Sami Damak

Tas1r3 , encoding a new candidate taste receptor, is allelic to the sweet responsiveness locus Sac

The ability to taste the sweetness of carbohydrate-rich foodstuffs has a critical role in the nutritional status of humans. Although several components of bitter transduction pathways have been identified, the receptors and other sweet transduction elements remain unknown. The Sac locus in mouse, mapped to the distal end of chromosome 4 (refs. 7–9), is the major determinant of differences between sweet-sensitive and -insensitive strains of mice in their responsiveness to saccharin, sucrose and other sweeteners. To identify the human Sac locus, we searched for candidate genes within a region of approximately one million base pairs of the sequenced human genome syntenous to the region of Sac in mouse. From this search, we identified a likely candidate: T1R3, a previously unknown G protein-coupled receptor (GPCR) and the only GPCR in this region. Mouse Tas1r3 (encoding T1r3) maps to within 20,000 bp of the marker closest to Sac (ref. 9) and, like human TAS1R3, is expressed selectively in taste receptor cells. By comparing the sequence of Tas1r3 from several independently derived strains of mice, we identified a specific polymorphism that assorts between taster and non-taster strains. According to models of its structure, T1r3 from non-tasters is predicted to have an extra amino-terminal glycosylation site that, if used, would interfere with dimerization.

Taste Preference for Fatty Acids Is Mediated by GPR40 and GPR120

The oral perception of fat has traditionally been considered to rely mainly on texture and olfaction, but recent findings suggest that taste may also play a role in the detection of long chain fatty acids. The two G-protein coupled receptors GPR40 (Ffar1) and GPR120 are activated by medium and long chain fatty acids. Here we show that GPR120 and GPR40 are expressed in the taste buds, mainly in type II and type I cells, respectively. Compared with wild-type mice, male and female GPR120 knock-out and GPR40 knock-out mice show a diminished preference for linoleic acid and oleic acid, and diminished taste nerve responses to several fatty acids. These results show that GPR40 and GPR120 mediate the taste of fatty acids.

The molecular physiology of taste transduction

Taste receptor cells use a variety of mechanisms to transduce chemical information into cellular signals. Seven-transmembrane-helix receptors initiate signaling cascades by coupling to G proteins, effector enzymes, second messengers and ion channels. Apical ion channels pass ions, leading to depolarizing and/or hyperpolarizing responses. New insights into the mechanisms of taste sensation have been gained from molecular cloning of the transduction elements, biochemical elucidation of the transduction pathways, and electrophysiological analysis of the function of taste cell ion channels.

Mouse taste cells with G protein-coupled taste receptors lack voltage-gated calcium channels and SNAP-25.

BackgroundTaste receptor cells are responsible for transducing chemical stimuli from the environment and relaying information to the nervous system. Bitter, sweet and umami stimuli utilize G-protein coupled receptors which activate the phospholipase C (PLC) signaling pathway in Type II taste cells. However, it is not known how these cells communicate with the nervous system. Previous studies have shown that the subset of taste cells that expresses the T2R bitter receptors lack voltage-gated Ca2+ channels, which are normally required for synaptic transmission at conventional synapses. Here we use two lines of transgenic mice expressing green fluorescent protein (GFP) from two taste-specific promoters to examine Ca2+ signaling in subsets of Type II cells: T1R3-GFP mice were used to identify sweet- and umami-sensitive taste cells, while TRPM5-GFP mice were used to identify all cells that utilize the PLC signaling pathway for transduction. Voltage-gated Ca2+ currents were assessed with Ca2+ imaging and whole cell recording, while immunocytochemistry was used to detect expression of SNAP-25, a presynaptic SNARE protein that is associated with conventional synapses in taste cells.ResultsDepolarization with high K+ resulted in an increase in intracellular Ca2+ in a small subset of non-GFP labeled cells of both transgenic mouse lines. In contrast, no depolarization-evoked Ca2+ responses were observed in GFP-expressing taste cells of either genotype, but GFP-labeled cells responded to the PLC activator m-3M3FBS, suggesting that these cells were viable. Whole cell recording indicated that the GFP-labeled cells of both genotypes had small voltage-dependent Na+ and K+ currents, but no evidence of Ca2+ currents. A subset of non-GFP labeled taste cells exhibited large voltage-dependent Na+ and K+ currents and a high threshold voltage-gated Ca2+ current. Immunocytochemistry indicated that SNAP-25 was expressed in a separate population of taste cells from those expressing T1R3 or TRPM5. These data indicate that G protein-coupled taste receptors and conventional synaptic signaling mechanisms are expressed in separate populations of taste cells.ConclusionThe taste receptor cells responsible for the transduction of bitter, sweet, and umami stimuli are unlikely to communicate with nerve fibers by using conventional chemical synapses.

