Our results illustrate that despite many commonalities in the sweet, umami, and bitter taste signaling pathways, different Ca2+ clearance/taste signal regulation mechanisms may be used by the TRCs for these three tastes. This notion is supported by a recent report describing the possible role of another calcium-signaling- related protein, calcium- and integrin-binding protein 1 (CIB1), in sweet TRCs [55]. It is also noteworthy that most of Serca2 proteins appeared to be located in the ER regions closer to the taste pore relative to the nucleus, which may have functional implications. It is possible that the proximately situated pumps can more rapidly respond to the signaling cascades initiated by the activated taste receptors at the taste pore. The exact contribution of Serca2 to taste signal processing in taste bud cells can be further revealed by studies with Serca2 KO mice. However, the systemic knockout of this gene turned out to be embryonic lethal [56]. Generation and characterization of a taste-cell-specific Serca2 KO mouse can further define Serca2’s function in taste perception and may provide novel insights into taste signal regulation mechanisms. In summary, our findings show that sweet, umami, and bitter TRCs possess different Ca2+ clearance machineries and that mutations in these proteins can alter taste perception. Materials and Methods Animals FVB/NJ (WT) mice and CB6-Tg(Gad1-EGFP)G42Zjh/J transgenic mice (referred to as Gad1-GFP mice throughout) were purchased from the Jackson Laboratory (Bar Harbor, ME). Serca3 knockout (KO) mice [47] and Trpm5-GFP transgenic mice [40] were bred and maintained at the Monell Chemical Senses Center animal facility. Trpm5-GFP transgenic mice carry a green fluorescent protein (GFP) transgene driven by Trpm5 (transient receptor potential cation channel, subfamily M, member 5) promoter. All procedures involving animals were approved by the Monell Chemical Senses Center Institutional Animal Care and Use Committee. Construction of the subtracted single-taste-cell library To isolate the genes that are selectively or preferentially expressed in sweet, umami, or bitter taste receptor cells (TRCs), we generated rat subtracted single-TRC cDNA libraries. First, we performed single-taste-cell transcriptome amplification as de- scribed previously [18]. Briefly, a rat tongue was removed, and Tyrode’s solution containing 2 mg/ml dispase II and 1 mg/ml collagenase was subepithelially injected into the tongue, which was then incubated at 25uC for 30 min. The lingual epithelium was peeled off from the rest of the tongue, and circumvallate (CV) and foliate taste papillae were excised and dissociated into individual cells. Taste bud cells were identified based on their bipolar shape and individually transferred into 0.5-ml tubes. First-strand cDNA was synthesized with reverse transcriptase (Invitrogen, Carlsbad, CA) and oligo(dT) primers, tailed with dATP and terminal transferase (Roche Applied Science, Indianapolis, IN), and then amplified with oligo(dT) primers. Aliquots of the amplified products were used for post hoc cell typing by quantitative real- time PCR (qPCR) with Power SYBR Green Master Mix (Applied Biosystems, Foster City, CA) and specific PCR primers for known cell-type marker genes (see Table S1): ectonucleoside triphosphate diphosphohydrolase 2 (Entpd2) for type I cells [4]; Trpm5 and Tas1r1 and Tas1r2 (taste receptor, type 1, member 1 and member 2) for all sweet/umami/bitter-, umami-, and sweet-sensing TRCs (or type II cells), respectively [9,10,12]; and Snap25 (synaptosomal- associated protein 25) for presynaptic (or type III) cells [57]. The Contribution of SERCA to Taste Perception We then subtracted the amplified products of Entpd2-expressing type I taste bud cells from those of a single cell expressing Trpm5 using a previously published method, with some modification [58]. Briefly, the driver cDNA pooled from rat type I cells was labeled with an equal amount of photobiotin (Sigma, St. Louis, MO) by ultraviolet irradiation in a Stratalinker 2400 crosslinker (Strata- gene, Cedar Creek, TX) and then hybridized with tracer cDNA from a single TRC at a ratio of 24:1 (cDNA amount of driver vs. tracer). The mixture was incubated in the hybridization buffer (12.5% PEG8000, 1.5 M NaCl, 10 mM EPPS, pH 8.25, 1 mM EDTA, pH 8.0, and 0.1% SDS) according to the following parameters: 5 min at 80uC, ramp 80uC to 68uC for 16 min, 90 min at 68uC, 10 min at 80uC. The hybrids were then removed with streptavidin and purified with phenol/chloroform extraction. After two rounds of subtraction, the resultant cDNA was further amplified using restriction-enzyme site-tagged oligo(dT) primers and then subcloned into the pBluescript II vector. The sequences of the insert DNA were determined by DNA sequencing analysis and BLAST-searched against the GenBank databases. Isolation and expression analysis of Serca3 cDNA One clone, A14, matched part of the Serca3 cDNA sequence (GenBank accession number NM_012914.1) [42]. To determine whether Serca3 is selectively expressed in taste tissue, rat and mouse lingual epithelia containing CV and foliate papillae or devoid of any taste buds as negative control were excised, and total RNA was prepared using Absolutely RNAH Miniprep Kit (Stratagene), which was reverse transcribed into first-strand cDNA using random primers and the Superscript III kit (Invitrogen). PCR amplification was performed with primers that covered the full coding regions of mouse and rat Serca3 transcripts (see Table S2A). To ensure detection of all expressed splice variants, additional PCR reactions were performed using primers specific for all known variants (see Table S2B). The PCR products were subcloned into pGEM-T Easy (Promega, Madison, WI) or pCR4-TOPO vector (Invitrogen), and the insert DNA was sequenced with the vector primers. Immunohistochemistry Mouse and rat tongue and soft palate were removed and fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 1 h on ice and cryoprotected in 20% sucrose in PBS at 4uC for 2 h and then in 30% sucrose in PBS at 4uC overnight. The regions containing taste papillae (fungiform on the dorsal surface of anterior one-third of the tongue, CV on the dorsal surface of posterior tongue, foliate at the lateral edge of posterior tongue, and soft palate) were excised and embedded in Tissue-TekH OCT compound (Sakura Finetek USA, Inc., Torrance, CA). Frozen sections (10 mm thickness) were obtained using a cryostat and were then permeabilized with PBS-T (0.3% Triton X-100 in PBS) and blocked with the blocking buffer (2% goat serum, 3% BSA, 0.3% Triton X-100, and 0.1% sodium azide in PBS) overnight at 4uC. The sections were incubated with properly diluted primary antibodies at 25uC for 4 h or at 4uC overnight. After six washes with PBS-T for 10 min each, the sections were incubated with secondary antibodies at 25uC for 1 h. The working concentrations and other information about all antibodies used in this study are summarized in Table S3. The sections were thoroughly washed with PBS-T again before being covered with Vectashield H-1000 (Vector Laboratories, Burlingame, CA). Fluorescent images were taken using a confocal microscope (Leica Microsystems Inc., Bannockburn, IL). Control experiments performed without primary antibodies, or with taste tissue sections from Serca3 KO mice, showed neither nonspecific staining of primary antibodies nor cross-reactivity between secondary antibodies, validating PLoS ONE | www.plosone.org 10 August 2011 | Volume 6 | Issue 8 | e23165