Single Ca²⁺-activated Cl⁻ channel currents recorded from toad olfactory cilia

Odor transduction is confined to the chemosensory cilia of olfactory sensory neurons (OSNs), where it gives origin to a depolarizing receptor potential. Odorants bind to G-protein coupled receptors, which activate adenylyl cyclase type III by coupling to a heterotrimeric G-protein (Golf), producing cyclic-AMP. This nucleotide directly opens non-selective cationic cyclic nucleotide gated (CNG) channels [1, 2]. Ca²⁺ ions entering the cilia through these channels in turn gate Ca²⁺-activated Cl⁻ channels (CaCCs) [3], generating a Cl⁻ efflux that amplifies the depolarization initiated by the CNG-dependent current [4, 5] (but see [6]). The CNG channel has been thoroughly characterized [7], but the CaCC channel has been difficult to assess, and has been focus of attention since it was discovered [3] and its physiological importance unraveled [4, 5]. Its electrophysiological characterization has been hampered by the extremely small conductances of the reported channels and the miniscule diameter of the olfactory cilia, bordering the limit of resolution of light microscopy [8]. For this reason, only macroscopic current measurements had been performed on OSNs by means of whole cell recording [6], excised entire individual cilium [3] and patch clamping the dendritic knob [9], an apical round-shape structure of the dendrite from where the olfactory cilia emanate. As the knob has a nearly tenfold larger diameter than a cilium (~2 vs. 0.2 µm), it is more amenable for patch clamping; nevertheless, the measurements obtained from the dendritic knob assume that the same channels of the cilia are also present in the knob. No single Ca²⁺-activated Cl⁻ currents have been directly recorded from the ciliary membrane, limiting the biophysical characterization of the olfactory Cl⁻ conductance.

Anoctamin-2 (ANO2), an ion channel belonging to the anoctamin family of CaCCs, has been proposed as the main contributor to the odor-dependent Ca²⁺-activated Cl⁻ conductance, as the Cl⁻ current is largely absent in the ANO2 knock-out mice. However, field potential recordings from the olfactory epithelium of these mutant mice are only partly reduced and these animals do not exhibit impairment in smell behavioral tests [10], suggesting that more than one CaCC species might be involved in odor transduction. A second CaCC channel type was found in the olfactory cilia, Best2 from the Bestrophin family [11], later considered not to be implicated in odor transduction because its genetic ablation had no influence in smelling behavior [12]. Both channels possess estimated conductances below 1 pS, too small for resolving unitary currents. Two additional CaCCs, ClCa4l and ClCa2 from the ClCa family, were recently reported in rat olfactory cilia by PCR, western blotting and immunochemical evidence, being ClCa4l much more abundant than ClCa2 [13]; none of these two channels have been characterized electrophysiologically. Direct patch-clamp recordings from the chemosensory cilia of Ca²⁺-activated Cl⁻ currents at the single-channel level would help to clarify this scenario, but they have not been performed for any of the aforementioned channels.

Here we explored the ciliary membrane for detectable single-channel currents by recording directly from inside-out membrane patches excised from toad olfactory cilia, taking advantage of their somewhat larger dimensions than those of mammals, and found a Ca²⁺-activated Cl⁻ channel not previously described. A complementary immunocytochemistry study with anti-ClCa and anti-ANO2 antibodies revealed co-immunoreactivity in the toad cilia, raising the possibility that the novel channel may belongs to the ClCa channel family.

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 [14], 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 [14] plasma membrane Ca²⁺ ATPase [15, 16] and Na⁺/Ca²⁺ exchanger [16] of olfactory cilia illustrate this fact. [15].

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 K_0.5 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, K_0.5 = 4.8 µM) [17].

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, P_Ac/P_Cl = 0.47, close to previously reported values for CaCCs [10].

The channel reported hereby possesses distinct properties compared with the two olfactory CaCCs previously reported in mice, namely ANO2 [10] and Best2 [11], 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 [13], 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 (K_0.5 = 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.

The reported channel suggests that the electrophysiological tools possessed by the olfactory cilia to carry out chemotransduction may be more complex than previously considered.

The aim of this study was to report a novel Ca²⁺-activated Cl⁻ channel discovered in olfactory cilia and explore its possible significance and identity, using electrophysiology and immunocytochemistry.