The stability of tastant detection by mouse lingual chemosensory tissue requires Regulator of G protein Signaling-21 (RGS21).

The T1R and T2R families of G protein-coupled receptors (GPCRs) initiate tastant perception by signaling via guanine nucleotide exchange and hydrolysis performed by associated heterotrimeric G proteins (Gαßγ). Heterotrimeric G protein signal termination is sped up by Gα-directed GTPase-accelerating proteins (GAPs) known as the Regulators of G protein Signaling (RGS proteins). Of this family, RGS21 is highly expressed in lingual epithelial cells and we have shown it acting in vitro to decrease the potency of bitterants on cultured cells. However, constitutive RGS21 loss in mice reduces organismal response to GPCR-mediated tastants-opposite to expectations arising from observed in vitro activity of RGS21 as a GAP and inhibitor of T2R signaling. Here, we show reduced quinine aversion and reduced sucrose preference by mice lacking RGS21 does not result from post-ingestive effects, as taste-salient brief-access tests confirm the reduced bitterant aversion and reduced sweetener preference seen using two-bottle choice testing. Eliminating Rgs21 expression after chemosensory system development, via tamoxifen-induced Cre recombination in eight week-old mice, led to a reduction in quinine aversive behavior that advanced over time, suggesting that RGS21 functions as a negative regulator to sustain stable bitter tastant reception. Consistent with this notion, we observed downregulation of multiple T2R proteins in the lingual tissue of Rgs21-deficient mice. Reduced tastant-mediated responses exhibited by mice lacking Rgs21 expression either since birth or in adulthood has highlighted the potential requirement for a GPCR GAP to maintain the full character of tastant signaling, likely at the level of mitigating receptor downregulation.

GAD65Cre Drives Reporter Expression in Multiple Taste Cell Types.

In taste buds, Type I cells represent the majority of cells (50-60%) and primarily have a glial-like function in taste buds. However, recent studies suggest that they have additional sensory and signaling functions including amiloride-sensitive salt transduction, oxytocin modulation of taste, and substance P mediated GABA release. Nonetheless, the overall function of Type I cells in transduction and signaling remains unclear, primarily because of the lack of a reliable reporter for this cell type. GAD65 expression is specific to Type I taste cells and GAD65 has been used as a Cre driver to study Type I cells in salt taste transduction. To test the specificity of transgene-driven expression, we crossed GAD65Cre mice with floxed tdTomato and Channelrhodopsin (ChR2) lines and examined the progeny with immunochemistry, chorda tympani recording, and calcium imaging. We report that while many tdTomato+ taste cells express NTPDase2, a specific marker of Type I cells, we see some expression of tdTomato in both Gustducin and SNAP25-positive taste cells. We also see ChR2 in cells just outside the fungiform taste buds. Chorda tympani recordings in the GAD65Cre/ChR2 mice show large responses to blue light. Furthermore, several isolated tdTomato-positive taste cells responded to KCl depolarization with increases in intracellular calcium, indicating the presence of voltage-gated calcium channels. Taken together, these data suggest that GAD65Cre mice drive expression in multiple taste cell types and thus cannot be considered a reliable reporter of Type I cell function.

Sugar causes obesity and metabolic syndrome in mice independently of sweet taste.

Intake of sugars, especially the fructose component, is strongly associated with the development of obesity and metabolic syndrome, but the relative role of taste versus metabolism in driving preference, intake, and metabolic outcome is not fully understood. We aimed to evaluate the preference for sweet substances and the tendency to develop metabolic syndrome in response to these sugars in mice lacking functional taste signaling [P2X2 (P2X purinoreceptor 2)/P2X3 (P2X purinoreceptor 3) double knockout mice (DKO)] and mice unable to metabolize fructose (fructokinase knockout mice). Of interest, our data indicate that despite their inability to taste sweetness, P2X2/3 DKO mice still prefer caloric sugars (including fructose and glucose) to water in long-term testing, although with diminished preference compared with control mice. Despite reduced intake of caloric sugars by P2X2/3 DKO animals, the DKO mice still show increased levels of the sugar-dependent hormone FGF21 (fibroblast growth factor 21) in plasma and liver. Despite lower sugar intake, taste-blind mice develop severe features of metabolic syndrome due to reduced sensitivity to leptin, reduced ability to mobilize and oxidize fats, and increased hepatic de novo lipogenesis. In contrast to P2X2/3 DKO and wild-type mice, fructokinase knockout mice, which cannot metabolize fructose and are protected against fructose-induced metabolic syndrome, demonstrate reduced preference and intake for all fructose-containing sugars tested but not for glucose or artificial sweeteners. Based on these observations, we conclude that sugar can induce metabolic syndrome in mice independently of its sweet properties. Furthermore, our data demonstrate that the metabolism of fructose is necessary for sugar to drive intake and preference in mice.

