Comparison of a Ca²⁺-Gated Conductance and a Second-Messenger-Gated Conductance in Rat Olfactory Neurons

Odorants bind to their specific receptor proteins on the cilia that extend from the dendrite of olfactory neurons, triggering membrane depolarization and a discharge of action potentials (for a review, see Schild and Restrepo, 1998). Biochemical studies have shown that stimulation of olfactory neurons with odorants results in a rise in the level of either adenosine 3′,5′-cyclic monophosphate (cAMP) or inositol 1,4,5-trisphosphate (1,4,5-InsP₃) (Pace et al., 1985; Sklar et al., 1986; Boekhoff et al., 1990). Odorants that elevate cAMP levels activate cAMP-gated cation channels in the cilia (Nakamura and Gold, 1987). Intracellular application of 1,4,5-InsP₃ also activates a cationic conductance, and this may be different from the cAMP-induced conductance (Restrepo et al., 1990; Miyamoto et al., 1992; Okada et al., 1994; Schild et al., 1995; Kashiwayanagi, 1996). Odorants elicit an influx of Ca²⁺ through cAMP- or 1,4,5-InsP₃-gated cation channels, resulting in a rise in intracellular Ca²⁺ levels ([Ca²⁺]_i) (Restrepo et al., 1990, 1993a,b). The increase in [Ca²⁺]_i may open Ca²⁺-activated Cl⁻ channels (Kleene and Gesteland, 1991; Kleene, 1993; Kurahashi and Yau, 1993; Lowe and Gold, 1993a; Zhainazarov and Ache, 1995) or Ca²⁺-activated K⁺ channels (Morales et al., 1995, 1997). While activation of K⁺ channels leads to hyperpolarization of the olfactory neurons, the effect of Cl⁻ channel opening has not been defined. This is because the probe for Cl⁻ was not very effective for measuring the normal intracellular Cl⁻ concentration, although the reversal potential of the Cl⁻ component in the odorant response suggested a high intracellular Cl⁻ concentration (Zhainazarov and Ache, 1995). In the present experiments, we show that an increase in intracellular [Ca²⁺] activates 5-nitro-2-(3-penylpropylamino)-benzoic acid (NPPB)-sensitive Cl⁻ channels in rat olfactory neurons, while 1,4,5-InsP₃ and cAMP induce the Ca²⁺-dependent Cl⁻ conductance and Ca²⁺-independent cation conductance in the cells.

Olfactory receptor neurons were isolated from adult Wistar rats. Prior to decapitation, animals were anaesthetized by intraperitoneal injection of pentobarbital (30 mg kg⁻¹). The olfactory epithelium and supporting cartilage were quickly removed and washed in Ca²⁺-, Mg²⁺-free saline containing 2 mmol l⁻¹ EDTA. In a previous study (Okada et al., 1994), olfactory epithelium was cut into small pieces together with the cartilage, but in the present study, the epithelium was completely separated from the cartilage. It was cut into small pieces and incubated for 10 min in 3 ml of the same saline containing 5 mmol l⁻¹ L-cysteine and 15 units ml⁻¹ papain (Sigma, St Louis, MO, USA). After removal of 2 ml of the dissociation solution, the tissue was gently triturated with a heat-polished pipette. Dissociation was terminated by the addition of 3 ml of normal saline solution containing 1 mmol l⁻ Ca²⁺. Olfactory receptor neurons were readily distinguished by their characteristic bipolar morphology. Trituration in the absence of cartilage resulted in the neurons maintaining their characteristics for at least 5 h.

