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• W1967425088 abstract "Cyclic nucleotide-gated (CNG) channels have been characterized as important factors involved in physiological processes including sensory reception for vision and olfaction. The possibility thus exists that a certain CNG channel functions in gustation as well. In the present study, we carried out reverse transcription-polymerase chain reaction and genomic DNA cloning and characterized a CNG channel (CNGgust) as a cyclic nucleotide-activated species expressed in rat tongue epithelial tissues where taste reception takes place. Several types of 5′-rapid amplification of cDNA ends clones of CNGgust cDNA were obtained with various 5′-terminal sequences. As the CNGgust gene was a single copy, the formation of such CNGgust variants should result from alternative splicing. The encoded protein was homologous to known vertebrate CNG channels with 50–80% similarities in amino acid sequence, and particularly homologous to bovine testis CNG channel and human cone CNG channel with 82% similarities. CNGgust was functional when expressed in human embryonic kidney cells, where it opened upon the addition of cGMP or cAMP. Immunohistochemical analysis using an antibody raised against a CNGgust peptide demonstrated the channel to be localized on the pore side of each taste bud in the circumvallate papillae, with no signal observed for degenerated taste buds after denervation of the glossopharyngeal nerve. All these results, together with the indication that cyclic nucleotides play a role gustatory signaling pathway(s), strongly suggest the involvement of CNGgust in taste signal transduction. Cyclic nucleotide-gated (CNG) channels have been characterized as important factors involved in physiological processes including sensory reception for vision and olfaction. The possibility thus exists that a certain CNG channel functions in gustation as well. In the present study, we carried out reverse transcription-polymerase chain reaction and genomic DNA cloning and characterized a CNG channel (CNGgust) as a cyclic nucleotide-activated species expressed in rat tongue epithelial tissues where taste reception takes place. Several types of 5′-rapid amplification of cDNA ends clones of CNGgust cDNA were obtained with various 5′-terminal sequences. As the CNGgust gene was a single copy, the formation of such CNGgust variants should result from alternative splicing. The encoded protein was homologous to known vertebrate CNG channels with 50–80% similarities in amino acid sequence, and particularly homologous to bovine testis CNG channel and human cone CNG channel with 82% similarities. CNGgust was functional when expressed in human embryonic kidney cells, where it opened upon the addition of cGMP or cAMP. Immunohistochemical analysis using an antibody raised against a CNGgust peptide demonstrated the channel to be localized on the pore side of each taste bud in the circumvallate papillae, with no signal observed for degenerated taste buds after denervation of the glossopharyngeal nerve. All these results, together with the indication that cyclic nucleotides play a role gustatory signaling pathway(s), strongly suggest the involvement of CNGgust in taste signal transduction. Vertebrate sensory systems comprise several distinct organs, cell types, and cellular signalings. These systems share many common features in their transmission and amplification of signals intercellularly or intracellularly leading to sense transduction to the central nervous system. Vision is the most characteristic sensory process in vertebrates. Vision involves membrane receptors (rhodopsins) catching light, signal transducers such as transducin and phosphodiesterase, a soluble second messenger (cGMP), and membrane components such as a cyclic nucleotide-gated (CNG) 1The abbreviations used are: CNG, cyclic nucleotide gated; nt, nucleotide; RACE, rapid amplification of cDNA ends; HEK, human embryonic kidney; RT, reverse transcription; PCR, polymerase chain reaction; PBS, phosphate-buffered saline; bp, base pair(s); kb, kilobase pair(s).1The abbreviations used are: CNG, cyclic nucleotide gated; nt, nucleotide; RACE, rapid amplification of cDNA ends; HEK, human embryonic kidney; RT, reverse transcription; PCR, polymerase chain reaction; PBS, phosphate-buffered saline; bp, base pair(s); kb, kilobase pair(s).channel, all of which cooperate in the transduction of light signals to the central nervous system (1Yau K.