Sour Transduction Involves Activation of NPPB-Sensitive Conductance in Mouse Taste Cells
Takenori Miyamoto,
Rie Fujiyama,
Yukio Okada, and
Toshihide Sato
Department of Physiology, Nagasaki University School of Dentistry, Nagasaki 852-8588, Japan
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ABSTRACT |
Miyamoto, Takenori, Rie Fujiyama, Yukio Okada, and Toshihide Sato. Sour transduction involves activation of NPPB-sensitive conductance in mouse taste cells. J. Neurophysiol. 80: 1852-1859, 1998. We examined the sour taste transduction mechanism in the mouse by applying whole cell patch-clamp technique to nondissociated taste cells from the fungiform papillae. Localized stimulation with 0.5 M NaCl and 25 mM citric acid (pH 3.0) of the apical membrane enabled us to obtain responses from single taste cells under a quasi-natural condition. Of 28 taste cells examined, 11 cells (39%) responded to 0.5 M NaCl alone and 2 cells (7%) responded to 25 mM citric acid alone, indicating the presence of salty- and sour-specific taste cells. Ten cells (36%) responded to both NaCl and citric acid and 5 cells (18%) responded to neither salt nor citric acid. Amiloride reversibly suppressed NaCl-induced responses in mouse taste cells but not citric acid-induced responses. On the other hand, a Cl
channel blocker, 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB), reversibly suppressed all the citric-acid-induced responses. Most of the NaCl-induced current responses displayed an inwardly rectifying property, whereas all the citric-acid-induced responses displayed an outwardly rectifying property. The reversal potential for NPPB-sensitive component in citric-acid-induced current responses was
2 ± 7 mV (mean ± SE, n = 4), which was close to the equilibrium potential of Cl
(ECl), whereas the reversal potential for NPPB-insensitive component was 34 ± 8 mV (n = 4). The reversal potential of citric-acid-induced current responses (19 ± 8 mV, n = 4) was mostly present at the middle point between reversal potentials of NPPB-sensitive and -insensitive current components. In some taste cells, an inorganic cation channel blocker, Cd2+, suppressed citric-acid-induced responses, but an inorganic stretch-activated cation channel blocker, Gd3+, did not affect these responses. These results suggest that salt- and acid-induced responses were mediated by differential transduction mechanisms in mouse taste cells and that NPPB-sensitive Cl
channels play a more important role to sour taste transduction rather than amiloride-sensitive Na+ channels. However, the fact that the reversal potentials of citric-acid-induced responses had more positive than ECl suggests that Ca2+ or H+ permeable and poorly selective cation channels, which should be amiloride insensitive, may be activated by citric acid.
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INTRODUCTION |
It has been proposed that acid responses in amphibian taste cells are mediated by a block of apical K+ channels by protons (Cummings and Kinnamon 1992
; Kinnamon et al. 1988
; Sugimoto and Teeter 1991
), activation of proton-gated cation channels (Miyamoto et al. 1988
; Okada et al. 1994
) and proton-transporters (Okada et al. 1993
). In the works using the microelectrode impalement, Ozeki (1971)
reported that HCl as well as NaCl induced an increase in the membrane conductance during the generation of the depolarizing receptor potential in the rat, whereas Sato and Beidler (1982)
showed a controversial result in which the input resistance of rat taste cells increased during the generation of HCl-induced depolarizing receptor potential. The result of latter authors fits with the block of a K+ channel mediating the acid-induced response. However, there appears to be no apical K+ channels in mammalian taste cells (Avenet and Lindemann 1991
; Miyamoto et al. 1996
; Ye et al. 1994
), and K+ channel blockers have little effect on sour responses (Gilbertson et al. 1992
) in the hamster, whereas a K+ channel blocker, 4-aminopyridine, suppressed potassium and alkali salts-induced responses in the rat (Kim and Mistretta 1993
).
