Chorda Tympani Responses Under Lingual Voltage Clamp: Implications for NH4 Salt Taste Transduction

Mamoun A. Kloub, Gerard L. Heck, and John A. Desimone

Department of Physiology, Virginia Commonwealth University, Richmond, Virginia 23298-0551

    ABSTRACT
Abstract
Introduction
Methods
Results
Discussion
References

Kloub, Mamoun A., Gerard L. Heck, and John A. DeSimone. Chorda tympani responses under lingual voltage clamp: implications for NH4 salt taste transduction. J. Neurophysiol. 77: 1393-1406, 1997. Rat chorda tympani (CT) responses to NH4Cl, ammonium acetate (NH4Ac), and ammonium hippurate (NH4Hp) were obtained during simultaneous current and voltage clamping of the lingual field potential. Although functional and developmental similarities for gustation have been reported for NH+4 and K+ salts, we report here that significant differences are discernible in the CT responses to both salts. Unlike neural responses to KCl, those to NH4Cl are voltage sensitive, enhanced by submucosa negative and suppressed by positive voltage clamp. In this regard, NH4Cl responses are qualitatively similar to NaCl responses; however, the magnitude of NH4Cl voltage sensitivity is significantly less than that of NaCl. The concentration dependence of the CT response to NH4Cl manifests a biphasic nonlinear relationship not observed with KCl or NaCl. Below 0.3 M, the CT response increases as if to approach a saturation value. However, beyond 0.3 M an inflection appears in the CT-concentration curve because of an abrupt increase in CT responses. This kinetic profile is Cl- dependent and is correlated with an increase in transepithelial conductance that displays similar NH4Cl concentration dependence. The biphasic relation to salt concentration is not observed when acetate or hippurate is substituted for Cl-. As with Na+ and K+ salts, less mobile anions than Cl- (Ac- and Hp-) lower the CT responses. However, like Na+ salts, but in contrast to K+ salts, the onset kinetics of CT responses to NH4Ac or NH4Hp remain rapid, even under positive voltage-clamp conditions. Amiloride (100 µM) partially suppresses CT responses within the concentration range of 0.05-0.3 M (48-20% suppression). Amiloride also suppresses the voltage sensitivity of NH4Cl CT responses, but does not eliminate that sensitivity as it does for Na+ salts. In conclusion, the data suggest that taste transduction for NH4 salts is mediated over two NH+4 conduction pathways in the taste bud. This is especially evident with NH4Cl, where the CT-concentration curves show two distinct kinetic regimes. Below 0.3 M the saturation with increasing concentration, clamp voltage response dependence, and amiloride sensitivity suggest an apical membrane transduction conductance. Above 0.3 M, the high anion dependence of the response and its amiloride insensitivity indicate participation of the paracellular pathway in transduction.

    INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References

NH4Cl has been widely used as a reference stimulus in recordings from different levels of the taste sensory system in various animal models. These include cortical taste areas (Ogawa et al. 1994), the nucleus tractus solitarius (Hill et al. 1983; Nakamura and Norgren 1993), and the chorda tympani (CT) nerve (Elliott and Simon 1990; Hill and Phillips 1994; Hyman and Frank 1980; Somenarain et al. 1992; Ye et al. 1994). Animal (Erickson 1963) and human (van der Klaauw and Smith 1995) psychophysical studies have suggested similarities in the taste quality profiles of NH4Cl and KCl. In addition, functional similarities at the level of nucleus tractus solitarius for NH+4 and K+ salts have been suggested (Nakamura and Norgren 1993). NH+4 and K+ ions have similar hydrated radii and ionic conductances in free solution (Knepper et al. 1989). These ions may also have similar properties at the cell membrane level, e.g., NH+4 has been found to substitute for K+ on transporters in many cell types (Amlal et al. 1994; Kinne et al. 1986; Tsuruoka et al. 1993).

Developmental studies of salt taste in neonatal rats show that CT responses to NH4Cl are fully developed before those to NaCl (Hill et al. 1982; Mistretta and Bradley 1980; Yamada 1980). Recordings from more central loci in the taste neuraxis, in the nucleus tractus solitarius (Hill et al. 1983), and in cortical taste areas (Ogawa et al. 1994) are generally consistent with this. The different maturation rates for NaCl and NH4Cl neural responses suggest that taste receptors for NH+4 and Na+ are, at least in part, functionally distinct. A recent study indicates that K+ salt CT responses are mediated by a single, diffusion-controlled voltage-insensitive transduction mechanism (Ye et al. 1994). K+ salt taste responses occur via a sub-tight junctional transducer for K+ ions with access limited by anion mobility. In contrast, NaCl CT responses consist of a voltage-dependent (amiloride-sensitive) component and smaller voltage-independent (amiloride-insensitive) component (Ye et al. 1993). The amiloride and voltage sensitivity suggests that this Na taste transducing element is an apical membrane ion channel (Avenet and Lindemann 1991; Garty and Benos 1988). The amiloride-insensitive component depends on the presence of Cl- in Na salt taste (Elliott and Simon 1990; Formaker and Hill 1988; Ye et al. 1993).

The main objective of this study is to investigate the transduction mechanisms involved in NH+4 salt taste perception. This was accomplished by comparing CT responses to NH+4 salts, with the use of the in situ lingual voltage-clamp method, with the responses to Na+ and K+ salts. The results indicate the presence of two transduction mechanisms for NH4Cl: an apical NH+4 ion conductance, dominant with NH4Cl concentrations below ~0.3 M, and a mechanism accessible via the paracellular pathway. The latter is especially prominant in the presence of Cl- and with NH+4 concentrations >0.3 M.

Portions of this work were presented in abstract form at the 18th annual meeting of the Association for Chemoreception Sciences, April 1996, Sarasota, FL, and at the 7th International Ammoniagenesis workshop: Nutritional and Acid-Base Aspects of Amino Acid Metabolism, May 20-23 1996, Galway, Ireland.

    METHODS
Abstract
Introduction
Methods
Results
Discussion
References

Surgical preparation

The surgical procedure has been described in detail (Heck et al. 1989; Ye et al. 1993). Spague-Dawley rats weighing 180-240 g were preanesthetized with ether and then given an intraperitoneal injection of pentobarbital sodium (65 mg/kg). Additional injections were administered as needed during the experiment. Rats were placed on an isothermal pad to maintain their body temperatures. The trachea was cannulated, and the head was immobilized with a nontraumatic head holder (Erickson 1966). The left CT nerve was surgically exposed, cut caudally, and placed on a platinum electrode. Petroleum jelly was placed around the CT and a platinum reference electrode was positioned nearby.

