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Ca2+-activated Cl currents are dispensable for olfaction

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Abstract

Canonical olfactory signal transduction involves the activation of cyclic AMP–activated cation channels that depolarize the cilia of receptor neurons and raise intracellular calcium. Calcium then activates Cl currents that may be up to tenfold larger than cation currents and are believed to powerfully amplify the response. We identified Anoctamin2 (Ano2, also known as TMEM16B) as the ciliary Ca2+-activated Cl channel of olfactory receptor neurons. Ano2 is expressed in the main olfactory epithelium (MOE) and in the vomeronasal organ (VNO), which also expresses the related Ano1 channel. Disruption of Ano2 in mice virtually abolished Ca2+-activated Cl currents in the MOE and VNO. Ano2 disruption reduced fluid-phase electro-olfactogram responses by only 40%, did not change air-phase electro-olfactograms and did not reduce performance in olfactory behavioral tasks. In contrast with the current view, cyclic nucleotide–gated cation channels do not need a boost by Cl channels to achieve near-physiological levels of olfaction.

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Figure 1: Immunoblot analysis of Ano2 expression.
Figure 2: Ano2 localizes to sensory protrusions in olfactory epithelia.
Figure 3: Ano2 and Ano1 are co-expressed in the VNO but not in the MOE.
Figure 4: The olfactory bulb in Ano2−/− and Ano2+/+ mice.
Figure 5: Effect of Ano2 disruption on Ca2+-activated Cl currents.
Figure 6: Response of isolated olfactory receptor neurons to photoreleased Ca2+ or 8-Br-cAMP.
Figure 7: EOGs from the MOE.
Figure 8: Ano2 disruption affected neither odor discrimination nor olfactory sensitivity.

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Change history

  • 08 May 2011

    In the version of this article initially published online, there were two errors in Figure 7 and one in the author affiliations. The units given in Figure 7e for the y axis and for the concentration of mix1 were incorrect; the correct units are µV and mM, respectively. The abbreviation in the affiliations for the Max-Delbrück-Centrum für Molekulare Medizin was incorrect; the correct abbreviation is MDC. The errors have been corrected for the print, PDF and HTML versions of this article.

References

  1. Kleene, S.J. The electrochemical basis of odor transduction in vertebrate olfactory cilia. Chem. Senses 33, 839–859 (2008).

    Article  CAS  Google Scholar 

  2. Munger, S.D., Leinders-Zufall, T. & Zufall, F. Subsystem organization of the mammalian sense of smell. Annu. Rev. Physiol. 71, 115–140 (2009).

    Article  CAS  Google Scholar 

  3. Bönigk, W. et al. The native rat olfactory cyclic nucleotide-gated channel is composed of three distinct subunits. J. Neurosci. 19, 5332–5347 (1999).

    Article  Google Scholar 

  4. Kleene, S.J. Origin of the chloride current in olfactory transduction. Neuron 11, 123–132 (1993).

    Article  CAS  Google Scholar 

  5. Kurahashi, T. & Yau, K.W. Co-existence of cationic and chloride components in odorant-induced current of vertebrate olfactory receptor cells. Nature 363, 71–74 (1993).

    Article  CAS  Google Scholar 

  6. Lowe, G. & Gold, G.H. Nonlinear amplification by calcium-dependent chloride channels in olfactory receptor cells. Nature 366, 283–286 (1993).

    Article  CAS  Google Scholar 

  7. Kleene, S.J. & Gesteland, R.C. Calcium-activated chloride conductance in frog olfactory cilia. J. Neurosci. 11, 3624–3629 (1991).

    Article  CAS  Google Scholar 

  8. Reisert, J., Bauer, P.J., Yau, K.W. & Frings, S. The Ca-activated Cl channel and its control in rat olfactory receptor neurons. J. Gen. Physiol. 122, 349–363 (2003).

