Abnormal Taste Perception in Mice Lacking the Type 3 Inositol 1,4,5-Trisphosphate Receptor
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. 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. 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 (1Scott K. Curr. Opin. Neurobiol. 2004; 14: 423-427Crossref PubMed Scopus (72) Google Scholar, 2Scott K. Neuron. 2005; 48: 455-464Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). For perception of sweet, bitter, and umami taste, phospholipase C β2 (PLCβ2) 3The abbreviations used are:PLCβ2phospholipase C β2WTwild-typeCTchorda tympaniMSGmonosodium glutamateNGglossopharyngeal nerveIP3Rinositol 1,4,5-trisphosphate receptorTRPM5transient-receptor potential receptor M5PBSphosphate-buffered salineRTreverse transcription. activation through G-protein-coupled receptor (sweet, T1R2 + T1R3; umami, T1R1 + T1R3; bitter, T2Rs) (1Scott K. Curr. Opin. Neurobiol. 2004; 14: 423-427Crossref PubMed Scopus (72) Google Scholar, 3Adler E. Hoon M.A. Mueller K.L. Chandrashekar J. Ryba N.J. Zuker C.S. Cell. 2000; 100: 693-702Abstract Full Text Full Text PDF PubMed Scopus (1059) Google Scholar, 4Chandrashekar J. Mueller K.L. Hoon M.A. Adler E. Feng L. Guo W. Zuker C.S. Ryba N.J. Cell. 2000; 100: 703-711Abstract Full Text Full Text PDF PubMed Scopus (1104) Google Scholar, 5Nelson G. Hoon M.A. Chandrashekar J. Zhang Y. Ryba N.J. Zuker C.S. Cell. 2001; 106: 381-390Abstract Full Text Full Text PDF PubMed Scopus (1406) Google Scholar, 6Nelson G. Chandrashekar J. Hoon M.A. Feng L. Zhao G. Ryba N.J. Zuker C.S. Nature. 2002; 416: 199-202Crossref PubMed Scopus (1168) Google Scholar, 7Zhao G.Q. Zhang Y. Hoon M.A. Chandrashekar J. Erlenbach I. Ryba N.J. Zuker C.S. Cell. 2003; 115: 255-266Abstract Full Text Full Text PDF PubMed Scopus (994) Google Scholar, 8Damak S. Rong M. Yasumatsu K. Kokrashvili Z. Varadarajan V. Zou S. Jiang P. Ninomiya Y. Margolskee R.F. Science. 2003; 301: 850-853Crossref PubMed Scopus (501) Google Scholar) and the subsequent activation of PLCβ2 and transient-receptor potential receptor M5 (TRPM5) are necessary (8Damak S. Rong M. Yasumatsu K. Kokrashvili Z. Varadarajan V. Zou S. Jiang P. Ninomiya Y. Margolskee R.F. Science. 2003; 301: 850-853Crossref PubMed Scopus (501) Google Scholar, 9Zhang Y. Hoon M.A. Chandrashekar J. Mueller K.L. Cook B. Wu D. Zuker C.S. Ryba N.J. Cell. 2003; 112: 293-301Abstract Full Text Full Text PDF PubMed Scopus (1019) Google Scholar), but the molecular mechanism by which PLCβ2 activation leads to TRPM5 in vivo is still unclear (2Scott K. Neuron. 2005; 48: 455-464Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). Several reports have suggested the possible involvement of Ca2+, probably released from the intracellular stores, in the activation of TRPM5 in heterologously expressed cells (10Ullrich N.D. Voets T. Prenen J. Vennekens R. Talavera K. Droogmans G. Nilius B. Cell Calcium. 2005; 37: 267-278Crossref PubMed Scopus (188) Google Scholar, 11Perez C.A. Huang L. Rong M. Kozak J.A. Preuss A.K. Zhang H. Max M. Margolskee R.F. Nat. Neurosci. 2002; 5: 1169-1176Crossref PubMed Scopus (484) Google Scholar, 12Perez C.A. Margolskee R.F. Kinnamon S.C. Ogura T. Cell Calcium. 2003; 33: 541-549Crossref PubMed Scopus (78) Google Scholar, 13Prawitt D. Monteilh-Zoller M.K. Brixel L. Spangenberg C. Zabel B. Fleig A. Penner R. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 15166-15171Crossref PubMed Scopus (291) Google Scholar, 14Hofmann T. Chubanov V. Gudermann T. Montell C. Curr. Biol. 2003; 13: 1153-1158Abstract Full Text Full Text PDF PubMed Scopus (326) Google Scholar) and in taste cells (15Zhang Z. Zhao Z. Margolskee R. Liman E. J. Neurosci. 2007; 27: 5777-5786Crossref PubMed Scopus (158) Google Scholar); however this remains controversial (9Zhang Y. Hoon M.A. Chandrashekar J. Mueller K.L. Cook B. Wu D. Zuker C.S. Ryba N.J. Cell. 2003; 112: 293-301Abstract Full Text Full Text PDF PubMed Scopus (1019) Google Scholar). 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 (16Futatsugi A. Nakamura T. Yamada M.K. Ebisui E. Nakamura K. Uchida K. Kitaguchi T. Takahashi-Iwanaga H. Noda T. Aruga J. Mikoshiba K. Science. 2005; 309: 2232-2234Crossref PubMed Scopus (255) Google Scholar). 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. phospholipase C β2 wild-type chorda tympani monosodium glutamate glossopharyngeal nerve inositol 1,4,5-trisphosphate receptor transient-receptor potential receptor M5 phosphate-buffered saline reverse transcription. Mice—IP3R3- and IP3R2-deficient mice were generated as described previously (16Futatsugi A. Nakamura T. Yamada M.K. Ebisui E. Nakamura K. Uchida K. Kitaguchi T. Takahashi-Iwanaga H. Noda T. Aruga J. Mikoshiba K. Science. 