-W. Baylor D.A. Annu. Rev. Neurosci. 1989; 12: 289-327Crossref PubMed Scopus (432) Google Scholar). Although this molecular apparatus is thought to be specific to vision, it is believed that similar signaling pathways exist for other sensory processes such as olfaction and gustation (2Buck L.B. Annu. Rev. Neurosci. 1996; 19: 517-544Crossref PubMed Scopus (444) Google Scholar, 3Kinnamon S.C. Margolskee R.F. Curr. Opin. Neurobiol. 1996; 6: 506-513Crossref PubMed Scopus (139) Google Scholar). In olfactory neuron cells, olfactory receptors encoded by more than 1000 genes (4Ressler K.J. Sullivan S.L. Buck L.B. Curr. Opin. Neurobiol. 1994; 4: 588-596Crossref PubMed Scopus (108) Google Scholar), a specific GTP-binding protein (G-protein) (Golf) (5Jones D.T. Reed R.R. Science. 1989; 244: 790-795Crossref PubMed Scopus (636) Google Scholar), and a CNG channel (6Dhallan R.S. Yau K.-W. Schrader K.A. Reed R.R. Nature. 1990; 347: 184-187Crossref PubMed Scopus (510) Google Scholar) have been identified. One intriguing fact is that each olfactory neuron expresses one or a few receptor genes from a very large gene family, which strongly suggests that a certain receptor molecule determines the specificity for ligands (odorants) (4Ressler K.J. Sullivan S.L. Buck L.B. Curr. Opin. Neurobiol. 1994; 4: 588-596Crossref PubMed Scopus (108) Google Scholar). A similar situation has been implicated in taste chemoreception. We have identified multiple seven-transmembrane receptors similar to olfactory receptors that may participate in gustatory signaling (7Abe K. Kusakabe Y. Tanemura K. Emori Y. Arai S. FEBS Lett. 1993; 316: 253-256Crossref PubMed Scopus (40) Google Scholar). We have also demonstrated by in situ hybridization (8Abe K. Kusakabe Y. Tanemura K. Emori Y. Arai S. J. Biol. Chem. 1993; 268: 12033-12039Abstract Full Text PDF PubMed Google Scholar) and immunostaining (9Kusakabe Y. Abe K. Tanemura K. Emori Y. Arai S. Chem. Senses. 1996; 21: 335-340Crossref PubMed Scopus (15) Google Scholar) that GUST27, a representative receptor, is closely related to known olfactory receptors with a similarity of about 60% (8Abe K. Kusakabe Y. Tanemura K. Emori Y. Arai S. J. Biol. Chem. 1993; 268: 12033-12039Abstract Full Text PDF PubMed Google Scholar) and is expressed exclusively in taste buds and surrounding sites. An independent group has reported similar receptors expressed in taste cells (10Matsuoka I. Mori T. Aoki J. Sato T. Kurihara K. Biochem. Biophys. Res. Commun. 1993; 194: 504-511Crossref PubMed Scopus (40) Google Scholar, 11Tal M. Ammar D.A. Karpuj M. Krizhanovsky V. Naim M. Thompson D.A. Biochem. Biophys. Res. Commun. 1995; 209: 752-759Crossref PubMed Scopus (89) Google Scholar, 12Thomas M.B. Haines S.L. Akeson R.A. Gene ( Amst. ). 1996; 178: 1-5Crossref PubMed Scopus (32) Google Scholar). McLaughlin et al. (13McLaughlin S.K. McKinnon P.J. Margolskee R.F. Nature. 1992; 357: 563-569Crossref PubMed Scopus (542) Google Scholar) and Ruiz-Avilaet al. (14Ruiz-Avila L. McLaughlin S.K. Wildman D. McKinnon P.J. Robichon A. Spickofsky N. Margolskee R.F. Nature. 1995; 376: 80-85Crossref PubMed Scopus (173) Google Scholar) have confirmed the expression of gustducin, a taste bud-specific G-protein, and transducin in taste cells. Although many of the molecules present in olfactory and/or gustatory cells have not yet been proved to be involved directly in the intracellular signaling cascade, those that are homologous to the visual signaling process may give rise to olfactory and gustatory sensations. Recently, the functions of various specific molecules have been revealed. Knockout mice lacking the olfactory CNG channel are insensitive to most odors (15Brunet L.J. Gold G.H. Ngai J. Neuron. 