    Article  CAS  Google Scholar 

  9. Reisert, J., Lai, J., Yau, K.W. & Bradley, J. Mechanism of the excitatory Cl response in mouse olfactory receptor neurons. Neuron 45, 553–561 (2005).

    Article  CAS  Google Scholar 

  10. Kaneko, H., Putzier, I., Frings, S., Kaupp, U.B. & Gensch, T. Chloride accumulation in mammalian olfactory sensory neurons. J. Neurosci. 24, 7931–7938 (2004).

    Article  CAS  Google Scholar 

  11. Nickell, W.T., Kleene, N.K., Gesteland, R.C. & Kleene, S.J. Neuronal chloride accumulation in olfactory epithelium of mice lacking NKCC1. J. Neurophysiol. 95, 2003–2006 (2006).

    Article  CAS  Google Scholar 

  12. Boccaccio, A. & Menini, A. Temporal development of cyclic nucleotide-gated and Ca2+-activated Cl currents in isolated mouse olfactory sensory neurons. J. Neurophysiol. 98, 153–160 (2007).

    Article  CAS  Google Scholar 

  13. Hengl, T. et al. Molecular components of signal amplification in olfactory sensory cilia. Proc. Natl. Acad. Sci. USA 107, 6052–6057 (2010).

    Article  CAS  Google Scholar 

  14. Reuter, D., Zierold, K., Schröder, W.H. & Frings, S. A depolarizing chloride current contributes to chemoelectrical transduction in olfactory sensory neurons in situ. J. Neurosci. 18, 6623–6630 (1998).

    Article  CAS  Google Scholar 

  15. Chiu, D., Nakamura, T. & Gold, G.H. Ionic composition of toad olfactory mucus measured with ion selective microelectrodes. Chem. Senses (Abstract) 13, 677–678 (1988).

    Google Scholar 

  16. Nickell, W.T., Kleene, N.K. & Kleene, S.J. Mechanisms of neuronal chloride accumulation in intact mouse olfactory epithelium. J. Physiol. (Lond.) 583, 1005–1020 (2007).

    Article  CAS  Google Scholar 

  17. Smith, D.W., Thach, S., Marshall, E.L., Mendoza, M.G. & Kleene, S.J. Mice lacking NKCC1 have normal olfactory sensitivity. Physiol. Behav. 93, 44–49 (2008).

    Article  CAS  Google Scholar 

  18. Yang, Y.D. et al. TMEM16A confers receptor-activated calcium-dependent chloride conductance. Nature 455, 1210–1215 (2008).

    Article  CAS  Google Scholar 

  19. Caputo, A. et al. TMEM16A, a membrane protein associated with calcium-dependent chloride channel activity. Science 322, 590–594 (2008).

    Article  CAS  Google Scholar 

  20. Schroeder, B.C., Cheng, T., Jan, Y.N. & Jan, L.Y. Expression cloning of TMEM16A as a calcium-activated chloride channel subunit. Cell 134, 1019–1029 (2008).

    Article  CAS  Google Scholar 

  21. Stephan, A.B. et al. ANO2 is the cilial calcium-activated chloride channel that may mediate olfactory amplification. Proc. Natl. Acad. Sci. USA 106, 11776–11781 (2009).

    Article  CAS  Google Scholar 

  22. Stöhr, H. et al. TMEM16B, a novel protein with calcium-dependent chloride channel activity, associates with a presynaptic protein complex in photoreceptor terminals. J. Neurosci. 29, 6809–6818 (2009).

    Article  Google Scholar 

  23. Rasche, S. et al. Tmem16b is specifically expressed in the cilia of olfactory sensory neurons. Chem. Senses 35, 239–245 (2010).

    Article  CAS  Google Scholar 

  24. Schwenk, F., Baron, U. & Rajewsky, K. A cre-transgenic mouse strain for the ubiquitous deletion of loxP-flanked gene segments including deletion in germ cells. Nucleic Acids Res. 23, 5080–5081 (1995).

