A Ca2+-activated Cl− current constitutes a large part of the transduction current in olfactory sensory neurons. The binding of odorants to olfactory receptors in the cilia produces an increase in cAMP concentration; Ca2+ enters into the cilia through CNG channels and activates a Cl− current. In intact mouse olfactory sensory neurons little is known about the kinetics of the Ca2+-activated Cl− current. Here, we directly activated CNG channels by flash photolysis of caged cAMP or 8-Br-cAMP and measured the current response with the whole cell voltage-clamp technique in mouse neurons. We measured multiphasic currents in the rising phase of the response at −50 mV. The current rising phase became monophasic in the absence of extracellular Ca2+, at +50 mV, or when most of the intracellular Cl− was replaced by gluconate to shift the equilibrium potential for Cl− to −50 mV. These results show that the second phase of the current in mouse intact neurons is attributed to a Cl− current activated by Ca2+, similarly to previous results on isolated frog cilia. The percentage of the total saturating current carried by Cl− was estimated in two ways: 1) by measuring the maximum secondary current and 2) by blocking the Cl− channel with niflumic acid. We estimated that in the presence of 1 mM extracellular Ca2+ and in symmetrical Cl− concentrations the Cl− component can constitute up to 90% of the total current response. These data show how to unravel the CNG and Ca2+-activated Cl− component of the current rising phase.
The binding of odorant molecules to olfactory receptors in the cilia of olfactory sensory neurons initiates a transduction cascade that leads, by G-protein and adenylyl cyclase activation, to an increase in the concentration of cyclic adenosine monophosphate (cAMP). Cyclic nucleotide-gated (CNG) channels in the ciliary plasma membrane are directly gated by cAMP (Nakamura and Gold 1987), allowing the entry of Na+ and Ca2+ ions. However, the odorant-induced inward transduction current has been shown to be composed not only of a cation influx through CNG channels, but also of a Cl− efflux through Cl− channels activated by Ca2+. In fact, Ca2+-activated Cl− channels in the cilia are activated by a rise in ciliary Ca2+ concentration and, because olfactory sensory neurons maintain an elevated intracellular Cl− concentration (Kaneko et al. 2004; Nickell et al. 2006; Reisert et al. 2005; Reuter et al. 1998), Cl− ions will exit from the cilia. Thus the depolarizing response to odorants is generated by both cation influx and Cl− efflux (Firestein 2001; Frings et al. 2000; Matthews and Reisert 2003; Menini 1999; Menini et al. 2004; Pifferi et al. 2006a; Schild and Restrepo 1998).
The Ca2+-activated Cl− conductance was first discovered in frog olfactory cilia (Kleene 1993; Kleene and Gesteland 1991) and its involvement in the response to odorants has been demonstrated in isolated olfactory sensory neurons from amphibians (Firestein and Shepherd 1995; Kurahashi and Yau 1993; Zhainazarov and Ache 1995) and from rats and mice (Lowe and Gold 1993a; Reisert et al. 2005).
The presence of a pair of cationic and anionic currents has been suggested to be useful to allow depolarizing current responses in a variety of extracellular ionic environments (Kurahashi and Yau 1993). In fact, provided that external Ca2+ is present, the transduction current persists in a wide range of salinities, indicating that amphibians can smell both in salt and fresh water (Kleene and Pun 1996). Another advantage of the combination of cationic and anionic currents is that the Cl− current produces a large amplification of the primary cationic CNG current (Lowe and Gold 1993a) and that the amplified signal has a higher signal-to-noise ratio than does the primary signal (Kleene 1997).