Voltage-clamp recording was performed in the whole-cell configuration (Hamill et al., 1981) using a CEZ 2300 patch-clamp amplifier (Nihon Kohden, Tokyo, Japan). The patch pipettes were pulled from Pyrex glass capillaries containing a fine filament with a two-stage puller (Narishige PD-5, Tokyo, Japan). The tips of the electrodes were heat-polished with a microforge (Narishige MF-83). The resistance of the resulting patch electrodes was 5–10 MΩ when filled with internal solution. The formation of 5–20 GΩ seals between the patch pipette and the cell surface was facilitated by applying weak suction to the interior of the pipette. The patch membrane was broken by applying strong suction, resulting in a sudden increase in capacitance. Amphotericin B (133 μg ml⁻¹, Sigma) or gramicidin (100 μg ml⁻¹, Sigma) was added to the pipette solution when using the perforated method (Rae et al., 1991; Akaike, 1997). The perforated whole-cell condition was obtained within 5 min of the establishment of a gigaohm seal. Recordings were made from olfactory neurons that had been allowed to settle on the bottom of a chamber placed on the stage of an inverted microscope (Olympus IMT-2, Tokyo, Japan). The recording pipette was positioned with a hydraulic micromanipulator (Narishige WR-88). The current signal was low-pass-filtered at 5 kHz, digitized at 125 kHz using a TL-1 interface (Axon Instruments, Foster City, CA, USA), acquired at a sampling rate of 0.25-5 kHz using the pCLAMP 5.5 software (Axon Instruments) and stored on the hard disk of an IBM-compatible personal computer running the pCLAMP. This was also used to control the digital/analogue converter for the generation of the clamp protocol. The indifferent electrode was a chlorided silver wire. All voltages were corrected for the liquid junction potential. Capacitance and series resistance were compensated for, as appropriate. The whole-cell current/voltage (I/V) relationship was obtained from the current generated by a 167 mV s⁻¹ voltage ramp from −100 to +100 mV. Input resistance was calculated from the current generated by the voltage ramp from −100 to −50 mV.

Normal saline solution consisted of (in mmol l⁻¹): NaCl, 145; KCl, 5; CaCl₂, 1; MgCl₂, 1; sodium pyruvate, 1; Tris-Hepes, 20; glucose, 5; pH 7.2. For stock solutions, BaCl₂ (500 mmol l⁻¹) and CdCl₂ (100 mmol l⁻¹) were dissolved in deionized water, and ionomycin (2 mmol l⁻¹; Calbiochem, La Jolla, CA, USA), 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB, 100 mmol l⁻¹; Calbiochem) and thapsigargin (10 mmol l⁻¹; Calbiochem) were dissolved in dimethyl sulphoxide (DMSO). Samples of the stock solutions were added to normal saline solution to give the desired final concentration. Niflumic acid (0.3 mmol l⁻¹, Sigma) was dissolved in normal saline solution. The standard-K⁺,Cl⁻ (137 mmol l⁻¹) internal solution contained (in mmol l⁻): KCl, 135; CaCl₂, 0.1; MgCl₂, 1; EGTA, 1; Tris-Hepes, 10; pH 7.2. In some experiments, KCl was replaced with potassium gluconate [standard-K+, low-Cl⁻ (10 mmol l⁻¹) internal solution]. Stock solutions of 1,4,5-InsP₃ (1 mmol l⁻¹; Sigma) and cyclic AMP (10 mmol l⁻¹; Sigma) were made up in the internal solution and were further diluted in this solution to give an appropriate working concentration. For Ca²⁺ dialysis, CaCl₂ was made up in deionized water and was added to the pipette solution without EGTA. All experiments were carried out at room temperature (20–25 °C). Values are presented as means ± S.E.M.

Under conventional whole-cell mode in standard-K⁺,Cl⁻ (137 mmol l⁻¹) internal solution, rat olfactory neurons displayed resting potentials of −43 to −58 mV (−54±1 mV, N=17). The input resistance ranged of 1.5 to 8.3 GΩ (3.4±0.5 GΩ, N=17), and the membrane capacitance averaged 4.2±0.2 pF (N=17). Even when the Cl⁻ concentration in the pipette was decreased from 137 to 10 mmol l⁻¹, the cells maintained resting potentials of −54±2 mV (N=10). The perforated whole-cell recording using amphotericin B (133 μg ml⁻¹) in standard-K⁺, low-Cl⁻ (10 mmol l⁻¹) internal solution also maintained resting potentials of −50±3 mV (N=11). The presence of external NPPB (0.1 mmol l⁻¹, a blocker of the Cl⁻ channel) did not change the resting potentials under the perforated condition in standard-K⁺, low-Cl⁻ internal solution. All cells displayed transient inward currents followed by sustained outward currents in response to depolarizing voltage steps from a holding potential of −100 mV (data not shown).

