Odorants Suppress a Voltage-Activated K+ Conductance in Rat Olfactory Neurons

Fritz W. Lischka,1 John H. Teeter,1,2 and Diego Restrepo3

 1Monell Chemical Senses Center;  2Department of Physiology, University of Pennsylvania, Philadelphia, Pennsylvania 19104; and  3Department of Cellular and Structural Biology, University of Colorado Health Sciences Center, Denver, Colorado 80262


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Lischka, Fritz W., John H. Teeter, and Diego Restrepo. Odorants Suppress a Voltage-Activated K+ Conductance in Rat Olfactory Neurons. J. Neurophysiol. 82: 226-236, 1999. Stimulation of olfactory receptor neurons (ORNs) with odors elicits an increase in the concentration of cAMP leading to opening of cyclic nucleotide-gated (CNG) channels and subsequent depolarization. Although opening of CNG channels is thought to be the main mechanism mediating signal transduction, modulation of other ion conductances by odorants has been postulated. To determine whether K+ conductances are modulated by odorants in mammalian ORNs, we examined the response of rat ORNs to odors by recording membrane current under perforated-patch conditions. We find that rat ORNs display two predominant types of responses. Thirty percent of the cells responded to odorants with activation of a CNG conductance. In contrast, in 55% of the ORNs, stimulation with odorants inhibited a voltage-activated K+ conductance (IKo). In terms of pharmacology, ion permeation, outward rectification, and time course for inactivation, IKo resembled a delayed rectifier K+ conductance. The effect of odorants on IKo was specific (only certain odorants inhibited IKo in each ORN) and concentration dependent, and there was a significant latency between arrival of odorants to the cell and the onset of suppression. These results indicate that indirect suppression of a K+ conductance (IKo) by odorants plays a role in signal transduction in mammalian ORNs.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Olfactory receptor neurons (ORNs) typically respond to odor stimulation with an increase in the frequency of firing of action potentials caused by a depolarizing odor-induced receptor potential (Getchell 1986). Depolarization of ORNs during stimulation with odorants is due to opening of second-messenger-gated nonspecific cation channels and the subsequent opening of Ca2+-activated Cl- channels (cf. Brunet et al. 1996; Fadool and Ache 1992; Firestein et al. 1991; Kleene and Gesteland 1991; Kurahashi 1990; Lowe et al. 1989; Nakamura and Gold 1987; Restrepo et al. 1990; Zufall et al. 1991). It is clear that, at least for certain odorants, stimulation of olfactory receptors elicits a G protein-mediated increase in cAMP concentration that in turn induces opening of cyclic nucleotide-gated nonspecific cation channels (CNG channels). There is evidence that other odorants open an InsP3-gated nonspecific cation conductance, but the precise role of this conductance as a mediator in olfactory transduction remains controversial (Schild and Restrepo 1998).

Although there is a consensus that the primary response of ORNs to odorants is mediated by second-messenger-activated cation channels, it is clear that, at least in amphibians and invertebrates, there is involvement of other conductances. In particular, odorants elicit opening of K+-selective channels leading to ORN hyperpolarization in lobster (Hatt and Ache 1994; Michel et al. 1991) and Chilean toad (Morales et al. 1994, 1995) ORNs. In lobster this effect is mediated by cAMP-gated K+ channels, whereas in Chilean toad the effect is mediated by opening of apically localized Ca2+-activated K+ channels. In addition, in newt (Kawai et al. 1997) and Necturus (Dionne 1992; Dubin and Dionne 1993) ORNs odorants inhibit voltage-activated K+ conductances thereby modifying the shape and frequency of firing of action potentials. These data indicate that the action of odorants on K+ conductances may play an important role in ORN function.

Although effects of odorants on K+ conductances have been demonstrated in amphibians and lobster, little is known about the modulation of K+ conductances by odorants in mammalian ORNs. In this paper we examined the effects of odorants on slowly inactivating voltage-dependent outward currents in rat ORNs. We show that odors block a K+ conductance with properties resembling a delayed rectifier. The effect of odorants appears to be specific (not all odorants elicit the response in a given ORN). Odorants also elicited an increase in K+ outward current in rat ORNs in a small fraction of the receptor cells.


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Solutions

Dissociation solution contained (in mM) 145 NaCl, 5 KCl, 2 EDTA, 20 HEPES, and 1 Na-pyruvate. Cells were maintained in standard Ringer (in mM): 145 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 20 Na-HEPES, 1 Na-pyruvate, and 5 glucose. During experiments the cells were perfused with standard Ringer without CaCl2 (bath solution). CaCl2 was omitted to abolish contributions by Ca2+-activated Cl- channels to the odorant responses (Kleene and Gesteland 1991; Kurahashi and Yau 1993). Unless specified in the figure legends, bath solution was used in all experiments shown. KCl Ringer solution was the same as bath solution except that all NaCl was replaced with KCl. The pseudointracellular solution contained (in mM) 110 K-aspartate, 36 KCl, 1 MgCl2, 1 CaCl2, and 10 K-HEPES. In some records potassium was replaced by cesium (indicated in the figure legends). All solutions had a pH of 7.2, adjusted with the hydroxide of the main cation and an osmolarity of 300 mOsmol. All salts, enzymes, and reagents were from Sigma (St. Louis, MO) unless otherwise stated.