The properties of ciliary Cl− channels have been investigated in isolated frog olfactory cilia (Kleene 1993, 1997; Kleene and Pun 1996) and in excised inside-out patches from knob/cilia of olfactory sensory neurons from rat (Hallani et al. 1998; Reisert et al. 2003) and from mouse (Pifferi et al. 2006b; Reisert et al. 2005). Moreover, it has been recently demonstrated that mouse bestrophin-2 (mBest2) (Kramer et al. 2004) is expressed in the cilia of olfactory sensory neurons and that it is a good candidate for the Ca2+-activated Cl− channel involved in olfactory transduction (Pifferi et al. 2006b). However, a detailed understanding of the functional properties of Ca2+-activated Cl− currents in intact mouse olfactory sensory neurons is still missing and this information is necessary for a complete molecular identification of the channel protein and its possible modulators.
To our knowledge, there are no reports studying the kinetics of Ca2+-activated Cl− currents in intact olfactory sensory neurons.
Here we recorded the current in intact mouse olfactory sensory neurons in response to fast photolysis of caged cyclic nucleotides. By analyzing the rising phase of the response we observed the presence of multiphasic currents at negative potentials in the presence of symmetrical Cl− and with external Ca2+. We showed that the secondary current was eliminated by removing external Ca2+, replacing intracellular Cl− with the impermeant anion gluconate and by the presence of the Cl− channel inhibitor niflumic acid. All of these properties, measured in intact mouse olfactory neurons, are similar to those measured by Kleene (1993) in frog excised cilia and are consistent with the secondary current being a Ca2+-activated Cl− current.
Preparation of olfactory sensory neurons
Olfactory sensory neurons were dissociated enzymatically, with a papain–cystein treatment, from the olfactory epithelium of 4- to 8-wk-old mice of the BALB/c strain, as previously described (Boccaccio et al. 2006; Lagostena and Menini 2003). All experiments were carried out in accordance with the Italian Guidelines for the Use of Laboratory Animals (Decreto Legislativo 27/01/1992, no. 116). Cells were then plated in a Ringer solution (see Solutions) on glass coverslips coated with concanavalin A and poly-l-lysine to favor cell adhesion.
Olfactory sensory neurons were observed with an inverted microscope (Olympus IX70) with an oil-immersion ×100 objective (Olympus or Zeiss) and identified by their characteristic bipolar shape. Only olfactory sensory neurons with clearly visible cilia were used for the experiments.
Currents were measured with an Axopatch 200B patch-clamp amplifier (Axon Instruments, Union City, CA) in the whole cell voltage-clamp mode. Patch pipettes were made using borosilicate capillaries (WPI, Sarasota, FL) and pulled with a Narishige PP83 puller (Narishige, Tokyo, Japan), using a double-stage pull. The diameter of the tip was about 1 μm and the pipette resistances were 2–7 MΩ when filled with the standard intracellular solution. Currents were low-pass filtered at 1 kHz and acquired at 2 kHz by the A/D interface Digidata 1322A (Axon Instruments). Acquisition and storage of data were performed with a pClamp 8.2 software (Axon Instruments).
All experiments were carried out at room temperature (20–22°C).
Photolysis of caged compounds
For flash photolysis of the caged compounds, we used a xenon flash-lamp system JML-C2 (Rapp OptoElectronic, Hamburg, Germany) coupled with the epifluorescence port of the microscope with a quartz light guide (Boccaccio et al. 2006). The spot of light had a diameter of about 15 μm that could cover only the ciliary region and, in some cases, also part of the dendrite.
The flash duration was <1.5 ms and was kept constant during each experiment. The light intensity was controlled by neutral-density filters (Omega Optical, Brattleboro, VT or Glen Spectra, Stanmore, Middlesex, UK). The interval between experiments was ≥2 min to allow the cell to recover completely from adaptation.
The extracellular mammalian Ringer solution contained (in mM): 140 NaCl, 5 KCl, 1 CaCl2, 1 MgCl2, 10 HEPES, 10 glucose, and 1 sodium pyruvate (pH 7.4). The composition of the nominally 0 Ca2+ extracellular solution was similar, except that it contained 10 mM EGTA and no added Ca2+. Whole cell pipette solution contained (in mM): 145 KCl, 4 MgCl2, 0.5 EGTA, 10 HEPES, 1 MgATP, and 0.1 GTP (pH 7.4). In some experiments, 0.24 CaCl2 was added to the intracellular solution, to have about 80 nM free Ca2+ (calculated with MaxChelator, http://www.stanford.edu/∼cpatton/maxc.html).