    Article  CAS  Google Scholar 

  25. Brunet, L.J., Gold, G.H. & Ngai, J. General anosmia caused by a targeted disruption of the mouse olfactory cyclic nucleotide–gated cation channel. Neuron 17, 681–693 (1996).

    Article  CAS  Google Scholar 

  26. Nakamura, T. & Gold, G.H. A cyclic nucleotide-gated conductance in olfactory receptor cilia. Nature 325, 442–444 (1987).

    Article  CAS  Google Scholar 

  27. Romanenko, V.G. et al. Tmem16A encodes the Ca2+-activated Cl channel in mouse submandibular salivary gland acinar cells. J. Biol. Chem. 285, 12990–13001 (2010).

    Article  CAS  Google Scholar 

  28. Rock, J.R. et al. Transmembrane protein 16A (TMEM16A) is a Ca2+-regulated Cl secretory channel in mouse airways. J. Biol. Chem. 284, 14875–14880 (2009).

    Article  CAS  Google Scholar 

  29. Keller, A. & Margolis, F.L. Immunological studies of the rat olfactory marker protein. J. Neurochem. 24, 1101–1106 (1975).

    Article  CAS  Google Scholar 

  30. Lin, W., Arellano, J., Slotnick, B. & Restrepo, D. Odors detected by mice deficient in cyclic nucleotide-gated channel subunit A2 stimulate the main olfactory system. J. Neurosci. 24, 3703–3710 (2004).

    Article  CAS  Google Scholar 

  31. Zheng, C., Feinstein, P., Bozza, T., Rodriguez, I. & Mombaerts, P. Peripheral olfactory projections are differentially affected in mice deficient in a cyclic nucleotide-gated channel subunit. Neuron 26, 81–91 (2000).

    Article  CAS  Google Scholar 

  32. Zou, D.J. et al. Absence of adenylyl cyclase 3 perturbs peripheral olfactory projections in mice. J. Neurosci. 27, 6675–6683 (2007).

    Article  CAS  Google Scholar 

  33. Pifferi, S., Dibattista, M. & Menini, A. TMEM16B induces chloride currents activated by calcium in mammalian cells. Pflugers Arch. 458, 1023–1038 (2009).

    Article  CAS  Google Scholar 

  34. Sagheddu, C. et al. Calcium concentration jumps reveal dynamic ion selectivity of calcium-activated chloride currents in mouse olfactory sensory neurons and TMEM16B/anoctamin2-transfected HEK 293T cells. J. Physiol. (Lond.) 588, 4189–4204 (2010).

    Article  CAS  Google Scholar 

  35. Pifferi, S. et al. Calcium-activated chloride currents in olfactory sensory neurons from mice lacking bestrophin-2. J. Physiol. (Lond.) 587, 4265–4279 (2009).

    Article  CAS  Google Scholar 

  36. Frings, S., Reuter, D. & Kleene, S.J. Neuronal Ca2+ -activated Cl channels–homing in on an elusive channel species. Prog. Neurobiol. 60, 247–289 (2000).

    Article  CAS  Google Scholar 

  37. Pinato, G. et al. Electroolfactogram responses from organotypic cultures of the olfactory epithelium from postnatal mice. Chem. Senses 33, 397–404 (2008).

    Article  Google Scholar 

  38. Bodyak, N. & Slotnick, B. Performance of mice in an automated olfactometer: odor detection, discrimination and odor memory. Chem. Senses 24, 637–645 (1999).

    Article  CAS  Google Scholar 

  39. Kelliher, K.R., Ziesmann, J., Munger, S.D., Reed, R.R. & Zufall, F. Importance of the CNGA4 channel gene for odor discrimination and adaptation in behaving mice. Proc. Natl. Acad. Sci. USA 100, 4299–4304 (2003).