Stimulation chamber and recording

The stimulation chamber allowed delivery of stimulus solution to a 7 mm diam section of the anterior tongue containing an average of 25 fungiform papillae (Miller 1976). Aliquots (3 ml) of stimulating and rinse solutions were injected into the chamber at 1 ml/s, and the solutions were kept in the chamber for 1 min. The whole CT neural activity was detected with a battery-operated differential amplifier (Ye et al. 1993). The amplified signal was recorded on a modified Toshiba DX-900 VCR, filtered by a band-pass filter (cutoff frequencies 40 Hz to 3 kHz), and fed to an oscilloscope. To obtain integrated CT responses, the signal was full wave rectified and integrated with a time constant of 1.0 s and displayed on a Linseis TYP 7045 strip chart recorder.

Current and voltage clamp

Transepithelial voltage or current clamp was maintained with a model VCC600 voltage-clamp amplifier (Physiological Instruments, San Diego, CA). An Ag/AgCl current-passing electrode and a voltage-sensing salt bridge were placed noninvasively beneath the tongue. A second Ag/AgCl current-passing electrode, positioned inside the stimulating inflow tubing, was in electrical contact with solutions at all times and acted as virtual ground. A second voltage-sensing salt bridge was placed in the chamber. The clamp could be operated in either voltage- or current-clamp mode. In voltage-clamp mode, the clamp drove sufficient current so that the differential voltage (Vvc) matched a programmed reference voltage. All voltages were referenced to the mucosal side, and the direction of positive current was taken as the direction of the cation flow from mucosa to submucosa. The potential at zero current clamp (Vcc) yielded the equivalent of an open circuit potential. The voltage-clamp values in our experiments were measured relative to the current-clamp potential (Vcc; Delta V = Vvc - Vcc). A periodic (15 s) bidirectional pulse of either 1 µA (current clamp) or 20 mV (voltage clamp) was generated to measure the transepithelial resistance and transepithelial conductance (TC).

Data analysis

Integrated CT responses were analyzed off-line as previously described (Ye et al. 1993). The area under the integrated CT response curve for 1 min from the onset of chemically evoked neural activity was used as the numerical value of an integrated CT response. Areas were calculated with the use of the computer software AutoCad (Autodesk, Sausalito, CA). To detect changes in the responsiveness of the CT, 0.1 M NaCl was applied at the beginning and end of each experiment as a reference. Only preparations that maintained a stable baseline throughout the experiment were used. A series of neural responses was included for analysis only when the initial and final NaCl responses differed by <20%. In any given experiment the initial/final NaCl response difference was used to correct all other responses by assuming that the rate of change in neural activity during the course of the experiment was linear. All responses for a given animal were normalized to that of 0.1 M NaCl. Phasic responses were observed to be sensitive to nonchemosensory factors, such as stimulus flow rate, and therefore were not analyzed in this study. In experiments with amiloride, 0.3 M KCl was used as a second reference, because NaCl CT responses are amiloride sensitive and recover slowly after repeated amiloride application (DeSimone and Ferrell 1985). To quantify the effect of the amiloride on NH4Cl CT responses, the normalized CT responses for a given concentration of NH4Cl were compared in the presence and absence of the drug, as illustrated
[NH<SUB>4</SUB>Cl]



[NH<SUB>4</SUB>Cl+amiloride]
 


1 min 
Rinse



1 min



Rinse

Statistics

Numerical results are expressed as means ± SE. Statistical significance was determined by paired Student's t-test or by one-way analysis of variance. P values < 0.05 were considered statistically significant.

Solutions and chemicals

STIMULUS SALTS. NaCl, KCl, NH4Cl, NH4Ac, and NH4Hp were obtained from Mallinckrodt Chemical (Paris, KY). All chemicals were reagent grade and were prepared in distilled water. A 1-min rinse consisting of 15 mM KHCO3 and 15 mM KCl (pH 8.3) preceded and followed each test stimulus. To maintain stable recording conditions, NaCl-depleted Krebs-Henseleit buffer was periodically applied to the tongue as an artificial saliva. The buffer consisted of (in mM) 6 KCl, 2 CaCl2, 1.2 MgSO4, 1.3 NaH2PO4, 25 NaHCO3, and 5.6 glucose, pH 7.5. Test stimuli had pH values between 5.1 and 7 depending on the stimulus and its concentration (NH4Cl, 0.05-0.6 M, pH 5.92-5.1; NH4Ac, 0.1-1 M, pH 6.5-7; NH4Hp, 0.1-0.5 M, pH 5.49-6). To test for a specific effect of pH on the CT response, 0.5 M NH4Cl solutions were adjusted to pH 5.1, 5.3, 5.6, and 5.8 with tris(hydroxymethyl)aminomethane base. A typical stimulation sequence was as follows: test 0; test I. . .test n; test 0. Here test 0 represents 0.1 M NaCl (used as a reference), and test I (I = 2,3, . . . . ,n) are particular stimuli under study. The stimulation sequence was run under current- and voltage-clamp conditions. The agents tested for their ability to block the CT response to NH+4 salts [amiloride, BaCl2, and amiloride 5-(N-methyl-N-isobutyl) (MIA)] were applied in the rinse and/or with the test salts.

    RESULTS
Abstract
Introduction
Methods
Results
Discussion
References

pH effects

As stated in the METHODS, changes in the NH4Cl concentration over the range of 0.05-0.6 M were accompanied by a monotonic decrease in pH from 5.9 to 5.1. We tested the effect of pH over this range with a fixed concentration of ammonium ion to determine the extent of contribution of pH to the observed CT response. The integrated CT responses to 0.5 M NH4Cl at pH 5.1, 5.3, 5.6, and 5.8 did not differ significantly from each other (data not shown). pH changes within this range are evidently not a factor in the magnitude of the ammonium response (see also DISCUSSION).

Voltage sensitivity of NH4Cl CT responses

Figure 1, top, shows the CT response to 0.1 M NaCl at zero current clamp and ±50 mV. In accordance with earlier results, positive voltage perturbation of the lingual epithelium caused marked suppression and negative voltage perturbation enhanced the CT response (Ye et al. 1993). Modulation of the CT response through voltage perturbation follows from the conduction properties of the amiloride blockable epithelial Na+ channels in taste cell apical membranes (Avenet and Lindemann 1991; Ye et al. 1993). In contrast, CT responses to 0.25 M KCl were insensitive to voltage perturbation (Fig. 1, middle). As previously reported (Ye et al. 1994), the absence of the voltage-dependent modulation of the CT response to KCl is probably caused by the absence of an apical membrane K+ transduction site. Figure 1, bottom, also shows a voltage-dependent modulation of the CT response to 0.2 M NH4Cl. Clamping at negative voltage enhanced the CT response; positive voltage suppressed it. In this regard, NH4Cl responses are qualitatively similar to NaCl responses, but the voltage sensitivity for NH4Cl is less than that for NaCl. The sensitivity of NaCl, KCl, and NH4Cl CT responses to voltage perturbation is apparent when expressed as the voltage sensitivity index (VSI), defined as
VSI = <IT>R</IT>(<IT>c</IT>, −50) − <IT>R</IT>(<IT>c</IT>, +50) (1)
where R is the CT response to a salt stimulus at a concentration c and clamp voltage V (here, ±50 mV). Table 1 shows that VSI for 0.1 M NaCl is significantly greater than for NH4Cl. Although the VSI for NH4Cl at a higher concentration increased (e.g., VSI for 0.2 M NH4Cl in Table 1), it never exceeded that for 0.1 M NaCl. VSI values for KCl were <= 0.1 and did not vary significantly with concentration (Ye et al. 1994). One can summarize the voltage sensitivity of NaCl, KCl, and NH4Cl as follows
VSI(NaCl, 0.1 M) > VSI(NH<SUB>4</SUB>Cl, <IT>c</IT>) > VSI(KCl, <IT>c</IT>) (2)