2005; 309: 2232-2234Crossref PubMed Scopus (255) Google Scholar), 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 × 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 (17Sugiyama T. Yamamoto-Hino M. Miyawaki A. Furuichi T. Mikoshiba K. Hasegawa M. FEBS Lett. 1994; 349: 191-196Crossref PubMed Scopus (57) Google Scholar). 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). 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. Two-bottle Preference Tests—Two-bottle preference tests were performed as described previously (18Wong G.T. Gannon K.S. Margolskee R.F. Nature. 1996; 381: 796-800Crossref PubMed Scopus (568) Google Scholar). 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. Electrophysiological Recordings—Whole-nerve responses of the CT and glossopharyngeal nerves (NGs) to lingual application of tastants were recorded as described previously (19Kawai K. Sugimoto K. Nakashima K. Miura H. Ninomiya Y. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 11044-11049Crossref PubMed Scopus (257) Google Scholar). 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. Taste Bud Morphology and the Expression of Taste-related Proteins in IP3R3KO Mice—We first examined the of and taste in WT and IP3R3-deficient mice. shown in we all three of in WT taste lysates by Western with the data using in and M.A. K. Y. 2001; PubMed Google Scholar, Margolskee R.F. Kinnamon S.C. Neurosci. 2001; PubMed Scopus Google Scholar), the of IP3R3 is the three of IP3R the which all of at a similar M. T. H. T. Nakamura T. T. Mikoshiba K. J. Biol. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar), the for in WT taste lysates but not in IP3R3-deficient taste lysates The of PLCβ2 and in IP3R3-deficient taste buds were to in WT taste buds studies also indicated that and which are crucial for taste were expressed in IP3R3-deficient mice at with in WT mice for IP3R3 were in IP3R3-deficient mice, whereas IP3R3 signal was in WT taste buds the of the We also to the for and in taste buds, but they were under the detection not the were in the taste buds of IP3R3-deficient mice as compared with WT mice by staining We further examined the taste in electron microscopy The taste buds in the IP3R3 KO mice, as as in the mice, were by of cells, each of which from the to the taste and described by M. PubMed Scopus (57) Google Scholar), the three major of cells (17Sugiyama T. Yamamoto-Hino M. Miyawaki A. Furuichi T. Mikoshiba K. Hasegawa M. FEBS Lett. 1994; 349: 191-196Crossref PubMed Scopus (57) Google Scholar) were by their the type I a with and of high electron the type II with an and of endoplasmic reticulum C and and the type that is by a and in the electron of the membrane and of In both IP3R3 KO and WT mice, the type II and cells with nerve that were in and and and type II taste cells and in the and Kinnamon J. PubMed Scopus Google Scholar). Because taste through the from the to the at least two type II cells and a type with their in IP3R3 KO mice as similar to WT mice, the number of these cells each taste not to be largely IP3R3 KO and WT a be for of in Taste of and in IP3R3KO Mice—We examined the taste perception of IP3R3-deficient mice using the preference were provided with two sipper bottles bottle contained distilled water and the other contained a tastant and the preference of tastant solution to total was calculated for each tastant solution at various We found an in taste perception in the IP3R3-deficient mice as shown in for IP3R3 showed responses to and umami and bitter and whereas WT mice to the tastes in a In contrast, the behavioral response of IP3R3-deficient mice to salty taste was normal IP3R3-deficient mice the sour solution in a similar to the WT mice. By contrast, mutant mice lacking largely not show in taste perception, was a that the mice showed a and and responses compared with WT mice. Because we the in taste buds at a be in the signal transduction for these tastants. our data indicated a specific for IP3R3 in in vivo taste We could not similar experiments in mice of their and M. T. T. E. K. H. J. S. Yamada M. H. Miyawaki A. Y. Furuichi T. H. Mikoshiba K. Noda T. Nature. 