Optogenetic Activation of Type III Taste Cells Modulates Taste Responses.

Studies have suggested that communication between taste cells shapes the gustatory signal before transmission to the brain. To further explore the possibility of intragemmal signal modulation, we adopted an optogenetic approach to stimulate sour-sensitive (Type III) taste cells using mice expressing Cre recombinase under a specific Type III cell promoter, Pkd2l1 (polycystic kidney disease-2-like 1), crossed with mice expressing Cre-dependent channelrhodopsin (ChR2). The application of blue light onto the tongue allowed for the specific stimulation of Type III cells and circumvented the nonspecific effects of chemical stimulation. To understand whether taste modality information is preprocessed in the taste bud before transmission to the sensory nerves, we recorded chorda tympani nerve activity during light and/or chemical tastant application to the tongue. To assess intragemmal modulation, we compared nerve responses to various tastants with or without concurrent light-induced activation of the Type III cells. Our results show that light significantly decreased taste responses to sweet, bitter, salty, and acidic stimuli. On the contrary, the light response was not consistently affected by sweet or bitter stimuli, suggesting that activation of Type II cells does not affect nerve responses to stimuli that activate Type III cells.

Is the Amiloride-Sensitive Na+ Channel in Taste Cells Really ENaC?

Among the 5 taste qualities, salt is the least understood. The receptors, their expression pattern in taste cells, and the transduction mechanisms for salt taste are still unclear. Previous studies have suggested that low concentrations of NaCl are detected by the amiloride-sensitive epithelial Na+ channel (ENaC), which in other systems requires assembly of 3 homologous subunits (α, ß, and γ) to form a functional channel. However, a new study from Lossow and colleagues, published in this issue of Chemical Senses, challenges that hypothesis by examining expression levels of the 3 ENaC subunits in individual taste cells using gene-targeted mice in combination with immunohistochemistry and in situ hybridization. Results show a lack of colocalization of ENaC subunits in taste cells as well as expression of subunits in taste cells that show no amiloride sensitivity. These new results question the molecular identity of the amiloride-sensitive Na+ conductance in taste cells.

Function, Innervation, and Neurotransmitter Signaling in Mice Lacking Type-II Taste Cells.

The Skn-1a transcription factor (Pou2f3) is required for Type II taste cell differentiation in taste buds. Taste buds in Skn-1a-/- mice lack Type II taste cells but have a concomitant expansion of Type III cells, providing an ideal model to determine the relative role of taste cell types in response specificity. We confirmed that chorda tympani responses to sweet, bitter, and umami stimuli were greatly reduced in the knock-outs (KOs) compared with wild-type (WT) littermates. Skn-1a-/- mice also had reductions to NaCl that were partially amiloride-insensitive, suggesting that both Type II and Type III cells contribute to amiloride-insensitive salt detection in anterior tongue. We also confirmed that responses to sour stimuli are equivalent in the KOs, despite the large increase in the number of Type III taste cells. To examine their innervation, we crossed the Htr3a-GFP (5-HT3A-GFP) reporter mouse with the Skn-1a-/- mice and examined geniculate ganglion neurons for GFP expression and responses to 5-HT. We found no change in the number of 5-HT3A-expressing neurons with KO of Skn-1a Calcium imaging showed that only 5-HT3A-expressing neurons respond to exogenous 5-HT, while most neurons respond to ATP, similar to WT mice. Interestingly, despite loss of all Type II cells, the P2X3 antagonist AF353 blocked all chorda tympani responses. These data collectively raise questions pertaining the source of ATP signaling in the absence of Type II taste cells and whether the additional Type III cells are innervated by fibers that would have normally innervated Type II cells.

Physiological and Behavioral Responses to Optogenetic Stimulation of PKD2L1+ Type III Taste Cells.

