The olfactory system detects a large variety of volatile molecules. Odorant molecules enter into the nasal cavity and bind to odorant receptors in the cilia of olfactory sensory neurons, activating a transduction cascade that involves the opening of cyclic nucleotide-gated channels and the entry of Ca²⁺ in the cilia. Ca²⁺ activates Cl^- channels that, given the unusually elevated intracellular Cl^- concentration, produce an efflux of Cl^- ions and amplify the depolarization. Ca²⁺-activated Cl^- channels contribute up to 90% of the total current but their molecular identity is still unknown. Some members of the protein family named transmembrane 16 (TMEM16; also known as anoctamin) have been recently proposed to function as Ca²⁺-activated Cl^- channels. At least one member of this family, TMEM16B, is present in the mouse olfactory ciliary proteome (Mayer et al. Proteomics, 2009; Stephan et al. PNAS, 2009). We investigated the functional properties of the mouse TMEM16B (mTMEM16B) after expression in human embryonic kidney (HEK) 293T cells. Currents were measured with the patch-clamp technique both in the whole-cell configuration and in inside-out excised patches.

In whole-cell, a current induced by mTMEM16B was activated by intracellular Ca²⁺ diffusing from the patch pipette, released from intracellular stores through activation of a G-protein coupled receptor, or photoreleased from caged Ca²⁺ inside the cell. In inside-out excised patches, bath application of different Ca²⁺ concentrations rapidly activated a current, indicating that mTMEM16B is directly gated by Ca²⁺. The Ca²⁺-induced current was anion selective, displayed a Ca²⁺-dependent rectification, and was blocked by the chloride channel blocker niflumic acid. In inside-out patches, the Ca²⁺ concentration for half-maximal current activation decreased from 4.9 mM at -50 mV to 3.3 mM at +50 mV, while the Hill coefficient was >2. Currents showed a time-dependent decrease (inactivation) at -50 mV in the presence of a constant high Ca²⁺ concentration and, moreover, an irreversible rundown, not observed in whole-cell recordings, indicating that some unknown modulator was lost upon patch excision. We compared the electrophysiological properties of native Ca²⁺-activated Cl^- currents in mouse olfactory sensory neurons with those of mTMEM16B-induced currents in HEK cells. We found that dose-response relations for Ca²⁺ activation, estimated single channel conductance, rectification properties, Ca²⁺-dependent inactivation and irreversible rundown are remarkably similar indicating that, at present, mTMEM16B is the best candidate for being the main molecular component of the native Ca²⁺-activated Cl^- channel in the cilia of vertebrate olfactory sensory neurons.