In this work we are presenting the first electrophysiological recordings of a Ca2+-activated Cl− channel directly obtained from vertebrate olfactory cilia. Based on the conserved morphological and functional characteristics of the olfactory sensory neurons between amphibians and mammals studied so far , and in particular the similarity of their transduction cascades, it is plausible that orthologs of the toad CaCC cilia channels are shared with mammals. Often proteins from evolutionarily distant species cross-react to antibodies developed to one of them; amphibian and mammalian CNG  plasma membrane Ca2+ ATPase [15, 16] and Na+/Ca2+ exchanger  of olfactory cilia illustrate this fact. .
The channel exhibits a steep sigmoidal dose–response relation, revealing a pronounced Ca2+ dependence in a narrow range of Ca2+ concentrations (below 0.5 µM) within physiological levels. Notably, although the curve presents a similar shape than those of ANO2 and Best2, it is shifted to lower Ca2+ concentrations; its Ca2+ affinity is considerably higher than these two other channels, as indicated by its K0.5 of ~0.38 µM, compared to 1.8 µM for ANO2 and 4.7 µM for Best2 [10, 11]. Previous estimates from macroscopic current measurements in excised intact frog cilia are within the same range of the two mammalian channels (γ = 0.8 pS, K0.5 = 4.8 µM) .
The evidence is consistent with a Cl− channel. This channel also conducts acetate, the anion used to replace Cl− in the pipette, as revealed by the ~40 mV reversal potential in the current–voltage relations. In agreement to the Goldman, Hodgkin and Katz equation, PAc/PCl = 0.47, close to previously reported values for CaCCs .
The channel reported hereby possesses distinct properties compared with the two olfactory CaCCs previously reported in mice, namely ANO2  and Best2 , suggesting that they correspond to a novel type of CaCC channel protein. Because of its relatively large unit conductance (γ > 10 pS), its single-channel currents could be clearly resolved; in contrast, those from the other two channels are below the resolution of patch clamp amplifiers, as their unitary conductances are in the sub-pS range, according to noise analysis estimates (0.8 and 0.26 pS, respectively [10, 11]).
The immunochemical data showing co-expression of ClCa4l and ANO2 in the cilia suggests that the toad CaCC may correspond to an ortholog of ClCa4l, but in the absence of molecular biology evidence this cannot be established. An electrophysiological correlation would have helped to support the presence of the channel reported hereby and ANO2 in the olfactory cilia such as observing the current events of the two different channels in the patches; however, this is not plausible mainly because of the size of the ANO2 unitary currents.
Why has this channel not been detected in mammals, in spite of its big conductance? Besides the trivial possibility of not being expressed in them, it is plausible that even though its single-channel currents are sufficiently large to be resolved, their overall number in the cilia could be much smaller than that of ANO2; in such a case, the ANO2 currents would dominate, masking the currents of the other channel in the macroscopic measurements.
Recordings from inside-out ciliary patches revealed transitions with complex kinetics. The data do not allow discriminating whether the recordings emerged from an individual channel with more than one conductance state or from more than one channel in the patches. Further investigation is necessary to elucidate this issue.
Our immunocytochemical evidence obtained with an anti-ClCa antibody is consistent with the presence in the toad olfactory cilia of an orthologous of the rat ClCa4l , which may have some role in chemotransduction. It is debated whether or not the CaCl family is comprised of functional ion channels or of accessory proteins that regulate the expression of other CaCCs [18, 19]. Further investigation is required to identify the molecular nature of the toad CaCC reported hereby and establish its functional role.
A channel with the relatively high Ca2+ affinity, strong Ca2+ dependence and comparatively large conductance such as the toad CaCC could play a relevant role in odorant responses. It is reasonable to imagine that as the CNG conductance develops, the membrane potential begins to depolarize and the Ca2+ level to gradually rise, opening the CaCCs. The first CaCC to open would be that with the highest Ca2+ affinity; the higher its conductance, the bigger would be its impact on the receptor potential. This would be the case for ClCa, which fulfills both characteristics (K0.5 = 0.38 µM, γ > 10 pS). The role of this channel could be to boost the receptor potential in its early phase, until Ca2+ levels reach the threshold for the activation of the lower Ca2+ affinity more abundant CaCCs, which would then gradually govern the receptor potential by allowing a relatively much larger Cl− current component.