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FIG. 1. Integrated chorda typmani (CT) records of responses to 0.1 M NaCl (top), 0.25 M KCl (middle), and 0.2 M NH4Cl (bottom) at 0 current clamp and ±50 mV. NaCl and NH4Cl CT responses were obtained from the same animal, whereas KCl responses were from a different animal. Note the sensitivity of NaCl and NH4Cl and contrasting insensitivity of KCl CT responses to voltage.

 
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TABLE 1. VSI

CT responses and TC

A typical CT recording for a series of NH4Cl concentrations (0.05-0.5 M) under zero current clamp is shown in Fig. 2, bottom. The concentration-CT response relation shows an unusual nonlinearity. Below ~0.3 M the CT response increased with concentration in a manner suggesting saturation kinetics. However, above 0.3 M an abrupt increase in CT response appeared. In fact, this nonlinearity of the CT responses as a function of concentration was observed in every experiment in which a range of NH4Cl concentration was used. Although the concentration at which the abrupt increase in CT response was observed varied somewhat among animals, the main inflection point occurred ~at 0.3 M. Figure 3A shows a plot of a concentration-CT response function for three individual experiments and the mean responses for all experiments conducted (Fig. 3B). The biphasic character of the concentration-CT relation was a dominant feature in all animals studied. This also implies that a small change in one variable (NH4Cl concentration) produces a large nonlinear change in the other variable (CT response). In fact such an interactive correlation is a characteristic of a positively cooperative process (Fromm 1975). Further demonstration of this phenomenon can be obtained by the use of Scatchard plot analysis. Figure 3C shows a Scatchard plot over NH4Cl concentration ranging from 0.05 to 0.7 M. Analysis of this plot indicates the presence of two process: one describes the CT responses <0.7 (which corresponds to 0.3 M NH4Cl concentration), with a linearity suggesting a single Michaelis-Menten-type process in this region. The other process is characterized by a pronounced convex geometry (Hill type). The latter finding is considered to be diagnostic of positive cooperativity (Boeynaems and Dumont 1975; Fromm 1975). The rising phase of this convex is expected with the Hill coefficient >1, and the falling phase is also expected when the final saturation region has been reached. At saturation, a further increase in one variable (NH4Cl concentration) produces no change in the other variable (CT response), and therefore the ratio diminishes.


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FIG. 2. Typical experiment showing the integrated CT and voltage responses, under 0 current clamp, for a series of NH4Cl concentrations ranging from 0.05 to 0.5 M. Pulses superimposed on the potential response were used as a measure of resistance. Voltage pulses were in response to a periodic (15 s) bidirectional current pulse (1 µA). Note how the neural responses (bottom) appear to reach a saturation limit at 0.3 M. Note also the abrupt increase in the CT response above 0.3 M. These manifestations in the CT response are also seen in the transepithelial resistance (top) at the same concentrations of NH4Cl.


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FIG. 3. A: 3 individual experiments showing the normalized CT response as a function of NH4Cl concentration. Each experiment consists of a concentration series; the response to 0.1 M NaCl was assigned a value of 1 and all other responses were normalized to it. B: normalized mean CT responses as a function of NH4Cl concentration. Each point represents the mean ± SE (n = 6-12 except for the last 2 points, where n = 3). Note that in A and B CT responses exhibit biphasic profiles with a mean inflection point at ~0.3 M. C: Scatchard plot for NH4Cl concentrations ranging from 0.1 to 0.7 M. Note that the plot contain evidence of 2 processes: noncooperative and cooperative.

Monitoring changes in TC along with the CT response provides some insight into the origin of the unusual NH4Cl CT response changes with concentration. As shown in Fig. 2, top, the abrupt increase in CT response at >0.3 M coincides with a sudden decrease in transepithelial resistance (i.e., an increase in TC). Figure 4 shows a plot of the normalized concentration-conductance functions for three individual experiments (top) and the mean normalized conductance for all experiments analyzed (bottom). The similarities between the two functions (concentration-CT and concentration-conductance) suggest a correlation between TC and NH4Cl-evoked neural responses. We note, however, that the CT response function saturates at lower NH4Cl concentrations than the TC function. This probably reflects a limitation on further increases in CT response by the cellular processes that determine maximum rate of firing of taste nerves. The latter play little if any role in determining the conductance of transepithelial ion shunts.


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FIG. 4. Top: 3 individual experiments showing the normalized conductances (relative to 0.1 M NaCl) at 0 current clamp as a function of NH4Cl concentration. Bottom: normalized mean conductances at 0 current clamp as a function of NH4Cl concentration. Note the similarities of these conductance-concentration profiles with those of their CT response-concentration profiles.

The mean CT responses as a function of NH4Ac concentration under zero current clamp are shown in Fig. 5A. The NH4Ac neural response is nearly a linear function of concentration. The absence of the biphasic character in the CT responses (Fig. 5A) and in the normalized conductances for NH4Ac (Fig. 5B) suggests that these response differences are Cl- dependent; the biphasic response profile seen with NH4Cl was observed neither for NH4Ac nor for NH4Hp (not shown). The similarities between the functional profiles of TC and CT response for NH4Cl suggest that the increase in CT response at >0.3 M is caused by a significant increase in TC. Because the paracellular ion pathways comprise a major part of the TC, it would appear that the NH4Cl-induced conductance increase is a highly cooperative process that takes place when the concentration of NH+4 ion exceeds a critical value (see DISCUSSION). Achieving the critical NH+4 ion concentration is evidently coion limited, because the biphasic conductance is observed for NH4Cl but not for NH4Ac or NH4Hp. The obvious correlation of the respective TC changes with the CT responses for NH4Cl and NH4Ac suggests that transport of ammonium salts through the paracellular shunts plays a role in taste transduction for NH4 salts, especially for NH4Cl at concentrations >0.3 M.


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FIG. 5. A: normalized mean CT responses as a function of NH4Ac concentration (n = 4). B: normalized mean conductances at 0 current clamp as a function of NH4Ac concentration. Note the absence of the biphasic profile in the CT and conductance curves, in contrast to NH4Cl responses.