1996; 17: 681-693Abstract Full Text Full Text PDF PubMed Scopus (405) Google Scholar), and those lacking gustducin show greatly reduced or sometimes a complete lack of reactivity toward sweet and bitter tastants (16Wong G.T. Gannon K.S. Margolskee R.F. Nature. 1996; 381: 796-800Crossref PubMed Scopus (545) Google Scholar). As described above, CNG channels may be generally positioned in the middle of the intracellular signaling pathway of a chemosensation. Despite that, there have been no reports of the existence or function of CNG channel(s) in the gustatory system, with the interesting exception that there is a cyclic nucleotide-suppressible conductance/channel in frog taste cells (17Kolesnikov S.S. Margolskee R.F. Nature. 1995; 376: 85-88Crossref PubMed Scopus (100) Google Scholar). In the present study, we carried out experiments aimed at finding and characterizing a mammalian gustatory CNG channel that is activated by cyclic nucleotides. The present report is the first that deals with a CNG channel occurring in taste buds. It also concerns the location of this gustatory CNG channel, CNGgust, on the pore side of each taste bud. Incidentally, it is noted that in this report CNGgust is sometimes specified as cyclic nucleotide-activated channel to distinguish it from the cyclic nucleotide-suppressible one (17Kolesnikov S.S. Margolskee R.F. Nature. 1995; 376: 85-88Crossref PubMed Scopus (100) Google Scholar). Poly(A+) RNA was isolated from the tongue epithelia of rat circumvallate papillae using oligo(dT)-cellulose. A first-strand cDNA was synthesized using oligo(dT) primer and a first-strand cDNA synthesis kit (Pharmacia). Two degenerate oligonucleotide primers, 5′-GGITCIATGAT(ATC)TCIAA(TC)ATGAA(TC)GC-3′ and 5′-AG(TC)TTICC(TC)TC(TC)TT(GAT)AT(GAT)AT(GA)TACAT(TC)TC(TC)TT-3′, were synthesized according to the amino acid sequences GSMISNMNA and KEMYIIKEGKL, respectively, which are commonly conserved in the COOH-terminal cytosolic region of known CNG channels. PCR amplification was performed using the primers (1 μm each) and rat tongue epithelial cDNA as a template (45 s at 96 °C, 2 min at 45 °C, 3 min at 72 °C, 50 cycles). The amplified DNA fragments of about 380 bp were excised and subcloned into pBluescript KS vector. To obtain clones for CNG channel, another oligonucleotide, 5′-GTCCAIA(GA)(GA)TA(GA)TC(GA)AACCA(TC)TT(GAT)ATIAC-3′, was prepared as a probe according to the sequence VIKWFDYLW. To identify the cDNA corresponding to the RT-PCR fragment encoding the CNG channel named CNGgust, genomic DNA cloning and 5′-RACE were carried out. One million plaques from a rat genomic DNA library (kindly supplied by Dr. E. Kominami, Juntendo University School of Medicine, Tokyo) were screened with the 32P-labeled RT-PCR fragment encoding CNGgust. Hybridization was performed at 65 °C and washing at 65 °C in 0.1 × SSC containing 0.1% SDS. The positive clone obtained, designated λ2, was subcloned into pUC18 vector and sequenced. 5′-RACE was carried out to determine the nucleotide sequence of the 5′-end of the CNGgust cDNA as follows. Poly(A+) RNA from rat tongue epithelia was reverse-transcribed into cDNA using the antisense oligonucleotide primer 5′-TGAAGTGCAAGGTC-3′ (+498 to +511 nt in Fig. 2), which is included in the λ2 genomic DNA fragment. An oligo(dA)-tail was added by terminal deoxynucleotidyltransferase. The oligo(dA)-tailed cDNA was amplified (30 s at 94 °C, 1 min at 50 °C, 1 min at 72 °C, 50 cycles) using an oligo(dT) primer, 5′-GAGTCGACTCGAGAATTCTTTTTTTTTTTTTTTTTTT-3′, and a specific primer according to the nucleotide sequence of the λ2 genomic DNA fragment, 5′-GTTTCCACAGCCTCTTGG-3′ (+470 to +487 nt, Fig. 2). The products were subcloned into a pUC 18 vector and sequenced. To obtain a full-length open reading frame of CNGgust cDNA, a random primed rat tongue epithelial cDNA was amplified using specific primers, 5′-TTCAGGATGCATCAGATG-3′ (−6 to +12 