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• W2065897911 abstract "Taste bud cells are epithelial cells with neuronal properties. Voltage-dependent ion channels have been physiologically described in these cells. Here, we report the molecular identification and functional characterization of a voltage-gated chloride channel (ClC-4) and its novel splice variant (ClC-4A) from taste bud cells. ClC-4A skipped an exon near its 5′-end, incurring the loss of 60 amino acids at the N terminus. In situ hybridization and immunohistochemistry localized these two channels' transcripts and proteins to a subset of taste bud cells. Electrophysiological recordings of the heterologously expressed channels in Xenopus oocytes showed that ClC-4 and ClC-4A have opposite sensitivity to pH and unique ion selectivity. The chloride channel blockers niflumic acid and 5-nitro-2-(3-phenylpropylamino)benzoic acid had a slight or no inhibitory effect on the conductance of ClC-4, but both blockers inhibited ClC-4A, suggesting that ClC-4A is a candidate channel for an acid-induced 5-nitro-2-(3-phenylpropylamino)benzoic acid-sensitive current. Furthermore, these two channels may play a role in bitter-, sweet-, and umami-mediated taste transmission by regulating transmitter uptake into synaptic vesicles. Taste bud cells are epithelial cells with neuronal properties. Voltage-dependent ion channels have been physiologically described in these cells. Here, we report the molecular identification and functional characterization of a voltage-gated chloride channel (ClC-4) and its novel splice variant (ClC-4A) from taste bud cells. ClC-4A skipped an exon near its 5′-end, incurring the loss of 60 amino acids at the N terminus. In situ hybridization and immunohistochemistry localized these two channels' transcripts and proteins to a subset of taste bud cells. Electrophysiological recordings of the heterologously expressed channels in Xenopus oocytes showed that ClC-4 and ClC-4A have opposite sensitivity to pH and unique ion selectivity. The chloride channel blockers niflumic acid and 5-nitro-2-(3-phenylpropylamino)benzoic acid had a slight or no inhibitory effect on the conductance of ClC-4, but both blockers inhibited ClC-4A, suggesting that ClC-4A is a candidate channel for an acid-induced 5-nitro-2-(3-phenylpropylamino)benzoic acid-sensitive current. Furthermore, these two channels may play a role in bitter-, sweet-, and umami-mediated taste transmission by regulating transmitter uptake into synaptic vesicles. Most mammals, including humans, are believed to possess at least five taste qualities: sour, salty, bitter, sweet, and umami (the latter being the taste for monosodium glutamate and certain 5′-ribonucleotides in humans while being a taste for a more diverse group of l-amino acids in several other species). Interactions between sapid molecules in food-stuffs and specific receptor cells in taste buds of the oral cavity initiate taste sensation. Bitter, sweet, and umami compounds stimulate G protein-coupled seven-transmembrane receptors (GPCRs), 2The abbreviations used are:GPCRsG protein-coupled seven-transmembrane receptorsIP3R3inositol 1,4,5-triphosphate receptor-3ClCchloride channelNPPB5-nitro-2-(3-phenylpropylamino)benzoic acidSNAPsoluble N-ethylmaleimide-sensitive factor attachment proteinMES4-morpholineethanesulfonic acid whereas ionic stimuli such as protons in acids and Na+ in salts either permeate or activate ion channels on the surface of taste receptor cells (1Lindemann B. 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Sci. 1998; 855: 398-406Crossref PubMed Scopus (67) Google Scholar) and -1, along with some of their downstream components, including G protein subunits α-gustducin, Gβ1,Gβ3, and Gγ13 (18McLaughlin S.K. McKinnon P.J. Spickofsky N. Danho W. Margolskee R.F. Physiol. Behav. 