The odor mixtures consisted of the following odorants (100 µM each) in bath solution. Mix A contained hedione, geraniol, phenethylalcohol, citralva, citronellal, eugenol, and menthone. Mix B contained lyral, lilial, triethylamine, ethylvanilin, isovaleric acid, and phenylethylamine. The mixtures A1 and A2 included two of the odorants of mix A, namely hedione and geraniol in mix A1 and phenethylalcohol and citralva in mix A2 (all odorants are a generous gift from Firmenich).

Isolation of rat ORNs

Isolated ORNs were obtained from the posterior part of the septum and the turbinates of both sides of the nasal cavity of male Sprague-Dawley rats. Animals were killed by exposure to 100% CO2, and the olfactory tissue was quickly removed. The tissue was cut in small pieces and incubated with dissociation solution containing 12 U/ml papain for 15 min. The suspension was triturated with a fire-polished Pasteur pipette (tip diameter ~1 mm), and Ringer solution with 1 mg/ml leupeptin (stop solution) was added. The cells were then filtered through a nylon mesh, and the solution was put onto a density gradient consisting of a bottom layer of Ringer solution containing 40% Percoll and a top layer with 20% Percoll solution. After centrifugation for 5 min at 500 times g, isolated ORNs could be harvested from the interface between the 40% and the 20% solution. Olfactory neurons were plated onto concanavalin A-coated micro cover glasses, kept in a moist chamber at room temperature, and could be subsequently used for up to 5 h.

Perforated-patch recordings and stimulation

Voltage-activated and odor-modulated currents were recorded under the perforated-patch configuration of the patch-clamp technique (Akaike and Harata 1994; Hamill et al. 1981; Zhainazarov and Ache 1995). We used gramicidin as ionophore. Gramicidin (2 mg) was dissolved in 33 µl DMSO as a stock solution. This stock solution (4 µl of it) was added to 1 ml pseudointracellular solution corresponding to a final concentration of 240 µg gramicidin in 1 ml solution.

The recording pipettes were pulled from Corning 7052 glass (World Precision Instruments, Sarasota, FL) using a vertical puller (Narishige, Japan) and were fire polished to a final resistance of 5-8 MOmega .

The tips of the pipettes were filled with gramicidin-free intracellular solution for ~0.5 mm and then backfilled with the ionophore-containing solution. Within 10-30 min after obtaining a GOmega -seal, access to the cell was gained as indicated by larger capacitative transients, a reduction in input resistance to ~0.5-2 GOmega , and the presence of voltage-activated inward currents in many of the ORNs on application of a voltage step from -80 to -30 mV. Voltage-dependent inward currents are not evident in the records shown in the figures in this manuscript because the sampling rate was slow enough to miss much of the transient inward current and because at a holding potential of -60 mV most of the sodium current was inactivated (Rajendra et al. 1992). To make sure that the measurements were performed under perforated-patch conditions rather than under whole cell conditions, we added 1 mM CaCl2 to the pipette solution (as suggested by Dr. Carol Deutsch, personal communication). High pipette calcium concentrations do not alter cytosolic Ca2+ because Ca2+ does not permeate gramicidin pores. Spontaneous rupture of the membrane leading to establishment of the whole cell configuration results in flooding of the cell with Ca2+, which is immediately noticed in electrophysiological measurements as a sudden decrease in membrane resistance.

The recording chamber was mounted on the stage of an inverted microscope (Zeiss IM 35). Currents were recorded under voltage clamp using a patch-clamp amplifier (Axopatch 200A, Axon Instruments, Foster City, CA). Records were low-pass filtered at 1 kHz (Bessel filter) and digitized at 2-10 kHz with a DigiData 1200 interface (Axon Instruments). Data were subsequently loaded into Origin (Microcal Software, Northampton, MA), which was used to perform the exponential fitting.

Stimuli were applied using a Pico Spritzer unit (General Valve, Fairfield, NJ) connected to a single-, triple-, or five-barrel pipette (WPI, Sarasota, FL), depending on the number of stimuli to be applied.


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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
REFERENCES

Suppression of a slowly inactivating voltage-activated K+ conductance (IKo) by stimulation of rat ORNs with odorants

Mammalian ORNs respond to cell depolarization with an outward K+ current comprised of rapidly inactivating (A type), delayed rectifier and Ca2+-activated K+ components (Liman and Corey 1996; Lynch and Barry 1991a-c; Maue and Dionne 1987; Okada et al. 1994; Trombley and Westbrook 1991). Although most rat ORNs express these three K+ conductances, the precise contribution of each of these conductances to depolarization-induced current varies from cell to cell. To determine the effect of odorants on the slowly inactivating component of the depolarization-induced outward current, we stimulated rat ORNs with odorant mixture A 1 s after a step change in membrane potential from a holding potential of -60 mV to potentials ranging from -120 to +80 mV (Fig. 1). Measurements were performed under perforated-patch conditions with pseudointracellular solutions containing either K+ or Cs+ in the pipette. As expected from the known effect of cAMP on CNG channels in rat ORNs (Lowe and Gold 1993), a substantial number of ORNs responded to the odorant mixture with activation of a nonspecific cation conductance with reversal potential near 0 mV (Fig. 1, A and B). The magnitude of the odor-induced current was estimated as the difference between the current at the peak or nadir minus the current before stimulation. This conductance could also be activated by addition of the cAMP phosphodiesterase inhibitor IBMX (1 mM) and by the adenylyl cyclase activator forskolin (20 µM, not shown) indicating that the odor-induced nonspecific cation current was mediated by opening of CNG channels. The nonspecific cation conductance was detected in ~30% of the ORNs tested with odorant mixture A (25 responses from a total of 86 cells).



