Calcium concentration jumps reveal dynamic ion selectivity of calcium-activated chloride currents in mouse olfactory sensory neurons and TMEM16b-transfected HEK 293T cells.
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.
Sagheddu
Claudia
C
International School for Advanced Studies, Scuola Internazionale Superiore di Studi Avanzati, SISSA, Trieste, Italy.
Boccaccio
Anna
A
Dibattista
Michele
M
Montani
Giorgia
G
Tirindelli
Roberto
R
Menini
Anna
A
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D000075368:Anoctamins; D002118:Calcium / Q000378:metabolism*; D018118:Chloride Channels / Q000037:antagonists & inhibitors / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D007668:Kidney / Q000166:cytology / Q000378:metabolism*; D051379:Mice; D008815:Mice, Inbred Strains; D023421:Models, Animal; D009544:Niflumic Acid / Q000494:pharmacology; D009579:Nitrobenzoates / Q000494:pharmacology; D009832:Olfactory Nerve / Q000166:cytology / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D011984:Sensory Receptor Cells / Q000166:cytology / Q000378:metabolism*; D014162:Transfection
C532161:ANO2 protein, mouse; D000075368:Anoctamins; D018118:Chloride Channels; D009579:Nitrobenzoates; C058176:5-nitro-2-(3-phenylpropylamino)benzoic acid; D009544:Niflumic Acid; D000262:Adenylyl Cyclases; C421784:adenylate cyclase 3; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D002118:Calcium