Calcium concentration jumps reveal dynamic ion selectivity of calcium-activated chloride currents in mouse olfactory sensory neurons and TMEM16b-transfected HEK 293T cells

Abstract

Ca2+ -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.

Four basic residues critical for the ion selectivity and pore blocker sensitivity of TMEM16A calcium-activated chloride channels

“…Although the mechanism by which anions permeate the pore requires additional investigation, it is tempting to speculate that the presence of the positively charged side chains of these residues might preferentially promote flux of the smaller and more electronegative chloride ions given that the crystal structure is in its calcium-bound and presumably, fully active state (27), where chloride ions tend to flow best. These results are also consistent with previous findings that permeant ions influence the gating process (25,26). If the open state of the pore at low Ca 2+ is more selective to thiocyanate, as shown in Fig.…”

“…In one of the original studies to identify TMEM16A as a CaCC, the selectivity of the Xenopus TMEM16A was dynamically modulated by the concentration of intracellular calcium, suggesting the possibility that multiple open states might exist with differentiable permeation properties (3). More recently, studies of TMEM16B have shown that anion permeation seems to be linked with calcium dependence (25,26). To test the contribution of intracellular calcium to the selectivity properties of TMEM16A, we performed ramp protocols similar to those used at 1.7 μM [Ca 2+ ] i (Fig.…”

Ca2+-activated Cl− currents are dispensable for olfaction

“…5d,f), but not at 13 µM free Ca 2+ (Fig. 5g,i) similar to data reported for Ca 2+ -activated Cl -currents for heterologously expressed Ano1 and Ano2 [20][21][22]33,34 and for frog OSNs 7 . We could not detect these Cl -currents in Ano2 -/-OSNs (Fig.…”

“…The superfusate could be switched to a solution containing niflumic acid (NFA). NFA has been widely used to block Ca 2+ -activated Cl -channels in OSNs and other 6 cells 4,6,9,11,16,34,36,37 and inhibits both Ano1 and Ano2 18,20,21,33,34 . The response to odorants was reduced by roughly 40% in Ano2 -/-EOGs compared to the wild type (Fig.…”

TMEM16F is a component of a Ca2+-activated Cl− channel but not a volume-sensitive outwardly rectifying Cl− channel

“…The TMEM16 protein family consists of 10 different proteins that possess 8 putative transmembrane domains with intracellular NH 2 -and COOH-terminal tails (37). In addition to TMEM16A, human and mouse TMEM16B has been reported to generate CaCC currents in mammalian cell expression systems (22,24,29,31). On the other hand, the functions of other TMEM16 proteins are poorly understood.…”