Recently, the contribution of amiloride-sensitive Na+ channels to sour-taste transduction has been postulated on the basis of permeation of protons through the amiloride-sensitive Na+ channel in taste cells of hamster fungiform papillae (Gilbertson et al. 1992
, 1993
). Amiloride inhibited the gustatory neural responses to both NaCl and HCl in hamsters and rats, but its inhibiting effect was restricted to the HCl response in sodium-selective nerve fibers carrying primarily information for salty taste (Hettinger and Frank 1990
; Ninomiya and Funakoshi 1988
). The hamster chorda tympani nerve responses to HCl are only slightly reduced by amiloride (Herness 1987
; Hettinger and Frank 1990
). The two-bottle preference tests show that the aversiveness of citric acid is reduced only partially by 30 µM amiloride (Gilbertson and Gilbertson 1994
). Thus the relationship between the amiloride-sensitive pathway of H+ permeation and sour-taste transduction is unclear even if there is a amiloride-sensitive pathway through which H+ can pass. In contrast, gustatory responses to HCl or citric acid in monkeys (Hellekant et al. 1988
) and humans (Ossebaard and Smith 1995
; Schiffman et al. 1983
; Tennissen and McCutcheon 1996
) were not affected by amiloride. It has been postulated in the rat that H+ does not flux through the apical membrane into taste cells but diffuses through tight junctions to transducer sites in the basolateral membrane of taste cells because chorda tympani responses to HCl barely displayed voltage sensitivity and were not inhibited by amiloride (DeSimone et al. 1995
). It also has been postulated that the potentiation of short-circuit current of the dog lingual epithelium induced by low pH results from Cl
efflux through amiloride- and ouabain-insensitive pathway (Simon and Garvin 1985
).
Most of the previous conclusions for salt- and sour-taste transduction have been based on either indirect lines of evidence (Avenet and Lindemann 1991
; DeSimone et al. 1995
; Gilbertson et al. 1992
; Mierson et al. 1996
; Simon and Garvin 1985
; Ye et al. 1994
) or the results obtained from the whole taste cell plasma membrane including the basolateral and apical receptive membrane (Doolin and Gilbertson 1996
; Gilbertson et al. 1993
). The purpose of the present study is to examine whether sour-taste transduction is mediated by amiloride-sensitive Na+ channels or other mechanisms in the mouse gustatory system. To obtain direct evidence for the mechanisms underlying sour transduction under the natural gustatory stimulation as much as possible, we applied a patch-clamp technique and a localized gustatory stimulation method to nondissociated taste cells from the taste buds in mouse fungiform papillae. Our data indicate that Cl
efflux through NPPB-sensitive pathways rather than H+ influx through amiloride-sensitive pathways plays an important role to sour-taste transduction in mouse taste cells. An abstract of these data has been published (Miyamoto et al. 1997
).
 |
METHODS |
Nondissociated cell preparation
Experiments were performed on nondissociated taste cells from the fungiform papillae of the mouse (C57BL/6 and BALB/c) using a combination of the whole cell patch-clamp recording technique and localized gustatory stimulation of apical receptive membrane (Miyamoto et al. 1996
). The mice were anesthetized by an intraperitoneal injection of pentobarbital (30 mg/kg) and killed by dislocating the cervical vertebra. After the tongue was removed quickly and washed with a normal extracellular solution (NES), 2 mg/ml elastase (Boehringer Mannheim) dissolved in NES was injected into the tongue (0.2-0.4 ml/tongue) from the cut end of the tongue. In this case, the tip of an injection needle was advanced carefully to the rostral end of the tongue along the space between the lingual epithelial and muscular layers. After the tongue was incubated in NES for 30 min at 26°C, the epithelial sheet was peeled free from the rest of the tongue. During incubation in NES, 50 µM amiloride (Sigma) was added to the bath to protect the amiloride-sensitive Na+ channels.
The epithelial sheet was pinned serosal side up in silicone-rubber molded on the bottom of a laboratory dish and was washed three times by a divalent cation-free extracellular solution containing 2 mM EDTA to exclude divalent cations in NES thoroughly and to prevent the extracellular solution from being acidified. An individual taste bud with an epithelial brim (Fig. 1) was obtained by sucking the fungiform papilla from its inside with a pipette (ca. 100 µm diam) after incubation in the divalent cation-free extracellular solution for 20 min in a refrigerator (4°C) and washing by NES three times. The remaining taste buds in the epithelium were stored in the refrigerator for another series of experiments.

