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, 558 (Pt 1), 147-59

The Mammalian Amiloride-Insensitive Non-Specific Salt Taste Receptor Is a Vanilloid receptor-1 Variant

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The Mammalian Amiloride-Insensitive Non-Specific Salt Taste Receptor Is a Vanilloid receptor-1 Variant

Vijay Lyall et al. J Physiol.

Abstract

The amiloride-insensitive salt taste receptor is the predominant transducer of salt taste in some mammalian species, including humans. The physiological, pharmacological and biochemical properties of the amiloride-insensitive salt taste receptor were investigated by RT-PCR, by the measurement of unilateral apical Na+ fluxes in polarized rat fungiform taste receptor cells and by chorda tympani taste nerve recordings. The chorda tympani responses to NaCl, KCl, NH4Cl and CaCl2 were recorded in Sprague-Dawley rats, and in wild-type and vanilloid receptor-1 (VR-1) knockout mice. The chorda tympani responses to mineral salts were monitored in the presence of vanilloids (resiniferatoxin and capsaicin), VR-1 antagonists (capsazepine and SB-366791), and at elevated temperatures. The results indicate that the amiloride-insensitive salt taste receptor is a constitutively active non-selective cation channel derived from the VR-1 gene. It accounts for all of the amiloride-insensitive chorda tympani taste nerve response to Na+ salts and part of the response to K+, NH4+ and Ca2+ salts. It is activated by vanilloids and temperature (> 38 degrees C), and is inhibited by VR-1 antagonists. In the presence of vanilloids, external pH and ATP lower the temperature threshold of the channel. This allows for increased salt taste sensitivity without an increase in temperature. VR-1 knockout mice demonstrate no functional amiloride-insensitive salt taste receptor and no salt taste sensitivity to vanilloids and temperature. We conclude that the mammalian non-specific salt taste receptor is a VR-1 variant.