The pipette gluconate solution contained (in mM): 12 KCl, 133 K-gluconate, 4 MgCl2, 0.24 CaCl2, 0.5 EGTA, 10 HEPES, 1 MgATP, and 0.1 GTP (pH 7.4).
Osmolarity was adjusted to 310 mOsm for the extracellular and to 290 mOsm for the intracellular solutions. Liquid junction potential was corrected off-line for the experiments performed with the gluconate intracellular solution.
We directly activated the CNG channels by photoreleasing the physiological activator cAMP or its poorly hydrolyzable analogue 8-bromoadenosine-3′,5′-cyclic monophosphate (8-Br-cAMP). The caged BCMCM-cAMP (Hagen et al. 2001) and BCMCM-8-Br-cAMP (Boccaccio et al. 2006) were dissolved in DMSO at 10 or 50 mM and stored at −20°C for up to 3 mo. The final concentration of 50 μM was obtained by diluting an aliquot of the stock solution into the pipette solution, kept refrigerated in the dark during the experimental session, and stored for a few days at −20°C. The intracellular recording solution for the photorelease of caged Ca2+ contained (in mM): 3 DMNP-EDTA, 1.5 CaCl2, 140 KCl, and 10 HEPES (pH 7.4). DMNP-EDTA was purchased from Molecular Probes–Invitrogen (West Eugene, OR), and CaCl2 was adjusted with a 0.1 M standard solution from Fluka (Deisenhofen, Germany).
The caged compounds were allowed to diffuse freely from the patch pipette into the cytoplasm of an olfactory sensory neuron for ≥2 min after establishment of the whole cell configuration.
Niflumic acid was dissolved in DMSO at 200 mM and, on the day of the experiment, an aliquot was added to the bath solution (Ringer or low extracellular Ca2+) to reach a final concentration of 500 μM.
Chemicals, except for caged compounds or otherwise stated, were purchased from Sigma.
Data analysis and figures were made by use of Igor software (Wavemetrics, Lake Oswego, OR). To measure the rising phase of the current we calculated the time in which the current was 50% of its maximal value (t50). A single exponential function was fitted to the rising phase of the current for monophasic responses.
Current amplitudes at each holding potential were calculated by subtracting the value of the baseline. Averages were shown ± SD and the total number of neurons (n).
Ca2+ dependency of the current rising phase
Figure 1, A–C shows the current responses to photorelease of various 8-Br-cAMP concentrations in an olfactory sensory neuron bathed in a nominally 0 Ca2+ extracellular solution. The rising phase of the current responses to each light intensity could be well fitted by a single exponential function (dotted lines in Fig. 1B), and therefore appeared to have only one component. When the same type of experiment was repeated in the presence of 1 mM extracellular Ca2+ in another olfactory sensory neuron (Fig. 1, D–F), the current rising phase could not be fitted by a single exponential function and had more than one component. A multiphasic rising phase was observed in 57 of 62 olfactory sensory neurons, whereas the remaining five neurons had a very fast monophasic response.