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| FIG. 1.
Schematic drawing of localized multiple gustatory stimulations and whole cell recording from nondissociated mouse taste cells. Scale bar: 50 µm.
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Solutions
NES contained (in mM) 140 NaCl, 5 KCl, 1 MgCl2, 1 CaCl2, 10 glucose, 10 sodium pyruvate, and 10 N-(2-hydroxyethyl)-piperazine-N'-(2-ethanesulfonic acid) (HEPES)-tris (hydroxymethyl) aminomethane (Tris) (pH 7.4). Divalent cation-free extracellular solution did not contain CaCl2 or MgCl2 but 2 mM EDTA. A high K+ pipette solution (K+ solution) contained (in mM) 120 KCl, 2 MgCl2, 1 CaCl2, 11 ethyleneglycol-bis-(
-aminoethylether) N,N,N',N'-tetraacetic acid (EGTA), and 10 HEPES-Tris (pH 7.2). KCl in the K+ solution was replaced by 20 mM KCl and 100 mM K-gluconate in the low Cl
and high K+ intracellular solution (low Cl
solution) and 120 mM CsCl in the high Cs+ intracellular solution (Cs+ solution). A pipette solution containing 250 µg/ml amphotericin B (Sigma) (Rae et al. 1991
) was employed for obtaining perforated patches. There was no difference between results obtained by conventional whole cell recordings and by recordings with perforated patch. In a Na+-free extracellular solution (Na+-free solution), N-methyl-D-glucamine+ (NMDG+) was substituted for Na+. Citric acid (25 mM) dissolved in Na+-free solution (pH 3.0) was employed as an acid stimulus. The salt stimulus, 0.5 M NaCl, was dissolved in deionized water. Amiloride-containing solution was freshly made. A 100 mM stock solution of 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB, Calbiochem) was prepared in dimethylsulfoxide and was diluted by Na+-free solution immediately before use.
Electrical recording and data analysis
Pipettes were fabricated from borosilicate glass capillaries with microfilament inside (Clark Electromedical Instruments) with an electrode puller (Narishige, PP-83). The electrode tip was heat-polished using a microforge (Narishige, PF-83) so that the resistance was between 10 and 20 M
when filled with the K+ solution. Whole cell currents were measured with a patch-clamp amplifier (List, EPC-7), and the current signals were filtered at 1 kHz, digitized at 125 kHz, and analyzed by a pCLAMP (Axon Instruments) and Origin (Microcal) software. Averaged values of experimental data are expressed as means ± SE. Differences between means were tested using a paired t-test. All the experiments were performed at 22-24°C.
Experimental setting up
An experimental chamber containing an isolated taste bud with the epithelial brim was mounted on the stage of an inverted microscope with fluorescent phase-contrast optics (Olympus, IMT-2). The epithelial brim surrounding the taste bud was held by a pipette >200 µm in diameter (pipette for holding in Fig. 1), which was subjected to continuously applied negative pressure (~100 mm H2O) for keeping the orientation of a taste bud in the bathing solution so that the taste pore was placed left side of the field. After the whole cell configuration was established in a nondissociated taste cell with a patch pipette (patch pipette in Fig. 1), which also operated as another holding pipette, taste stimuli (0.5 M NaCl and 25 mM citric acid) were applied from the third and fourth pipettes for stimulation (stim. 1 and stim. 2 in Fig. 1) to the taste pore region by pressure ejection. The bathing solution was flowed continuously with a flow rate of 14 µl/s from the serosal side of the preparation to the mucosal side to prevent taste stimuli ejected through the stimulating pipettes from spreading to the basolateral membrane of taste cells. Thus a quasi-natural condition of gustatory stimulation was attained, although the apical and basolateral membranes were not completely isolated. Delivery of a taste stimulus was regulated by a microinjector system (Narishige, IM-200) with a pClamp software. Because the background flow was slowed down at the taste pore region due to the barrier of the epithelial brim, the stimulus concentration ejected by constant pressure was maintained for
10 s (see Fig. 2 of Miyamoto et al. 1996
).

