Tmem16b is Specifically Expressed in the Cilia of Olfactory Sensory Neurons

Rasche, Sebastian; Toetter, Bastian; Adler, Jenny; Tschapek, Astrid; Doerner, Julia F.; Kurtenbach, Stefan; Hatt, Hanns; Meyer, Helmut E.; Warscheid, Bettina; Neuhaus, Eva M.

doi:10.1093/chemse/bjq007

Cited by 94 publications

(103 citation statements)

References 22 publications

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“…Consistent with previous data [21][22][23] , western blots indicated that Ano2 is most highly expressed in olfactory epithelium (MOE and VNO) and eye tissue (Fig. 1a,b).…”

Section: Disruption Of Ano2 and Ano2 Expression Patternsupporting

confidence: 92%

“…Our N-terminal antibody did not detect a truncated protein in Ano2 -/-tissue, suggesting the occurrence of nonsensemediated mRNA decay, instability of the truncated protein, or both. Ano2 -/-mice were born at Mendelian ratio; they grew, mated, and survived normally and lacked an immediately apparent phenotype.Consistent with previous data [21][22][23] , western blots indicated that Ano2 is most highly expressed in olfactory epithelium (MOE and VNO) and eye tissue (Fig. 1a,b).…”

supporting

confidence: 92%

“…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][22][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.…”

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confidence: 89%

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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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“…Consistent with previous data [21][22][23] , western blots indicated that Ano2 is most highly expressed in olfactory epithelium (MOE and VNO) and eye tissue (Fig. 1a,b).…”

Section: Disruption Of Ano2 and Ano2 Expression Patternsupporting

confidence: 92%

supporting

confidence: 92%

supporting

confidence: 89%

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Ca2+-activated Cl− currents are dispensable for olfaction

et al. 2011

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“…A molecular understanding of the dual regulation of CaCCs by voltage and Ca 2+ has recently become possible with the discovery that Ano1 (TMEM16a) is an essential subunit of CaCCs. Ano1 can be gated by Ca 2+ (1,2), including epithelial secretion (3,4), sensory transduction and adaptation (5)(6)(7)(8), regulation of smooth muscle contraction (9), control of neuronal and cardiac excitability (10), and nociception (11). This myriad of functions has attracted attention for more than 25 years (12,13), but a lack of consensus regarding their molecular composition has stymied a mechanistic understanding of their gating.…”

Section: +mentioning

confidence: 99%

Voltage- and calcium-dependent gating of TMEM16A/Ano1 chloride channels are physically coupled by the first intracellular loop

Xiao

Pérez‐Cornejo³

et al. 2011

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

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365

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Ca 2+-activated Cl − channels (CaCCs) are exceptionally well adapted to subserve diverse physiological roles, from epithelial fluid transport to sensory transduction, because their gating is cooperatively controlled by the interplay between ionotropic and metabotropic signals. A molecular understanding of the dual regulation of CaCCs by voltage and Ca 2+ has recently become possible with the discovery that Ano1 (TMEM16a) − channels (CaCCs) play manifold roles in cell physiology (1, 2), including epithelial secretion (3, 4), sensory transduction and adaptation (5-8), regulation of smooth muscle contraction (9), control of neuronal and cardiac excitability (10), and nociception (11). This myriad of functions has attracted attention for more than 25 years (12, 13), but a lack of consensus regarding their molecular composition has stymied a mechanistic understanding of their gating. Recently, two members of the TMEM16/anoctamin family (Ano1 and Ano2) were identified as CaCC channels (14-16) and shown to be essential for salivary exocrine secretion (14,17,18), gut slow-wave activity (18, 19), tracheal secretion (18,20,21), and olfactory transduction (5-7).Ano1 and Ano2 are well suited for their diverse roles because they are dually gated by voltage (V m ) and intracellular Ca 2+ concentration ([Ca 2+ ] i ), so that their activity is tuned by the interplay between metabotropic and ionotropic inputs (14,16,(22)(23)(24) and depolarization. In the absence of Ca 2+ , no current was evident at V m between −100 mV and +100 mV, but as [Ca 2+ ] i was increased, an outward current was activated by depolarization and deactivated by hyperpolarization (Fig. 1 A-D and F). As [Ca 2+ ] i was increased, outward rectification (Fig. 1F) and the fraction of total current exhibiting time-dependence (Fig. 1E) were reduced. V m -dependent activation of Ano1 was evaluated by plotting normalized conductance versus V m (G/G max vs. V m curves; Fig. 1G). The data were well fit by the Boltzmann equation,where G/G max is normalized conductance; z is the equivalent gating charge associated with voltage-dependent channel opening; V 0.5 is the membrane potential (V m ) where G/G max is halfmaximal and is related to the conformational energy associated with voltage-independent channel opening; and F/RT = 0.039 mV −1 . At 1 μM Ca 2+ , V 0.5 was 64 ± 0.9 mV (Fig. 1G, black squares); doubling [Ca 2+ ] to 2 μM shifted the G/G max vs. V m curve to the left by −145 mV (Fig. 1G, red circles) with no significant effect on z. Because Ca 2+ shifts the G/G max vs. V m curves so dramatically, a complete G/G max vs. V m curve could be recorded for only a narrow range of [Ca 2+ ] i . For these [Ca 2+ ], z was not obviously Ca 2+ -dependent (z = 0.40-0.46). This indicates that Ca 2+ does not change the V m sensitivity (z) of the Ano1 channel, but rather shifts V 0.5 , the energy associated with V m -independent gating.Although these data may imply that Ano1 is a simple ligandgated channel, Ano1 is actually more complicated, because Ca 2+ gating is st...