When the pipette was filled with a modified K⁺, low-Cl⁻ (10 mmol l⁻¹) internal solution (E_Cl=−69 mV) containing 1 mmol l⁻¹ Ca²⁺ without EGTA, all cells displayed an increase in outward current (Fig. 1). The outward current averaged 49±5 pA (N=13) at −50 mV and had a mean reversal potential of −70±1 mV (N=13). The response latency was less than 5 s, and the time constant (τ) for current activation was 10–30 s. The outward current was inhibited by external application of NPPB (b in Fig. 1B). External NPPB (0.1 mmol l⁻¹) decreased the magnitude of the outward current at −50 mV to 34±7 % (N=7) of the response in normal saline solution and depolarized the membrane potential from −73±2 mV to −66±2 mV (N=7). The inhibitory effect of NPPB was reversible in four cells and irreversible in three cells. Another Cl⁻ channel blocker, 0.3 mmol l⁻¹ niflumic acid, also inhibited the outward current when applied externally (Fig. 2), but the effect was irreversible in all three cells tested. Calcium dialysis in internal solution containing 137 mmol l⁻¹ Cl⁻ (E_Cl=−3 mV) induced an increase of inward current at −50 mV in three of five cells (data not shown). The magnitude of the current at −50 mV was −260±92 pA and the reversal potential was −16±7 mV (N=3). External Ba²⁺ (10 mmol l⁻¹, a blocker of K⁺ channel) also decreased the outward current (two cells) (c in Fig. 1B), suggesting the existence of a K⁺ current. Thus, Ca²⁺-dialyzed rat olfactory neurons appear to possess both K⁺ and Cl⁻ currents.

It is known that thapsigargin releases Ca²⁺ from endoplasmic reticulum by inhibiting the Ca²⁺-ATPase. External application of thapsigargin (5 μmol l⁻¹) had two effects on the membrane properties of rat olfactory neurons. Initially, the drug greatly inhibited the outward current at levels more positive than −50 mV, and subsequently caused an increase in another outward current at −50 mV (Fig. 3). The early effect did not affect the membrane potential (Fig. 3B), but the delayed effect hyperpolarized the cells from −53±1 mV to −68±1 mV (N=4) (Fig. 3C), which is close to the equilibrium potential of Cl⁻ (E_Cl). Thus, the depletion of Ca²⁺ stores elicited by thapsigargin did not induce an inward current in the cells, but did elicit a Ca²⁺-dependent Cl⁻ or K⁺ conductance.