Because the kinetics of the response can be very different from cell to cell (Boccaccio et al. 2006), it is necessary to obtain a quantitative estimation of the Ca2+ dependency of the kinetics from ion substitution experiments performed on the same neuron. This variability is likely partially attributable to different light illumination and morphology of the neurons examined. We restricted the illumination area to the ciliary region, but a partial illumination of the dendrite could not be avoided. For this reason we investigated the Ca2+ dependency of the rising phase by comparing responses to light flashes of the same maximal intensity in extracellular 0 Ca2+ or in 1 mM Ca2+ in the same neuron (Fig. 1, G–I). Figure 1G shows the current response elicited by the uncaging of 8-Br-cAMP at −50 mV in the 0 Ca2+ solution. The fast rising phase of the current was fitted with a single exponential function with a time constant τ of 1.3 ms. When the same neuron was bathed in Ringer containing 1 mM Ca2+, the rising phase was slower and could not be described by a single exponential function (Fig. 1H). The different rising components are illustrated in more detail in Fig. 1I, where responses in Ringer and in 0 Ca2+ from the same neuron are shown superimposed on an expanded timescale. We measured the time necessary for the current to reach 50% of its maximal response, t50, after the delivery of the light flash: t50 = 1.8 ms in 0 Ca2+ solution, whereas t50 became 9.0 ms, five times slower, in 1 mM external Ca2+. The peak amplitudes of current in 0 Ca2+ (Fig. 1G) and in Ringer (Fig. 1H) were similar, although their ionic composition is very different. In 0 Ca2+ the inward current is mainly carried by Na+ ions through CNG channels, partially blocked by Mg2+ ions in the external solution (Kleene 1995). In Ringer the inward current through CNG channels is carried not only by Na+ but also by Ca2+ ions, which produce an additional extracellular block (Kleene 1995), but also activate a Cl− current (Kleene 1993).
Similar results were obtained from several other cells with variable kinetics: the rising phase was always well described by a single exponential function in 0 Ca2+, whereas it was formed by more than one component in Ringer. The ratio between t50 measured in Ringer and in 0 Ca2+ at −50 mV was calculated per each cell and the average value was 7.7 ± 2.9 (n = 13). From experiments obtained with cAMP, the average value of the ratios between t50 measured in Ringer and in 0 Ca2+ was 5.7 ± 3.5 (n = 6), very similar to the value obtained in 8-Br-cAMP.
Voltage dependency of the current rising phase
Another way to look into Ca2+ effects on the rising phase of the response is to compare kinetics in Ringer at +50 or −50 mV in the same neuron. At +50 mV the influx of Ca2+ through CNG channels is greatly reduced and the outward current is mainly carried by K+ ions, whose permeation through CNG channels is similar to that of Na ions (reviewed in Kaupp and Seifert 2002). Figure 2 A shows currents elicited by photorelease of 8-Br-cAMP at +50 mV (gray lines) and −50 mV (black lines) on the same olfactory sensory neuron. The rising phase at +50 mV could be well described by a single exponential function (dotted line), whereas more than one current component was present at −50 mV, where a small current of about −60 pA was followed by a larger component that reached about −600 pA (Fig. 2, A and B). To better illustrate the rising phase, traces were normalized to their peak values and plotted superimposed on an expanded timescale in the right column (Fig. 2C). Similar results were obtained for several cells and we consistently found that the rising phase of the current response at +50 mV was monophasic. Moreover, the rising time of the response measured as t50 was faster at +50 than at −50 mV. The ratio between t50 at +50 and at −50 mV was calculated per each cell and the mean value was 0.32 ± 0.34 for 8-Br-cAMP (n = 7) and 0.44 ± 0.20 for cAMP (n = 5). In the same set of experiments the ratio between peak current amplitude at +50 and −50 mV was 0.24 ± 0.11 for 8-Br-cAMP and 0.33 ± 0.11 for cAMP.
We observed that, in about 10% of neurons, the current rising phase at −50 mV was composed of a primary current that reached a plateau lasting several milliseconds, followed by a clearly separated secondary current, as shown in Fig. 2, D–F (see also ⇓⇓⇓Fig. 6D). Figure 2E clearly shows that the rising phase is monoexponential at +50 mV, whereas at −50 mV it was composed of a primary current of about −42 pA followed by a larger secondary current component. The total current reached a maximal amplitude of −445 pA; thus the secondary current was about 90% of the total current.