NH4Cl response under voltage perturbation

Analysis of the biphasic character of the concentration-CT response, Scatchard Plot, and concentration-conductance functions suggests the presence of two types of processes: a Michaelis-Menten type and a Hill type. On this basis, and on the basis of other data presented latter in this paper, we have constructed a model that describes the two regimes of the CT-concentration response function and each of its transformations (see APPENDIX). The theoretical lines in Fig. 6A are derived from that model, and when the transepithelial potential is considered, the same model describes the CT-clamped voltage function at constant NH4Cl concentration (Fig. 6B). Figure 6A shows the change in CT response for a series of NH4Cl concentrations under zero-clamp and ±50-mV voltage-clamp conditions. When the concentration series was run at +50-mV voltage clamp, CT responses were generally suppressed and biphasic responses, seen under zero current clamp, were not observed. At -50-mV voltage clamp, responses were enhanced as indicated by a shift of the whole concentration-CT response relation to the left along the concentration axis. The -50-mV curve also showed evidence of saturation at >0.4 M. A more detailed plot of the CT response as a function of clamp voltage is shown in Fig. 6B. Here we display the voltage dependence of the response for two concentrations, one lower (0.2 M) than the inflection concentration (0.3 M) in the biphasic response curve and one higher (0.5 M). At 0.5 M, negative voltage perturbation does not result in a significant increase in CT response, i.e., true saturation of the NH+4 CT response has been achieved at or slightly above 0.5 M under zero current-clamp conditions. At some lower concentrations, sufficient negative voltage perturbation can result in response enhancement up to the true saturation point, e.g., 0.4 M NH4Cl at -50 mV (Fig. 6A). In contrast to 0.5 M NH4Cl, CT responses at 0.2 M NH4Cl can be significantly enhanced by negative voltage perturbation. Between 0 and +50 mV, the decline in the CT response per unit change in voltage for 0.5 M NH4Cl exceeds that for 0.2 M NH4Cl for any voltage interval (Fig. 6B).


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FIG. 6. A: CT responses over NH4Cl concentration ranges from 0.05 to 0.5 M under 0 current clamp (bullet ) and -50-mV (black-square) and +50-mV (black-triangle) voltage clamp. Each experiment represents the mean ± SE (n = 6-12). The theoretical lines were obtained with the use of Eq. A5 (see APPENDIX). The best fit was obtained with the following parameter values: Ram = 1.17, Kam = 183 mM, Rpm = 1.10, Kpm = 435 mM, and n = 10.6. Additional parameters were added under voltage-clamp conditions: at -50 mV delta  = 0.25, gamma  = 0.12, n = 4; at 50 mV delta  = 0.25, gamma  = 0.14, n = 13.8. B: CT responses to 0.2 M NH4Cl (a concentration below the CT response inflection at 0.3 M NH4Cl, bullet ) and 0.5 M NH4Cl (a concentration above the CT response inflection at 0.3 M NH4Cl, black-triangle) under voltage clamp. Each point represents the mean ± SE (n = 6-12). Note that clamping at negative voltage enhances the CT response for 0.2 M NH4Cl, but not for 0.5 M NH4Cl, whereas clamping at positive voltage suppresses the CT responses for NH4Cl at both concentrations, with the largest suppression for 0.5 M NH4Cl. The theoretical lines were obtained with the use of Eqs. A5 and A6 (see APPENDIX). The best fit was obtained with the following parameter values: for 0.2 M NH4Cl concentration delta  = 0.25, gamma  = 0.19; for 0.5 M delta  = 0.25, gamma  = 0.13.

Anion modulation of NH+4 response

The CT responses to NH4Cl at 0.2 M exceeded those to NH4Ac and NH4Hp under zero current clamp at the same concentration (Fig. 7, top). The values of Vcc for NH4Cl were typically less than those for the same concentration of NH4Ac and NH4Hp (Fig. 7, top). Figure 7 also shows that the TCs for NH4Cl exceed those for NH4Ac and NH4Hp, consistent with the higher mobility of Cl- than Ac- or Hp-. Consistent with previous results for NaCl and KCl (Ye et al. 1993, 1994), the Cl- salt gave the highest CT response at zero current clamp among the NH+4 salts (Fig. 7, top). At sufficiently high negative voltage clamp (in this case -75 mV for 0.2 M salts; Fig. 7, middle), all responses were enhanced and differences were completely compensated. At +75 mV (Fig. 7, bottom) the response for NH4Cl was significantly suppressed, whereas those to NH4Ac and NH4Hp were not. This probably reflects the fact that 0.2 M NH4Ac and NH4Hp responses were small in current-clamp mode; the responses show measurable suppression at higher concentrations (not shown). At +75 mV, the Cl- salt continued to give the largest response, as it did under current-clamp conditions. In this respect the NH+4 salts behave like the Na+ salts; responses to Cl- exceed those to other anions. This is also true for K+ salts, but NH+4 and Na+ salt responses share similar temporal characteristics that are different from K+ salt responses. Both Na+ and NH+4 salt responses show a rapid phasic first component in their CT responses irrespective of the anion. However, large anions such as gluconate eliminate the phasic response to K+ salts altogether and overall have a more profound inhibitory effect (Ye et al. 1994).


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FIG. 7. CT and electrical responses to 0.1 M NaCl and 0.2 M NH4Cl, NH4Ac, and NH4Hp. Top: CT responses under 0 current clamp. Middle: responses obtained under voltage clamp at -75 mV. Bottom: responses obtained under voltage clamp at +75 mV. Current pulses were in response to a periodic voltage pulse (20 mV). Note the similarities to Na+ salt CT responses.

Effect of various channel blockers

BaCl2 (5 mM), MIA (1 µM), and amiloride (100 µM) were tested as possible inhibitors of CT responses to NH4Cl. The tongue was first rinsed for 1 min in a solution containing either BaCl2 or MIA, and then a stimulus solution of either 0.2 or 0.5 M NH4Cl containing the same test agent was applied. No significant differences in CT responses were observed between control presentations and BaCl2- or MIA-treated cases (data not shown). However, a significant reduction in CT responses was observed with amiloride treatment. Further investigation of the amiloride effect on NH4Cl CT responses revealed that it suppressed CT responses in a concentration-dependent manner. Figure 8 shows CT responses as a function of NH4Cl concentration at zero current clamp with and without amiloride (100 µM). The application of amiloride did not change the biphasic profile for NH4Cl CT responses. Furthermore, the inhibitory effect of amiloride was nonuniform over the two domains of the NH4Cl CT responses. The percent suppression decreased with increasing NH4Cl concentration, with the largest percent suppression at <0.3 M. The percent suppression rangedfrom 48% for 0.05 M to 4% for 0.5 M NH4Cl. As previously shown (Ye et al. 1993), the voltage-sensitive component of NaCl CT responses is amiloride sensitive, i.e., amiloride eliminated the voltage-dependent modulation of NaCl CT responses. Because NH4Cl CT responses were also voltage sensitive, we tested the effect of amiloride on the voltage-sensitive component of the NH4Cl CT response. Figure 9 shows the CT response to 0.2 M NH4Cl under voltage clamp with or without 100 µM amiloride. This concentration was chosen as a representative of the lower concentration domain of NH4Cl CT responses, where the amiloride sensitivity is higher. After the application of amiloride, the CT responses were significantly less voltage sensitive. However, amiloride treatment did not completely eliminate the voltage sensitivity of NH4Cl CT responses, as it does for NaCl CT responses (Ye et al. 1993). Figure 9 shows that the suppression of the CT response by amiloride is also voltage dependent. The percent suppression is larger at negative voltage than at zero current clamp or positive voltage clamp (see DISCUSSION). On the other hand, the application of amiloride did not significantly affect the CT response for 0.5 M NH4Cl under any voltage-clamp condition (Fig. 9). These results suggest that the voltage-sensitive component of the high concentration domain is amiloride insensitive.