The results suggest that a rise in intracellular [Ca²⁺] activates a Ca²⁺-dependent Cl⁻ conductance. However, the normal concentration of intracellular Cl⁻ in rat olfactory neurons is not known. We examined the effect of the Ca²⁺ ionophore ionomycin using the gramicidin-perforated method, which maintained intracellular Cl⁻ at normal levels (Akaike, 1997). Only three of eight cells displayed an increase in inward current at −50 mV in response to 3 μmol l⁻¹ ionomycin (Fig. 4Ai,ii). On average, ionomycin increased the inward current by −143±46 pA (N=3) at −50 mV, depolarized the membrane potential from −49±3 mV to −23±4 mV (N=3), and decreased the input resistance from 2.2±1.0 GΩ to 0.2±0.1 GΩ (N=3). The steady state I/V relationship of the inward current was almost linear (b in Fig. 4Aii). External 2 mmol l⁻ Cd²⁺ (a blocker of cation channel) had little effect in inhibiting the inward current. The other five cells displayed an increase of outward current at −50 mV in response to ionomycin (Fig. 4Bi,ii). On average, the drug increased the outward current at −50 mV by 30±4 pA (N=5), hyperpolarized the membrane potential from −53±4 mV to −71±4 mV (N=5), and decreased the input resistance from 2.2±0.4 GΩ to 0.7±0.1 GΩ (N=5). The I/V relationship of the outward current after Ba²⁺ application was almost linear (c in Fig. 4Bii). The outward current at membrane potentials lower than 30 mV was considerably inhibited by 10 mmol l⁻¹ Ba²⁺, but the resting potential was almost the same as that in normal saline, suggesting that a Ba²⁺-insensitive conductance existed even after K⁺ channels were blocked by Ba²⁺. Under conventional whole-cell mode in standard-K⁺,Cl⁻ (137 mmol l⁻¹) internal solution (E_Cl=−3 mV), three of nine cells displayed an increase of inward current at −50 mV in response to 3 μmol l⁻¹ ionomycin, while two cells showed an increase of outward current at −50 mV.

When the pipette was filled with standard-K⁺, low-Cl⁻ (10 mmol l⁻¹) internal solution (E_Cl=−69 mV) containing 50 μmol l⁻¹ 1,4,5-InsP₃, seven of 13 cells displayed an increase in inward current (Fig. 5A). The inward current averaged −156±44 pA (N=7) at −50mV and had a mean reversal potential of −26±6 mV (N=7). External Cd2+ greatly decreased the inward current to 10±10 % (N=7) of the response in normal saline solution (b in Fig. 5Ai,ii). The I/V relationship was almost linear (a in Fig. 5Aii). The onset of the inward current was very fast. The other four cells showed an increase in outward current (a in Fig. 5Bi,ii) that was inhibited by external NPPB (b in Fig. 5Bi,ii). The current level at −50 mV was reversed from 22±22 pA to −40±26 pA (N=4). After the inhibition of the outward current by 0.1 mmol l⁻¹ NPPB (b in Fig. 5Bi,ii), the remained inward current was inhibited by external Cd2+ (c in Fig. 5Bi,ii).

When 500 μmol l⁻¹ cAMP was added to the pipette containing standard-K⁺, low-Cl⁻ (10 mmol l⁻¹) internal solution (E_Cl=−69 mV), six of 13 cells displayed an increase of inward current (Fig. 6A). The inward current averaged −174±37 pA (N=6) at −50 mV and had a mean reversal potential of −17±3 mV (N=6). The inward current gradually adapted (a and b in Fig. 6Ai,ii) and was decreased by external Cd²⁺ (c in Fig. 6Ai,ii) to 15±8 % (N=6) of the response in normal saline solution. The I/V relationship was almost linear (a in Fig. 6Aii). The onset of the inward current was very fast. The other three cells showed a gradual increase of outward current (a and b in Fig. 6Bi,ii) and a decrease of the current in response to external NPPB (c in Fig. 6Bi,ii). The current level at −50 mV was decreased from 24±6 pA to 8±8 pA (N=3) by NPPB. We never found the appearance of an inward current after application of NPPB, although the inward current was observed in 1,4,5-InsP₃ dialysis.

The present results show that in the whole-cell mode intracellular Ca²⁺ dialysis activates an NPPB-sensitive outward Cl⁻ current in all the rat olfactory neurons examined, causing hyperpolarization at low intracellular Cl⁻ levels. A similar conductance was activated by intracellular dialysis of 1,4,5-InsP₃ in 31 % of the neurons, and the outward current was reversed to an inward current at −50 mV by NPPB. This means that NPPB did not inhibit 1,4,5-InsP₃-gated inward currents. Cyclic AMP also induced the NPPB-sensitive outward Cl⁻ current in 23 % of the cells, but this outward current was not reversed by NPPB. It is known that cyclic-nucleotide-gated (CNG) conductance is blocked by a rise in intracellular [Ca²⁺] (Liu et al., 1994). Our present results also indicate that a rise in intracellular Ca²⁺ inhibits the CNG conductance, and induces the NPPB-sensitive conductance. In contrast, the increase in [Ca²⁺] elicited by 1,4,5-InsP₃ did not inhibit the 1,4,5-InsP₃-gated inward current, suggesting that this current is different from the CNG inward current.