Figures

Figure 1
Figure 1. Effect of VR-1 agonists (RTX and CAP) and CPC on the rat chorda tympani response to NaCl
The tongue was stimulated with a rinse solution (R; 10 mm KCl) and with 100 mm NaCl + 10 mm KCl (N) or with 100 mm NaCl + 10 mm KCl + 5 μm Bz (N + Bz). A, the Bz-insensitive NaCl chorda tympani response was enhanced (de) by 1 μm RTX and inhibited (jk) by 10 μm RTX. B, increasing concentrations of RTX (•), CAP (▪) and CPC (▴) produced biphasic changes in the Bz-insensitive NaCl chorda tympani response. Each point represents the mean ± s.e.m. of the normalized chorda tympani response from 3 animals.
Figure 2
Figure 2. Effect of elevated temperature on the rat chorda tympani response to NaCl
The tongue was stimulated with a rinse solution (R; 10 mm KCl) and with 100 mm NaCl + 10 mm KCl (N) or with 100 mm NaCl + 10 mm KCl + 5 μm Bz (N + Bz). A, at 23°C superfusing with N + Bz inhibited 70% of the chorda tympani response (bc) relative to ab. At 41°C the NaCl chorda tympani response was smaller (ef < ab) and Bz produced a much smaller decrease in the NaCl chorda tympani response (fg < bc) relative to 23°C. B, variation of the chorda tympani response to N + Bz with temperature between 23 and 55.5°C in the presence of RTX (0–10 μm; pH 6). In a typical experiment the rat tongue was rinsed with 10 mm KCl (pH 6) at 23°C followed by stimulation with N + Bz + RTX at a fixed concentration of RTX varying between 0 and 10 μm (pH 6) over a range of temperatures between 23 and 55.5°C. Fitted curves in each case were drawn using eqn (3).
Figure 3
Figure 3. Effect of VR-1 antagonists (CZP and SB-366791) on the rat chorda tympani response to NaCl
A, chorda tympani responses were recorded during superfusion of the tongue with a rinse solution (10 mm KCl) and a stimulating solution (100 mm NaCl + 5 μm Bz + 10 μm CZP) containing RTX (0–10 μm). B, chorda tympani responses were recorded during superfusion of the tongue with a rinse solution (10 mm KCl) and stimulating solutions containing 10 mm KCl + 100 mm NaCl + 5 μm Bz + 0.75 μm RTX + CZP (0–500 μm). C, chorda tympani responses were recorded during superfusion of the tongue with a rinse solution (10 mm KCl) and a stimulating solution (100 mm NaCl + 5 μm Bz) containing 0 (•), 0.1 μm (▪) and 1 μm (▴) SB-366791. Each point represents the mean ± s.e.m. of the normalized chorda tympani response from 3 animals. Fitted curves in each case were drawn using eqn (3).
Figure 4
Figure 4. Rat fungiform taste receptor cells contain a VR-1 variant transducer
A cDNA library from rat fungiform taste receptor cells was screened for VR-1 and its homologues (Liu & Simon, 2001) and yielded a single band of expected size (Lane 1; →). An identical PCR fragment was amplified from rat dorsal root ganglia cDNA (Lane 2). Lane 3: DNA ladder.
Figure 5
Figure 5. Chorda tympani responses in wild-type mice (WT) (A) and VR-1 knockout mice (KO) (B)
Tongues were stimulated with 100 mm NaCl (N23°, N42°; subscripts refer to 23°C and 42°C temperatures, respectively), 100 mm NaCl + 5 μm Bz (N + Bz23°; N + Bz42°), and 100 mm NaCl + 5 μm Bz + 0.75 μm RTX (N + Bz + RTX23°; N + Bz + RTX42°) at either 23 or 42°C. Data from 3 wild-type mice and 3 VR-1 knockout mice are summarized in C. Each bar represents the mean ± s.e.m. of the normalized chorda tympani response from 3 animals.
Figure 6
Figure 6. Cation selectivity and voltage sensitivity of the amiloride-insensitive channel
A, CPC induced biphasic changes in rat chorda tympani responses to 100 mm NaCl + 5 μm Bz (•), 100 mm NH4Cl (▴), and 100 mm KCl (▪). The CPC-sensitive chorda tympani responses to KCl and NH4Cl were obtained by subtracting the maximum suppression value at 10 mm CPC. Each point represents the mean ± s.e.m. of the normalized chorda tympani response from 3 animals. B, rat chorda tympani responses to 100 mm KCl at zero current clamp (0cc), −60 mV and +60 mV voltage clamp in the absence (left trace) and presence (right trace) of 0.25 mm CPC. C, rat chorda tympani responses to 500 mm KCl between −80 and +80 mV lingual voltage clamp in the absence (▪) and presence of 0.25 mm CPC (•). Each point represents the mean ± s.e.m. of the normalized chorda tympani response from 3 animals. D, relative changes in [Na+]i in polarized rat fungiform taste receptor cells loaded with sodium green. The changes in [Na+]i are expressed as the percentage change in fluorescence intensity (F490) of sodium green. Values are presented as mean ± s.e.m. from 6 regions of interest within the taste bud.
Figure 7
Figure 7. Effect of RTX, temperature and ATP on Tκ values of taste receptor cells in situ
A, rat chorda tympani responses were recorded during superfusion of the tongue with a rinse solution (10 mm KCl) and with stimulation solutions containing 100 mm NaCl + 5 μm Bz (•; NaCl + Bz) or 100 mm NaCl + 5 μm Bz + 0.5 μm RTX (▴; NaCl + Bz + RTX) at 23°C. In the absence of RTX, n = 8 and in the presence of RTX, n = 4. B, chorda tympani responses were recorded during superfusion of the tongue with a rinse solution (10 mm KCl + 10 mm Hepes; pH 6.0, 23°) and stimulating solutions (10 mm KCl + 100 mm NaCl + 5 μm Bz + 10 mm Hepes; pH 6.0) containing 0 (•), 0.25 μm (▴), and 0.5 μm RTX (▪). For each RTX concentration tested the stimulus solution temperature was varied between 23 and 55.5°C. For 0, 0.25 and 0.5 μm RTX, n = 6, 4 and 3, respectively. C, chorda tympani responses were recorded during superfusion of the tongue with a rinse solution (10 mm KCl + 10 mm Hepes; pH 6.0, 23°C) and stimulating solutions (10 mm KCl + 100 mm NaCl + 5 μm Bz + 10 mm Hepes; pH 6.0) containing 0.25 μm RTX (•), and 0.25 μm RTX + 500 μm ATP (▪). For each RTX and RTX + ATP concentration tested the stimulus solution temperature was varied between 23 and 55.5°C. For 0.25 μm RTX and 0.25 μm RTX + 0.5 mm ATP, n = 4 and 3, respectively. Chorda tympani responses were recorded at zero current clamp (0cc) and at ± 60 mV lingual voltage clamp. In each case the NaCl chorda tympani responses were normalized to the corresponding chorda tympani responses obtained with 300 mm NH4Cl. In B and C the Tκ (product of absolute temperature (K), T, with response conductance (κ) data points were plotted as a function of temperature ranging from 23 to 55.5°C. The Tκ data were fitted toeqn (4) as described in the text. Values are presented as the mean ± s.e.m. of n, where n = number of animals.
Figure 8
Figure 8. Effect of external pH (pHo) on the NaCl chorda tympani response
A, effect of pHo (2–10) on the rat chorda tympani response to 100 mm NaCl + 5 μm Bz + 0.5 μm RTX. Each point represents the mean ± s.e.m. of the normalized chorda tympani response from 3 animals. B, effect of pHo 4.7 (▴; n = 3), 6.0 (•; n = 9), 9.7 (▪; n = 6) and ATP (○; n = 4) on the temperature-induced chorda tympani response to 100 mm NaCl + 5 μm Bz + 0.25 μm RTX. Each point represents the mean ± s.e.m. of the normalized chorda tympani responses from n, number of animals. Fitted curves in each case were drawn using eqn (3).
Figure 9
Figure 9. Effect of RTX on rat chorda tympani responses to sucrose, quinine and HCl
Rat chorda tympani responses were recorded during superfusion of the tongue with a rinse solution (R; 10 mm KCl) and with stimulation solutions (arrows) containing 500 mm sucrose (S) (A), 10 mm quinine (Q) (B) and 20 mm HCl (H) (C) at 23°C with and without 1 μm and 10 μm RTX. Since RTX at 1 or 10 μm concentration induced no change in the chorda tympani responses to sucrose, quinine and HCl relative to control, the data for both concentrations of RTX were combined and are summarized in D. Each bar represents the mean ± s.e.m. of the normalized chorda tympani response in the presence of RTX relative to control from 3 animals. In the presence of RTX the chorda tympani responses to sucrose, quinine and HCl were not significantly different from control (P > 0.05).

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