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Originally published In Press as doi:10.1074/jbc.M705641200 on October 9, 2007

J. Biol. Chem., Vol. 282, Issue 51, 37225-37231, December 21, 2007
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Abnormal Taste Perception in Mice Lacking the Type 3 Inositol 1,4,5-Trisphosphate Receptor*Formula

Chihiro Hisatsune{ddagger}§1, Keiko Yasumatsu, Hiromi Takahashi-Iwanaga||, Naoko Ogawa{ddagger}, Yukiko Kuroda{ddagger}, Ryusuke Yoshida, Yuzo Ninomiya, and Katsuhiko Mikoshiba{ddagger}§**2

From the {ddagger}Laboratory for Developmental Neurobiology, RIKEN Brain Science Institute, 2-1 Hirosawa, Wako city, Saitama 351-0198, Japan, §Division of Molecular Neurobiology, Institute of Medical Science, University of Tokyo, 3-4-1, Shirokane-dai, Minato-ku, Tokyo 108-8639, Japan, Section of Oral Neuroscience, Graduate School of Dental Science, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan, ||Department of Anatomy, School of Medicine, Hokkaido University, Sapporo 060-8638, Japan, and **Calcium Oscillation Project, ICORP, Japan Science and Technology Agency, 3 Nibancho, Chiyoda-ku, Tokyo 102-0084, Japan

Received for publication, July 10, 2007 , and in revised form, September 14, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Inositol 1,4,5-trisphosphate receptor (IP3R) is one of the important calcium channels expressed in the endoplasmic reticulum and has been shown to play crucial roles in various physiological phenomena. Type 3 IP3R is expressed in taste cells, but the physiological relevance of this receptor in taste perception in vivo is still unknown. Here, we show that mice lacking IP3R3 show abnormal behavioral and electrophysiological responses to sweet, umami, and bitter substances that trigger G-protein-coupled receptor activation. In contrast, responses to salty and acid tastes are largely normal in the mutant mice. We conclude that IP3R3 is a principal mediator of sweet, bitter, and umami taste perception and would be a missing molecule linking phospholipase C β2 to TRPM5 activation.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Taste perception is a pivotal and primitive sensory system for survival in animals. By sensing taste, animals are provided with valuable information about foods (e.g. qualities and nature) and can choose the nutrient-rich foods necessary for living or avoid harmful and toxic substances. There are five taste categories (sweet, bitter, umami, sour, and salty), and recent studies have furthered our understanding of the molecular mechanisms of taste perception, especially for sweet, bitter, and umami tastes (1, 2).

For perception of sweet, bitter, and umami taste, phospholipase C β2 (PLCβ2)3 activation through G-protein-coupled receptor (sweet, T1R2 + T1R3; umami, T1R1 + T1R3; bitter, T2Rs) (1, 3-8) and the subsequent activation of PLCβ2 and transient-receptor potential receptor M5 (TRPM5) are necessary (8, 9), but the molecular mechanism by which PLCβ2 activation leads to TRPM5 in vivo is still unclear (2). Several reports have suggested the possible involvement of Ca2+, probably released from the intracellular stores, in the activation of TRPM5 in heterologously expressed cells (10-14) and in taste cells (15); however this remains controversial (9). Because PLCβ2 activation actually leads to production of both IP3 and diacylglycerol, it is an important issue to definitely determine, which is a major player for gustatory systems. To clarify whether IP3R is necessary for taste perception in vivo, we analyzed the taste signaling of IP3R-deficient mice in this study (16). We found that mice lacking IP3R3 showed altered taste recognition for sweet, bitter, and umami, whereas they were indistinguishable from wild-type (WT) mice in their recognition for salty and sour stimuli. However, they showed residual responses to high concentrations of sweets and bitter. Our data present the direct validation that IP3R3 is a key molecule in taste perception for sweet, bitter, and umami and also suggest the existence of IP3R3-independent taste signal transduction for recognition of high dose of these tastants.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Mice—IP3R3- and IP3R2-deficient mice were generated as described previously (16), and the mice intercrossed with C57BL/6 mice at least twelve times were used. WT C57BL/6 mice were littermates or purchased from SLC (Shizuoka, Japan). All experiments were performed in accordance with the Animal Experiment Committee of RIKEN Brain Science Institute.