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| FIG. 2.
Membrane potential changes evoked by citric acid and NaCl in mouse taste cells under current clamp. A and B: effects of 5 µM amiloride and removal of Na+-free solution on the membrane potential in an amiloride-sensitive cell (A) and an amiloride-insensitive cell (B). Cells were adapted to the normal extracellular solution (NES) at the beginning of the recording. C: responses to 25 mM citric acid (pH 3.0) (CA) and 0.5 M NaCl (Na) before, during, and after 5 µM amiloride application in the amiloride-sensitive cell (the same cell as in A). Bath, Na+-free solution; pipette, low Cl solution. Insets in this and other figures show configurations for stimulation, recording, and perfusion.
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RESULTS |
The response characteristics of 28 mouse taste cells to 0.5 M NaCl and 25 mM citric acid of mouse taste cells are summarized in Table 1. Taste cells responded to 0.5 M NaCl alone and 25 mM citric acid alone were 39 and 7%, respectively. Taste cells sensitive to both stimuli were 36% and taste cells insensitive to both stimuli was 18%.
Amiloride-sensitivity of NaCl-induced and citric-acid-induced responses
Amiloride (5 µM) added to the bath induced a hyperpolarization of 5-40 mV (21 ± 5 mV, n = 7) from the zero current membrane potential, which ranged from
10 to
27 mV (
14 ± 2 mV, n = 7), in amiloride-sensitive taste cells (Fig. 2A). Replacement of NES with Na+-free solution induced more hyperpolarization of 8-52 mV (25 ± 6 mV, n = 7) in these taste cells under the current clamp (Fig. 2A). Under the voltage clamp, stationary inward currents were blocked by amiloride or Na+-free solution in amiloride-sensitive cells. In amiloride-insensitive taste cells, amiloride barely affected the membrane potential under the current clamp as shown in Fig. 2B. However, replacement of NES with Na+-free solution induced hyperpolarization of 5-52 mV (28 ± 4 mV, n = 11) from the zero-current potential, which ranged from
10 to
44 mV (
21 ± 3 mV, n = 11). Application of 25 mM citric acid and 0.5 M NaCl to the amiloride-sensitive taste cells bathed in Na+-free solution elicited a depolarization of 10-30 mV (Fig. 2C, left). When 5 µM amiloride was added to Na+-free solution, the NaCl-induced responses were suppressed but the acid-induced responses were barely affected (Fig. 2C, middle). After the amiloride was washed by Na+-free solution, the NaCl-induced responses were recovered (Fig. 2C, right).
In amiloride-sensitive cells, the salt-induced response was completely (Fig. 2C) or partially (Fig. 3) blocked by 5 µM amiloride. In amiloride-insensitive cells, both NaCl- and citric-acid-induced responses were obtained and were not suppressed by 5 µM amiloride (Fig. 3). Of five taste cells responsive to both 0.5 M NaCl and 25 mM citric acid, NaCl-induced responses were suppressed in four cells by 5 µM amiloride, but any citric-acid-induced responses were not affected by amiloride. The same magnitude of inhibition of 0.5 M NaCl-induced responses was produced by 5 and 50 µM amiloride (data not shown), indicating that the magnitude of inhibition is maximal and the inhibitory constant (Ki) may be in the submicromolar range as reported in hamster (Gilbertson et al. 1993
) and rat taste cells (Doolin and Gilbertson 1996
). In the example of Fig. 2C, citric-acid-induced responses were much larger than NaCl-induced responses. However, in other taste cells, NaCl-induced responses were inversely larger than citric-acid-induced responses. Thus the magnitudes of responses induced by NaCl and citric acid were approximately the same as each other on an average as seen in Fig. 3.

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| FIG. 3.
Summarized effects of 5 µM amiloride on 0.5 M NaCl- and 25 mM citric-acid-induced responses. Left: 0.5 M NaCl-induced amiloride-sensitive responses (n = 6). Middle: 0.5 M NaCl-induced amiloride-insensitive responses (n = 4). Right: 25 mM citric-acid-induced responses (n = 5) including both amiloride-sensitive and -insensitive cells. Error bars: ±SE. Bath, Na+-free solution; pipette, low Cl solution.