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FIG. 8. CT responses as a function of NH4Cl concentration at 0 current clamp with or without 100 µM amiloride treatment. Each point represents the mean ± SE (n = 6). Note that the amiloride treatment suppresses CT responses in a concentration-dependent manner. Suppression decreases with increasing concentration, with the largest suppression at <0.3 M NH4Cl (see inset).


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FIG. 9. CT response to 0.2 M NH4Cl under voltage clamp with (black-square) or without (bullet ) 100 µM amiloride treatment (n = 6). Note that after the application of 100 µM amiloride, the CT responses become less voltage sensitive. Note also that suppression by amiloride is voltage dependent; % suppression is larger at negative voltage than at 0 current clamp or positive voltage clamp. CT response to 0.5 M NH4Cl under voltage clamp with (square ) or without (open circle ) 100 µM amiloride is also shown (n = 4). Note that amiloride treatment had little effect on the CT responses to 0.5 M NH4Cl under all voltage conditions.

Amiloride suppression of the CT response of NH4Ac under zero current-clamp condition was also observed (data not shown). Amiloride suppressed the CT response to 0.5 M by 33%. This is a higher percent suppression than was observed for 0.5 M NH4Cl (4%, Fig. 8). This is probably because NH4Ac responses show less of the relatively amiloride-insensitive higher concentration domain, i.e., 0.5 M NH4Ac responses appear qualitatively similar to the lower concentration domain of NH4Cl responses.

    DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References

Ionic transport of NH4Cl

Ammonium permeation across most cell membranes has been assumed to occur mainly by diffusion of uncharged NH3 through the lipid phase of the membrane (nonionic diffusion). Recently, however, cell types have been observed that acidify immediately on NH4Cl exposure and thus appear to be more permeable to the charged NH+4 than to the uncharged NH3 (Amlal et al. 1994; Burckhardt and Fromter 1992; Grandin and Charbonneau 1989; Hall et al. 1992; Kikeri et al. 1989; Lee and Steinhardt 1981; Wall and Koger 1994). Our data indicate that taste receptor cells (TRCs) have high permeability to NH+4 ion. Observations in support of this are as follows.

Changing the NH4Cl extracellular pH over the range encountered with NH+4 salt did not affect the neural response. This could occur if NH3 and NH+4 enter the taste cells at the same rate, thereby preserving the ratio of NH3 concentration to NH+4 concentration, keeping the intracellular pH constant. It is also possible that TRCs have a high-capacity buffering system that eliminates intracellular pH changes due to NH4Cl and thus any pH-mediated CT responses. If TRC permeability to NH3 were significantly greater than that to NH+4, NH4Cl exposure ought to result in transient alkalinization and NH4Cl removal in acidification (Roos and Boron 1981). However, preliminary studies by Lyall et al. (1996) on isolated TRCs show that NH4Cl exposure causes little change in intracellular pH, suggesting that NH+4 enters TRCs at least as fast as NH3. In addition, we show here that apical application of MIA, a potent inhibitor for the Na+/H+ exchanger, had no effect on NH4Cl CT responses. This eliminates the direct involvement of the apical MIA-sensitive Na+/H+ exchanger in NH4Cl taste transduction. The voltage and amiloride sensitivity of NH4Cl CT responses are similar to those of NaCl responses. This suggests that ammonium is also probably entering the TRCs in the ionic form, because both manipulations (voltage perturbation and amiloride application) exert their effects on ion transport through various conducting pathways.

Two transduction mechanisms for NH4Cl

The possibility that NH4Cl transduction involves more than a single mechanism was suggested by Beidler (1961) on the basis of the nonlinearity of the Scatchard plot of the CT response over a range of concentration. Beidler concluded that NH4Cl taste sensing involved both a high-affinity/low-capacity receptor site and a low-affinity/high-capacity site. Our data support the concept of two NH+4 taste receptor mechanisms. This is immediately suggested by the biphasic nature of the CT response curves as a function of the NH4Cl concentration (cf. Fig. 3, A and B) and by Scatchard plot analysis (cf. Fig. 3C). The CT response-concentration curve for NH4Cl can be divided operationally into low and high concentration domains with the concentration at the inflection point (0.3 M) chosen as a convenient boundary.

LOW CONCENTRATION DOMAIN (<0.3 M). Below 0.3 M NH4Cl, responses appear to be approaching a saturation limit with a half-maximal concentration of ~0.15 M (cf. Fig. 3B), and amiloride significantly inhibited the NH4Cl CT response (cf. Fig. 8), both similar to NaCl responses (Ye et al. 1993). The CT responses in both concentration domains were affected by perturbations in lingual field potential (Fig. 6A). However, the effect of amiloride on the voltage sensitivity is different in the two domains. Amiloride significantly reduced the enhancing effect of negative clamp voltage on the response to 0.2 M NH4Cl (Fig. 9), again reminiscent of the effect of amiloride on the NaCl response. In contrast, amiloride had practically no effect at 0.5 M NH4Cl at negative clamp voltage (Fig. 9). There are two possible sources of voltage sensitivity in the CT response. The first, as with Na+, is that NH+4 ions enter taste cells through apical membrane ion channels. A second possibility is that NH+4, like Na+ and K+, can be transported by electrophoresis through the paracellular shunts. Of course, these are not mutually exclusive, as shown by voltage-clamp studies on the NaCl response (Ye et al. 1993, 1994). This appears also to be true for NH4Cl responses, but with different fractions of the response attributable to each pathway compared with NaCl and KCl. This becomes clear in a comparison of the effects of amiloride on the NH4Cl CT response at different clamp voltages (Fig. 9). Amiloride suppressed the CT response of 0.2 M NH4Cl at -50-mV voltage clamp by 35% (cf. Fig. 9), consistent with a partial blockage of an apical membrane conductance. On the other hand, amiloride did not significantly suppress the CT response to 0.5 M NH4Cl under the same conditions (cf. Fig. 9). This suggests that an amiloride-sensitive apical membrane conductance is not a major factor in the NH4Cl response at high concentration, i.e., the voltage sensitivity represents mainly paracellular electrophoresis of NH+4 ions.