1994; 56: 1157-1164Crossref PubMed Scopus (62) Google Scholar, 19Wong G.T. Gannon K.S. Margolskee R.F. Nature. 1996; 381: 796-800Crossref PubMed Scopus (558) Google Scholar, 20Huang L. Shanker Y.G. Dubauskaite J. Zheng J.Z. Yan W. Rosenzweig S. Spielman A.I. Max M. Margolskee R.F. Nat. Neurosci. 1999; 2: 1055-1062Crossref PubMed Scopus (285) Google Scholar, 21Rossler P. Boekhoff I. Tareilus E. Beck S. Breer H. Freitag J. Chem. Senses. 2000; 25: 413-421Crossref PubMed Google Scholar, 22Caicedo A. Pereira E. Margolskee R.F. Roper S.D. J. Neurosci. 2003; 23: 9947-9952Crossref PubMed Google Scholar, 23Ruiz C.J. Wray K. Delay E. Margolskee R.F. Kinnamon S.C. Chem. Senses. 2003; 28: 573-579Crossref PubMed Scopus (77) Google Scholar); phospholipase Cβ2 (24Yan W. Sunavala G. Rosenzweig S. Dasso M. Brand J.G. Spielman A.I. Am. J. Physiol. 2001; 280: C742-C751Crossref PubMed Google Scholar, 25Rossler P. Kroner C. Freitag J. Noe J. Breer H. Eur. J. Cell Biol. 1998; 77: 253-261Crossref PubMed Scopus (183) Google Scholar); inositol 1,4,5-triphosphate receptor-3 (IP3R3) (26Clapp T.R. Stone L.M. Margolskee R.F. Kinnamon S.C. BMC Neuroscience. 2001; http://www.biomedcentral.com/1471-2202/2/6PubMed Google Scholar); and the transient receptor ion channel TRPM5 (27Perez 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 (476) Google Scholar, 28Zhang 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 (999) Google Scholar, 29Liu D. Liman E.R. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 15160-15165Crossref PubMed Scopus (353) Google Scholar, 30Prawitt 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 (289) Google Scholar, 31Hofmann T. Chubanov V. Gudermann T. Montell C. Curr. Biol. 2003; 13: 1153-1158Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar), have been identified. Activation of these taste GPCRs presumably leads to changes in intracellular calcium activity and influx of ions through TRPM5. Opening of the TRPM5 channel is critical to the transmission of gustatory signals to the peripheral nerves. However, an increase in [Ca2+]i alone is not sufficient to trigger the full taste response because TRPM5-null animals in which phospholipase Cβ2 and IP3R3 are intact have markedly reduced behavioral and integrated nerve responses to sweet, bitter, and umami stimuli. TRPM5-mediated ion influx is very likely a major contributor to taste receptor cell membrane depolarization; however, the underlying mechanisms remain to be uncovered. Sour and salty tastes apparently use several ion channel types to initiate their transduction, including amiloride-sensitive and -insensitive epithelial sodium channels (32Lin W. Finger T.E. Rossier B.C. Kinnamon S.C. J. Comp. Neurol. 1999; 405: 406-420Crossref PubMed Scopus (174) Google Scholar), acid-sensing ion channels (33Ugawa S. Minami Y. Guo W. Saishin Y. Takatsuji K. Yamamoto T. Tohyama M. Shimada S. Nature. 1998; 395: 555-556Crossref PubMed Scopus (150) Google Scholar, 34Liu L. Simon S.A. Brain Res. 2001; 923: 58-70Crossref PubMed Scopus (71) Google Scholar, 35Lin W. Ogura T. Kinnamon S.C. J. Neurophysiol. 2002; 88: 133-141Crossref PubMed Scopus (93) Google Scholar), hyperpolarization- and cyclic nucleotide-gated channels (36Stevens D.R. Seifert R. Bufe B. Muller F. Kremmer E. Gauss R. Meyerhof W. Kaupp U.B. Lindemann B. Nature. 2001; 413: 631-635Crossref PubMed Scopus (189) Google Scholar), and proton-gated potassium channels (37Kinnamon S.C. Dionne V.E. Beam K.G. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 7023-7027Crossref PubMed Scopus (134) Google Scholar). In addition, acid-induced chloride currents (38Miyamoto T. Fujiyama R. Okada Y. Sato T. J. Neurophysiol. 1998; 80: 1852-1859Crossref PubMed Scopus (47) Google Scholar) and intracellular acidification (39Lyall V. Alam R.I. Phan T.H. Phan D.Q. Heck G.L. DeSimone J.A. J. Neurophysiol. 