Type III taste cells in mammalian taste buds are implicated in the detection and communication of sour and some salty stimuli, as well as carbonation and water. With this variety of proposed roles, it is unclear what information activated type III cells are communicating to the CNS. To better elucidate the role of type III cells in the taste bud, we use a type III cell-specific protein (polycystic kidney disease 2-like 1) to drive Cre-dependent expression of light-sensitive channelrhodopsin (Ai32) in mouse type III taste cells. Activation of these cells with light produces a taste nerve response in both the chorda tympani and glossopharyngeal nerves, and elicits a slight but significant aversion in two-bottle preference tests in both male and female mice. Unlike previous reports (Zocchi et al., 2017), our mice did not react to blue light stimulation with sustained drinking responses. These data suggest that type III cells are capable of communicating the presence of aversive stimuli in the oral cavity, which is in line with their responsiveness to sour and high concentrations of salt stimuli.

Development of Full Sweet, Umami, and Bitter Taste Responsiveness Requires Regulator of G protein Signaling-21 (RGS21).

The mammalian tastes of sweet, umami, and bitter are initiated by activation of G protein-coupled receptors (GPCRs) of the T1R and T2R families on taste receptor cells. GPCRs signal via nucleotide exchange and hydrolysis, the latter hastened by GTPase-accelerating proteins (GAPs) that include the Regulators of G protein Signaling (RGS) protein family. We previously reported that RGS21, uniquely expressed in Type II taste receptor cells, decreases the potency of bitter-stimulated T2R signaling in cultured cells, consistent with its in vitro GAP activity. However, the role of RGS21 in organismal responses to GPCR-mediated tastants was not established. Here, we characterized mice lacking the Rgs21 fifth exon. Eliminating Rgs21 expression had no effect on body mass accumulation (a measure of alimentation), fungiform papillae number and morphology, circumvallate papillae morphology, and taste bud number. Two-bottle preference tests, however, revealed that Rgs21-null mice have blunted aversion to quinine and denatonium, and blunted preference for monosodium glutamate, the sweeteners sucrose and SC45647, and (surprisingly) NaCl. Observed reductions in GPCR-mediated tastant responses upon Rgs21 loss are opposite to original expectations, given that loss of RGS21-a GPCR signaling negative regulator-should lead to increased responsiveness to tastant-mediated GPCR signaling (all else being equal). Yet, reduced organismal tastant responses are consistent with observations of reduced chorda tympani nerve recordings in Rgs21-null mice. Reduced tastant-mediated responses and behaviors exhibited by adult mice lacking Rgs21 expression since birth have thus revealed an underappreciated requirement for a GPCR GAP to establish the full character of tastant signaling.

Glutamate: Tastant and Neuromodulator in Taste Buds.

In taste buds, glutamate plays a double role as a gustatory stimulus and neuromodulator. The detection of glutamate as a tastant involves several G protein-coupled receptors, including the heterodimer taste receptor type 1, member 1 and 3 as well as metabotropic glutamate receptors (mGluR1 and mGluR4). Both receptor types participate in the detection of glutamate as shown with knockout animals and selective antagonists. At the basal part of taste buds, ionotropic glutamate receptors [N-methyl-d-aspartate (NMDA) and non-NMDA] are expressed and participate in the modulation of the taste signal before its transmission to the brain. Evidence suggests that glutamate has an efferent function on taste cells and modulates the release of other neurotransmitters such as serotonin and ATP. This short article reviews the recent developments in the field with regard to glutamate receptors involved in both functions as well as the influence of glutamate on the taste signal.

FGF21 Mediates Endocrine Control of Simple Sugar Intake and Sweet Taste Preference by the Liver.

The liver is an important integrator of nutrient metabolism, yet no liver-derived factors regulating nutrient preference or carbohydrate appetite have been identified. Here we show that the liver regulates carbohydrate intake through production of the hepatokine fibroblast growth factor 21 (FGF21), which markedly suppresses consumption of simple sugars, but not complex carbohydrates, proteins, or lipids. Genetic loss of FGF21 in mice increases sucrose consumption, whereas acute administration or overexpression of FGF21 suppresses the intake of both sugar and non-caloric sweeteners. FGF21 does not affect chorda tympani nerve responses to sweet tastants, instead reducing sweet-seeking behavior and meal size via neurons in the hypothalamus. This liver-to-brain hormonal axis likely represents a negative feedback loop as hepatic FGF21 production is elevated by sucrose ingestion. We conclude that the liver functions to regulate macronutrient-specific intake by producing an endocrine satiety signal that acts centrally to suppress the intake of "sweets."