A comparison of these results with NaCl under the same conditions shows that at 0.2 M, amiloride suppressed ~80% of the response at -50 mV and ~75% at 0.5 M (Ye et al. 1993). It seems reasonable to conclude that an amiloride-sensitive apical membrane conductance plays a role in gustatory transduction for NH+4 ions in the low concentration domain. However, the affinity of the NH+4 binding site for amiloride is much less than affinity of the Na+ site for this blocker. Whether the different binding affinities for amiloride reflect a population of two channel types, one Na+ conducting and one NH+4 conducting, or different amiloride binding domains on a single channel is unclear. However, the epithelial sodium channel appears to have a single voltage-dependent amiloride binding affinity (Benos 1982; Kleyman and Cragoe 1988; Palmer 1986). Amiloride blockage of the Na+ conductance of taste cells (Gilbertson et al. 1993) and the NaCl CT response (Ye et al. 1993) is fully consistent with this model. Thus it is unlikely that the taste cell Na+ conductance has dual amiloride binding sites. A further possibility is the presence of channels inhibitable by amiloride in a voltage-dependent manner but not strictly Na+ selective. Light et al. (1988) have described an amiloride-sensitive cation channel with these properties in the apical membrane of cells from the inner medullary collecting duct of the kidney. If there is a similar channel in rat taste cells, it must be more selective for NH+4 than to Na+ so that it plays a significant role in NH+4 but not Na+ transduction. In contrast, a nonspecific inhibition by amiloride of canine CT responses to various salts, including NaCl and NH4Cl, has been reported (Nakumara and Kurihara 1990). This suggests that a common transduction pathway is utilized for Na+, NH+4, and other cations in the dog, indicating the involvement of a nonspecific cation channel in the transduction mechanism of these salts for the dog. However, in the rat, the rates of recovery from amiloride inhibition of the NaCl and NH4Cl CT responses show that NaCl response recovery lags that of NH4Cl recovery (unpublished observations). This makes it unlikely that, for the rat, recovery represents the time course of amiloride dissociation from and consequent increase in the conductance of a single channel species.

The major differences observed in rat Na+ and NH+4 CT responses are fully consistent with single-unit recordings (Erickson et al. 1980) and behavioral studies (Nowlis and Frank 1981). Although most of the information the rat receives about Na+ is through amiloride-sensitive ion channels (Hill et al. 1990; Spector et al. 1996), the larger part of the NH+4 response is amiloride insensitive. In this respect the CT responses of K+ and NH+4 show greater similarity to one another. It is perhaps, therefore, not wholly unexpected that single-unit recordings and behavioral studies involving KCl and NH4Cl show a high degree of similarity. Yet, certain differences between KCl and NH4Cl responses have also been noted in single-unit recordings and psychophysical studies. CT single units responsive to both K+ and NH+4 generally responded more vigorously to NH+4 (Erickson et al. 1980). In human psychophysical studies NH4Cl responses are of higher intensity and salty quality than KCl responses (van der Klaauw and Smith 1995). These differences are consistent with our findings that NH4Cl whole nerve responses are generally greater than those to KCl. Yet, evidence suggests that both salts are similar in that a major part of their responses involves transduction mechanisms depending on ion transport across paracellular shunts in the taste buds. Thus it seems that both similarities and differences as seen in the periphery for NH4Cl and KCl responses are translated during taste encoding.

HIGH CONCENTRATION DOMAIN (>0.3 M). Figure 3B shows that CT responses more than double in magnitude between 0.3 and 0.5 M NH4Cl after appearing to approach a saturation limit with increasing concentration at <0.3 M. This is an unusual CT response-concentration function, thus far observed only for NH4Cl. Insight into the origin of the increased CT responses at >0.3 M is obtained from the fact that the TC of NH4Cl, measured simultaneously with the CT response, showed a similar biphasic form (Fig. 4). Because the TC primarily reflects the ionic traffic across the relatively lower-resistance paracellular shunt (DeSimone et al. 1984; Holland et al. 1991; Simon et al. 1988), the increased CT responses are probably related to increased NH+4 transport through the shunts. Involvement of the shunts is also indicated by the amiloride insensitivity of the NH4Cl response in this concentration range (cf. Figs. 8 and 9) and by the anion dependence of the increased responses at >0.3 M, i.e., the highly mobile Cl- ion is obligatory. As seen in Fig. 5, NH4Ac shows neither the biphasic pattern in CT response nor TC. The higher Cl- permeability implied by the higher TC of NH4Cl than NH4Ac suggests that a major part of NH+4 taste transduction can occur in the taste cell submucosa. The relative contribution of submucosal sites apparently depends on their accessibility. This in turn depends on the ability of the salt stimulus to permeate the tight junctions. This appears to be an anion diffusion-controlled process for the NH4 salts, much as it is for the Na and K salts (Ye et al. 1994). Relative ion permeability of tight junctions in leaky epithelia indicates that permeability decreases with increasing molecular size (Moreno and Diamond 1975). This would make the relatively smaller Cl- ion more efficient than the larger Ac- or Hp- ions in providing NH+4 access to the submucosa, accounting for the decreased CT response on substitution for Cl-. In this respect the effect of anion substitution for NH+4 salt stimuli resembles that already demonstrated for Na+ and K+ salts (Elliot and Simon 1990; Rehnberg et al. 1993; Ye et al. 1993, 1994), where anion effects are mainly paracellular (Elliot and Simon 1990; Harper 1987; Ye et al. 1991, 1993, 1994).

Positive cooperativity in the TC and CT response

The CT response-concentration curves for Na+ and K+ salts have slopes that decrease monotonically with increasing concentration, approaching zero in the limit (Ye et al. 1994). The CT response concentration for NH4Cl is uniquely different. As seen in Fig. 3B, the slope begins by decreasing, but at ~0.3 M it increases sharply again. The same behavior is observed in the TC for NH4Cl, suggesting that the critical events responsible for this NH+4-induced increase in NH+4 response take place in the paracellular shunts. The necessary conditions that seem to be required for this autoaccelerating process are 1) the presence of NH+4, 2) a critical concentration, and 3) the presence of Cl-.

The Cl- requirement may derive from its higher mobility among the anions studied. Because the rate of diffusion through the cation exchanger shunts is controlled by the anion, Cl- will confer the highest mobility to NH4Cl. This probably allows NH+4 ions to achieve the requisite critical concentration in the controlling paracellular region (probably the tight junctions). The question becomes: what does the critical NH+4 concentration do to cause an increase in NH+4 conductance? Some insight into this is gained from the work of Moreno and Diamond (1975) on the ion permeability of gallbladder tight junctions. Those studies indicate that a cation's permeability increases with its proton donor ability. Hydrogen bonding solutes (such as NH+4) in an hydrogen bonding microenvironment behave as if they were smaller than their van der Waals radii and therefore smaller than similar-sized cations without hydrogen donor ability. Other cases of a strong correlation between hydrogen bond formation and ion permeability are well established (Hille 1971).