We estimated that the contribution of the secondary to the total current in saturating conditions at −50 mV was 90 ± 3% (n = 7). The mean maximum current in these cells was −667 ± 431 pA (n = 7).
The delayed component is a Cl− current
To evaluate the contribution of the Cl− current to the rising phase of the inward current response we reduced the inward Cl− current by decreasing the intracellular Cl− concentration. In this set of experiments, most of the intracellular Cl− was replaced by gluconate to shift the equilibrium potential for Cl− to −50 mV. Figure 3, A and B shows the responses to identical light flashes applied to photorelease 8-Br-cAMP at various holding potentials. At −64 mV, below the equilibrium potential for Cl−, the rising phase of the current clearly had more than one component, whereas at −54 and +46 mV the rising phase was well described by a single exponential function (Fig. 3B). To better compare the rising phase, some traces were normalized to their peak values and plotted superimposed on an expanded timescale in Fig. 3C. The observation that a secondary current was present at −64 mV, whereas it disappeared at −54 mV, close to the equilibrium potential for Cl−, indicates that the delayed current component is a Cl− current.
Figure 3, D–F shows the results of an experiment in the same ionic conditions of Fig. 3A on a different cell at the holding potentials of +46 or −54 mV. The rising phase of the current response at both holding potentials was well described by a single exponential function with τ = 23 ms at +46 mV and 13 ms at −54 mV (Fig. 3E). At +46 mV the peak current was 105 pA, 5.8-fold larger than the value of only −18 pA measured at −54 mV. On average, the ratio between current amplitude at +46 and −54 mV was 4.5 ± 3 (14 measurements from six neurons). This rectification is in agreement with the known blocking properties of CNG channels; in fact, in the presence of 1 mM Ca2+ in the external solution, CNG channels are more strongly inhibited by Ca2+ at negative than at positive potentials (reviewed in Kaupp and Seifert 2002). Current–voltage relation properties of CNG channels in frog olfactory cilia have been carefully investigated by Kleene (1995). From Fig. 1 of Kleene (1995) it can be estimated that, in the presence of 1 mM Ca2+, the ratio between the CNG current at +50 and −50 mV was about 4, very similar to the value of 4.5 measured in our experimental conditions.
Therefore experiments illustrated in Fig. 3 show that the slower component of the rising phase of the inward current is abolished when the Cl− current component is strongly reduced by changing the equilibrium potential for Cl−. Moreover, the rectification properties of the remaining current are consistent with those of CNG channels.
Another way to unravel the current components of the rising phase of the response is to combine photorelease of 8-Br-cAMP with a voltage protocol (Fig. 4). 8-Br-cAMP was photoreleased while neurons were held at a positive holding potential, where Ca2+ influx through CNG channels is much smaller than at −50 mV and therefore intraciliary Ca2+ is not expected to increase to a level sufficient to activate Cl− channels. The voltage then suddenly jumped to a negative value, producing a rapid change in the permeation properties through the activated CNG channels, including a larger Ca2+ entry into the cilia. Experiments were obtained both in low intracellular Cl− (Fig. 4, A and B) and in symmetrical Cl− solutions (Fig. 4, C and D).
Figure 4, A and B illustrates the results of an experiment in low intracellular Cl−: the gray trace represents the current elicited by a flash applied at time 0 to photorelease 8-Br-cAMP at a holding potential of +26 mV. The outward current reached a maximum value, where it remained for several milliseconds, and then returned to baseline levels. The experiment was repeated with the modified voltage protocol: during the outward current plateau the voltage was suddenly jumped to −54 mV, close to the equilibrium potential for Cl−. An inward current rapidly developed with the voltage jump and then slowly returned to baseline. The response is shown in Fig. 4B on an expanded timescale to illustrate the time course of the current: the rising phase of the inward current appeared to have only one component and the current amplitude was smaller than that at +26 mV. Figure 4, C and D shows results from a different neuron: when the equilibrium potential for Cl− was set to be close to zero, by using symmetrical Cl− solutions, a similar experimental protocol gave origin to a more complex current shape. From a holding potential of +50 mV a flash was applied at time 0 to photorelease 8-Br-cAMP and, during the outward current plateau, the voltage suddenly jumped from +50 to −50 mV. An inward current rapidly developed with the voltage jump. The response is shown in Fig. 6D on an expanded timescale to illustrate the time course of the current induced by the voltage jump: in symmetrical Cl−, the inward current developed more than one current components. About 20 ms after the voltage step the inward current was about −60 pA and subsequently increased to about −530 pA with a slower time course. The secondary current was therefore about 89% of the total current.