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“…Its molecular correlate has recently been identified as TMEM16A/anoctamin1 (Amjad et al 2015), the founding member of the anoctamin family of Ca 2+ -activated Cl − channels (Caputo et al 2008;Schroeder et al 2008;Yang et al 2008). While different groups have demonstrated expression of both anoctamin1 and anoctamin2 in VSNs (Rasche et al 2010;Billig et al 2011;Dauner et al 2012;Dibattista et al 2012), vomeronasal Ca 2+ -activated Cl − currents were abolished in conditional anoctamin1 null mice (Amjad et al 2015). For interpretation of these results, however, it is essential to determine whether vomeronasal Cl − channels contribute a strongly depolarizing current or, by contrast, if anoctamin1 activation mediates membrane hyperpolarization or shunting inhibition.…”

Section: +mentioning

confidence: 99%

Elevated Cytosolic Cl⁻Concentrations in Dendritic Knobs of Mouse Vomeronasal Sensory Neurons

Untiet¹,

Moeller²,

Ibarra-Soria³

et al. 2016

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In rodents, the vomeronasal system controls social and sexual behavior. However, several mechanistic aspects of sensory signaling in the vomeronasal organ remain unclear. Here, we investigate the biophysical basis of a recently proposed vomeronasal signal transduction component-a Ca 2+ -activated Cl − current. As the physiological role of such a current is a direct function of the Cl − equilibrium potential, we determined the intracellular Cl − concentration in dendritic knobs of vomeronasal neurons. Quantitative fluorescence lifetime imaging of a Cl − -sensitive dye at the apical surface of the intact vomeronasal neuroepithelium revealed increased cytosolic Cl − levels in dendritic knobs, a substantially lower Cl − concentration in vomeronasal sustentacular cells, and an apparent Cl − gradient in vomeronasal neurons along their dendritic apicobasal axis. Together, our data provide a biophysical basis for sensory signal amplification in vomeronasal neuron microvilli by opening Ca 2+ -activated Cl − channels.

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Tmem16b is Specifically Expressed in the Cilia of Olfactory Sensory Neurons

Cited by 94 publications

References 22 publications

Ca2+-activated Cl− currents are dispensable for olfaction

Ca2+-activated Cl− currents are dispensable for olfaction

Voltage- and calcium-dependent gating of TMEM16A/Ano1 chloride channels are physically coupled by the first intracellular loop

Elevated Cytosolic Cl⁻Concentrations in Dendritic Knobs of Mouse Vomeronasal Sensory Neurons

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Tmem16b is Specifically Expressed in the Cilia of Olfactory Sensory Neurons

Cited by 94 publications

References 22 publications

Ca2+-activated Cl− currents are dispensable for olfaction

Ca2+-activated Cl− currents are dispensable for olfaction

Voltage- and calcium-dependent gating of TMEM16A/Ano1 chloride channels are physically coupled by the first intracellular loop

Elevated Cytosolic Cl−Concentrations in Dendritic Knobs of Mouse Vomeronasal Sensory Neurons

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Elevated Cytosolic Cl⁻Concentrations in Dendritic Knobs of Mouse Vomeronasal Sensory Neurons