2002; 87: 399-408Crossref PubMed Scopus (32) Google Scholar, 40Lyall V. Alam R.I. Phan D.Q. Ereso G.L. Phan T.H. Malik S.A. Montrose M.H. Chu S. Heck G.L. Feldman G.M. DeSimone J.A. Am. J. Physiol. 2001; 281: C1005-C1013Crossref PubMed Google Scholar) have been recorded for taste bud cells. Like taste GPCR-mediated transduction, stimulation of channel receptors by ionic taste stimuli also triggers changes in membrane potential. It remains to be understood how receptor potentials encode and transmit the identity of gustatory stimuli to afferent neural fibers. Taste bud cells are epithelial cells with neuronal properties. A curious and not well understood property of these cells is their electrical excitability (41Roper S. Science. 1983; 220: 1311-1312Crossref PubMed Scopus (95) Google Scholar, 42Kashiwayanagi M. Miyake M. Kurihara K. Am. J. Physiol. 1983; 244: C82-C88Crossref PubMed Google Scholar). The passing of current or chemical stimulation by appropriate taste substances evokes action potentials in these cells, but not in surrounding non-gustatory lingual epithelial cells. Electrophysiological studies have characterized various voltage-gated currents, including tetrodotoxin-sensitive Na+ currents, tetraethylammonium-sensitive transient and sustained K+ currents, inward rectifier K+ currents, outward rectifier Cl– currents, and low/high voltage-activated Ca2+ currents (43Chen Y. Sun X.D. Herness S. J. Neurophysiol. 1996; 75: 820-831Crossref PubMed Scopus (58) Google Scholar, 44Behe P. DeSimone J.A. Avenet P. Lindemann B. J. Gen. Physiol. 1990; 96: 1061-1084Crossref PubMed Scopus (161) Google Scholar, 45Herness M.S. Sun X.D. J. Membr. Biol. 1995; 146: 73-84Crossref PubMed Scopus (53) Google Scholar, 46Chen Y. Herness M.S. Pfluegers Arch. Eur. J. Physiol. 1997; 434: 215-226Crossref PubMed Scopus (33) Google Scholar, 47Sun X.D. Herness M.S. Am. J. Physiol. 1996; 271: C1221-C1232Crossref PubMed Google Scholar, 48Herness M.S. Sun X.D. J. Neurophysiol. 1999; 82: 260-271Crossref PubMed Scopus (44) Google Scholar). The distribution of these currents across cells within a taste bud is heterogeneous. However, little is known about these channels' molecular identities and their expression and interaction with other taste signaling molecules. Two types of action potentials have been recorded: 1) fast action potentials with shorter duration and larger inward and outward currents and 2) slow action potentials with longer duration and smaller inward and outward currents. It is unclear how these two types of action potentials may contribute to gustatory signal transmission and peripheral coding. Recently, analyses of the action potential activity of a population of taste buds in response to taste stimuli using an artificial neural network suggested that patterns of action potentials may be the source of taste quality coding (49Varkevisser B. Peterson D. Ogura T. Kinnamon S.C. Chem. Senses. 2001; 26: 499-505Crossref PubMed Scopus (17) Google Scholar). In an attempt to identify signaling molecules in taste bud cells and to gain insight into taste transduction and peripheral coding in the taste end organ, we have employed a single cell strategy and bioinformatics tools to isolate cell type-specific genes that are involved in defining particular physiological functions of each taste bud cell type. This approach has resulted in the identification of several key taste signaling molecules, including the taste receptor T1R3; G protein subunits Gβ1, Gβ3, and Gγ13; and the ion channel TRPM5 (7Max M. Shanker Y.G. Huang L. Rong M. Liu Z. Campagne F. Weinstein H. Damak S. Margolskee R.F. Nat. Genet. 2001; 28: 58-63Crossref PubMed Scopus (462) Google Scholar, 20Huang L. Shanker Y.G. Dubauskaite J. Zheng J.Z. Yan W. Rosenzweig S. Spielman A.I. Max M. Margolskee R.F. Nat. Neurosci. 