Although the composition of the dense fibrous material of tight junctions is not yet fully characterized (Madara 1992), their ion exchanger properties are well established. When enough hydrogen bonds have been formed in the controlling paracellular regions, i.e., when the local NH+4 concentration is at a critical level, a conformational change resulting in increased NH+4 permeability may occur; thus the increase in TC. Although there is no direct evidence that a conformational change occurs in tight junctional protein, some observations on lingual epithelia support the concept of modulation of TC by ion exchange reaction (DeSimone et al. 1995). The junctional polyelectrolytes may normally be relatively condensed because of Ca2+ ions. If an ion exchange reaction can occur in which NH+4 ions displace Ca2+ ions, the polyelectrolyte structures could expand (Katchalsky and Oplatka 1971). In fact, they may undergo sudden expansion when they interact with particular electrolytes at a critical concentration. Such structural transformations often resemble phase transitions, or highly cooperativeorder-disorder transitions (Flory 1956; Katchalsky andOplatka 1970).

Chloride versus acetate or hippurate

The slope of the CT response for NH4Cl decreased markedly at >0.5 M, where the response approaches a second plateau (Fig. 3B). This is the system's maximal NH+4 response intensity; responses to 0.5 M NH4Cl at negative voltage clamp were not significantly larger (cf. Fig. 6A). Figure 5A shows that NH4Ac response was still only 50% of the system's maximal NH+4 response at 1 M. As might be expected, these submaximal NH+4 responses can be increased significantly under negative voltage clamp (unpublished observations). The second plateau in the NH4Cl response probably reflects a saturation in the NH+4 transduction mechanism below the tight junctions, as a consequence of NH+4 ion reaching a saturating concentration in the intercellular space. This probably explains the ineffectiveness of negative voltage perturbation in increasing the response to 0.5 M NH4Cl beyond its current-clamp value. On the other hand, as little as 25-mV positive voltage clamp suppressed the response by 30% (cf. Fig. 6B). This decline reflects, in turn, a decline in the influx of NH+4 ions in the current and probably the decline in NH+4 ion concentration in the tight junctions below the critical value required for the TC to be increased. Both effects acting in concert will significantly reduce the NH+4 ion available to the submucosal transduction mechanism. A 25-mV positive perturbation has a smaller, graded effect on the CT response to 0.2 M NH4Cl (Fig. 6B). This may be because paracellular transport may not involve cooperative processes under these circumstances. The failure of acetate or hippurate to evoke the paracellular cooperative process required to increase TC means that, for these anions, a smaller proportion of the net NH+4 CT response will occur through paracellular NH+4 transport. A larger proportion should occur through the apical membrane transduction route. Consequently a higher proportion of the NH4Ac response at concentrations >0.3 M should be amiloride sensitive relative to NH4Cl. The results support this expectation. This is further confirmation of our conclusion that, as with Na salts, NH4 salt taste transduction involves an NH+4 conducting channel in the taste cell apical membrane and a submucosal transduction site accessible through the tight junctions.

Membrane conductive pathways for NH+4 ion

The search for an NH+4 ion conducting channel has been and continues to be concentrated in a system in which ammonium (NH+4 and NH3) plays an important role in maintaining the acid-base balance, the nephron. Several lines of evidence, in different segments of the nephron, have shown that NH+4 may be accepted by the K+ site of the luminal Na+-K+-2Cl- cotransport system, the basolateral Na+/K+ATPase (Kinne et al. 1986), the K+/H+ antiport system (Amlal et al. 1994), and the Ba2+-sensitive K+ channel (Tsuruoka et al. 1993). On the other hand, there is evidence for NH+4 conductances distinct from K+ conductances (Bichara et al. 1990; Burckhardt and Fromter 1992; Tsuruoka et al. 1993; Volkl and Lang 1991). Amiloride-sensitive pathways mediating NH+4 ion conductance have been reported (Amlal et al. 1994; Light et al. 1988; Tsuruoka et al. 1993; Volkl and Lang 1991). In the sensory epithelia, the ionic conductance for the guanosine 3',5'-cyclic monophosphate-gated channels in the rod outer segment is 3 times as high for NH+4 ion as for Na+ or K+ ions (Kaupp et al. 1989). The higher ammonium conductance is also observed through the adenosine 3',5'-cyclic monophosphate-gated channels in olfactory receptor neurons (Balasubramanian et al. 1995). Why might NH+4 have a higher conductance than the usual cations present in a particular physiological system, especially if NH+4 has no functional and homeostatic role in that system? The functional importance of an ammonium conducting channel in the taste system is not immediately apparent. It is unclear whether this conductance functions as a specific sensor for NH+4 ion or whether ammonium happens to be a probe ion for a taste cell conductance of as yet unknown physiological significance.

In conclusion, the data are consistent with the presence of two transduction pathways for NH4Cl in taste cells: an apical NH+4 ion conductance and one accessible via the paracellular pathway. The latter is especially dominant in the presence of Cl- and when NH+4 concentration exceeds0.3 M.

    ACKNOWLEDGEMENTS

  This research was supported by National Institute of Deafness and Other Communications Disorders Grant DC-00122 to J. A. DeSimone and G. L. Heck.

    APPENDIX

Fit of the CT response data to a cooperative model

The data in Fig. 3, A and B, can be accounted for by a straightforward cooperative model. The biphasic behavior of the NH4Cl CT response may be regarded as arising from the sum an apical membrane conductance and a paracellular shunt conductance in parallel with it. The apical membrane component will be expected to have the form of the Beidler equation as adapted by Ye et al. (1993, 1994) for an ion, viz.
<IT>R</IT><SUB>a</SUB>(<IT>c</IT><SUB>a</SUB>) = <IT>R</IT><SUB>am</SUB><IT>c</IT><SUB>a</SUB>/(<IT>K</IT><SUB>am</SUB><IT>+ c</IT><SUB>a</SUB>) (A1)
where Ra is the part of the response, R, due to the apical membrane conductance, Ram is the maximum response from the apical membrane component, Kam is the concentration at which Ra is half maximal, and ca is the electrochemical concentration of NH+4 across the apical membrane conductance. The latter is given by
<IT>c</IT><SUB>a</SUB><IT>= c</IT>exp[−δφ] (A2)
where c is the stimulating concentration of NH4Cl, phi  is the normalized clamp potential, FDelta V/RT, and delta  is the fraction of the clamp voltage (phi ) dropped across the apical membrane conductances.