1996; PubMed Scopus Google Scholar). the of to taste signal transduction remains unknown. of Taste in IP3R3KO further the of taste signaling in the IP3R3-deficient mice, we recordings of the CT of the nerve and the which taste buds in the and the of the with the behavioral data described the CT and NG responses in IP3R3-deficient mice to glucose, and and bitter and and umami tastes were but not compared with of the WT mice, as shown in the were observed IP3R3-deficient mice and WT mice in the CT and NG responses to sour and salty which was with the behavioral data described in these that IP3R3 is a crucial mediator of sweet, bitter, and umami taste Here, we have presented from the behavioral experiments and recordings that mice lacking IP3R3 show abnormal taste perception for sweet, bitter, and umami with of sour and salty Because the taste of IP3R3 KO mice was normal both in and electron and and the taste cells expressed other IP3R3 at an to WT cells by and and and the abnormal of taste buds in IP3R3 KO mice not the of taste with the that IP3R3 was the the three of in taste buds the of taste perception was specific for IP3R3-deficient mice, and IP3R2-deficient mice showed normal taste perception IP3R3-deficient mice showed a residual behavioral response to and bitter substances at high recordings of taste nerve responses further the behavioral and showed a in the of the in responses from the two taste for the taste, CT responses were whereas NG responses were but were still with a (e.g. and at Electrophysiological responses of both CT and NG nerves to bitter substances and were but were not in response to high for the umami taste CT nerve responses were in IP3R3-deficient mice, whereas responses were observed in IP3R3-deficient similar residual responses to a dose of tastants were found in mice S. Rong M. Yasumatsu K. Kokrashvili Z. C.A. R. B. Ninomiya Y. Margolskee R.F. PubMed Scopus Google Scholar). In taste recognition of high of and but not in mice was also by 2005; PubMed Scopus (72) Google Scholar). The for the residual behavioral and electrophysiological responses to sweet, bitter, and umami tastes in IP3R3-deficient mice is however our the of signal we the involvement of a residual of of IP3R1, IP3R2, or other of on taste from the that IP3R3-deficient but not mice showed residual responses to high concentrations of produced by PLCβ2 play an important in taste perception for at a high By contrast, taste recognition of high of and to be from the signal both and mice showed behavioral responses to the bitter of taste cells through activation of a T. Y. Y. S. K. Y. Acad. Sci. PubMed Scopus Google Scholar, Margolskee R.F. Nature. PubMed Scopus Google Scholar) or of channels P. F. B. Nature. PubMed Scopus (158) Google Scholar, Kinnamon S.C. J. PubMed Scopus (72) Google Scholar) in the of taste receptor activation the residual response to high of bitter stimuli. Although our behavioral data were with the electrophysiological is a the and the responses in IP3R3 KO the mutant mice showed residual nerve response to CT nerve responses and the NG but their preference for The similar for is also in and double KO mice W. Yasumatsu K. Varadarajan V. Yamada A. J. Ninomiya Y. Margolskee R.F. S. J. Neurosci. 2004; PubMed Scopus Google Scholar). In mice, umami taste is to both CT and NG nerve responses Y. Nakashima K. A. H. T. A. V. G. J. 2000; PubMed Google Scholar). CT nerve response is to the preference for whereas the NG response is to at concentrations W. Yasumatsu K. Varadarajan V. Yamada A. J. Ninomiya Y. Margolskee R.F. S. J. Neurosci. 2004; PubMed Scopus Google Scholar). one is that the from the two nerves are in the two each in their responses to be electrophysiological not be to the behavioral response in the preference on our data and the that TRPM5 in cells (10Ullrich N.D. Voets T. Prenen J. Vennekens R. Talavera K. Droogmans G. Nilius B. Cell Calcium. 2005; 37: 267-278Crossref PubMed Scopus (188) Google Scholar, 12Perez C.A. Margolskee R.F. Kinnamon S.C. Ogura T. Cell Calcium. 2003; 33: 541-549Crossref PubMed Scopus (78) Google Scholar, 13Prawitt D. Monteilh-Zoller M.K. Brixel L. Spangenberg C. Zabel B. Fleig A. Penner R. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 15166-15171Crossref PubMed Scopus (291) Google Scholar, 14Hofmann T. Chubanov V. Gudermann T. Montell C. Curr. Biol. 2003; 13: 1153-1158Abstract Full Text Full Text PDF PubMed Scopus (326) Google Scholar) and in taste cells (15Zhang Z. Zhao Z. Margolskee R. Liman E. J. Neurosci. 2007; 27: 5777-5786Crossref PubMed Scopus (158) Google Scholar), we the IP3 produced by PLCβ2 in response to G-protein-coupled receptor through IP3R3 from the endoplasmic which in TRPM5 in vivo IP3R3 is a key molecule to with TRPM5 and an in taste perception for sweet, bitter, and umami We Robert F. Margolskee and Charles S. Zuker for their gifts of anti-TRPM5 and antibodies, We also all of our A. for mice and Dr. T. for with