    Article  CAS  Google Scholar 

  40. Kleene, S.J. & Pun, R.Y. Persistence of the olfactory receptor current in a wide variety of extracellular environments. J. Neurophysiol. 75, 1386–1391 (1996).

    Article  CAS  Google Scholar 

  41. Pifferi, S. et al. Bestrophin-2 is a candidate calcium-activated chloride channel involved in olfactory transduction. Proc. Natl. Acad. Sci. USA 103, 12929–12934 (2006).

    Article  CAS  Google Scholar 

  42. Gribkoff, V.K. et al. Effects of channel modulators on cloned large-conductance calcium-activated potassium channels. Mol. Pharmacol. 50, 206–217 (1996).

    CAS  PubMed  Google Scholar 

  43. Vogalis, F., Hegg, C.C. & Lucero, M.T. Ionic conductances in sustentacular cells of the mouse olfactory epithelium. J. Physiol. (Lond.) 562, 785–799 (2005).

    Article  CAS  Google Scholar 

  44. Lindemann, B. Predicted profiles of ion concentrations in olfactory cilia in the steady state. Biophys. J. 80, 1712–1721 (2001).

    Article  CAS  Google Scholar 

  45. Schneppenheim, R. et al. A common 253-kb deletion involving VWF and TMEM16B in German and Italian patients with severe von Willebrand disease type 3. J. Thromb. Haemost. 5, 722–728 (2007).

    Article  CAS  Google Scholar 

  46. Gomez-Pinilla, P.J. et al. Ano1 is a selective marker of interstitial cells of Cajal in the human and mouse gastrointestinal tract. Am. J. Physiol. Gastrointest. Liver Physiol. 296, G1370–G1381 (2009).

    Article  CAS  Google Scholar 

  47. Shimazaki, R. et al. Electrophysiological properties and modeling of murine vomeronasal sensory neurons in acute slice preparations. Chem. Senses 31, 425–435 (2006).

    Article  Google Scholar 

  48. Cygnar, K.D., Stephan, A.B. & Zhao, H. Analyzing responses of mouse olfactory sensory neurons using the air-phase electroolfactogram recording. J. Vis. Exp. (2010).

  49. Windmüller, O. et al. Ion changes in spreading ischaemia induce rat middle cerebral artery constriction in the absence of NO. Brain 128, 2042–2051 (2005).

    Article  Google Scholar 

  50. Slotnick, B. & Restrepo, D. Olfactometry with mice. in Current Protocols in Neuroscience (eds. Crawley, J.N. et al.) Chapter 8, Unit 8 20 (2005).

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Acknowledgements

We thank P. Mombaerts (Max Planck Institute for Biophysics) for providing Cnga2, P2-IRES-tauLacZ and M72-IRES-tauLacZ mice, V. Hagen (FMP) for caged 8-Br-cAMP, I. Zarour and U. Heinemann (Charité) for help with ion-selective microelectrodes, S. Jabs and L. Leisle for help with flash photolysis, P. Mombaerts and B. Schroeder (MDC) for helpful discussions and N. Krönke and F. Binder for technical assistance. This work was supported, in part, by the Prix Louis-Jeantet de Médecine to T.J.J.

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G.M.B. generated Ano2−/− mice and Ano2 antibodies, designed, performed and evaluated expression analysis and immunohistochemistry, olfactometry and mouse behavioral studies and wrote the paper. B.P. designed, performed and evaluated patch-clamp analysis, EOG and ion-selective microelectrode measurements and wrote the paper. B.P. and P.F. designed, performed and evaluated flash-photolysis experiments. T.J.J. initiated the project, designed and evaluated experiments and wrote the paper.

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Correspondence to Thomas J Jentsch.

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Billig, G., Pál, B., Fidzinski, P. et al. Ca2+-activated Cl currents are dispensable for olfaction. Nat Neurosci 14, 763–769 (2011). https://doi.org/10.1038/nn.2821

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