Detection of Taste-related Proteins in Mouse Taste Buds—Two mouse tongues were removed and a protease solution (140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 10 mM Hepes, 10 mM glucose, 10 mM sodium pyruvate, pH 7.4) containing 0.25 mg/ml elastase and 2 mg/ml collagenase type I was injected under the circumvallate papilla, and the epithelium was peeled away after 15 min. The peeled epithelium was then incubated in the enzyme solution (0.25 mg/ml elastase, 2.0 mg/ml collagenase, and 1.6 mg/ml dispase) for 5 min at room temperature and further incubated in Ca2+-free solution of tyrode. After 30 min, taste buds became to be loosely attached to the epithelium, and we gently detached all taste buds using forceps in the buffer as possible as we could. The buffer containing all taste buds of two mice circumvallate papilla were centrifuged at 1000 x g for 5 min at 4 °C, and the precipitated taste buds were lysed with 50 µl of sample buffer. Fifteen µl of the lysate per lane was used for Western blotting analysis. Although we could not count the accurate number of taste buds, re-immunoblotting of β-actin of the same membrane helped us to confirm that lysates of WT and IP3R3-deficient samples largely contained similar numbers of taste buds. Proteins were separated by 7.5% SDS-polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane. The membrane was treated with the blocking solution (0.05% Tween/PBS) containing 5.0% skim milk for 1 h and probed with the indicated primary antibodies. Monoclonal mouse antibodies, KM1112, KM1083, and KM1082 were used for detection of IP3R1, IP3R2, and IP3R3, respectively (17). Rabbit polyclonal anti-gustducin and -PLCβ2 antibodies were purchased from Santa Cruz Biotechnology, Santa Cruz, CA. Monoclonal mouse anti-β-actin antibody was from Sigma. After incubation with horseradish peroxidase-labeled secondary antibodies, the immobilized specific antigen was visualized with ECL plus detection kit (GE Healthcare).


Figure 1
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  		FIGURE 1.
Taste bud morphology and the expression of taste signaling molecules in IP3R3-deficient mice. A, the expression of IP3R subtypes and the taste-related proteins in taste buds from WT and IP3R3-deficient mice. B, immunohistochemistry of the expression of several proteins important for taste transduction in taste buds in circumvallate papillae from WT and IP3R3-deficient mice. a) gustducin; b) IP3R3; c) T1R3; d) PLCβ2; e) TRPM5. Scale bar, 20 µm. All experiments were performed three times, and the representative data were shown. C, morphology of taste buds of circumvallate papillae from WT and IP3R3-deficient mice stained with hematoxylin-eosin. Scale bar, 50 µm.

 
Immunocytochemistry—Mouse tongue was removed and fixed with 4.0% formaldehyde/PBS for 3 h at 4°C, then immerged in 30% sucrose/PBS for overnight. The tissues were embedded in tissue compound, and the frozen sections at 12-µm thickness were created using cryostat. Frozen sections of mouse lingual tissue were permeabilized with 0.2% Triton/PBS for 10 min and blocked with 1.0% goat serum or 3.0% skim milk/PBS for 1 h. Then, sections were incubated with primary antibodies in blocking solution for 1 h at room temperature. The antibodies used for the experiments were: rabbit anti-IP3R3 antibody (raised in rabbit using amino acid 2391-2463 peptide of mIP3R3 for an antigen), rabbit anti-PLCβ2 antibody (Santa Cruz Biotechnology), rabbit anti-gustducin (Santa Cruz Biotechnology), rabbit anti-TRPM5 (kind gifts from Dr. Robert F. Margolskee), and rabbit anti-T1R3 (kind gifts from Dr. Charles S. Zuker). After washing with PBS for 15 min, sections were incubated with fluorescence-conjugated secondary antibodies (Alexa 488-conjugated goat anti-mouse IgG and Alexa 594-conjugated goat anti-rabbit IgG) (Invitrogen) for 1 h at RT. After washing with PBS for 15 min, the sections were mounted with Vectashield (Vector Laboratories, Burlingame, CA) and observed under IX-70 confocal fluorescence microscopy (Olympus, Tokyo, Japan).