The Role of 5-HT3 Receptors in Signaling from Taste Buds to Nerves.

Activation of taste buds triggers the release of several neurotransmitters, including ATP and serotonin (5-hydroxytryptamine; 5-HT). Type III taste cells release 5-HT directly in response to acidic (sour) stimuli and indirectly in response to bitter and sweet tasting stimuli. Although ATP is necessary for activation of nerve fibers for all taste stimuli, the role of 5-HT is unclear. We investigated whether gustatory afferents express functional 5-HT3 receptors and, if so, whether these receptors play a role in transmission of taste information from taste buds to nerves. In mice expressing GFP under the control of the 5-HT(3A) promoter, a subset of cells in the geniculate ganglion and nerve fibers in taste buds are GFP-positive. RT-PCR and in situ hybridization confirmed the presence of 5-HT(3A) mRNA in the geniculate ganglion. Functional studies show that only those geniculate ganglion cells expressing 5-HT3A-driven GFP respond to 10 µM 5-HT and this response is blocked by 1 µM ondansetron, a 5-HT3 antagonist, and mimicked by application of 10 µM m-chlorophenylbiguanide, a 5-HT3 agonist. Pharmacological blockade of 5-HT3 receptors in vivo or genetic deletion of the 5-HT3 receptors reduces taste nerve responses to acids and other taste stimuli compared with controls, but only when urethane was used as the anesthetic. We find that anesthetic levels of pentobarbital reduce taste nerve responses apparently by blocking the 5-HT3 receptors. Our results suggest that 5-HT released from type III cells activates gustatory nerve fibers via 5-HT3 receptors, accounting for a significant proportion of the neural taste response.

Mice Lacking Pannexin 1 Release ATP and Respond Normally to All Taste Qualities.

Adenosine triphosphate (ATP) is required for the transmission of all taste qualities from taste cells to afferent nerve fibers. ATP is released from Type II taste cells by a nonvesicular mechanism and activates purinergic receptors containing P2X2 and P2X3 on nerve fibers. Several ATP release channels are expressed in taste cells including CALHM1, Pannexin 1, Connexin 30, and Connexin 43, but whether all are involved in ATP release is not clear. We have used a global Pannexin 1 knock out (Panx1 KO) mouse in a series of in vitro and in vivo experiments. Our results confirm that Panx1 channels are absent in taste buds of the knockout mice and that other known ATP release channels are not upregulated. Using a luciferin/luciferase assay, we show that circumvallate taste buds from Panx1 KO mice normally release ATP upon taste stimulation compared with wild type (WT) mice. Gustatory nerve recordings in response to various tastants applied to the tongue and brief-access behavioral testing with SC45647 also show no difference between Panx1 KO and WT. These results confirm that Panx1 is not required for the taste evoked release of ATP or for neural and behavioral responses to taste stimuli.

Postsynaptic P2X3-containing receptors in gustatory nerve fibres mediate responses to all taste qualities in mice.

Taste buds release ATP to activate ionotropic purinoceptors composed of P2X2 and P2X3 subunits, present on the taste nerves. Mice with genetic deletion of P2X2 and P2X3 receptors (double knockout mice) lack responses to all taste stimuli presumably due to the absence of ATP-gated receptors on the afferent nerves. Recent experiments on the double knockout mice showed, however, that their taste buds fail to release ATP, suggesting the possibility of pleiotropic deficits in these global knockouts. To test further the role of postsynaptic P2X receptors in afferent signalling, we used AF-353, a selective antagonist of P2X3-containing receptors to inhibit the receptors acutely during taste nerve recording and behaviour. The specificity of AF-353 for P2X3-containing receptors was tested by recording Ca(2+) transients to exogenously applied ATP in fura-2 loaded isolated geniculate ganglion neurons from wild-type and P2X3 knockout mice. ATP responses were completely inhibited by 10 µm or 100 µm AF-353, but neither concentration blocked responses in P2X3 single knockout mice wherein the ganglion cells express only P2X2-containing receptors. Furthermore, AF-353 had no effect on taste-evoked ATP release from taste buds. In wild-type mice, i.p. injection of AF-353 or simple application of the drug directly to the tongue, inhibited taste nerve responses to all taste qualities in a dose-dependent fashion. A brief access behavioural assay confirmed the electrophysiological results and showed that preference for a synthetic sweetener, SC-45647, was abolished following i.p. injection of AF-353. These data indicate that activation of P2X3-containing receptors is required for transmission of all taste qualities.