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Fig. 1. Current responses of rat olfactory receptor neurons to stimulation with odorant mixture A. A and B: odorant mixture elicited a current that reversed near 0 mV in this rat olfactory receptor neuron (ORN). A: traces of currents during a step change in membrane potential. The membrane potential was stepped from a holding potential of -60 mV to various potentials (-120 to 80 mV) 256 ms after the beginning of acquisition. Pressure was applied to the stimulus pipette the time interval depicted by the solid bar. This figure is representative of 4 experiments with voltage pulses in the range from -120 to +80 mV and 3 experiments in the range from -60 to +80 mV. B: current-voltage relationship for the peak current elicited by odorants in the traces shown in A. C-E: odorant mixture A blocked the depolarization-induced outward current. C: examples of current traces for a step depolarization to +40 mV from -60 mV. Odorant mixture A or Ringer were applied during the time intervals depicted by the solid bars below the traces. Expanded time courses are shown below each trace. The dotted line is a fit of a single exponential decay to the current trace for the 1-s period preceding stimulation (tau  = 129 ms for mixture A and 131 ms Ringer). D and E: response of a different rat ORN to odorant mixture A after step changes in the membrane potential to holding potentials in the range from -120 to + 80 mV. D. Current traces. Figure is representative of 4 experiments with pulses in the range from -120 to +80 mV and 14 experiments in the range from -60 to +80 mV. E: current-voltage relationship for the outward current blocked by odorant mixture A. The magnitude of the current blocked by mixture A was estimated at each holding potential by subtracting the mean of the current during the last 200 ms of stimulation from the mean of the current calculated in the same time interval from the exponential fit to the current for the 1 s preceding stimulation. For the trace in C, this would correspond to the difference between the dotted line and the solid line in the bottom left panel. F: response of a different ORN to odorant mixture A after step changes in the membrane potential to +40 or -120 mV. Holding potential was changed from -60 mV to either +40 or -120 mV 1 s before odorant application. The voltage step protocols are shown in the insets. Solid lines are fits to a single exponential to the current decay before stimulation.

In contrast, in approximately one-half of the ORNs (47 of 86) odorant mixture A suppressed the depolarization-induced outward current (Fig. 1, C-E). Figure 1C shows the odorant-induced block in outward current in a cell stimulated with odorant mixture A 1 s after a step depolarization from a holding potential of -60 mV to +40 mV. Addition of the odorant mixture resulted in a decrease in the outward current significantly below the level predicted by a fit of the decay of the outward current with a single exponential for the 1 s before stimulation (dotted line). Stimulation of the cell with Ringer solution elicited no change in the time course of the decay of the depolarization-induced outward current (Fig. 1C, right). The difference in average current levels during the last 200 ms of stimulation between the exponential fit (dotted line) and the actual current trace (solid line) was used as a measure of the magnitude of the odorant-induced block of outward current. Figure 1D shows the odorant-induced current suppression for step changes in membrane potential from a holding potential of -60 mV to voltages ranging from -120 to 80 mV, and Fig. 1E shows the magnitude of the odorant-induced current block as a function of membrane potential. The conductance blocked by the odorants was outwardly rectifying in the positive range of membrane potentials and appeared to reverse at or near EK (approximately -80 mV under these conditions). However, the conductance suppressed by odorants at potentials negative to -80 mV was different from the conductance blocked at potentials positive to -80 mV evidenced by the fact that in some ORNs odor-induced suppression occurred only for voltage steps to potentials below -80 mV (Fig. 1F, representative of 3 ORNs). We also found one ORN that responded to odorant mixture A with a robust suppression of the outward current elicited by depolarization to +40 mV, but did not respond to the same odorant on hyperpolarization to -120 mV (not shown). The effect of odors on the inward current elicited by voltage steps to potentials more negative than -80 mV is likely to be an effect of odorants on an inward rectifier conductance. We did not further characterize this effect.

The current-voltage relationship shown in Fig. 1E resembles that of a delayed rectifier conductance. The K+ selectivity of the conductance was confirmed by a shift in reversal potential of the odorant-suppressed conductance to ~0 mV with replacement of K+ by Cs+ in the pipette (Fig. 2, performed in different cells), and by pharmacological experiments shown below. The channel is permeant to Cs+ with a relative permeability for Cs+ with respect to K+ of 0.03 calculated from the reversal potential (Fig. 2). This value is similar to the permeability ratio measured for the delayed rectifier in squid ORNs (Lucero and Chen 1997). We labeled this odorant-modulated K+ conductance IKo.



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Fig. 2. Odorant-induced suppression of voltage-dependent currents with K+ replaced by Cs+ in the pipette. A: traces of currents elicited by depolarization from a holding potential of -60 mV to potentials between -60 and +80 mV (protocol is shown in inset). Odorant mixture A was applied to the cell during the interval illustrated by the solid bar above the traces. B: current-voltage relationship for the current blocked by odorant stimulation. Notice reversal of the current near 0 mV. These current traces are representative of 4 experiments with Cs+ in the pipette.