RT-PCR—Total RNA was extracted from taste cells of three WT and IP3R3KO mice using TRIzol reagent according to the manufacturer's instructions (Invitrogen). First strand cDNA was produced from the total RNA using reverse transcriptase Superscript II (Invitrogen) and oligonucleotide (dT) primers. The cDNAs were amplified with specific primers for mT2R108: sense 5'-ggcaccaaacgaggaaagatg-3', antisense 5'-tcaggaccaaagaggctactaacg-3'; mT2R138: sense 5'-atgctgagtctgactcctgtcttaac-3', antisense 5'-gcaggagagaagaagaacaactag-3'; and glyceraldehyde-3-phosphate dehydrogenase (GAPDH): sense 5'-atggtgaaggtcggtgtgaaccg-3', antisense 5'-aaacatgggggcatcggcagaa-3'. After an initial cycle of 2 min at 95 °C, the reaction was cycled 30 times for 30 s at 95 °C, 30 s at 55 °C, and 1 min at 72 °C. The PCR products were separated by electrophoresis in 2.0% agarose gel and stained with ethidium bromide.

Electron Microscopy—Young adult KO mice and their littermates of wild-type mice, 4 in number for each group, were examined. Under an inhalation anesthesia with diethyl ether, the animals were perfused transcardially with Ringer's solution saturated with O2 and subsequently with a mixture of 2.5% glutaraldehyde and 0.5% paraformaldehyde buffered at pH 7.3 with 0.1 M phosphate. The circumvallate papilla was excised, halved on the midline, and kept overnight in the same fixative. The tissue pieces were postfixed in 1.0% OsO4, buffered at pH 7.2 with 0.1 M phosphate for 2 h at 4 °C, dehydrated through a series of ethanol, and embedded in Epon-812. Ultrathin sections were examined in a Hitachi H-7100 transmission electron microscope after double staining with uranyl acetate and lead citrate.


Figure 2
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  		FIGURE 2.
Ultrastructure of taste buds from WT and IP3R3-deficient mice. Taste buds of WT (A) and IP3R3KO (B) mice. Type I, II, and III taste cells are labeled with corresponding numbers. Arrows indicate taste pores. Bars, 10 µm. Basal portions of type II taste cells (II) in WT (C) and IP3R3 KO mice (D). C corresponds to the square area in A. In both animals, the cells are equipped with a considerable amount of smooth endoplasmic reticulum (stars) and brought into direct contact with intragemmal nerve fibers (N) with subsurface cisterns extended along the contacting area (arrowheads). Mitochondria in the IP3R3 KO cell, as well as those in the wild type cell, were normal with no pathological signs of swelling or enlargement (arrows). I, type I cells; bars, 1.0 µm. The square areas in C and D are presented in higher magnification by E and F, respectively. Nerve fibers (N) contain clear vesicles (short arrows) and a dense-cored vesicle (long arrow). Cytoplasm of type II cells appears spongy because of labyrinthine lumina of smooth endoplasmic reticulum. The subsurface cisterns are separated from the taste cell membrane by a 20-nm gap, which contains an electron-dense material (arrowheads). M, mitochondria; bars, 500 nm.

 
Two-bottle Preference Tests—Two-bottle preference tests were performed as described previously (18). In brief, mice were individually housed and given to food ad libitum. Two sipper bottles, one containing distilled water and the other containing tastant solution were presented. After 24 h, intakes of each solution were recorded, and the positions of the two sipper bottles were replaced to reduce the preference of the position of the bottle in a cage. Tastants were presented in an ascending order. Preference ratios were calculated as the intake of tastant compared with total intake.


Figure 3
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  		FIGURE 3.
Mean taste preference ratios of WT, IP3R2-, and IP3R3-deficient mice using 48-hour two-bottle (tastant versus distilled water) preference tests. A, behavioral responses to saccharin (0.25, 0.5, 1.0, 4.0, 16, and 32 mM). B, behavioral responses to sucrose (3, 15, 30, 60, 120, 300 mM) of WT, IP3R2-deficient, and IP3R3-deficient mice. C, behavioral responses to MSG (3, 10, 30, 100, and 300 mM). D, behavioral responses to cycloheximide (0.1, 0.5, 1.0, and 3.0 µM). E, behavioral responses to quinine sulfate (0.001, 0.01, 0.1, 0.3, 0.6, and 1.0 mM). F, behavioral responses to denatonium benzoate (0.01, 0.05, 0.3, 0.6, 1.0, 5.0, and 10 mM). G, behavioral responses to HCl (0.1, 1.0, 10, and 100 mM). H, behavioral responses to NaCl (75, 150, 300, and 600 mM). The values are means ± S.E. (n = 5). *, p < 0.05 (Student's t test).