Role of the ectonucleotidase NTPDase2 in taste bud function.

Taste buds are unusual in requiring ATP as a transmitter to activate sensory nerve fibers. In response to taste stimuli, taste cells release ATP, activating purinergic receptors containing the P2X2 and P2X3 subunits on taste nerves. In turn, the released ATP is hydrolyzed to ADP by a plasma membrane nucleoside triphosphate previously identified as nucleoside triphosphate diphosphohydrolase-2 (NTPDase2). In this paper we investigate the role of this ectonucleotidase in the function of taste buds by examining gene-targeted Entpd2-null mice globally lacking NTPDase2. RT-PCR confirmed the absence of NTPDase2, and ATPase enzyme histochemistry reveals no reaction product in taste buds of knockout mice, suggesting that NTPDase2 is the dominant form in taste buds. RT-PCR and immunocytochemistry demonstrated that in knockout mice all cell types are present in taste buds, even those cells normally expressing NTPDase2. In addition, the overall number and size of taste buds are normal in Entpd2-null mice. Luciferin/luciferase assays of circumvallate tissue of knockout mice detected elevated levels of extracellular ATP. Electrophysiological recordings from two taste nerves, the chorda tympani and glossopharyngeal, revealed depressed responses to all taste stimuli in Entpd2-null mice. Responses were more depressed in the glossopharyngeal nerve than in the chorda tympani nerve and involved all taste qualities; responses in the chorda tympani were more depressed to sweet and umami stimuli than to other qualities. We suggest that the excessive levels of extracellular ATP in the Entpd2-knockout animals desensitize the P2X receptors associated with nerve fibers, thereby depressing taste responses.

A2BR adenosine receptor modulates sweet taste in circumvallate taste buds.

In response to taste stimulation, taste buds release ATP, which activates ionotropic ATP receptors (P2X2/P2X3) on taste nerves as well as metabotropic (P2Y) purinergic receptors on taste bud cells. The action of the extracellular ATP is terminated by ectonucleotidases, ultimately generating adenosine, which itself can activate one or more G-protein coupled adenosine receptors: A1, A2A, A2B, and A3. Here we investigated the expression of adenosine receptors in mouse taste buds at both the nucleotide and protein expression levels. Of the adenosine receptors, only A2B receptor (A2BR) is expressed specifically in taste epithelia. Further, A2BR is expressed abundantly only in a subset of taste bud cells of posterior (circumvallate, foliate), but not anterior (fungiform, palate) taste fields in mice. Analysis of double-labeled tissue indicates that A2BR occurs on Type II taste bud cells that also express Gα14, which is present only in sweet-sensitive taste cells of the foliate and circumvallate papillae. Glossopharyngeal nerve recordings from A2BR knockout mice show significantly reduced responses to both sucrose and synthetic sweeteners, but normal responses to tastants representing other qualities. Thus, our study identified a novel regulator of sweet taste, the A2BR, which functions to potentiate sweet responses in posterior lingual taste fields.

Capacitance measurements of regulated exocytosis in mouse taste cells.

Exocytosis, consisting of the merger of vesicle and plasma membrane, is a common mechanism used by different types of nucleated cells to release their vesicular contents. Taste cells possess vesicles containing various neurotransmitters to communicate with adjacent taste cells and afferent nerve fibers. However, whether these vesicles engage in exocytosis on a stimulus is not known. Since vesicle membrane merger with the plasma membrane is reflected in plasma membrane area fluctuations, we measured membrane capacitance (C(m)), a parameter linearly related to membrane surface area. To investigate whether taste cells undergo regulated exocytosis, we used the compensated tight-seal whole-cell recording technique to monitor depolarization-induced changes in C(m) in the different types of taste cells. To identify taste cell types, mice expressing green fluorescent protein from the TRPM5 promoter or from the GAD67 promoter were used to discriminate type II and type III taste cells, respectively. Moreover, the cell types were also identified by monitoring their voltage-current properties. The results demonstrate that only type III taste cells show significant depolarization-induced increases in C(m), which were correlated to the voltage-activated calcium currents. The results suggest that type III, but neither type II nor type I cells exhibit depolarization-induced regulated exocytosis to release transmitter and activate gustatory afferent nerve fibers.

Evidence for a role of glutamate as an efferent transmitter in taste buds.