In addition to the odorant-induced cation conductance and the odorant-suppressed K+ conductance described above, we also detected an odorant-induced increase in outward current in one ORN. This cell responded to stimulation with either mixture A (86 cells tested) or mixture B (21 cells tested) with an increase in the K+ outward current (not shown). Therefore, although suppression of the outward current was frequently observed, odorant-induced increases in outward current was observed once.

Suppression of IKo is elicited by only certain odorant mixtures in each rat ORN

To determine whether the odor-induced blockage of IKo in rat ORNs was a nonspecific response to all odorants, rat ORNs were stimulated with different odorant mixtures.

Figure 3 shows representative data from two experiments in which rat ORNs were stimulated with two different odorant mixtures. The stimuli (odorant mixtures A and B) contained odorants known to stimulate either cAMP (mixture A) or InsP3 (mixture B) formation in isolated rat olfactory cilia (Breer and Boekhoff 1991). Although three cells (from a total of 21 cells tested) responded to both odorant mixtures with suppression of depolarization-induced outward current (Fig. 3, A-D), a substantial number of cells (9 of 21) responded to mixture A, but not to mixture B (Fig. 3, E and F). The rest of the cells (9 of 21) did not respond to either mixture (not shown). The voltage dependence of the current suppressed by mixture B was similar to that of the current blocked by mixture A (Fig. 3D).



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Fig. 3. Response of 2 ORNs to stimulation with odorant mixtures A and B. Cells were held at -60 mV and depolarized to +40 mV. One second after the step depolarization the cells were stimulated with odorant mixtures A or B for the time depicted by the solid bar (protocol is shown in A, inset). A-D: this ORN responded to both odorant mixtures. A: stimulation with mixture A. B: stimulation with Ringer. C: stimulation with odorant mixture B. D: current-voltage relationship for the odor-suppressed current. , odorant mixture A; , odorant mixture B. E and F: this ORN responded to mixture A only. E: odorant mixture A. F: odorant mixture B. Smooth solid lines in A-C, E, and F are least-squares fit of a single exponential decay component to the current for the 1st second after depolarization.

In another series of experiments, we stimulated the ORNs with odorant mixture A, and with two subsets of the odorants constituting mixture A (mixtures A1 and A2, see METHODS for composition). All cells that responded to mixture A with suppression of IKo (5 of 8 cells tested) responded to either mixture A1 or mixture A2. Interestingly, although two of these cells responded to both A1 and A2, three cells responded to A2 only (not shown).

These experiments indicate that, unlike voltage-dependent currents in newt ORNs, those in rat ORNs respond to odorants with blockage of IKo in an odorant-specific fashion. That some ORNs respond to some odorants, but not others, suggests that the response is not a direct effect of the odorants on the voltage-dependent ion channels, but that it is receptor mediated.

Odor-induced suppression of IKO is concentration dependent

To obtain information on the concentration dependence of the suppression of IKo by odorants, we stimulated rat ORNs with a single odorant (citralva). Figure 4 shows the response of an ORN to puffs of odorant with stimulus pipettes containing 1, 10, or 100 µM citralva dissolved in Ringer. Because of the fact that there is a dilution of the odorant, the concentration of citralva at the cell was ~10-fold lower (estimated from OD measurements of dye diffusion from the stimulus pipette). As shown, the response is concentration dependent. Similar results were obtained in two other cells. All cells responded to 10 µM citralva in the stimulus pipette, one responded to 1 µM. The estimated K1/2 for the response was 2, 2.7, and 2 µM for the three different cells (corrected for dilution). These numbers should be taken as order of magnitude estimations because the concentration of the odorant at the cell was not estimated directly (Firestein et al. 1991) and because estimation of a K1/2 from three points is inaccurate. These experiments show that the odor-induced suppression of IKo is dose dependent in a concentration range that falls within the range of responsiveness to odorants determined for isolated olfactory neurons in other studies (Firestein et al. 1993; Sato et al. 1994).



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Fig. 4. Odorant-induced suppression of IKo is concentration dependent. Cells were held at -60 mV and depolarized to +40 mV. One second after the step depolarization the cells were stimulated with citralva dissolved in Ringer for the time depicted by the solid bar. Concentration of citralva at the stimulus pipette was 1 µM (A), 10 µM (B), and 100 µM (C). Solid line is best fit of an exponential decay function to the current decay before the odor pulse.

Onset of odorant-induced suppression of IKo occurs with a finite characteristic latency

The current resulting from the opening of CNG channels via an odor-induced increase in intracellular cAMP displays a significant latency (50 ms to 1 s, depending on the species) because the effect of odorants on CNG channels is indirect, involving several intermediate enzymatic and binding steps (Firestein et al. 1990, 1993; Kurahashi et al. 1994) and possibly because of the postulated suppression of the CNG channel by odorants (Kurahashi et al. 1994). In contrast, odorant suppression of voltage-gated conductances (Kawai et al. 1997) and CNG channels (Kurahashi et al. 1994) by odorants in newt ORNs are postulated to be direct effects of odorants on the channels and occur with very short latency (<20 ms). We have estimated the latency of the odorant block of the depolarization-induced outward current in rat ORNs. Latency was measured as the time difference between the onset of the current response to odorant mixture A and the response to puffs of KCl Ringer or IBMX (1 mM). KCl Ringer and IBMX were used to estimate the time of arrival of stimuli at the cell, which varied considerably from experiment to experiment because of differences in the diameter and shape of the stimulus pipette and its position relative to the cell. These stimuli can be used to determine the precise time of arrival of odorant solution to the cell because they elicit nearly instantaneous changes in current due to their direct effect on K+ channels (KCl) and cAMP phosphodiesterase (IBMX) (Firestein et al. 1990).