At +50 mV only CNG channels are activated and therefore the current measured at the time of the sudden voltage change to −50 mV is expected to arise only from the CNG channels. As time passed, Ca2+ entry through the CNG channels could reach an intraciliary concentration sufficient to activate Cl− channels.
Thus the experiments shown in Fig. 4 further indicate that the delayed component of the rising phase is a Cl− current.
We also examined the possibility that the delayed Cl− current component could be a result of the slow kinetics of channel activation by Ca2+. We directly photoreleased Ca2+ inside the cilia of intact olfactory sensory neurons (Fig. 5 A) and measured the rising time. The rising phase of the Ca2+-activated current at various light intensities was well described by a single exponential function with time constants varying from 3.8 to 5 ms. Similar measurements were obtained in a total of five cells with a mean value of 5.5 ± 2.0 ms. The mean saturating current value was 866 ± 410 pA (n = 5), showing that a large current can, in fact, be activated by Ca2+.
These experiments demonstrate that the current activation by Ca2+ occurs in a few milliseconds and therefore it is not responsible for the slow activation of the secondary current. The delay in activation of the secondary current observed in Figs. 1–4 is likely to arise from the time necessary for Ca2+ to increase to concentrations high enough to activate the Cl− channels.
Blockage by niflumic acid
The Cl− current component was further investigated by using niflumic acid, one of the most commonly used blockers of Ca2+-activated Cl− channels in olfactory cilia (Kleene 1993; Lowe and Gold 1993a). Figure 6A shows the blockage by 500 μM niflumic acid of the current elicited at −50 mV by photorelease of 8-Br-cAMP in a neuron with fast kinetics. In this experiment, the maximum inward current amplitude was reduced from −310 to −60 pA, corresponding to a blockage of about 80%. On average, peak currents in the presence of 500 μM niflumic acid at −50 mV were 24 ± 15% (range 8–40%) of the control value. After removal of niflumic acid the current amplitude recovered, on average, to 60% of the control value.
Because niflumic acid selectively inhibits Ca2+-activated Cl− channels and does not inhibit CNG channels (Kleene 1993), the blocked current provides an estimate of the Cl− current. It is of interest to note that, in the presence of niflumic acid, the current at +50 mV was 1.7-fold greater than at −50 mV (Fig. 6B). Results of experiments in low intracellular Cl− showed that the current at +46 mV was on average 4.5-fold greater than at −54 mV (Fig. 3D), whereas in symmetrical Cl−, the current at +50 mV was on average 0.24 ± 0.11 times smaller than that at −50 mV (Fig. 2). These results are consistent with a reduction of Ca2+-activated Cl− currents in experimental conditions of low intracellular Cl− (Fig. 3) and in the presence of niflumic acid (Fig. 6) compared with symmetrical Cl− in the absence of a blocker.
We have also investigated the blockage by niflumic acid in some of the neurons that presented a primary current component reaching a plateau of several milliseconds. Figure 6, C and D shows that the addition of 500 μM niflumic acid almost completely blocked the secondary current. Therefore given the selective blockage of the Cl− current by niflumic acid (Kleene 1993), these results further indicate that the secondary current component responsible for the larger fraction of the inward current is carried by Cl−.