The contribution of the paracellular shunt is assumed to have a cooperative aspect described by a Hill-type equation, viz.
<IT>R</IT><SUB>p</SUB>(<IT>c</IT><SUB>p</SUB>) = <IT>R</IT><SUB>pm</SUB><IT>c</IT><SUP>n</SUP><SUB>p</SUB>/(<IT>K</IT><SUP>n</SUP><SUB>pm</SUB>+ <IT>c</IT><SUP>n</SUP><SUB>p</SUB>) (A3)
where Rp is the part of the response, R, due to the paracellular shunt conductance, Rpm is the maximum response from the paracellular shunt component, Kpm is the concentration at which Rp is half maximal, and cp is the electrochemical concentration of NH+4 across the paracellular shunt conductance. The latter is given by
<IT>c</IT><SUB>p</SUB><IT>= c</IT>exp[−γφ] (A4)
where gamma  is the fraction of clamp voltage dropped across the barrier to conductance through the paracellular shunt. The Hill coefficient, n, should be regarded as a mathematical device permitting the representation of a physical process in which a small change in one variable (concentration here) produces a large change in another variable (CT response), i.e., a cooperative process. Our data indicate that the cooperativity manifested here operates through the shunt conductance (see DISCUSSION). This is represented here empirically by n values much larger than unity that are not necessarily integral. It should be clear that at this level of analysis n does not represent any one physical process or the order of any stoichiometric reaction. In fact, n turns out to be a function of clamp voltage, phi , as described below.

The overall response, RT, is then given by
<IT>R</IT><SUB>T</SUB><IT>= R</IT><SUB>a</SUB><IT>+ R</IT><SUB>p</SUB> (A5)
Although the number of parameters seems large, there are some constraints that one might reasonably expect. For example, the values of Ram, Kam, Rpm, and Kpm obtained from fitting the CT response versus concentration for the zero current-clamp curve (i.e., phi  = 0) should apply then to all the other curves under any voltage-clamp condition. With the use of this strategy we obtained the CT response versus concentration at zero current clamp. The fit required an n value of 10.6, and of course, gamma  and delta  were not involved (a similar n value was found in other systems) (Yonekura et al. 1991). Fitting the CT response-concentration curve for -50-mV voltage clamp with the same R and K parameters required an n value of 4.0, whereas at +50 mV n was 13.8. When delta  was treated as a fit parameter in each the voltage-clamp conditions, a value of ~0.2 was obtained at -50 mV. At +50 mV the fit produced a delta  of nearly the same value (0.3). To conserve the number of parameters, we chose the average value, delta  = 0.25, for use under all voltage-clamp conditions. Similarly the value of gamma  was preserved between the two voltage conditions, i.e., at -50 mV gamma  = 0.13, whereas at +50 mV gamma  = 0.15. The constancy of gamma  is expected and is one test of the internal consistency of the model.

Fitting the CT response versus clamp voltage at fixed concentration required a functional representation of the voltage dependence of the Hill coefficient, n. In fitting CT response versus concentration we obtained the following pairs, (Delta V, n); (-50, 4.0), (0, 10.6), (+50, 13.8). Assuming that n changes continuously with voltage over the range of -50 to +75 mV, we represented n approximately as follows
<IT>n</IT>= (58φ + 179.8)/(φ + 19.1) (A6)
To be consistent we refitted the CT response-concentration curves with the use of Eq. A6 to represent n. The resulting curves are shown in Fig. 6A, and these curves do not differ significantly from those generated with n as a free parameter. With the use of Eq. A6 we obtained the CT response-clamp voltage curves in Fig. 6B. In these curves the single fit parameter was gamma . For 0.5 M NH4Cl the CT response-voltage curve was fit with gamma  = 0.14, and for 0.2 M NH4Cl with gamma  = 0.21. These are again consistent with the results from fitting CT response versus concentration (see above).

We note that the apparent dependence of n on Delta V suggests that voltage affects the CT response in a more complex manner than is described merely through the electrochemical concentration. This probably means that key control parameters change with concentration and/or voltage, and these are not fully represented in a simple model. This contrasts sharply with the case of Na salts, where the CT response is uniquely described by the stimulus electrochemical concentration. This implies that the critical transducing conductances for Na salts are not voltage dependent. This does not seem to be the case for NH4Cl.

Analysis of Scatchard plot The same type of analysis for the concentration-response function can be applied for Scatchard plot analysis. Here the total CT response, RT, is plotted as a function of RT/C. With the use of the same parameters in Eqs. A1 and A3, we can obtain the value of C in terms of Ra, i.e.
<IT>C = R</IT><SUB>a</SUB><IT>K</IT><SUB>am</SUB>/(<IT>R</IT><SUB>am</SUB>− <IT>R</IT><SUB>a</SUB>) (A7)
Substituting this value of C in Eq. A5, RT is now defined as follows
<IT>R</IT><SUB>T</SUB>= <IT>R</IT><SUB>a</SUB>+ <IT>R</IT><SUB>pm</SUB>(<IT>K</IT><SUB>am</SUB><IT>R</IT><SUB>a</SUB>)<SUP>n</SUP>/[<IT>K</IT><SUP>n</SUP><SUB>pm</SUB>(<IT>R</IT><SUB>am</SUB>− <IT>R</IT><SUB>a</SUB>)<SUP>n</SUP>+ (<IT>K</IT><SUB>am</SUB><IT>R</IT><SUB>a</SUB>)<SUP>n</SUP>] (A8)
dividing the value of RT by C, we obtain
<IT>R</IT><SUB>T</SUB>/<IT>C = R</IT><SUB>pm</SUB>(<IT>K</IT><SUB>am</SUB><IT>R</IT><SUB>a</SUB>)<SUP>n−1</SUP>(<IT>R</IT><SUB>am</SUB>− <IT>R</IT><SUB>a</SUB>)/
[<IT>K</IT><SUP>n</SUP><SUB>pm</SUB>(<IT>R</IT><SUB>am</SUB>− <IT>R</IT><SUB>a</SUB>)<SUP>n</SUP>+ (<IT>K</IT><SUB>am</SUB><IT>R</IT><SUB>a</SUB>)<SUP>n</SUP>] + (<IT>R</IT><SUB>am</SUB>− <IT>R</IT><SUB>a</SUB>)/<IT>K</IT><SUB>am</SUB> (A9)
The results of such analysis can be seen in Fig. A1, in which same values of the parameters obtained earlier are used. A comparison between the experimental (Fig. 3C) and the theoretical data (Fig. A1) shows that it is important to include both noncooperative and cooperative processes in representing the data.


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FIG. A1. Scatchard plot obtained by the use of Eqs. A8 and A9 with the same values of parameters used in Fig. 6A. Note that the plot indicates the presence of 2 processes: noncooperative and cooperative.

    FOOTNOTES

  Address for reprint requests: M. A. Kloub, Dept. of Physiology, Virginia Commonwealth University, Box 980551, Richmond, VA 23298-0551.

  Received 5 September 1996; accepted in final form 14 November 1996.

    REFERENCES
Abstract
Introduction
Methods
Results
Discussion
References

0022-3077/97 $5.00 Copyright ©1997 The American Physiological Society