As expected for the indirect effect of odorants on CNG channels, the nonspecific cation responses to odorant mixture A occurred with a significant latency (875 ± 235 ms, mean ± SD, n = 3; see Fig. 5A). Note that the onset of the responses to K+ and IBMX occurred at the same time after application of pressure to the stimulus pipette. Odorant suppression of IKo also occurred with a substantial, but somewhat shorter, latency (274 ± 169 ms, n = 8; see Fig. 5B), suggesting that the odorant block of depolarization-induced outward current is not a direct effect of odorants on K+ ion channels. Occasionally ORNs responded to odor stimulation with both activation of the CNG nonspecific cation conductance and blockage of IKo (Fig. 6A, representative of responses in 9 cells). This provided an opportunity to compare the relative latency for these two processes. As shown in the figure, the onset of suppression of IKo appeared to precede the onset of the CNG channel response, as predicted from the latency measurements. Because the odor-induced block of IKo was not always complete by the time that the CNG conductance started to activate, the relative speed of the onset of the two responses could not be determined with absolute certainty in these experiments.



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Fig. 5. Latency of odor-induced current responses. A: latency of the odorant-induced nonselective cation conductance in a rat ORN in response to odorant mixture A. The latency was measured with respect to the onset of the response to a puff of either K+ or IBMX, which elicited immediate responses due to direct effects on K+ channels and phosphodiesterase, respectively. In this particular neuron the odorant-induced current occurred with a latency of 1,070 ms. This result is representative of 3 independent experiments. B: latency of the onset of the block of outward current in a rat ORN that responded to odorant mixture A with suppression of the depolarization-induced outward current. The latency for this particular cell was 102 ms. Figure is representative of 8 independent experiments. The membrane potential was stepped from -60 mV to +40 mV for both traces, and the voltage and stimulation protocols are shown in B, inset. Dashed line is drawn at the point where current begins to activate in response to stimulation with K+ or IBMX. Dotted line is drawn at the point where the current begins to respond to the odor mixture.



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Fig. 6. Tetraethylammonium (TEA) block of odorant-suppressed voltage-activated K+ conductance (IKo). A: whole cell current from an ORN depolarized to steady potentials between -60 and +80 mV (20-mV steps, see protocol in inset). The ORN was stimulated with odorant mixture A during the period depicted by the solid bar above the traces. This cell responded to odorant mixture A with a triphasic current consisting of a rapid suppression of the depolarization-induced outward current (IKo) and a somewhat slower activation of a nonspecific cation current (CNG conductance). Similar triphasic responses to odorant mixture A were obtained in 5 independent experiments. B: current traces under the same conditions as A except that 10 mM TEA was present in the bath. TEA inhibited IKo, but did not affect the CNG conductance appreciably. Figure is representative of 2 experiments at 10 mM TEA; addition of 2 mM TEA induced 50% inhibition of IKo in another independent experiment. C: Current traces at +40 mV holding potential are shown in an expanded time scale. Top trace is current before addition of TEA, and bottom trace is current in the presence of 10 mM TEA. Dashed line is placed at the time of onset of odorant-induced block of IKo, and dotted line is placed at the point of onset of odor-activated CNG current.

Inhibition of IKo by tetraethylammonium (TEA)

Selective inhibition of IKo by TEA provided the opportunity to more accurately test the relative latencies of these two odor-activated processes. The two types of response to odors could be dissociated pharmacologically by addition to the bath of the K+ blocker TEA, which at 10 mM abolished IKo (Fig. 6, A and B). Figure 6A shows records from a rat ORN that responded to stimulation with odorant mixture A with both suppression of IKo and activation of the CNG conductance. The current traces are triphasic at positive potentials due to the rapid onset of the suppression of IKo causing an initial decrease in outward current followed by stimulation of CNG channels causing an increase in outward current. The third phase is the return of the current trace to baseline following removal of the odorant mixture. In contrast, at negative potentials the current traces are biphasic because at potentials below the reversal potential for the CNG conductance (0 mV) suppression of IKo and stimulation of CNG channels both result in apparent inward current. As a result of addition of TEA, the current deflections elicited by the odorant mixture became biphasic at all membrane potentials with a reversal at 0 mV (Fig. 6B) as expected for odor-induced opening and subsequent closing of the CNG conductance, which was not affected by TEA. Addition of 2 mM TEA induced a partial (54 ± 9%, mean ± SE, n = 3) inhibition of IKo (not shown).

As indicated above, the onset of the odor-induced blockage of IKo and of the activation of the CNG conductance both occurred with a significant latency. The block of IKo, but not the CNG conductance by TEA, made it possible to estimate the relative latencies of these two processes. As shown in Fig. 6C, the onset of the block of IKo occurred before the onset of the activation of the CNG conductance.