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I-V relationships of citric-acid-induced current responses
An example of current response to 25 mM citric acid at the holding potential of
80 mV in an amiloride-sensitive taste cell is shown in Fig. 4A, where the pipette was filled with a Cs+ solution to eliminate the influence of K+ channel. I-V relationships evoked by a voltage ramp from
80 to 80 mV (100 mV/s) before (Fig. 4A, a) and during gustatory stimulation with 25 mM citric acid (Fig. 4A, b) are shown in Fig. 4B. In this taste cell, the reversal potential for the acid response was quite positive to 0 mV (Fig. 4B, b), and the I-V relationship shows an outwardly rectifying property. The I-V relationships of salt- and acid-induced current responses differed from each other; the I-V relationships of salt-induced currents showed an inward rectification (Miyamoto et al. 1996
), whereas those of the acid-induced currents showed an outward rectification (Fig. 4B).

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| FIG. 4.
Current responses evoked by 25 mM citric acid under voltage clamp. A: currents induced by 25 mM citric acid (CA) at a holding potential of 80 mV. B: I-V relationships of currents induced by the voltage ramps from 80 to +80 mV (100 mV/s) before (A, a) and during (A, b) 25 mM citric acid stimulation. Bath, Na+-free solution; pipettes, Cs+ solution.
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Suppression of citric-acid-induced responses by NPPB
Although amiloride did not affect the citric-acid-induced responses (Figs. 2C and 3), one of the Cl
channel blockers, NPPB applied to the bath reversibly suppressed the citric-acid-induced responses in all of 11 taste cells tested. Typical examples and the dose-response curve of NPPB-inhibition are shown in Fig. 5. The Hill coefficient was 1.4 and Ki was 12.0 µM. Averaged I-V relationships for the total current (b-a), NPPB-sensitive current (b-d), and NPPB-insensitive current (d-c) induced by 25 mM citric acid were obtained from the experiments as shown in Fig. 6A. The total current at negative potentials was approximately sum of NPPB-sensitive and -insensitive currents (Fig. 6B). In paired experiments with four taste cells, the reversal potential of total current induced by 25 mM citric acid (19 ± 8 mV) was close to the middle point between the reversal potentials for NPPB-sensitive current (
2 ± 7 mV), which was close to ECl (
4.6 mV) and that for NPPB-insensitive currents (34 ± 8 mV) (Figs. 6 and 7). In these experiments, Cs+ solution was employed as pipette solution to eliminate influence of K+ current. The 100 µM NPPB applied to the mucosal side only slightly suppressed the citric-acid-induced responses (Fig. 8A), whereas the NPPB applied to serosal side greatly suppressed the citric-acid-induced responses (Fig. 8B, n = 3).

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| FIG. 5.
Reversible suppression of the citric-acid-induced response by 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB). A: depolarizing responses induced by 25 mM citric acid before (top), during (middle), and after (bottom) application of 100 µM NPPB. Bath, Na+-free solution; pipette, K+ solution. B: dose-dependent suppression by NPPB of responses induced by 25 mM citric acid. Bath, Na+-free solution; pipette, low Cl solution. C: dose-response curve for NPPB inhibition of responses induced by 25 mM citric acid. Each point represents the average (±SE) of 3 ~ 5 cells. , fit of the data using a sigmoidal binding equation, with Ki of 12 µM and Hill coefficient of 1.4. Bath, Na+-free solution; pipette, low Cl solution.
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| FIG. 6.
Effect of NPPB on citric-acid-induced current responses. A: current responses induced by 25 mM citric acid (CA) with and without 100 µM NPPB applied to the bath. Holding potential: 80 mV. B: averaged I-V relationships (n = 4) of total (b-a, ), NPPB-sensitive (b-d, ), and NPPB-insensitive (d-c, ) currents estimated from currents evoked by voltage ramps from 80 to 80 mV (100 mV/s) at times a-d in A. Bath, Na+-free solution; pipette, Cs+ solution.