1999; 2: 1055-1062Crossref PubMed Scopus (285) Google Scholar, 27Perez 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 (476) Google Scholar). In this study, we describe a voltage-gated, pH-sensitive chloride channel (ClC-4) and its novel splice variant (ClC-4A); their coexpression with other taste GPCR-mediated signal transduction components; and their unique pharmacological properties, pH sensitivities, and ion selectivity. Although the specific functions and activities of taste cell-associated chloride channels are still not well understood at this point, chloride currents have been previously recorded from taste bud cells. Some of these currents are modulated by adrenergic agonists, activated by hypoosmotic stimuli, or inhibited by the chloride channel blocker 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) (38Miyamoto T. Fujiyama R. Okada Y. Sato T. J. Neurophysiol. 1998; 80: 1852-1859Crossref PubMed Scopus (47) Google Scholar, 48Herness M.S. Sun X.D. J. Neurophysiol. 1999; 82: 260-271Crossref PubMed Scopus (44) Google Scholar, 50Gilbertson T.A. Chem. Senses. 2002; 27: 383-394Crossref PubMed Scopus (28) Google Scholar). During mouse postnatal development, Cl– currents appear in a subset of excitable taste bud cells only after postnatal day 8, which coincides with the maturation of taste bud cells (51Ghiaroni V. Fieni F. Pietra P. Bigiani A. Chem. Senses. 2003; 28: 827-833Crossref PubMed Scopus (6) Google Scholar). Molecular identification of these chloride channels is important for eventual determination of their contributions to action potential waveforms, firing properties, and ultimately to the strength of transduction. The data from existing literature combined with the characterization studies of taste ClC-4 and ClC-4A presented here suggest that these two channels may play a role in the taste stimulus-induced depolarization of membrane potential and signal transmission. Isolation of ClC-4 and Splice Variant ClC-4A cDNAs from Mouse Taste Bud Cells—Isolation of ClC-4 and ClC-4A cDNAs from taste bud cells was accomplished in two steps. 1) A 3′-end cDNA fragment was isolated from a single taste cell cDNA library by differential screening with cDNAs from gustatory and non-gustatory lingual epithelia. Construction and differential screening of single taste cell cDNA libraries were described previously (20Huang L. Shanker Y.G. Dubauskaite J. Zheng J.Z. Yan W. Rosenzweig S. Spielman A.I. Max M. Margolskee R.F. Nat. Neurosci. 1999; 2: 1055-1062Crossref PubMed Scopus (285) Google Scholar, 27Perez 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 (476) Google Scholar). Briefly, taste papillae were separated from the rest of a mouse tongue and enzymatically dissociated into individual cells. Taste bud cells were identified by their characteristic bipolar shape and transferred individually into Eppendorf tubes. First-strand cDNAs were synthesized from single cells with oligo(dT) primers, tailed with dATP and terminal transferase, and amplified by PCR. The PCR products were ligated into the λZapII vector to construct single taste cell cDNA libraries. Individual phage plaques were picked, and their insert cDNAs were amplified by PCR with vector-specific primers. The amplified products were size-fractionated by electrophoresis, transferred onto a nylon membrane, and screened with 32P-labeled cDNAs prepared from non-gustatory lingual epithelium devoid of taste buds. Insert cDNAs that were not hybridized with the non-gustatory probe were presumed to be expressed selectively in taste cells, and their sequences were analyzed and searched against genome and expressed sequence tag data bases. One of the clones matched the 3′-end of human ClC-4. 