 
Electrophysiological Recordings—Whole-nerve responses of the CT and glossopharyngeal nerves (NGs) to lingual application of tastants were recorded as described previously (19). Briefly, tastants were applied to the tongue for 30 (CT) and 60 s NG at a regular flow rate. Integrated whole-nerve response magnitudes (time constant, 1 s) were measured 5, 10, 15, 20, and 25 s (for CT) and 5, 10, 20, 30, and 40 s (for NG) after stimulus onset. These data were averaged, normalized to the responses to 0.1 M NH4Cl, and analyzed with the general linear model multiple measures of the statistics package SPSS. For nerve recordings to monosodium glutamate (MSG), we performed the experiments in the presence of 10 µM amiloride.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Taste Bud Morphology and the Expression of Taste-related Proteins in IP3R3KO Mice—We first examined the expression level of several taste-related proteins and taste bud morphology in WT and IP3R3-deficient mice. As shown in Fig. 1A, we detected all three subtypes of IP3Rs in WT taste bud lysates by Western blotting. Consistent with the previous data using in situ hybridization and RT-PCR (20, 21), the expression of IP3R3 is predominant among the three subtypes of IP3R because the pan-IP3R antibody, which recognizes all types of IP3Rs at a similar level (22), detected the bands for IP3Rs in WT taste bud lysates but not in IP3R3-deficient taste bud lysates (Fig. 1A). The expression levels of PLCβ2 and gustducin in IP3R3-deficient taste buds were equivalent to those in WT taste buds (Fig. 1A). Immunohistochemical studies also indicated that PLCβ2, gustducin, T1R3, and TRPM5, which are crucial molecules for taste signaling, were normally expressed in IP3R3-deficient mice at levels comparable with those in WT mice (Fig. 1B). Immuno-signals for IP3R3 were completely abolished in IP3R3-deficient mice, whereas IP3R3 signal was detected in WT taste buds (Fig. 1B), confirming the specificity of the antibody. We also tried to detect the immunosignals for IP3R1 and IP3R2 in taste buds, but they were under the detection level (data not shown).

At the morphological level, no apparent alterations were detected in the taste buds of IP3R3-deficient mice as compared with WT mice by hematoxylin-eosin staining (Fig. 1C). We further examined the taste bud morphology in electron microscopy (Fig. 2). The taste buds in the IP3R3 KO mice, as well as those in the control mice, were represented by tight aggregates of spindle-shaped cells, each of which extended from the base to the taste pore (Fig. 2, A and B). As originally described by Takeda (23), the three major types of intragemmal cells (17) were identifiable by their cytoplasmic constituents: the type I cell possessing a nucleus with deep indentations and apical granules of high electron density, the type II cell with an oval nucleus and considerable amounts of smooth endoplasmic reticulum (Fig. 2, C and D), and the type III cell that is characterized by a round clear nucleus and synaptic specialization; elevation in the electron density of the cell membrane and accumulation of granules. In both IP3R3 KO and WT mice, the taste-sensing type II and III cells frequently contacted with nerve endings that were rich in mitochondria and dense and clear vesicles, and type II taste cells displayed subsurface cisterns and atypical mitochondria in the contacting area (Fig. 2, E and F) (24). Because taste bud sectioned through the axis, passing from the base to the pore, exhibited at least two type II cells and a single type III cell with their typical features in IP3R3 KO mice as similar to WT mice, the number of these cells constituting each taste bud did not seem to be largely different between IP3R3 KO and WT mice; a more detailed analysis might be required for evaluation of subtle changes in ultrastructure.


Figure 4
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  		FIGURE 4.
Whole-nerve recordings from CT and NG taste nerves of WT and IP3R3-deficient mice upon lingual application of taste stimuli. A, dose-dependent response of the CT nerve from WT and IP3R3-deficient mice to various tastants: sweet (sucrose, SC45647, acesulfame potassium), bitter (denatonium, quinine sulfate, quinine-HCl), sour (HCl), salty (NaCl), and umami (MSG). B, whole-nerve responses from the CT nerve of WT and IP3R3-deficient mice to sweet (sorbitol, sucralose, saccharin, glucose, maltose, fructose), bitter (caffeine, cycloheximide), and umami (L-proline, L-alanine, glycine, D-alanine, D-tryptophan) tastes. C, dose-dependent response of NG nerve from WT and IP3R3-deficient mice to various taste stimuli. D, whole-nerve responses from the NG nerve of WT and IP3R3-deficient mice to sweet (sorbitol, sucralose, saccharin, glucose, maltose, fructose), bitter (caffeine, cycloheximide), and umami (L-proline, L-alanine, glycine, D-alanine, D-tryptophan) tastes. Data are means ± S.E. The number of experiments was at least five. *, p < 0.05 (Student's t test).