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| FIG. 7.
Comparison of reversal potentials for total and NPPB-sensitive and -insensitive currents. Number of taste cells tested was 4. Error bars: ±SE. Bath, Na+-free solution; pipette, Cs+ solution.
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| FIG. 8.
Localization of NPPB-sensitive conductance. A: slight suppression of responses induced by 25 mM citric acid immediately after an apical application of 100 µM NPPB for 20 s. B: large suppression of responses induced by 25 mM citric acid immediately after a basolateral application of 100 µM NPPB for 20 s. Bath, Na+-free solution; pipette, low Cl solution.
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An inorganic cation channel blocker, Cd2+, suppressed some citric-acid-induced responses, whereas an inorganic blocker of stretch-activated cation channel, Gd3+, did not affect citric-acid-induced responses (data not shown). In paired experiments with four taste cells, responses induced by 25 mM citric acid were reduced from 27 ± 5 mV to 19 ± 6 mV by Cd2+ though the suppression was not significant.
 |
DISCUSSION |
In the present experiments, no acid responses in mouse taste cells were suppressed by amiloride, which is an effective blocker of salt-induced responses in the mouse (Miyamoto et al. 1996
, 1997
) and the rat (Doolin and Gilbertson 1996
) taste cells. The I-V relationships of the acid-induced current responses showed an outward rectification in contrast to an inward rectification observed in the I-V relationships of salt-induced responses (Miyamoto et al. 1996
), although the reversal potentials for current responses induced by both salt and acid showed a similar value to each other. These results suggest that the transduction mechanism underlying the acid-induced response is different from the mechanism underlying the salt-induced responses in mouse taste cells. On the other hand, Gilbertson et al. (1993)
reported that citric-acid-induced responses were suppressed by amiloride in hamster taste cells. However, as indicated by recent reviews (Lindemann 1996
; Sato et al. 1994
; Stewart et al. 1997
), the amiloride-blockable sour transduction mechanism must be special in the hamster and does not contribute to the sour transduction in the rat.
The resting membrane potential of taste cells responding to 0.5 M NaCl and/or 25 mM citric acid was relatively low in NES in the present experiment. The mean resting membrane potential value (approximately
35 mV), which includes data (ranged from
10 to
85 mV) obtained from all taste cells regardless of responsivity to NaCl and/or citric acid, was similar to the previously reported values of the mean resting membrane potential (Herness and Sun 1995
; Miyamoto et al. 1996
). However, the value is lower than that obtained in adaptation of the apical membrane to deionized water (Furue and Yoshii 1997
). These facts indicate that taste cells responding to NaCl and/or citric acid have lower resting membrane potential than those not responding to NaCl and/or citric acid due to the existence of larger stationary conductances in the responsive taste cells. Both amiloride-sensitive and -insensitive cation conductances at the basolateral membrane as well as the apical membrane may be included in the stationary conductances, which play important roles for salty and sour transduction. The magnitude of depolarization induced by apically applied 0.5 M NaCl was lower than the magnitude of hyperpolarization induced by Na+-free solution, indicating that there is much larger stationary conductance to cation at the basolateral membrane than at the apical membrane. Furthermore, in the preliminary experiment, we found evidence of a contribution of Cl
conductance to salt transduction, which may reduce the depolarization induced by Na+ (Miyamoto et al. 1997
).
We found in the present experiment that citric-acid-induced responses of the mouse taste cells were suppressed effectively by the Cl
channel blocker, NPPB (Wangemann et al. 1986
). A contribution of Cl
channels to sour taste transduction already has been suggested in dog tongue epithelium (Simon and Garvin 1985
). However, apically applied different Cl
channel blockers, anthracene-9-carboxylic acid and diisothiocyanate-stilbene-2,2'-disulfonic acid did not affect citric-acid-induced action currents (Gilbertson et al. 1992
), so that it was concluded that Cl
channels may not be localized at the apical membrane. In the present experiment, NPPB applied to the basolateral membrane greatly suppressed the citric-acid-induced responses but apically applied NPPB only slightly suppressed the citric acid responses. This result suggests that most NPPB-sensitive Cl
channels localize at the basolateral membrane though the presence at the apical membrane is not excluded.