BACKGROUND: Glutamate has been proposed as a transmitter in the peripheral taste system in addition to its well-documented role as an umami taste stimulus. Evidence for a role as a transmitter includes the presence of ionotropic glutamate receptors in nerve fibers and taste cells, as well as the expression of the glutamate transporter GLAST in Type I taste cells. However, the source and targets of glutamate in lingual tissue are unclear. In the present study, we used molecular, physiological and immunohistochemical methods to investigate the origin of glutamate as well as the targeted receptors in taste buds. RESULTS: Using molecular and immunohistochemical techniques, we show that the vesicular transporters for glutamate, VGLUT 1 and 2, but not VGLUT3, are expressed in the nerve fibers surrounding taste buds but likely not in taste cells themselves. Further, we show that P2X2, a specific marker for gustatory but not trigeminal fibers, co-localizes with VGLUT2, suggesting the VGLUT-expressing nerve fibers are of gustatory origin. Calcium imaging indicates that GAD67-GFP Type III taste cells, but not T1R3-GFP Type II cells, respond to glutamate at concentrations expected for a glutamate transmitter, and further, that these responses are partially blocked by NBQX, a specific AMPA/Kainate receptor antagonist. RT-PCR and immunohistochemistry confirm the presence of the Kainate receptor GluR7 in Type III taste cells, suggesting it may be a target of glutamate released from gustatory nerve fibers. CONCLUSIONS: Taken together, the results suggest that glutamate may be released from gustatory nerve fibers using a vesicular mechanism to modulate Type III taste cells via GluR7.

Nasal chemosensory cells use bitter taste signaling to detect irritants and bacterial signals.

The upper respiratory tract is continually assaulted with harmful dusts and xenobiotics carried on the incoming airstream. Detection of such irritants by the trigeminal nerve evokes protective reflexes, including sneezing, apnea, and local neurogenic inflammation of the mucosa. Although free intra-epithelial nerve endings can detect certain lipophilic irritants (e.g., mints, ammonia), the epithelium also houses a population of trigeminally innervated solitary chemosensory cells (SCCs) that express T2R bitter taste receptors along with their downstream signaling components. These SCCs have been postulated to enhance the chemoresponsive capabilities of the trigeminal irritant-detection system. Here we show that transduction by the intranasal solitary chemosensory cells is necessary to evoke trigeminally mediated reflex reactions to some irritants including acyl-homoserine lactone bacterial quorum-sensing molecules, which activate the downstream signaling effectors associated with bitter taste transduction. Isolated nasal chemosensory cells respond to the classic bitter ligand denatonium as well as to the bacterial signals by increasing intracellular Ca(2+). Furthermore, these same substances evoke changes in respiration indicative of trigeminal activation. Genetic ablation of either G alpha-gustducin or TrpM5, essential elements of the T2R transduction cascade, eliminates the trigeminal response. Because acyl-homoserine lactones serve as quorum-sensing molecules for gram-negative pathogenic bacteria, detection of these substances by airway chemoreceptors offers a means by which the airway epithelium may trigger an epithelial inflammatory response before the bacteria reach population densities capable of forming destructive biofilms.

Receptors and transduction of umami taste stimuli.

L-glutamate and 5'-ribonucleotides, such as GMP and IMP, elicit the "umami" taste, also known as the fifth taste. This review will highlight recent advancements in our understanding of umami taste receptors and their downstream signaling effectors in taste receptor cells. Several G protein-coupled receptors that bind umami stimuli have been identified in taste buds, including the heterodimer T1R1/T1R3, truncated and brain forms of mGluR4 and mGluR1, brain mGluR2, and brain mGluR3. Further, ionotropic glutamate receptors are expressed in taste cells and may play a role in glutamate transduction or signaling between taste cells and/or nerve fibers. Knockout of T1R1 or T1R3 reduces, but does not eliminate, responses to umami stimuli, suggesting that multiple receptors contribute to umami taste. The signaling effectors downstream of umami G protein-coupled receptors involve Gbetagamma activation of PLCbeta2 to elicit Ca(2+) release from intracellular stores and activation of a cation channel, TRPM5. In fungiform and palatal taste buds, T1R1/T1R3 is co-expressed with Galpha gustducin and transducin, but the Galpha proteins involved in circumvallate taste buds have not been identified. In most taste fields, however, cAMP antagonizes responses to umami stimuli, suggesting that the Galpha subunit serves to modulate umami taste sensitivity.