 
Abnormal Taste Perception of Sweet, Bitter, and Umami in IP3R3KO Mice—We next examined the taste perception of IP3R3-deficient mice using the 48-hour two-bottle preference tests. Mice were provided with two sipper bottles (one bottle contained distilled water and the other contained a tastant solution), and the preference ratio (intake of tastant solution to total liquid intake) was calculated for each tastant solution at various concentrations. We found an obvious abnormality in taste perception in the IP3R3-deficient mice as shown in Fig. 3. Mice deficient for IP3R3 showed decreased responses to sweet (sucrose and saccharin), umami (monosodium glutamate), and bitter (quinine sulfate, denatonium benzoate, and cycloheximide) tastes, whereas WT mice responded to the tastes in a dose-dependent manner. In contrast, the behavioral response of IP3R3-deficient mice to salty taste (NaCl) was normal (Fig. 3). Likewise, IP3R3-deficient mice avoided the sour solution (HCl) in a dose-dependent manner, similar to the WT mice. By contrast, mutant mice lacking IP3R2 largely did not show any apparent abnormalities in taste perception, although there was a tendency that the mice showed a slightly reduced (sucrose, HCl) and stronger (denatonium, quinine sulfate, and NaCl) behavior responses compared with WT mice. Because we detected the IP3R2 expression in taste buds at a lower level (Fig. 1A), IP3R2 may be partially involved in the signal transduction for these tastants. Even if, our data strongly indicated a specific role for IP3R3 in in vivo taste signaling. We could not perform similar experiments in IP3R1-deficient mice because of their epilepsy and postnatal death within 21 days (25). Thus, the contribution of IP3R1 to taste signal transduction remains unknown.


Figure 5
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  		FIGURE 5.
A schematic model of taste signaling for bitter, sweet, and umami. Taste stimuli (sweet, bitter, and umami) trigger the activation of PLCβ2 through the taste receptors (T1R and T2R), resulting in the production of IP3 and diacylglycerol (DAG). Then, IP3 binds to IP3R3, and IP3R3 releases Ca2+ from the endoplasmic reticulum (ER). The elevation of intracellular Ca2+ triggers TRPM5 activation, which induces the depolarization of taste cells. In addition to this main signal pathway, the unknown signal pathways may also contribute to depolarization of taste cells for the detection of taste stimuli at the high concentration (dotted line).

 
Electrical Recordings of Taste Nerve Responses in IP3R3KO Mice—To further test the deficit of peripheral taste signaling in the IP3R3-deficient mice, we finally took electrical recordings of the CT branch of the facial nerve and the NG, which innervated taste buds in the anterior and the posterior part of the tongue, respectively. Consistent with the behavioral data described above, the CT and NG electrical responses in IP3R3-deficient mice to sweet (sucrose, glucose, sorbitol, sucralose, maltose, and fructose, SC45647, acesulfame K, and saccharin), bitter (denatonium, cycloheximide, and quinine), and umami tastes were greatly reduced but not abolished compared with those of the WT mice, as shown in Fig. 4. On the contrary, almost no significant differences were observed between IP3R3-deficient mice and WT mice in the CT and NG responses to sour and salty tastes, which again was consistent with the behavioral data described in Fig. 3. Taken together, these results indicate that IP3R3 is a crucial mediator of sweet, bitter, and umami taste perception.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Here, we have presented conclusive evidence from the behavioral experiments and electrical recordings that mice lacking IP3R3 show abnormal taste perception for sweet, bitter, and umami tastes, with gustation of sour and salty compounds being well preserved. Because the taste bud morphology of IP3R3 KO mice was apparently normal both in light and electron microscopic observation (Figs. 1C and 2), and the taste cells normally expressed several taste-related proteins other than IP3R3 at an equivalent level to WT cells by immunoblotting, immunohistochemistry, and RT-PCR (Fig. 1, A and B and supplemental Fig. 1), the abnormal development of taste buds in IP3R3 KO mice did not explain the deficit of taste recognition. Consistent with the finding that IP3R3 was the dominant isoform among the three types of IP3Rs in taste buds (Fig. 1A), the abnormality of taste perception was specific for IP3R3-deficient mice, and IP3R2-deficient mice generally showed normal taste perception (Fig. 3). Interestingly, however, IP3R3-deficient mice showed a residual behavioral response to sweet and bitter substances at high concentration (Fig. 3). Electrical recordings of taste nerve responses further supported the behavioral results and showed a slight difference in the degree of the reduction in responses from the two taste nerves. As for the sweet taste, CT responses were almost completely diminished, whereas NG responses were reduced but were still weak with a dose-dependent increase (e.g. sucrose (>0.3 mM) and acesulfame potassium at 0.3 M). Electrophysiological responses of both CT and NG nerves to bitter substances (quinine sulfate and denatonium) were generally reduced but were not diminished in response to high concentrations. As for the umami taste response, CT nerve responses were significantly reduced in IP3R3-deficient mice, whereas considerable responses were observed in IP3R3-deficient NGs. Recently, similar residual responses to a higher dose of tastants were found in TRPM5-deficient mice (26). In addition, taste recognition of high doses of quinine and denatonium but not sucrose in PLCβ2-deficient mice was also reported by Dotson et al. (27). The reason for the residual behavioral and electrophysiological responses to sweet, bitter, and umami tastes in IP3R3-deficient mice is unknown; however our results indicate the contribution of IP3R-independent signal transduction; although we cannot rule out the involvement of a residual amount of expression of IP3R1, IP3R2, or other subtypes of PLC on taste signaling. Judging from the evidence that IP3R3-deficient but not PLCβ2-deficient mice showed residual responses to high concentrations of sucrose, diacylglycerol produced by PLCβ2 may play an important role in taste perception for sucrose at a high concentration (Fig. 5). By contrast, taste recognition of high doses of denatonium and quinine seems to be completely independent from the PLCβ2-IP3R3 signal pathway because both IP3R3- and PLCβ2-deficient mice showed behavioral responses to the bitter compounds. Excitation of taste cells through activation of a cyclic nucleotide-gated channel (28, 29) or inactivation of K+ channels (30, 31) in the downstream events of taste receptor activation may explain the PLCβ2-independent residual response to high doses of bitter stimuli.