The reversal potential of acid-induced current responses was ~20 mV, which is not close to ECl (
4.6 mV); but the reversal potential of NPPB-sensitive current was closer to 0 mV than that of total current, suggesting that NPPB suppressed outwardly rectifying Cl
currents, as reported in human submandibular gland duct cell line (Ishikawa and Cook 1994
), but did not affect any other channels, such as nonselective cation channels or K+ channels, because the reversal potentials of those channels are expected to be more negative than
80 mV in adaptation to Na+-free solution with an assumption of no permeation of Ca2+ or H+ through nonselective cation channels. However, the reversal potentials of the total current induced by citric acid and the residual NPPB-insensitive current component are approximately +20 and +35 mV, respectively. This result suggests that the citric-acid-induced current may consist of NPPB-sensitive Cl
conductance and NPPB-insensitive cationic conductance. Therefore, sour-taste transduction is mediated by multiple pathways. Recently, it has been reported that not only nucleotide-gated cation channels but also Ca2+-activated Cl
channels contribute to generation of the odorant-induced current in vertebrate olfactory receptor cells (Kurahashi and Yau 1993
; Lowe and Gold 1993
) and that Xenopus olfactory receptor cells maintain high [Cl
]i (Zhainazarov and Ache 1995
). Therefore, a citric-acid-induced Cl
current could play an excitatory role in sour-taste transduction if high [Cl
]i also would be maintained in the mouse taste cells.
The reversal potential of the NPPB-insensitive component was approximately +35 mV. The stimulating solution, 25 mM citric acid, was dissolved in Na+-free solution containing (in mM) 1 H+, 5 K+, 1 Ca2+, 1 Mg2+, and 140 NMDG+ other than Cl
and citrate
. Among them, H+, K+, and Ca2+ in the bathing solution and Cs+ in the pipette solution are candidates for the charge carrier passing through nonselective cation channels if those channels exist. In cultured intestinal myocyte, the presence of poorly selective cation channels, which are equally permeable to K+, Na+, and Ca2+ but are blocked by intracellular Cs+, has been reported to exist (Nouailhetas et al. 1994
). Because the reversal potential of NPPB-insensitive component was +35 mV, pCs+/pCa2+ and pCs+/pH+ (or pK+) are calculated to be 0.002 and 0.001 with assumption that permeable ions are only Cs+, Ca2+, H+, or K+. Therefore the presence of amiloride-insensitive, poorly selective cation channels, which may trigger the sour-taste transduction, is expected at the apical receptive membrane of the mouse taste cells.
Using the microelectrode impalement, Ozeki (1971)
reported that HCl increased the membrane conductance during the generation of the depolarizing receptor potential in the rat taste cells, whereas Sato and Beidler (1982)
showed a controversial result in which the input resistance of rat taste cells increased during the generation of HCl-induced depolarizing receptor potential. Tonosaki and Funakoshi (1984)
observed both types of responses evoked by HCl in mouse taste cells using the microelectrode impalement. Recently Lyall et al. (1997)
reported that changes in external pH induced parallel changes in internal pH, suggesting that intracellular concentration of protons increases during sour stimulation and the protons may internally reduce stationary conductance in the plasma membrane of taste cells. Furthermore, external protons may directly block amiloride-sensitive Na+ channels of apical membrane at extremely low pH (2.0-3.0) as reported in Necturus gastric mucosa (Mustonen and Kivilaakso 1997
). Therefore the possibility that the pathway mediated by blocking channels with large open probability at the resting potential level contributes to sour taste transduction is not excluded at present.
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ACKNOWLEDGEMENTS |
This work was supported in part by a grant from the Human Frontier Science Program Organization of France and Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan.
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FOOTNOTES |
Address for reprint requests: T. Miyamoto, Dept. of Physiology, Nagasaki University School of Dentistry, 1-7-1 Sakamoto, Nagasaki 852-8588, Japan.
Received 16 March 1998; accepted in final form 1 July 1998.
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