Suppression of IKo by odorants is not mediated by an increase in intracellular cAMP

The odor-induced suppression of IKo occurred with a significant latency suggesting an indirect mechanism. One possibility is that the odorant-induced increase in cAMP mediates the block of IKo. To test this possibility, we stimulated rat ORNs with IBMX (1 mM, Fig. 7), which inhibits the cAMP phosphodiesterase, causing a rapid monotonic increase in intracellular cAMP concentration. Although IBMX activated a nonspecific cation conductance in 73% of the cells tested (8 of 11), it never elicited suppression of IKo (Fig. 5B, representative of 11 ORNs). The lack of suppression of the outward current by IBMX was not due to the absence of IKo in these cells because 6 of these 11 ORNs responded to odorant mixture A with suppression (Fig. 7B). This result rules out mediation of IKo suppression through a cAMP-dependent mechanism with a cAMP concentration dependence different from that of the CNG conductance. In this case, because the concentration of cAMP increases monotonically in response to IBMX, the mechanism with highest affinity for cAMP would be expected to dominate shortly after addition of IBMX, resulting in a biphasic response to IBMX. Suppression of IKo by a cAMP-dependent mechanism with nearly identical cAMP concentration dependence to that of the CNG conductance would still be consistent with the results shown in Fig. 7B, if the magnitude of the CNG current was always larger than the magnitude of the odorant-blocked IKo current. However, because the majority of ORNs possess a CNG conductance (73%), if suppression of the outward current were mediated by cAMP, it would be expected that a large fraction of the cells responding with suppression should also display a nonspecific cation conductance as well. This prediction is contradicted by our data showing that a large percent of ORNs respond to odorants with suppression of IKo in the absence of activation of a nonselective cation conductance (45%, n = 86; e.g., Fig. 1D). These results indicate that increases in cAMP concentration do not mediate the odorant-induced block of IKo. They are also consistent with the shorter latency for the onset of the odor-induced block of IKo compared with the latency for the odor-induced activation of the CNG conductance, because, if both block and activation were mediated by cAMP, latencies should be identical. Taken together, the lack of mediation of the odor-induced block of IKo by cAMP and the shorter latency for IKo suppression indicate that block of IKo by odorants is mediated by transduction events that take place before the production of cAMP (perhaps at the level of the G protein).



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Fig. 7. Response of a rat ORN to stimulation with IBMX (1 mM). A: current traces for holding potentials between -60 and +80 mV (20-mV steps, protocol is shown in inset). The cell was stimulated with 1 mM IBMX for the duration depicted by the solid line. Figure is representative of 8 independent experiments. B: stimulation of the same ORN with odorant mixture A elicited block of the depolarization-induced current. Pipette contained pseudointracellular solution with Cs+.

Ca2+-activated K+ channels do not contribute to the odorant-suppressed voltage-dependent K+ current (IKO)

To determine whether Ca2+-activated K+ channels contribute to IKo, we used pharmacological agents and varied the extracellular calcium concentration. ORNs were stimulated in the presence of charibdotoxin and apamin, inhibitors of large conductance (BK) and small conductance (SK) Ca2+-activated K+ channels, respectively (Sah 1996). We also examined the effects of Co2+, an inhibitor of voltage-dependent Ca2+ channels and 4-aminopyridine (4-AP), which blocks some K+ channels but does not inhibit Ca2+-activated K+ channels.

Addition of Co2+ would be expected to abolish any contribution by Ca2+-activated K+ channels activated by a rise in intracellular Ca2+ resulting from opening of voltage-dependent Ca2+ channels. As shown in Fig. 8C, Co2+ enhanced IKo.1 In addition, neither the BK channel inhibitor charibdotoxin nor the SK channel inhibitor apamin blocked the odorant-modulated outward current (Fig. 8, E and F), whereas 4-AP completely blocked IKo (Fig. 8B). These experiments indicate Ca2+-activated K+ conductances do not contribute to IKo.



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Fig. 8. Pharmacological tests for the involvement of Ca2+-activated K+ conductances in the odor-suppressed voltage-activated K+ current (IKo). A-C: current traces from an ORN (holding potential +40 mV) that responded to odorant mixture A with suppression of IKo. A: control. B: 10 mM 4-aminopyridine (4-AP) in bath. C: 10 mM Co2+ in bath. D-F: current traces for a different ORN. D: control. E: 1 µM apamin. F: 200 nM charibdotoxin. The pharmacological treatments were tested in the following number of independent experiments: 4-AP (3), Co2+ (3), apamin (1), and charibdotoxin (3).

To further examine the dependence of IKo on extracellular Ca2+, we stimulated ORNs in the presence of varying amounts of extracellular Ca2+. As shown in Fig. 9, IKo was only slightly dependent on extracellular Ca2+, with a decrease in [Ca2+]o from 1 mM to below 10 nM, eliciting no significant change in the magnitude of the odorant-suppressed current. This further supports the conclusion that IKo is not mediated by Ca2+-activated K+ channels.