The transduction current in olfactory sensory neurons is attributed to Na+ and Ca2+ entry through CNG channels and the subsequent activation of a Ca2+-activated Cl− current that constitutes a large part of it.
CNG channels allow Ca2+ entry into the cilia not only for excitatory but also for inhibitory effects (Matthews and Reisert 2003; Menini 1999). In fact, the complex Ca2+-calmodulin activates a phosphodiesterase that hydrolizes cAMP and also produces a negative feedback effect on the CNG channel itself, which has been shown to mediate olfactory adaptation (Boccaccio et al. 2006; Kurahashi and Menini 1997). The secondary Ca2+-activated Cl− current in olfactory transduction plays the key role of high-gain and low-noise amplifier of the primary CNG current because the unusually high concentration of ciliary Cl− produces the outflow of Cl−, contributing to the inward current.
The presence of a depolarizing Cl− current is not unique to olfactory neurons. It is well known that in the immature brain the neurotransmitter γ-aminobutyric acid (GABA) is excitatory as a result of a high intracellular concentration of Cl− (for review, see Ben-Ari 2002). Several developing neurons of some brain structures, including the hippocampus, the neocortex, and the hypothalamus, have a higher intracellular concentration of Cl− than adult neurons. Intracellular Cl− accumulation in some immature neurons is caused by the Na-K-2Cl cotransporter NKCC1 (Fukuda et al. 1998; Yamada et al. 2004). In adult neurons K-Cl cotransporters lower intracellular Cl− below its equilibrium potential, modifying GABA action from excitatory to inhibitory. In olfactory sensory neurons NKCC1 has been shown to be involved in the maintenance of a high intracellular Cl− (Reisert et al. 2005), although a subsequent study indicates the possibility that NKCC1 is not the only component involved in this process (Nickell et al. 2006).
The aim of this study was to investigate the kinetics of activation of the CNG and Ca2+-activated Cl− current and to provide an estimate of the Ca2+-activated Cl− current contribution in isolated olfactory sensory neurons in mice. An understanding of the functional properties of Ca2+-activated Cl− currents in mouse is necessary for a complete molecular identification of the channel protein and of its possible modulators. Recently bestrophin-2 has been identified as a candidate Ca2+-activated Cl− channel involved in olfactory transduction. Pifferi et al. (2006b) demonstrated that mouse bestrophin-2 (mBest2) is expressed in the cilia of olfactory sensory neurons. The comparison of electrophysiological properties of Ca2+-activated Cl− currents from native channels in dendritic knob/cilia of mouse olfactory sensory neurons (OSNs) with those induced by the expression of mBest2 in HEK-293 cells indicated that mBest2 is a good candidate for being a molecular component of the olfactory Ca2+-activated Cl− channel. However, even though functional properties of native Ca2+-activated Cl− currents and mBest2 currents are similar, nonetheless they are not identical, and the existing differences indicate the possibility that native channels may be composed of additional molecular components and/or modulators. It is therefore necessary to establish the functional properties of the native channels in intact olfactory sensory neurons from mice for future comparisons between native and cloned channel properties.
We controlled the production of cyclic nucleotides by flash photolysis of caged compounds and showed that, by a fast photorelease of cyclic nucleotides in the cilia of mouse olfactory sensory neurons, it is possible to measure the temporal development of various components in the rising phase of the current response. We used a flash lamp that allows a very fast photorelease of caged compounds and, in the absence of extracellular Ca2+, we measured monophasic current responses that had an exponential rise with a time constant as fast as 1.3 ms at a holding potential of −50 mV (Fig. 1G). When the same experiment was repeated in the presence of Ca2+, we could observe the appearance of multiple current components in the rising phase of the response. We showed that the rising phase of the current was multiphasic in 92% of OSNs measured at negative potentials in the presence of 1 mM extracellular Ca2+ and symmetrical Cl− concentrations.