nt, Fig.2) of the 5′-RACE clone and 5′-GGATCCAACAACACTCTC-3′ (+2183 to +2200 nt, Fig. 2) of the λ2 genomic DNA fragment (30 s at 94 °C, 1 min at 50 °C, 3 min at 72 °C, 50 cycles). A 10-μg portion of rat genomic DNA (CLONTECH) was digested with restriction endonuclease, EcoRI, BamHI, or PstI, electrophoresed, and transferred to a nylon filter. The filter was hybridized at 65 °C with the 32P-labeled RT-PCR fragment encoding CNGgust (+961 to +1343 nt in Fig. 2) and then washed at 65 °C in 0.1 × SSC containing 0.1% SDS. CNGgust expression plasmid pSRD-CNG was used for transient expression. pSRD-CNG was constructed as follows. pCNGg1 containing a 2.5 kb-BamHI fragment of λ2 was digested withStuI and SalI, and ligated with a StuI and SalI fragment of a 5′-RACE clone, pCNG5′-1, to generate a 2.2-kb insert encoding the full length of the coding region. The insert (−6 to +1836 nt in Fig. 2) was amplified and subcloned into the pSRD vector, yielding pSRD-CNG. The transient expression of CNGgust was performed by transfecting the expression plasmid pSRD-CNG into human embryonic kidney (HEK) 293 cells using LipofectAMINE (Life Technologies, Inc.). pAdVAntage (Promega) and a green fluorescent protein-expressing vector, pS64T-C1 (CLONTECH) were co-transfected with the expression plasmid. Cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum for 2 or 3 days. Electrical recordings were performed using green fluorescent protein-expressing cells. The current was recorded from inside-out patches using an EPC-7 amplifier (List Electronic) with the current filtered at 2 kHz. The pipette and bath solution used was 140 mm NaCl, 5 mm KCl, 0.5 mm EDTA, 0.5 mm EGTA, and 10 mm HEPES (pH 7.4). The inside-out patch was held at 0 mV in the bath solution containing cGMP or cAMP and stepped from −75 mV to +90 mV for 30 ms, increasing the voltage by 15 mV every 1 s. The current was measured 5 ms before the ending of the voltage pulse. The COOH-terminal 15-mer peptide of CNGgust, FSPDRENSEDASKAD, was synthesized, and a cysteine residue was added to its NH2 terminus to conjugate the peptide to keyhole limpet hemocyanin. Rabbit antiserum was prepared by immunizing the synthetic peptide conjugated to hemocyanin. An anti-CNGgust antibody was affinity-purified by a peptide column. Rats were anesthetized and their tongues were rapidly excised. The excised tongues were immediately embedded in OCT compound (Tissue-Tek, Miles Labs.), frozen in liquid nitrogen, and serially sectioned at 5-μm thickness. Sections containing circumvallate papillae were thaw-mounted onto glass slides, air-dried, and fixed for 10 min in 10% formaldehyde/PBS. The sections were preincubated with normal goat serum and subsequently immunoreacted with a diluted solution of the affinity-purified anti-CNGgust antibody (1:100 in PBS) overnight. After washing with PBS, the slide was incubated with an fluorescein isothiocyanate-conjugated secondary antibody (1:100 in PBS) for 30 min and observed using a fluorescent microscope. To promote degeneration of the taste buds in circumvallate papillae, the glossopharyngeal nerve was removed. This treatment was performed on 5-week-old male rats by the method described previously (18Ninomiya Y. Kajiura H. Naito Y. Mochizuki K. Katsukawa H. Torii K. Physiol. Behav. 1994; 56: 1179-1184Crossref PubMed Scopus (20) Google Scholar). Sections containing circumvallate papillae were excised 6 weeks after denervation and used for immunostaining as described above. We first tried to obtain cDNA fragments encoding a CNG channel by RT-PCR with mRNA prepared from the epithelia of rat circumvallate papillae. Degenerated oligonucleotide primers corresponding to highly conserved sequences in the COOH-terminal cytosolic regions of known CNG channels were used for the RT-PCR. Resulting products with the expected nucleotide lengths of about 380 bp were subcloned and screened with an internal oligonucleotide probe corresponding to another conserved CNG channel sequence. By this method, we isolated a clone, designated pch 