transduction pathways eventually converges at increases in [Ca2+] , suggesting the importance of Ca2+ in taste signal c transduction and transmission. To utilize Ca2+ effectively as a signaling molecule, taste cells must tightly regulate [Ca2+]c at both increasing and decreasing steps. Disruption of the exquisite [Ca2+]c regulation in taste cells could cause nonspecific or inappropriate responses and, in severe cases, lead to cell death because of adverse effects of high [Ca2+]c [32]. Thus, taste cells must have mechanisms to clear Ca2+ from cytosol to ensure the effectiveness of intracellular Ca2+ signaling mechanisms. To date, although many studies have focused on [Ca2+]c elevation, Ca2+ clearance mechanisms in taste cells—how taste cells deplete Ca2+ from cytoplasm and restore [Ca2+]c to prestimulation levels—are not well understood [17,20,33,34,35,36]. Recent reports indicate that Na+/Ca2+ exchangers and mitochondrial Ca2+ transporters clear Ca2+ in presynaptic taste bud cells that express voltage-gated Ca2+ channels (VGCCs) [34,35,37,38,39,40,41]. The Ca2+ clearance mechanisms utilized by other types of taste cells, however, remain to be determined; moreover, how Ca2+ clearance mechanisms affect taste detection in vivo needs to be characterized. In the present study, we hypothesized that different types of taste bud cells may employ different mechanisms in the restoration of [Ca2+]c following responses to taste stimuli to reset the cells for the next taste stimulus. To test this hypothesis, we generated and analyzed rat subtracted single-TRC cDNA libraries, and identified one of the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) family genes, Serca3 (Atp2a3) [42], that is expressed exclusively in sweet/umami/bitter TRCs. Serca3-knockout (KO) mice showed altered behavioral and gustatory nerve responses to bitter substances. In addition, we also found that another SERCA family member, Serca2, is largely expressed in T1R3-expressing taste bud cells and may compensate for the Serca3 loss of function in the mutant taste buds. Taken together, our results illustrate that unique sets of Ca2+ clearance proteins are utilized by different types of taste bud cells, and that malfunction of these proteins can alter taste perception. Results Mammalian taste papillae express Serca3 cDNAs To identify unique molecules possibly involved in [Ca2+]c homeostasis in TRCs, we isolated individual taste bud cells from rat circumvallate (CV) and foliate papillae and reverse transcribed and amplified all transcripts from these single cells. A portion of the amplified single-cell transcriptomes was used for post hoc gene expression profiling by quantitative real-time PCR (qPCR) with primers listed in Table S1 for the following cell-type marker genes: Entpd2 for type I cells, Trpm5 for type II TRCs, Tas1r1 for umami TRCs, Tas1r2 for sweet TRCs, and Snap25 for type III/ presynaptic cells. Another portion of the remaining amplified products for TRCs was subtracted with cDNA for type I cells to facilitate the isolation of genes specifically or more abundantly expressed in TRCs than in type I taste bud cells. The subtracted cDNAs were further amplified, subcloned into plasmid vectors, and sequenced. A BLAST search of the insert DNA sequences against the GenBank database indicated that one clone, A14, completely matched a 0.3-kb segment of the 39 end of the rat Serca3a sequence, a splice variant of sarco/endoplasmic reticulum Ca2+-ATPase 3 (Serca3/Atp2a3, GenBank accession number NM_012914.1) [42]. Previous studies have shown that rodents and humans express splice variants that differ at the C-terminus: all variants except Serca3a, which is present in all human and rodent species, are species specific [43]. To investigate which isoform is expressed in The Contribution of SERCA to Taste Perception rat and mouse taste tissue, we performed reverse-transcription PCR (RT-PCR) with variant-specific primer sets (Table S2). All known Serca3 isoforms—two in rats (Serca3a and 3b/c) and three in mice (Serca3a, 3b, and 3c)—were detected in taste papillae (T), but not in lingual epithelium devoid of taste buds (N) (Fig. 1). To isolate the full-length coding cDNAs, sets of PCR primers were designed to cover the entire coding sequences (Table S2). PCR with the first-strand cDNAs from rat and mouse CV and foliate taste papillae, and subsequent insert DNA sequencing analysis, revealed no additional splice variants, and the full-length coding sequences for all mouse and rat isoforms except mouse Serca3c were detected. Sweet/umami/bitter TRCs express Serca3 proteins To investigate Serca3 expression in taste papillae at the protein level, we performed immunohistochemistry against mouse lingual tissue sections with an antibody that recognizes all Serca3 isoforms. The robust signals were detected in a large subset of mouse taste bud cells obtained from four different oral taste fields (CV, foliate, and fungiform papillae and soft palate) (Fig. 2). Immunoreactivity extended throughout the cytoplasm of the labeled taste bud cells, which was not detected in taste tissues from Serca3 knockout (KO) mice, validating the specificity of the antibody as well as the absence of Serca3 proteins in the mutant mice (see below). Next, we explored which type of cells express Serca3 protein in taste buds by using anti-Serca3 antibody to immunostain taste papillae from two different transgenic mouse lines that expressed green fluorescent protein (GFP) under the promoter of either Trpm5 (Trpm5-GFP) or glutamic acid decarboxylase 1 (Gad1- GFP). The GFP expression of these two transgenic lines in sweet/ umami/bitter TRCs and in the majority of presynaptic cells, respectively, was previously verified [31,40,44], and further confirmed by our immunocytochemical studies with antibodies against PLCb2, Trpm5, and dopa decarboxylase (DDC), another marker for presynaptic cells [17,45,46] (data not shown). We found nearly perfect overlap of cell populations exhibiting GFP fluorescence and Serca3 immunoreactivity in the Trpm5-GFP mice, suggesting that Trpm5 and Serca3 are fully co-expressed in TRCs (Fig. 3, upper panels). In the contrast, Serca3 antibody staining on taste tissue sections of Gad1-GFP transgenic mice Figure 1. Selective expression of Serca3 splice variants in rat and mouse gustatory lingual epithelium. RT-PCR was performed with variant-specific primers, and RNA samples were prepared from rat (A) and mouse (B) taste-bud-containing papillae (T) or lingual epithelium devoid of taste buds (N). All known variants were detected. PCR reactions for Trpm5, a taste-specific marker, and b-actin (Actb), a general ‘‘housekeeping’’ gene, were included as controls. doi:10.1371/journal.pone.0023165.g001 PLoS ONE | www.plosone.org 2 August 2011 | Volume 6 | Issue 8 | e23165