Intracellular Ca²⁺ dialysis without EGTA for Ca²⁺ buffering induced the NPPB-sensitive Cl⁻ current in all cells, but 1,4,5-InsP₃ or cAMP dialysis with EGTA elicited the Cl− current in only 20–30 % of the cells. In rat olfactory neurons dialysed with a lower concentration of EGTA (0.1 mmol l⁻¹), a large Cl⁻ current was induced by odorants and cAMP (Lowe and Gold, 1993a). Higher intracellular [EGTA] (5 mmol l⁻¹), or natural intracellular condition using the perforated technique, decreased the fraction of Cl⁻ current in the transduction current of amphibian olfactory neurons (Kurahashi and Yau, 1993). It was reported that the threshold level of intracellular Ca²⁺ for Cl⁻ current activation was approximately 1 μmol l⁻¹ in frog olfactory cilia (Kleene and Gesteland, 1991). The natural capacity of intracellular Ca²⁺ buffering in the olfactory neuron is unknown. Kurahashi and Yau (1993) observed an odorant-induced inward Cl⁻ current using a perforated pipette without Cl⁻. Zhainazarov and Ache (1995) were also able to record odorant-induced inward Cl⁻ current using the gramicidin patch technique, which maintained the naturally occurring intracellular Cl⁻ level. These experiments suggested that the intracellular Cl⁻ concentration in olfactory neurons is high. However, the present experiments indicate that although some olfactory neurons maintain a higher concentration of intracellular Cl⁻, other cells have lower Cl⁻ level. Using a fluorescent probe, Nakamura et al. (1997) measured an intracellular Cl⁻ concentration of approximately 40 mmol l⁻¹ in the soma of newt olfactory neurons, but they also claimed that the intracellular concentration of Cl⁻ in the olfactory knob was lower than 10 mmol l⁻¹ (Kaneko et al., 1998). Another study reported a positive E_Cl of 6 mV in rat olfactory neurons (Reuter et al., 1998). It is, therefore, interesting to ascertain whether or not odorant-responsive cells can maintain a high level of intracellular Cl⁻. In many cell types, Cl⁻ enters the cells through the Na⁺/K⁺/2Cl⁻ cotransporter.

The olfactory signal-transduction apparatus, which contains putative odour receptors, G_olfα, type III adenylyl cyclase and CNG channels, localizes in the olfactory cilia (Nakamura and Gold, 1987; Menco et al., 1992, 1997). Recently, a knockout mouse lacking olfactory CNG channels proved unable to produce excitatory responses to both cAMP- and 1,4,5-InsP₃-producing odorants (Brunet et al., 1996). These results suggest that cAMP may be the sole second messenger in olfactory transduction. Other studies, however, indicated a ciliary localization for G_qα/G_11α and for the 1,4,5-InsP₃ receptor (Cunningham et al., 1993; Menco et al., 1994; DellaCorte et al., 1996; Smutzer et al., 1997). The results of our present experiments together with previous experiments have demonstrated an 1,4,5-InsP₃-activated conductance in olfactory cells (Miyamoto et al., 1992; Okada et al., 1994; Schild et al., 1995; Kashiwayanagi, 1996), consistent with the presence of 1,4,5-InsP₃ receptor in the cell membrane; however, other studies have reported that 1,4,5-InsP₃ dialysis has no effect in olfactory neurons (Firestein et al., 1991; Lowe and Gold, 1993b).

This work was supported by a Grant-in-Aid (no. 10671743) for Scientific Research from the Ministry of Education, Science, Sports and Culture in Japan.