12, encoding a part of the cyclic nucleotide-binding domain (Figs.1 and 2). From a data base search, the deduced amino acid sequence was found to be part of a CNG channel as a cyclic nucleotide-activated one, and so was named CNGgust. Next, we tried to obtain a full-length cDNA for CNGgust. Because no appreciable expression of CNGgust mRNA was observed by Northern blot analysis even using 10 μg of poly(A+) RNA extracted from rat tongue epithelial tissues (data not shown), it was considered very difficult to obtain any positive cDNA clone from a conventional cDNA library. Therefore, we obtained a genomic DNA fragment to determine a part of the CNGgust cDNA and then carried out 5′-RACE and RT-PCR. Since it is known that the 3′ two-thirds of the cDNA for the human rod CNG channel consists of a single exon (19Dhallan R.S. Macke J.P. Eddy R.L. Shows T.B. Reed R.R. Yau K.-W. Nathans J. J. Neurosci. 1992; 12: 3248-3256Crossref PubMed Google Scholar), a genomic DNA fragment containing a sequence of the RT-PCR clone (pch 12) was expected to code for the greater part of CNGgust. We then isolated a genomic DNA clone, termed λ2, by screening with pch 12 as a probe. Restriction mapping, Southern hybridization, and sequence analysis indicated that the 2.5-kbBamHI fragment contained a pch 12 sequence (Fig. 1) and that a single exon in this fragment encoded the COOH-terminal part of CNGgust, Phe147–Asp611 (Fig. 2). It was also suggested that the fragment contained at least the 3′-noncoding sequence and that exon(s) encoding the NH2-terminal part should be split by intron(s). To determine the sequence of the 5′-terminal region of the CNGgust cDNA, we next carried out 5′-RACE using a specific antisense primer corresponding to +470 to +487 nt (Fig. 2). As a result, several 5′-RACE clones were obtained. All had the same 3′-regional 446-bp sequence, but their 5′-regional sequences varied (data not shown). This variation was thought to result from alternative splicing, since the CNGgust gene is a single copy as revealed by genomic Southern analysis which yielded a single band under stringent conditions for digestion with EcoRI,BamHI, and PstI using the pch 12 insert (+961 to +1343 nt in Fig. 2) as a probe (Fig. 3). The lengths of the DNA fragments detected by genomic Southern analysis were consistent with the restriction map of λ2 (Figs. 1 and 3). A comparison of the nucleotide sequence of the clone λ2 with that of 5′-RACE clones showed the partial organization of CNGgust gene. Two exons, 108 bp (+222 to +329 nt) and 107 bp (+330 to +436 nt) in length, are located separately (Fig. 1). Fig. 2 shows the nucleotide and deduced amino acid sequences of CNGgust cDNA. The nucleotide sequence was based on the λ2 genomic DNA clone and the most abundant 5′-RACE clones (−13 to +487 nt). Since the 5′-terminal CNGgust cDNA has not been definitely elucidated, the initiation methionine codon could not be assigned. The methionine residue in the most 5′-terminal region was taken as the starting position for nucleotide and amino acid numbering. Although the 3′-end of the cDNA has not been elucidated, the 2206-nt sequence (−6 to +2200 nt) (Fig. 2) containing the termination codon and part of the 3′-noncoding region was contained in one of the mRNA species, because we obtained a 2206-bp product by RT-PCR using a primer set (−6 to +12 nt and +2183 to +2200 nt in Fig. 2) and random primed cDNA from rat circumvallate papillae. CNGgust shares an overall structural similarity with other CNG channels. It contains 1) a central hydrophobic region comprising six membrane-spanning segments, 2) a pore-forming region that may determine ion permeability, and 3) a cyclic nucleotide-binding domain near the COOH terminus that locates in the cytosolic region. CNGgust also contains two potentialN-glycosylation sites (Fig.4). Fig. 4 shows an amino acid alignment of CNGgust with known CNG channels. Since the NH2-terminal sequence of CNGgust has not been definitely determined, a precise comparison of the NH2-terminal regions is not achieved. In terms of overall homology, CNGgust shows the highest similarity (82%) to bovine testis CNG channel (20Weyand I. Godde M. Frings S. Weiner J. Müller F. Altenhofen W. Hatt H. Kaupp U.B. Nature. 