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Fig. 9. Extracellular Ca2+ concentration ([Ca2+]o) dependence of suppression of IKo by odorant mixture A. Rat ORNs were depolarized from a holding potential of -60 mV to +40 mV, and cells were stimulated with odorant mixture A for an interval of 1 s. The extracellular medium was standard Ringer with varying amounts of [Ca2+]o spanning the concentration range from 1 mM to <10 nM (1 mM EGTA, no added Ca2+). A and B: current traces; the duration of the stimulus is depicted by the solid bar. A: 1 mM EGTA in bath. B: 1 mM CaCl2 in bath. A and B are representative of 5 independent experiments. C: average of the magnitude of the suppression of IKo eilicted by odorant mixture A in 3 independent experiments. Bars are means ± SE.

Steady-state inactivation of the transient component (IKA) does not affect odor-induced suppression of IKo

Lynch and Barry (1991b) have provided a thorough characterization of the transient (A-type) component of depolarization-induced K+ outward current (IKA) in rat ORNs. They found a rapid decay of IKA that could be described as the sum of two exponential components with average time constants of 22 and 143 ms. Although we did not examine this issue in detail, our data are generally in agreement with those reported by Lynch and Barry. In most ORNs tested in this study, depolarization-induced outward currents decayed during the first second following a single exponential component with time constants ranging from 40 to 600 ms (Fig. 10A). In ~10% of the cells the decay followed a two exponential component time course (Fig. 10B). In addition, a few cells did not display fast inactivation of the K+ current (Fig. 10C, representative of ~5% of the ORNs).



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Fig. 10. Effect of steady-state inactivation of the transient component of depolarization-induced outward current (IKA) on odor-suppressed outward current. A-C: examples of suppression of depolarization-induced K+ outward current by odorant mixture A in ORNs that displayed different behaviors for decay of transient component of the outward current. Cells were held at a potential of -60 mV and were depolarized to +40 mV. One second after depolarization, the cells were stimulated with odorant mixture A for the period depicted by the solid bar (protocol shown in A, inset). A: during the 1st second after depolarization, the decay of the transient component of the depolarization-induced outward current could be fit with a single exponential with time constant of 130 ms in this ORN. B: in contrast, in a different ORN the decay of the outward current was not adequately fit by a single exponential component. The fit shown in the figure is to 2 exponential components with time constants of 28 and 433 ms. C: this ORN did not display a fast (A-type) transient outward current. The fit shown is for a single exponential with a time constant of 11 s. D and E: steady-state inactivation of the transient (A-type) outward K+ current. These traces are representative of recordings in ORNs that were depolarized from a holding potential of -60 mV to prepulse potentials ranging from -60 to +30 mV. Prepulse duration was 2 s. Following the prepulse, the cells were stepped to a test potential of +40 mV. Odorant mixture A was applied to the cell from a micropipette by pressure ejection 1 s after the start of the test step depolarization. D: current traces for prepulses to -60, -30, and 0 mV. Notice that the fast transient component was abolished by the prepulse to 0 mV. E: prepulse potential dependence of the transient (a-d) and odorant-suppressed (c-b) components of the depolarization-induced outward K+ current. Inset: current levels used to estimate the magnitude of the transient and odor-suppressed components. The peak outward current (a) is saturated for prepulses in the range from -60 to -40 mV. Because of this, these points are shown in parentheses in the graph.

Because of our choice to apply stimuli 1 s after the depolarizing pulse (Fig. 8, A and B), most of the transient component of IK had already decayed before the onset of stimulation. This suggests that the suppressive effect of odorants described in this manuscript was mainly an effect on the sustained or slowly inactivating K+ outward current. To study this issue more carefully, we elicited steady-state inactivation of the transient component of the depolarization-induced outward current by subjecting the ORNs to a 2-s prepulse to different conditioning potentials from an initial holding potential of -60 mV. The cell was then stepped to +40 mV (Fig. 10, D and E). Consistent with the data of Lynch and Barry (Lynch and Barry 1991b), the transient component was inactivated by prepulses to potentials more depolarized than about -40 mV. In contrast, the magnitude of the odor-suppressed current was not significantly altered, suggesting that under our experimental conditions the suppressive effect of odorants was mainly on a sustained or slowly inactivating K+ current (IKo). The sensitivity of IKA to odorants was not assessed in our experiments.


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The data presented in this manuscript show that odorants suppress a voltage-activated K+ conductance in rat ORNs. Steady-state inactivation of the transient depolarization-induced outward current had little effect on the odor-suppressed K+ outward current (Fig. 10), indicating that there was not a substantial involvement of the fast-inactivating A-type K+ current in the observed odor suppression. In addition, pharmacological experiments (Fig. 8) and the lack of a substantial effect of removal of extracellular calcium (Fig. 9) precluded the participation of Ca2+-activated K+ channels in the response. Involvement of inwardly rectifying ATP-sensitive K+ channels (K-ATP) is ruled out by the data shown in Fig. 1E because K-ATP channels are inward rectifiers (Nichols and Lopatin 1997). The pharmacology, time course for inactivation, outward rectification, and ionic permeablity suggest that the odor-modulated K+ conductance (IKo) could be mediated by delayed rectifier channels. IKo activates at -50 mV and is sensitive to TEA (K1/2 ~2 mM) but is not inhibited by charibdotoxin or apamin. These characteristics are similar to those of some cloned delayed rectifier channels (Chandy and Gutman 1995).

