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Calcium concentration jumps reveal dynamic ion selectivity of calcium‐activated chloride currents in mouse olfactory sensory neurons and TMEM16b‐transfected HEK 293T cells
Author(s) -
Sagheddu Claudia,
Boccaccio Anna,
Dibattista Michele,
Montani Giorgia,
Tirindelli Roberto,
Menini Anna
Publication year - 2010
Publication title -
the journal of physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.802
H-Index - 240
eISSN - 1469-7793
pISSN - 0022-3751
DOI - 10.1113/jphysiol.2010.194407
Subject(s) - niflumic acid , chloride channel , hek 293 cells , biophysics , chemistry , extracellular , ion channel , microbiology and biotechnology , patch clamp , transduction (biophysics) , intracellular , biochemistry , membrane potential , biology , receptor
Ca 2+ ‐activated Cl − channels play relevant roles in several physiological processes, including olfactory transduction, but their molecular identity is still unclear. Recent evidence suggests that members of the transmembrane 16 (TMEM16, also named anoctamin) family form Ca 2+ ‐activated Cl − channels in several cell types. In vertebrate olfactory transduction, TMEM16b/anoctamin2 has been proposed as the major molecular component of Ca 2+ ‐activated Cl − channels. However, a comparison of the functional properties in the whole‐cell configuration between the native and the candidate channel has not yet been performed. In this study, we have used the whole‐cell voltage‐clamp technique to measure functional properties of the native channel in mouse isolated olfactory sensory neurons and compare them with those of mouse TMEM16b/anoctamin2 expressed in HEK 293T cells. We directly activated channels by rapid and reproducible intracellular Ca 2+ concentration jumps obtained from photorelease of caged Ca 2+ and determined extracellular blocking properties and anion selectivity of the channels. We found that the Cl − channel blockers niflumic acid, 5‐nitro‐2‐(3‐phenylpropylamino)benzoic acid (NPPB) and DIDS applied at the extracellular side of the membrane caused a similar inhibition of the two currents. Anion selectivity measured exchanging external ions and revealed that, in both types of currents, the reversal potential for some anions was time dependent. Furthermore, we confirmed by immunohistochemistry that TMEM16b/anoctamin2 largely co‐localized with adenylyl cyclase III at the surface of the olfactory epithelium. Therefore, we conclude that the measured electrophysiological properties in the whole‐cell configuration are largely similar, and further indicate that TMEM16b/anoctamin2 is likely to be a major subunit of the native olfactory Ca 2+ ‐activated Cl − current.