Serca3c full-length coding sequence was likely due to its low expression level in taste buds. The presence of multiple Serca3 isoforms may allow taste bud cells to precisely regulate their focal and/or global Ca2+ signals in a cell-specific manner. For example, a previous study showed that some human SERCA3 protein isoforms have distinct Ca2+ pumping properties but similar affinities toward Ca2+ [48]. Further investigation into the exact distribution of each Serca3 isoform among different types of taste bud cells can provide additional information on the fine control of Ca2+ signals in these cells. Immunohistochemical studies with an antibody that recognizes all known Serca3 isoforms in mice showed that a subset of taste bud cells from all examined taste tissue—CV, foliate, and fungiform papillae and soft palate—express this Ca2+ pump, whereas extragemmal epithelial cells showed no detectable signals (Fig. 2). Further, Serca3-expressing taste bud cells are the same as those expressing Trpm5 but different from those expressing Gad1 (Fig. 3), indicating that Serca3 protein is expressed exclusively in sweet/umami/bitter TRCs but not in presynaptic cells. Functionally, the activation of sweet, umami, and bitter taste receptors eventually leads to the Ca2+ release from the ER into the cytosol. The increased cytosolic Ca2+ opens the Trpm5 channel, resulting in the depolarization of the receptor membrane potential and release of the transmitters onto the afferent nerve fibers. The Serca3 pump may not be active in the resting TRCs because the apparent Ca2+ affinity of Serca3 is low, with a K0.5 of ,1.1 mM, and the normal basal [Ca2+]c is about 0.1 mM, well below its K0.5 [37,48,49,50]. Once activated by the rising [Ca2+] , however, c Serca3 has a high uptake rate [49], and is able to sequester Ca2+ back into the ER, which modulates the net amount of [Ca2+]c and affects downstream signal transduction. The events also could lead to the termination of taste signal transduction. The complete co- expression of Serca3 and Trpm5 in TRCs suggests that Serca3 functions to reverse increased [Ca2+]c induced by sweet, umami, and bitter taste-receptor-mediated PLCb2-IP3R3 signaling. The sequestration of Ca2+ also enables TRCs to maintain a constant net total amount of Ca2+ within a cell (cytoplasm+cellular organelle) over a long period. This mechanism seems to differ from that used by presynaptic cells, which depend on Ca2+ influx to initiate synaptic transmission and utilize Na+/Ca2+ exchangers to transport Ca2+ across the plasma membrane back into the extracellular space [38,39]. To demonstrate for the first time how malfunction of the Ca2+ clearance mechanism affects taste perception, we analyzed the behavioral responses of mice lacking Serca3 to tastants represen- tative of five primary taste qualities (Figs. 5 and 6). The deficiency of Serca3 did not affect the animals’ preferences for various concentrations of sour and salty stimuli, which agrees with our immunostaining data showing no expression of Serca3 in presynaptic cells or in cells other than TRCs. In tests with sweet, umami, and bitter taste stimuli, we observed significant differences between the KO and WT mice in aversive responses to the bitter taste substances denatonium (Figs. 5D and 6C) in both the 48-h two-bottle preference assays and brief-access tests and to quinine in the brief-access tests (Fig. 5E). Although statistically insignifi- cant, the KO mice appeared to display an increased aversion to quinine as well, compared with WT mice (Fig. 6D) in brief-access tests. Electrophysiological data from the gustatory nerve responses indicate that the altered behavioral responses to bitter taste in Serca3 KO mice are contributable to the deficiency in the peripheral taste system (Fig. 7). Electrical recordings from the glossopharyngeal nerve that innervates the taste buds with relatively abundant expression of bitter taste receptors showed The Contribution of SERCA to Taste Perception significantly stronger responses for bitter taste compounds in Serca3 KO mice than in WT control mice, whereas the responses to sweet, umami, salty, and sour substances were indistinguishable between these two groups of mice. Intriguingly, the Serca3 gene deletion seemed to have no significant effect on preference or gustatory nerve responses for sweet and umami tastes (Figs. 5–7). We observed a trend toward stronger preferences in the KO mice for sweet and umami taste substances in behavioral tests, suggesting a perceptible but minor contribution of Serca3 in these tastes. To address the disparity in the behavioral responses of the Serca3 KO mice to bitter versus sweet and umami taste substances, we examined the expression of Serca2 in taste tissue (Fig. 8). Double immunolabeling of rat CV taste buds showed that Serca2 is expressed in a subset of Trpm5- expressing cells and is almost completely co-expressed with T1R3 in the same population of cells. Previous studies have shown that Trpm5 is present in all sweet, umami, and bitter TRCs, while T1R3 occurs only in sweet and umami TRCs. Thus, our results suggest that Serca2 is present in sweet and umami TRCs and is likely absent in the bitter receptor cells. Double-immunostaining data with antibodies against Serca2 and a-gustducin also support this conclusion: the cells expressing these two proteins only partially overlap (Fig. 8), since a-gustducin is present in most bitter taste receptor cells but in only a small fraction of T1R3-expressing cells in rodent CV papillae [13,51,52]. Therefore, we speculate that the absence of significant alterations in behavioral and nerve responses to sweet and umami taste stimuli in Serca3 KO mice can be attributed to Serca2, which compensates for the lack of Serca3 function in mutant sweet and umami TRCs. From our data, we propose the following model. First, bitter taste stimuli activate the taste receptor-G protein-PLCb2-IP3R3 signaling cascade, as well described previously [2,17,20]. Upon activation, IP3R3 releases Ca2+ into the cytoplasm from the ER, where Serca3 is localized, which leads to an increase of [Ca2+] , c especially in the immediate vicinity of the ER in a short time. This increased [Ca2+]c activates Serca3, which begins Ca2+ uptake into the ER, causing a net decrease in the amount of cytoplasmic Ca2+ that can be used to activate the downstream signaling molecules, such as Trpm5, thus modulating the activity of the downstream taste signaling cascade [12,21]. In Serca3 deficient bitter TRCs, without such a decrease in [Ca2+]c, higher levels of Ca2+ may be available for the downstream signaling cascade. The process can increase transmitter release onto the nerve fibers and enhance bitter taste sensation, which in turn engenders a stronger aversive response to the tastants. The stronger aversion to these bitter compounds by the KO mice clearly demonstrates the contribution of a Ca2+ clearance mechanism in animal taste perception. In sweet and umami TRCs, both Serca2 and Serca3 can be involved in cytoplasmic Ca2+ uptake. And Serca2 may partially compensate the role of Serca3 in these cells, leading to statistically insignificant but perceptible increases in the responses of the KO mice to these palatable taste stimuli. Further studies on taste bud cell responses upon taste stimuli can quantitatively characterize the effect of Serca3 mutation on kinetics of calcium responses in taste bud cells and its correlation with the altered gustatory nerve responses and, eventually, with the perception and behavioral responses. Unlike other KO mouse models such as T1R3 or Trpm5 KO that abolish nearly all responses to specific taste qualities [12,17,53,54], Serca3 KO mice provide a distinct model with a gradual change in taste reception. Additional data from such mouse models may further our understanding of how the central nervous system translates qualitative and quantitative sensory information into perception. PLoS ONE | www.plosone.org 9 August 2011 | Volume 6 | Issue 8 | e23165