1994; 368: 859-863Crossref PubMed Scopus (230) Google Scholar, 21Biel M. Zong X. Distler M. Bosse E. Klugbauer N. Murakami M. Flockerzi V. Hofmann F. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3505-3509Crossref PubMed Scopus (124) Google Scholar) and the same degree of similarity to human cone CNG channel (22Yu W.-P. Grunwald M.E. Yau K.-W. FEBS Lett. 1996; 393: 211-215Crossref PubMed Scopus (52) Google Scholar). However, it shows lower similarities (50–70%) to other CNG channels: 66% to rat olfactory CNG channel (6Dhallan R.S. Yau K.-W. Schrader K.A. Reed R.R. Nature. 1990; 347: 184-187Crossref PubMed Scopus (510) Google Scholar) and 63% to rat rod CNG channel (23Barnstable C.J. Wei J.-Y. J. Mol. Neurosci. 1995; 6: 289-302Crossref PubMed Scopus (19) Google Scholar). As for the central part ranging from the predicted first transmembrane region to the cyclic nucleotide-binding domain, where the highest similarity has generally been observed among known CNG channels, CNGgust has a similarity as high as 85% to the bovine testis and human cone CNG channels, and a similarity of 60–70% to other vertebrate CNG channels. In the NH2-terminal region, on the other hand, CNGgust lacks the 20–40 amino acids between Gly74 and Arg75 when compared with bovine testis and human cone CNG channels (Fig. 4). It should be noted that an intron is inserted in this position of the CNGgust cDNA (Fig.2). Since known cloned CNG channels are functional when expressed in cultured cells, we expressed CNGgust in HEK 293 cells and investigated CNG channel properties. As shown in Fig.5, electrophysiological recording of CNGgust-expressing cells showed that the expressed protein in inside-out patches, although it may be missing the NH2-terminal region, can open when a cyclic nucleotide, cGMP and cAMP, is added. The current-voltage relationship was approximately linear through the change in membrane potential (+90 to −75 mV) at every nucleotide concentration tested (Fig. 5 A). The half-maximal activation constant (K½) at −60 mV was about 3 μm for cGMP, whereas it was about 300 μm for cAMP (Fig. 5 B). The sensitivity to cGMP is about 100 times higher than it is to cAMP. Further, the current observed with saturating cAMP (10 mm) was 85% of that observed with saturating cGMP (100 μm) (Fig.5 B). This ratio is similar to that reported by Biel et al. (21Biel M. Zong X. Distler M. Bosse E. Klugbauer N. Murakami M. Flockerzi V. Hofmann F. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3505-3509Crossref PubMed Scopus (124) Google Scholar) for the bovine testis CNG channel. We observed the expression of CNGgust mRNA in the circumvallate papillae by 5′-RACE and RT-PCR. To determine the localization of CNGgust in tissue, immunostaining experiments were carried out using a polyclonal antibody raised against the COOH-terminal peptide. Intense fluorescence was observed in each of the taste buds, especially on the pore side of the apical region where taste reception followed by initial intracellular signaling may occur (Fig. 6, B and C). To examine whether or not the expression of CNGgust depends on the presence of taste buds, tongue sections from rats whose glossopharyngeal nerves connecting the tongue to the central nervous system had been destroyed were immunostained. The removal of this nerve is known to promote the degeneration of taste buds in the circumvallate papillae since regeneration no longer occurs (24Guth L. Anat. Rec. 1957; 128: 715-731Crossref PubMed Scopus (144) Google Scholar). The taste buds disappeared 6 weeks after denervation, and no CNGgust protein signal was observed in the corresponding region (Fig. 6 D). These results indicate that our cloned CNGgust is expressed in taste buds. In this study, we report the cloning of a CNG channel (CNGgust) as a cyclic nucleotide-activated one from rat tongue