There are two previous reports of suppression of K+ outward currents in ORNs. In newt ORNs, odorants suppressed voltage-dependent K+ currents in what appeared to be a nonspecific manner, in that all ORNs were affected by the odorants and the latency of the response was short (<20 ms) (Kawai et al. 1997). In contrast, the odor-induced suppression of IKo observed in the present study occurred with a significant latency (Fig. 5), was elicited by certain odorants in each ORN tested (Fig. 3), and was concentration-dependent in the physiologically relevant concentration range (Fig. 4). This indicates that odor-induced suppression of IKo is an indirect effect that could be receptor mediated. The odor-suppressed current characterized in this manuscript resembles the delayed rectifier current that is suppressed by the odorant taurine in mudpuppy olfactory neurons because that response is elicited by taurine in only some ORNs (Dubin and Dionne 1993). A similar stimulus-suppressed K+ current has been characterized in taste cells (Cummings and Kinnamon 1992). There are no reports of suppression of a K+ conductance in mammalian ORNs. It is not clear why this effect was not detected in previous studies with mammalian ORNs, but there are several potential reasons. 1) The odor-induced suppression of IKo could only be detected under perforated-patch conditions. Most measurements of odor responses have been previously done under whole cell conditions. 2) Usually ORNs are stimulated with odorants after a long (seconds) conditioning voltage pulse. Under these conditions the K+ current we report here would be largely inactivated. 3) Many measurements of odor-induced responses are performed with Cs+ in the pipette, which greatly reduces the magnitude of the odor-suppressed conductance.

Although we did not investigate the mechanism that mediates the odorant-induced suppression of IKo in detail, our experiments (Figs. 5-7) suggest that cAMP is not directly involved in this response. The latency for the odor-induced suppression of IKo was shorter than the onset for odor activation of the CNG conductance, suggesting that the effector(s) for the suppressive effect of odorants on IKo are upstream of the adenylyl cyclase, perhaps at the level of the G protein. Involvement of G protein alpha -subunits has been suggested in the muscarinic suppression of a delayed rectifier K+ current in rat ventromedial hypothalamic neurons, although it was not determined in those experiments whether the effects of the alpha -subunit were directly on the K+ channels (ffrench-Mullen et al. 1994). Alternatively, an unidentified second messenger eliciting actions faster than cAMP could mediate the suppression of IKo.

In summary, we have demonstrated that a majority of rat ORNs respond to odorant stimulation with suppression of a voltage-activated K+ current that resembles a delayed rectifier (IKo). It is likely that, as proposed for rat ventromedial hypothalamic neurons (ffrench-Mullen et al. 1994), depression of total K+ current by odorants would shift the cell into a prolonged excitable state and/or would result in prolonged duration of the action potential. However, the precise physiological role for suppression of IKo depends critically on whether a single odorant can simultaneously suppress IKo and activate the CNG channel pathway, and whether odorants suppress voltage-gated sodium channels (Kawai et al. 1997). We cannot answer this question at this time because, although we find that in some cells both processes were activated simultaneously, we do not know whether the two processes were activated by the same odorant because we stimulated the rat ORNs with odorant mixtures. If suppression of IKo and stimulation of CNG channels are activated simultaneously by a single odorant, suppression of IKo could play a role in enhancing depolarization via second-messenger-mediated pathways. This would result in lower thresholds and higher sensitivity for the system. However, if IKo and CNG channels were affected by different odorants in the same ORN, the physiological effect of suppression of IKo would be to affect quality coding (Ache 1994). Finally, if single odorants affect exclusively either IKo or CNG channels in each ORN, suppression of IKo could affect the basal rate of firing for the ORN. Such a response to odorants mediated exclusively by agonist effects on voltage-gated conductances without participation of a receptor potential have been observed in Necturus ORNs (Dubin and Dionne 1993) and have been termed "silent responses" (Dionne and Dubin 1994). Regardless of the precise physiological role for suppression of IKo, it is clear that apical stimulation of rat ORNs by odorants suppresses a voltage-activated K+ conductance. The fact that suppression of IKo is odorant specific and that it is faster when the apical compartments of the ORN are stimulated suggest that odor-induced suppression of IKo plays a physiological role in olfaction. Future work will define the physiological role of IKo in mammalian olfaction.


    ACKNOWLEDGMENTS

We thank Dr. Carol Deutsch for advice on the implementation of perforated-patch recordings with rat ORNs, Firmenich S.A. and Takasago Corporation for the gift of odorants, and Drs. Rona Delay and Angeles Ribera for comments on the manuscript.

This work was funded by Grant DC-00566 from the National Institute of Deafness and Other Communication Disorders.


    FOOTNOTES

Address for reprint requests: D. Restrepo, Dept. of Cellular and Structural Biology, University of Colorado Health Sciences Center, Campus Box B111, 4200 E. Ninth Ave., Denver, CO 80262.

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.

1 Because the mechanism for enhancement of IKo by Co2+ (Fig. 8C) is not central to the issues discussed in this manuscript, we did not study the effect of Co2+ in detail. However, the fact that addition of 1 mM Ca2+ to the bath solution (Fig. 9) also elicits enhancement of IKo suggests that Co2+ and Ca2+ might enhance IKo through the same mechanism. This enhancing effect of millimolar concentrations of Ca2+ and Co2+ added to the bath could be specific but may also be simply due to changes in the surface potential of the cell membrane.

Received 18 June 1998; accepted in final form 10 March 1999.


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0022-3077/99 $5.00 Copyright © 1999 The American Physiological Society