2) Isolation of full-length cDNAs was carried out with mouse taste tissue cDNA. PCR primers were designed to encompass the entire mouse ClC-4 coding region (sense, 5′-AGGAGGATGATCTAGGACGCTGTC-3′; and antisense, 5′-TCTCAAAATAATGCCCATCTTATTGCT-3′). Two fragments were obtained and subcloned into the pCR-BluntII-TOPO vector. DNA sequence analysis and sequence alignment showed that the long fragment was the same as mouse ClC-4 (GenBank™ accession number XM_193014), whereas the short fragment was a splice variant of ClC-4, which we designated ClC-4A (GenBank™ accession number DQ186662). To determine whether these two isoforms are expressed in all three types of gustatory lingual papillae, a new set of PCR primers (sense, 5′-AGGAGGATGATCTAGGACGCTGTC-3′; and antisense, 5′-CATGCACCAGTGGGCCCTCTTTG-3′) was designed and synthesized to encompass the differentially spliced region, and PCRs were performed with first-strand cDNAs from circumvallate, foliate, and fungiform papillae and non-gustatory lingual epithelium. In Situ Hybridization—Digoxigenin-labeled RNA probes (ClC-4, 2.2 kb) were used for in situ hybridization on fresh frozen sections (14 μm) as described (52Schaeren-Wiemers N. Gerfin-Moser A. Histochemistry. 1993; 100: 431-440Crossref PubMed Scopus (1085) Google Scholar). An alkaline phosphatase-conjugated anti-digoxigenin antibody in the presence of nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate was used for detection. Immunohistochemistry—Polyclonal antiserum against a keyhole limpet hemocyanin-conjugated 13-amino acid peptide near the C termini of mouse, rat, and human ClC-4 and ClC-4A was raised in rabbits (Alpha Diagnostic International, Inc.). Frozen sections (10 μm) of murine lingual tissue (previously fixed in 4% paraformaldehyde and cryoprotected in 20% sucrose) were blocked in 3% bovine serum albumin, 0.3% Triton X-100, 2% goat serum, and 0.1% sodium azide in phosphate-buffered saline for 1 h at room temperature and then incubated overnight at 4 °C with the polyclonal antiserum (1:1000 dilution). The secondary antibody used was Cy3-conjugated goat anti-rabbit Ig (Jackson ImmunoResearch Laboratories, Inc.). To determine the type of cells expressing ClC-4/ClC-4A, double immunostaining was carried out on taste sections with the rabbit polyclonal antibody against ClC-4/ClC-4A and the mouse monoclonal antibodies against IP3R3 (1:50 dilution; BD Biosciences) and SNAP-25 (1:1000 dilution; Sternberger Monoclonals Inc., Lutherville, MD). The secondary antibodies used were fluorescein isothiocyanate-conjugated anti-mouse and Cy3-conjugated anti-rabbit antibodies. Fluorescent signals were viewed under a Leica confocal microscope. Heterologous Expression in Xenopus Oocytes and Electrophysiological Recordings—Murine ClC-4 and ClC-4A cDNAs and human ClC-4 (GenBank™ accession number AB019432) were subcloned into the pCR-BluntII-TOPO expression vector. Capped sense cRNA was synthesized from the linearized expression constructs by T7 RNA polymerase using the mMessage mMachine in vitro transcription kit and tailed with a poly(A) tailing kit (Ambion Inc.). The synthesized cRNA products were phenol-extracted, ethyl alcohol-precipitated, and then dissolved in nuclease-free water at ∼0.5 ng/nl for injection. Dumont stage V or VI oocytes were obtained from adult female laboratory-bred Xenopus laevis, and their follicles were removed by collagenase digestion. Oocytes were injected with 50 nl of 0.5 ng/nl cRNA and maintained at 18 °C for 4–6 days in modified Barth's solution supplemented with 5 mm sodium pyruvate (53Goldin A.L. Sumikawa K. Methods Enzymol. 