Proceedings of The Physiological Society

University of Oxford (2011) Proc Physiol Soc 23, PC69

Poster Communications

Calcium- and voltage-dependence of the anoctamin2/TMEM16b-chloride channel: a candidate calcium-activated chloride channel in olfactory transduction.

V. Cenedese1,2, F. Celsi1,2, S. Pifferi1,2, A. Menini1,2

1. Neurobiology Sector, International School for Advanced Studies, Scuola Internazionale Superiore di Studi Avanzati, SISSA, Trieste, Italy. 2. Italian Institute of Technology, SISSA Unit, Trieste, Italy.


Olfactory transduction in vertebrates involves a depolarizing chloride current activated by intracellular calcium. Indeed, the binding of odorants to receptors in the cilia of olfactory sensory neurons activates a transduction cascade via a cAMP-signaling, which results in the opening of cyclic nucleotide-gated channels (CNG). The consequent Ca2+ influx through CNG channels produces an increased Ca2+ concentration in the cilia, which activates Ca2+-activated Cl- channels (CaCCs). Since olfactory sensory neurons maintain an unusually high intracellular Cl- concentration, CaCCs produce an efflux of Cl-. Therefore the Cl- current, contributing up to 90% of the total transduction current, amplifies the primary depolarization due to CNG current. Recent molecular and electrophysiological data strongly support that anoctamin2 (ano2, also called TMEM16b) is the molecular counterpart of CaCC in olfactory sensory neurons. Channel gating is both Ca2+- and voltage-dependent, but the mechanisms underlying the activation are still unknown. Ano2 does not have any apparent canonical Ca2+-binding site, but the first putative intracellular loop contains a series of negatively charged aminoacids. To test the hypothesis that this region is involved in channel activation, mutants of these residues were obtained and the functional properties of wild-type and mutant channels were measured and compared. After expression of wild-type and mutants of the mouse ano2 in a heterologous system (HEK293T cells), electrophysiological recordings with the patch-clamp technique in the inside-out configuration were performed. The exposure of the intracellular side of the channel to various Ca2+ concentrations rapidly activated a current both in wild-type and in mutant channels, indicating a direct activation by Ca2+. Currents showed a time-dependent decrease (inactivation) at -50 mV in the presence of a constant high Ca2+ concentration and an irreversible rundown. To determine the voltage dependence of the calcium sensitivity the wild-type and the mutant’s dose response relationships were compared. The Ca2+ concentrations for half-maximal current activation were: 4.6 ± 0.4 μM in the wild type and 6.5 ± 0.5 μM in one of the mutant at -50 mV, 2.8 ± 0.2 μM in the wild type and 4 ± 0.3 μM in the mutant at +50 mV (n=6, means ± s.d.). The Hill coefficients were between 1 and 2. The rectification properties of current-voltage relations were calcium dependent both in wild type and in mutant channels with a strong inward rectification at high concentrations and an outward rectification at low Ca2+ concentrations. These preliminary results indicate that the region rich in negatively charged aminoacids, located in the first putative intracellular loop, could be involved in Ca2+- and voltage-dependence of the anoctamin2/TMEM16b-chloride channel.

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