Kleene (1993), in isolated frog olfactory cilia, previously measured multiphasic currents in response to the application of cAMP. He showed that the current during the first phase arises from cationic influx through CNG channels, whereas the secondary current arises from Cl− channels activated by Ca2+ that accumulates in the cilium with time. Here, recording from intact OSNs from mice, we could also observe the temporal development of a primary and secondary current. Although the presence of a Ca2+-activated Cl− current in the response to odorants in the newt (Kurahashi and Yau 1993), rat (Lowe and Gold 1993a), and frog (Zhainazarov and Ache 1995) has been well established, multiphasic currents have not been previously characterized in intact neurons.
To unravel the Ca2+-activated Cl− from the CNG current component we varied the entry of Ca2+ into the cilia, changed the Cl− equilibrium potential, and added the Cl− current blocker niflumic acid. Ca2+ entry was reduced by removing Ca2+ from the extracellular solution or by holding the cell at positive potentials, whereas it was increased with sudden voltage jumps from positive to negative potentials or by photolysis of intracellular caged Ca2+. We showed that the secondary current was absent in low extracellular Ca2+ or at positive potentials, whereas it was seen in the presence of 1 mM Ca2+ at negative potentials. Thus the appearance of the secondary current requires the influx of Ca2+. Moreover the secondary current is a Cl− current because it could be reduced by changing the equilibrium potential for Cl− or by adding niflumic acid.
The possibility that the delayed Cl− current component could be the result of a slow kinetics of channel activation by Ca2+ was excluded by directly photoreleasing Ca2+ that produced a fast and monophasic current (Fig. 5).
In a previous analysis of the rising phase of the response to photolysis of caged cAMP, Lowe and Gold (1993b; Fig. 1B) noticed the existence of two components in the photolysis response: a fast component with a small amplitude and a slow component with a larger amplitude. However, because the Cl− component was still undiscovered when this observation was published, Lowe and Gold (1993b) attributed the origin of the two components to different photolysis reactions occurring in the lipid membrane and in the intraciliary solution. On the basis of the results from Kleene (1993) on excised cilium preparation and on our data on intact neurons, it is likely that the first component illustrated in Fig. 1B of Lowe and Gold (1993b) arose from the activation of CNG channels and the second, larger component, from the additional development of the Ca2+-activated Cl− current.
We estimated in two different ways the fraction of the total saturating current carried by Cl− at −50 mV in the presence of 1 mM Ca2+ in symmetrical Cl−. In OSNs displaying multiphasic saturating currents we estimated the Cl− current from the amplitude of the early and of the peak current component. On average, the contribution of the secondary current to the total current was 90%. In another set of experiments we blocked the Cl− component with niflumic acid. The total current amplitude was reduced, on average, by 76%, with values ranging from 60 to 92%. Previous experiments (Reisert et al. 2005) measured the odorant-induced current from isolated mouse OSNs with the suction pipette and calculated that, on average, 80% of the response was blocked by niflumic acid, and therefore was carried by Cl−. Another recent study in mouse olfactory epithelium has shown that >80% of the electroolfactogram is caused by a Cl− current (Nickell et al. 2006). However, it is likely that the Cl− current component is greater than the values estimated from the blockage by niflumic acid. In fact, it has been shown that niflumic acid blocks only ≤90% of the Cl− current in frog olfactory cilia (Kleene 1993).
Our results indicate that, in intact isolated mouse OSNs, the secondary component is a Ca2+-activated current that can constitute up to 90% of the total current response, therefore providing a large amplification to the primary CNG current response.
This work was supported by the Italian Institute of Technology and European Community Grant NFG-503221 to A. Menini.
We thank L. Lagostena for participating in some experiments, V. Hagen for kindly providing the cyclic nucleotides caged compounds, S. Pifferi and L. Lagostena for helpful discussions, L. Masten for the preparation of dissociated mouse olfactory sensory neurons, and M. Schipizza-Lough for checking the English.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
- Copyright © 2007 by the American Physiological Society