epithelia. This is the first study identifying a CNG channel in mammalian gustatory organs. CNGgust is expressed in taste buds including the taste cells where gustatory reception takes place. Our findings thus strongly indicate the possible involvement of CNGgust in the intracellular signal transduction of taste cells. CNGgust may consist of several variants judging from the results of 5′-RACE. Since the CNGgust gene is a single copy gene (Fig. 3), this variation probably results from alternative splicing that gives rise to different 5′-coding regions. Since the CNGgust derived from the most abundant type of 5′-RACE clones was shorter than other CNG channels in the NH2-terminal region (Fig. 4), a full-length CNGgust species with an additional NH2 sequence must exist. However, the CNGgust expressed in HEK cells, although not full-length, was functional as a cyclic nucleotide-gated channel (Fig. 5). Electrophysiological information concerning cloned vertebrate CNG channels is available. It has been reported that cAMP is not capable of activating rod CNG channels even when a high concentration of cAMP (1 mm) is applied (23Barnstable C.J. Wei J.-Y. J. Mol. Neurosci. 1995; 6: 289-302Crossref PubMed Scopus (19) Google Scholar, 25Kaupp U.B. Niidome T. Tanabe T. Terada S. Bönigk W. Stühmer W. Cook N.J. Kangawa K. Matsuo H. Hirose T. Miyata T. Numa S. Nature. 1989; 342: 762-766Crossref PubMed Scopus (502) Google Scholar). On the other hand, cAMP can activate olfactory CNG channels, but the sensitivity for cAMP is about 30 times lower than that for cGMP (6Dhallan R.S. Yau K.-W. Schrader K.A. Reed R.R. Nature. 1990; 347: 184-187Crossref PubMed Scopus (510) Google Scholar). In our study, we showed that CNGgust responded to cAMP as well as to cGMP, although the sensitivity for cGMP was about 100 times greater than that for cAMP in terms of theK½ value (Fig. 5 B). Recently, a CNG channel has been shown to function as a hetero-oligomer of α- and β-subunits. The α-subunit has been shown to function as a CNG channel in the absence of the β-subunit. On the other hand, the β-subunit, which has been cloned from rod outer segments, olfactory neurons, and testis, does not form any functional channel by itself (26Chen T.-Y. Peng Y.-W. Dhallan R.S. Ahamed B. Reed R.R. Yau K.-W. Nature. 1993; 362: 764-767Crossref PubMed Scopus (270) Google Scholar, 27Körschen H.G. Illing M. Seifert R. Sesti F. Williams A. Gotzes S. Colville C. Müller F. Dosé A. Godde M. Molday L. Kaupp U.B. Molday R.S. Neuron. 1995; 15: 627-636Abstract Full Text PDF PubMed Scopus (207) Google Scholar, 28Liman E.R. Buck L.B. Neuron. 1994; 13: 611-621Abstract Full Text PDF PubMed Scopus (211) Google Scholar, 29Bradley J. Li J. Davidson N. Lester H.A. Zinn K. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8890-8894Crossref PubMed Scopus (205) Google Scholar, 30Biel M. Zong X. Ludwig A. Sautter A. Hofmann F. J. Biol. Chem. 1996; 271: 6349-6355Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). However, it has also been shown that the β-subunit increases the sensitivity for cAMP when coexpressed with the α-subunit (28Liman E.R. Buck L.B. Neuron. 1994; 13: 611-621Abstract Full Text PDF PubMed Scopus (211) Google Scholar, 29Bradley J. Li J. Davidson N. Lester H.A. Zinn K. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8890-8894Crossref PubMed Scopus (205) Google Scholar). Thus the β-subunit is considered to be a modulatory subunit. It is probable that the β-subunit per se exists in taste cells to form an α-β hetero-oligomer with the α-subunit (CNGgust), and that the resulting oligomer shows a high sensitivity for cAMP, similar to that for cGMP. The involvement of CNGgust in the taste signaling process is strongly suggested by the following findings and knowledge. First, CNGgust is expressed specifically on the pore side of each taste bud in the circumvallate papilla (Fig. 6, B and C). In addition, this site-specific expression is dependent on the existence of taste buds as shown by the denervation experiments (Fig.6 D). Second, it has been reported that taste buds contain a series of intrinsic molecules participating in the signal transduction cascade involving cyclic nucleotides. These are exemplified by G-protein-coupled receptors (8Abe K. Kusakabe Y. Tanemura K. Emori Y. Arai S. J. Biol. Chem. 1993; 268: 12033-12039Abstract Full Text PDF PubMed Google Scholar, 10Matsuoka I. Mori T. Aoki J. Sato T. Kurihara K. Biochem. Biophys. Res. Commun. 