1992; 207: 279-297Crossref PubMed Scopus (58) Google Scholar). Membrane currents were recorded using the two-electrode voltage-clamp technique in ND96 solution (96 mm NaCl, 2 mm KCl, 1.8 mm CaCl2, 1 mm MgCl2, and 5 mm NaHEPES (pH 7.4)). To determine anionic selectivity, 80 mm Cl– was substituted with equivalent amounts of Br–, I–, or NO–3. In the study on the effect of pH on channel conductance, 5 mm HEPES (for pH 7.4) was replaced with 5 mm MES (for pH <7.0). For pharmacological analyses, inhibitors of niflumic acid (0.3 mm) or NPPB (at 0.1 mm) were dissolved in ND96 solution. Recording and current-passing micropipettes with tip resistances <5 megaohms when filled with 3 m KCl were pulled from glass capillaries (A-M Systems, Inc., Carlsborg, WA) using a horizontal puller (Sutter P-80/PC). Currents were recorded with a GeneClamp 500 amplifier, digitized with a Digidata 1200 A/DD/A system, and stored on a computer running pCLAMP 6 software (all from Axon Instruments, Foster City, CA). Currents were low pass-filtered at 2 kHz and are shown without subtraction of leakage currents. Isolation of ClC-4 cDNAs—To identify genes that are selectively expressed in taste bud cells, we isolated individual taste bud cells and constructed single cell cDNA libraries with the λZapII vector (20Huang L. Shanker Y.G. Dubauskaite J. Zheng J.Z. Yan W. Rosenzweig S. Spielman A.I. Max M. Margolskee R.F. Nat. Neurosci. 1999; 2: 1055-1062Crossref PubMed Scopus (285) Google Scholar, 27Perez 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 (476) Google Scholar). Individual phage plaques were picked, and their insert cDNAs were amplified by PCR with vector-specific primers. The amplified products were size-fractionated by electrophoresis and transferred onto a nylon membrane. Southern hybridization of the nylon membranes showed that many inserts from the single cell cDNA libraries could hybridize with the radiolabeled cDNAs prepared from non-gustatory lingual epithelium devoid of taste buds. The few that were not hybridized were considered taste bud-specific (Fig. 1); insert DNAs of these clones were sequenced and bioinformatically analyzed. Among ∼200 taste bud-specific clones analyzed, one clone (GA5508) with a 906-bp insert cDNA was 83% identical to the 3′-end sequence of the human voltage-gated, pH-sensitive chloride channel ClC-4 cDNA (GenBank™ accession number NM_001830.2). A subsequent search against the mouse genome data bases with the GA5508 insert sequence and the human ClC-4 cDNA sequence identified a corresponding mouse genomic sequence. Its putative cDNA sequence was predicted using GenScan software, encompassing partial mouse ClC-4 cDNA sequences deposited in the GenBank™ Data Bank. PCR primers were designed to cover the entire coding region of mouse ClC-4. Surprisingly, PCR amplification with taste circumvallate papilla cDNA yielded two fragments of 2.4 and 2.23 kb. DNA sequencing showed that the former is the same as the previously described mouse ClC-4 chloride channel with 747 amino acid residues (GenBank™ accession number XM_193014). However, the 2.23-kb fragment is a novel splice variant, lacking 155 bp near the 5′-end of ClC-4, including the presumed ClC-4 start codon (ATG). The amino acid sequence of the short form was deduced from the next in-frame start codon, which is probably the true start codon, there being no other start codon. A stop codon is present ∼140 bp upstream. Thus, ClC-4A putatively encodes a protein of 687 amino acid residues, lacking the N-terminal 60 amino acid residues of ClC-4. Sequence analysis indicated that the deletion of 155 bp is a result of exon skipping and that the new N terminus of the ClC-4A protein starts within a presumed helix domain of ClC-4 (helix A), which may be expected to significantly affect the channel's functions (Fig. 2). To determine whether these two isoforms are expressed in all three types of gustatory lingual papillae, a new set of PCR primers was designed and synthesized specifically for amplification of the differentially spliced region of the cDNAs. Reverse transcription-PCR detected both forms of the cDNA in all three types of taste papillae (circumvallate, foliate, and fungiform), but not in non-gustatory lingual epithelium (Fig. 3).FIGURE 3Expression of the chloride channels in three types of taste papillae. Both ClC-4 and ClC-4A were detected in all three types of taste papillae (fungiform, circumvallate (Vallate), and foliate), but not in non-taste lingual epithelium (NT). PCR amplification was carried out with primers covering the differentially spliced region and first-strand cDNAs prepared from taste and non-taste tissues. The expected PCR products for ClC-4 and ClC-4A are indicated.