors would negatively regulate both epithelial mucin secretion and ASM contraction. We used a high-throughput screen to identify small-molecule blockers of TMEM16A-CaCC channels. We show that inhibition of TMEM16A-CaCC significantly impairs mucus secretion in primary human airway surface epithelial cells. Furthermore, inhibition of TMEM16A-CaCC significantly reduces mouse and human ASM contraction in response to cholinergic agonists. TMEM16A-CaCC blockers, including those identified here, may positively impact multiple causes of asthma symptoms.A sthma is a significant cause of morbidity and mortality worldwide, and the prevalence of this disease is increasing among all age, sex, and racial groups. Characteristic features of asthma include inflammation, subepithelial fibrosis, hyperplasia of mucus-producing cells, accumulation of mucus within airway lumens, hyperplasia of airway smooth muscle (ASM), and ASM hyperresponsiveness. Together, these symptoms impair lung function by limiting the flow of gases to and from the alveoli in the distal lung.The current standard of care for asthma involves inhaled corticosteroids for the management of inflammation combined with long-acting agonists of β2-adrenergic receptors. Despite this treatment, lung function is not improved in 30-45% of asthmatic patients, and exacerbations continue to be a major problem (reviewed in ref. 1). Asthma can be divided into at least two distinct molecular phenotypes defined by the degree of Th2 inflammation (2, 3). Cytokines, including IL-4 and IL-13, promote airway epithelial mucous cell metaplasia, subepithelial fibrosis, and hyperplasia of smooth muscle in Th2-high asthmatics, and these patients generally show improved lung function with inhaled corticosteroid therapy. A greater understanding of this heterogeneity and the molecular and physiological events that lead to airway remodeling might lead to improved diagnosis and treatment.Calcium-activated chloride channels (CaCCs) have been ascribed numerous cellular functions (reviewed in refs. 4 and 5), among these are epithelial fluid secretion and smooth muscle contraction, both of which contribute to the progression and severity of asthma. Moreover, calcium-activated chloride currents in the airway epithelium are enhanced by the Th2 cytokines IL-4 and IL-13, as well as IFN-γ (6). For these reasons, CaCC is an attractive potential therapeutic target for asthma (7). However, the study of CaCC was impeded by lack of information about the gene(s) encoding this channel. It was only relatively recently that TMEM16A (transmembrane p...

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Tmem16A Encodes the Ca2+-activated Cl− Channel in Mouse Submandibular Salivary Gland Acinar Cells

Abstract: Activation of an apical

Cited by 182 publications

References 54 publications

Ca2+-activated Cl− currents are dispensable for olfaction

Ca2+-activated Cl− currents are dispensable for olfaction

Anoctamin 1 (Tmem16A) Ca ² ⁺ -activated chloride channel stoichiometrically interacts with an ezrin–radixin–moesin network

Calcium-activated chloride channel TMEM16A modulates mucin secretion and airway smooth muscle contraction

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Tmem16A Encodes the Ca2+-activated Cl− Channel in Mouse Submandibular Salivary Gland Acinar Cells

Abstract: Activation of an apical

Cited by 182 publications

References 54 publications

Ca2+-activated Cl− currents are dispensable for olfaction

Ca2+-activated Cl− currents are dispensable for olfaction

Anoctamin 1 (Tmem16A) Ca 2 + -activated chloride channel stoichiometrically interacts with an ezrin–radixin–moesin network

Calcium-activated chloride channel TMEM16A modulates mucin secretion and airway smooth muscle contraction

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Anoctamin 1 (Tmem16A) Ca ² ⁺ -activated chloride channel stoichiometrically interacts with an ezrin–radixin–moesin network