1993; 194: 504-511Crossref PubMed Scopus (40) Google Scholar, 11Tal M. Ammar D.A. Karpuj M. Krizhanovsky V. Naim M. Thompson D.A. Biochem. Biophys. Res. Commun. 1995; 209: 752-759Crossref PubMed Scopus (89) Google Scholar, 12Thomas M.B. Haines S.L. Akeson R.A. Gene ( Amst. ). 1996; 178: 1-5Crossref PubMed Scopus (32) Google Scholar), gustducin/transducin (13McLaughlin S.K. McKinnon P.J. Margolskee R.F. Nature. 1992; 357: 563-569Crossref PubMed Scopus (542) Google Scholar, 14Ruiz-Avila L. McLaughlin S.K. Wildman D. McKinnon P.J. Robichon A. Spickofsky N. Margolskee R.F. Nature. 1995; 376: 80-85Crossref PubMed Scopus (173) Google Scholar), phosphodiesterase (14Ruiz-Avila L. McLaughlin S.K. Wildman D. McKinnon P.J. Robichon A. Spickofsky N. Margolskee R.F. Nature. 1995; 376: 80-85Crossref PubMed Scopus (173) Google Scholar, 31McLaughlin S.K. McKinnon P.J. Spickofsky N. Danho W. Margolskee R.F. Physiol. Behav. 1994; 56: 1157-1164Crossref PubMed Scopus (60) Google Scholar), adenylyl cyclase/guanylyl cyclase (32Kurihara K. Koyama N. Biochem. Biophys. Res. Commun. 1972; 48: 30-34Crossref PubMed Scopus (77) Google Scholar, 33Asanuma N. Nomura H. Chem. Senses. 1995; 20: 231-237Crossref PubMed Scopus (12) Google Scholar), and rhodopsin kinase (34Premont R.T. Koch W.J. Inglese J. Lefkowitz R.J. J. Biol. Chem. 1994; 269: 6832-6841Abstract Full Text PDF PubMed Google Scholar). Third, it has been reported that the stimulation of taste cells with some sweet tastants increases their intracellular cAMP level (35Striem B.J. Naim M. Lindemann B. Cell. Physiol. Biochem. 1991; 1: 46-54Crossref Scopus (81) Google Scholar). Taken together, the CNGgust characterized here may be an essential mediator in cAMP/cGMP-dependent taste signaling. Recently, Kolesnikov and Margolskee (17Kolesnikov S.S. Margolskee R.F. Nature. 1995; 376: 85-88Crossref PubMed Scopus (100) Google Scholar) have electrophysiologically demonstrated the occurrence of a cyclic nucleotide-suppressible conductance/channel in frog taste cells. In contrast is our finding that CNGgust, as well as many other CNG channels, is activated in the presence of cyclic nucleotides. It may therefore be appropriate to distinguish CNGgust by designating it as a cyclic nucleotide-activated rather than gated channel. However, it remains unclear whether a similar suppressible channel occurs in rat taste cells or if it is just intrinsic to frogs. If such a channel resides in mammals as well, the possibility exists that both positively and negatively controlled pathways are together involved in mammalian taste signaling, and it will be extremely interesting to define each of them at the molecular level. We express our thanks to Dr. Y. Ninomiya, Asahi University, for performing the denervation treatments, to Dr. S. Seino, Chiba University, for advice concerning the electrophysiological study, and to Dr. K. Sugimoto, Tokyo Medical and Dental University, for pertinent discussion on the histology of taste cells." @default.
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• W1967425088 title "Taste Buds Have a Cyclic Nucleotide-activated Channel, CNGgust" @default.
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• W1967425088 cites W1983108533 @default.
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• W1967425088 cites W2036884791 @default.
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• W1967425088 cites W2048772732 @default.
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• W1967425088 cites W2065983999 @default.
• W1967425088 cites W2067346266 @default.
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