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Localization of the ClC-4/ClC-4A RNA Transcripts and Proteins to Taste Bud Cells—To localize the RNA transcripts to taste bud cells, in situ hybridization was carried out with a 2.2-kb probe common to both ClC-4 and ClC-4A. The results demonstrated that the ClC-4/ClC-4A transcripts were selectively expressed in taste bud cells, but were absent from the surrounding non-gustatory lingual epithelial cells (Fig. 4). Sense probe controls showed no nonspecific hybridization to lingual tissue. To determine the expression of the ClC-4/ClC-4A proteins in taste receptor cells, we used immunohistochemistry with antiserum to a peptide near the C termini of the ClC-4/ClC-4A proteins on sections of murine lingual tissue. This antibody was able to recognize both ClC-4 and ClC-4A. The immunostaining results indicated that the ClC-4/ClC-4A proteins were present on the cytoplasmic membrane. Some spotty staining seen in cells within the body of the bud suggested that the proteins could also be present in vesicles such as endosomes and synaptic vesicles (Fig. 5). To determine the type of cells expressing ClC-4/ClC-4A, double immunostaining was carried out on taste bud-containing sections with the anti-ClC-4/ClC-4A antibody (produced from rabbit) and mouse monoclonal antibodies against IP3R3 and SNAP-25, which are cellular markers for Type II and III taste bud cells, respectively. Confocal laser scanning microscope images showed that nearly all IP3R3-expressing cells also expressed ClC-4/ClC-4A (Fig. 6). Interestingly, many, if not all, SNAP-25-expressing cells also expressed these ClC channels. Because Type II cells are taste receptor cells and because Type III cells make synapses with afferent neurons, the expression of ClC-4/ClC-4A in these two types of cells suggests that these chloride channels may play a major role in bitter, sweet, and umami taste signal transduction, modulation, and transmission. Functional Characterization of the ClC-4 and ClC-4A Channels—To characterize the function of ClC-4 and ClC-4A, we subcloned their cDNAs into the pCR-BluntII-TOPO expression vector, synthesized the capped sense cRNA with in vitro transcription, and tailed the cRNA with poly(A). The cRNA was then purified and injected into Xenopus oocytes. Membrane currents were recorded using the two-electrode voltage-clamp technique 4–6 days after injection. Strong outward currents (which were absent in the control oocytes) were recorded in the ClC-4 cRNA-injected oocytes. The regulation of human ClC-4 activity by external pH has been reported, although the results appear contradictory to each other (54Kawasaki M. Fukuma T. Yamauchi K. Sakamoto H. Marumo F. Sasaki S. Am. J. Physiol. 1999; 277: C948-C954Crossref PubMed Google Scholar, 55Friedrich T. Breiderhoff T. Jentsch T.J. J. Biol. Chem. 1999; 274: 896-902Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar). To examine the effect of external pH on these chloride channels in our system and to determine whether pH exerts any differential effect on mouse ClC-4 and" @default.
• W2065897911 created "2016-06-24" @default.
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• W2065897911 date "2005-10-01" @default.
• W2065897911 modified "2023-09-28" @default.
• W2065897911 title "Identification and Functional Characterization of a Voltage-gated Chloride Channel and Its Novel Splice Variant in Taste Bud Cells" @default.
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• W2065897911 doi "https://doi.org/10.1074/jbc.m507706200" @default.
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