Ca2+-activated Cl− currents are dispensable for olfaction

Abstract

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...

Preassociated apocalmodulin mediates Ca 2+ -dependent sensitization of activation and inactivation of TMEM16A/16B Ca 2+ -gated Cl − channels

“…There is universal agreement that TMEM16A, and the closely related TMEM16B, are bona fide CaCCs (2,(12)(13)(14)19). Consistent with this, TMEM16A knockout mice displayed defective CaCC activity in a variety of epithelia (20)(21)(22), and the olfactory CaCC current was completely abolished in TMEM16B knockout mice (23). Hydropathy analyses suggest TMEM16 proteins have a similar topology with cytosolic N and C termini and eight predicted transmembrane helices (2,19).…”

Activation of Ca2+‐activated Cl− channel ANO1 by localized Ca2+ signals

“…2010; Billig et al . 2011) and photoreceptors (Stohr et al . 2009), apparently has a different Ca 2+ source preference as compared to ANO1.…”

“…2009; Billig et al . 2011). It has to be pointed out that some native CaCC currents may be mediated by other channels not related to ANO family (e.g.…”

Smelling better with chloride

“…All looked well for the electrical-amplification hypothesis until it was put to the test in a genetic ablation study. The Ca 2+ -activated Cl − channels of OSNs were identified on a molecular level as TMEM16B (alias Anoctamin 2) proteins (12)(13)(14), and a TMEM16B knockout mouse was generated whose OSNs lacked all Ca 2+ -induced Cl − current (15). Within the framework of the electricalamplification hypothesis, the expectation was that the TMEM16B-knockout mouse would display a reduced odor sensitivity.…”