Although our behavioral data were generally consistent with the electrophysiological data, there is a dichotomy between the behavior and the neural responses in IP3R3 KO mice: the mutant mice showed residual nerve response to MSG (the reduced CT nerve responses and the considerable NG responses) but abolished their preference for MSG. The similar dichotomy for MSG is also reported in {alpha}-transducin and -gustducin double KO mice (32). In mice, umami taste is known to elicit both CT and NG nerve responses (33). CT nerve response is related to the preference for MSG, whereas the NG neural response is related to aversion at higher concentrations (32). Therefore, one explanation is that if the inputs from the two nerves are equal in net, the two signals may cancel each other, resulting in their behavior responses to be indifferent. Alternatively, electrophysiological signals may not be enough to elicit the behavioral response in the two-bottle preference tests.

Based on our data and the previous findings that Ca2+ activates TRPM5 in heterologous cells (10, 12-14) and in taste cells (15), we propose the following model: IP3 produced by PLCβ2 in response to G-protein-coupled receptor stimulation triggers Ca2+ release through IP3R3 from the endoplasmic reticulum, which in turn activates TRPM5 in vivo (Fig. 5). Thus, IP3R3 is a key molecule to couple PLC with TRPM5 and plays an indispensable role in taste perception for sweet, bitter, and umami tastes.


   FOOTNOTES
 
* This work was supported by grants from the Ministry of Education, Science, and Culture of Japan (to K. M. and Y. N.), a grant-in-aid for young scientists (to C. H.), and the Japan Science and Technology Agency. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1. Back

1 To whom correspondence may be addressed: Tel.: 81-48-467-9745; Fax: 81-48-467-9744; E-mail: chihiro{at}brain.riken.go.jp.

2 To whom correspondence may be addressed: E-mail: mikosiba{at}brain.riken.go.jp.

3 The abbreviations used are: PLCβ2, phospholipase C β2; WT, wild-type; CT, chorda tympani; MSG, monosodium glutamate; NG, glossopharyngeal nerve; IP3R, inositol 1,4,5-trisphosphate receptor; TRPM5, transient-receptor potential receptor M5; PBS, phosphate-buffered saline; RT, reverse transcription. Back


   ACKNOWLEDGMENTS
 
We thank Drs. Robert F. Margolskee and Charles S. Zuker for their kind gifts of anti-TRPM5 and -T1R3 antibodies, respectively. We also thank all members of our laboratories, especially, A. Terauchi for breeding mice and Dr. T. Inoue for experimental advice.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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