Umami Taste Responses Are Mediated by α-Transducin and α-Gustducin

The sense of taste comprises at least five distinct qualities: sweet, bitter, sour, salty, and umami, the taste of glutamate. For bitter, sweet, and umami compounds, taste signaling is initiated by binding of tastants to G-protein-coupled receptors in specialized epithelial cells located in the taste buds, leading to the activation of signal transduction cascades. α-Gustducin, a taste cell-expressed G-protein α subunit closely related to the α-transducins, is a key mediator of sweet and bitter tastes. α-Gustducin knock-out (KO) mice have greatly diminished, but not entirely abolished, responses to many bitter and sweet compounds. We set out to determine whether α-gustducin also mediates umami taste and whether rod α-transducin (αt-rod), which is also expressed in taste receptor cells, plays a role in any of the taste responses that remain in α-gustducin KO mice. Behavioral tests and taste nerve recordings of single and double KO mice lacking α-gustducin and/or αt-rod confirmed the involvement of α-gustducin in bitter (quinine and denatonium) and sweet (sucrose and SC45647) taste and demonstrated the involvement ofα-gustducin in umami [monosodium glutamate (MSG), monopotassium glutamate (MPG), and inosine monophosphate (IMP)] taste as well. We found that αt-rod played no role in taste responses to the salty, bitter, and sweet compounds tested or to IMP but was involved in the umami taste of MSG and MPG. Umami detection involving α-gustducin and αt-rod occurs in anteriorly placed taste buds, however taste cells at the back of the tongue respond to umami compounds independently of these two G-protein subunits.

Murine intestinal cells expressing Trpm5 are mostly brush cells and express markers of neuronal and inflammatory cells.

To determine the role in chemosensation of intestinal solitary cells that express taste receptors and Trpm5, we carried out a microarray study of the transcriptome of FACS‐sorted transgenic mouse intestinal cells expressing enhanced green fluorescent protein (eGFP) under the control of the Trpm5 promoter and compared it with that of intestinal cells that do not express eGFP. The findings of the study are: 1) Morphology and expression of markers show that most eGFP+ cells are brush cells. 2) The majority of proteins known to be involved in taste signal transduction are expressed in the eGFP+ cells, although the isoforms are not always the same. 3) eGFP+ cells express pre‐ and postsynaptic markers and nerves are often found in close proximity. 4) Several genes that play a role in inflammation are expressed specifically in eGFP+ cells. Furthermore, these cells express the entire biosynthesis pathway of leucotriene C4, an eicosanoid involved in modulation of intestinal smooth muscle contraction. 5) Angiotensinogen, renin, and succinate receptor genes are expressed in the eGFP+ cells, suggesting a role in the regulation of water and sodium transport, vasomotricity, and blood pressure. These data suggest that the Trpm5‐expressing cells integrate many signals, including chemical signals from ingested food, and that they may regulate several physiological functions of the gastrointestinal tract. J. Comp. Neurol. 509:514–525, 2008.

Dominant loss of responsiveness to sweet and bitter compounds caused by a single mutation in α-gustducin

Biochemical and genetic studies have implicated α-gustducin as a key component in the transduction of both bitter or sweet taste. Yet, α-gustducin-null mice are not completely unresponsive to bitter or sweet compounds. To gain insights into how gustducin mediates responses to bitter and sweet compounds, and to elicit the nature of the gustducin-independent pathways, we generated a dominant-negative form of α-gustducin and expressed it as a transgene from the α-gustducin promoter in both wild-type and α-gustducin-null mice. A single mutation, G352P, introduced into the C-terminal region of α-gustducin critical for receptor interaction rendered the mutant protein unresponsive to activation by taste receptor, but left its other functions intact. In control experiments, expression of wild-type α-gustducin as a transgene in α-gustducin-null mice fully restored responsiveness to bitter and sweet compounds, formally proving that the targeted deletion of the α-gustducin gene caused the taste deficits of the null mice. In contrast, transgenic expression of the G352P mutant did not restore responsiveness of the null mice to either bitter or sweet compounds. Furthermore, in the wild-type background, the mutant transgene inhibited endogenous α-gustducins interactions with taste receptors, i.e., it acted as a dominant-negative. That the mutant transgene further diminished the residual bitter and sweet taste responsiveness of the α-gustducin-null mice suggests that other guanine nucleotide-binding regulatory proteins expressed in the α-gustducin lineage of taste cells mediate these responses.

Transsynaptic transport of wheat germ agglutinin expressed in a subset of type II taste cells of transgenic mice

BackgroundAnatomical tracing of neural circuits originating from specific subsets of taste receptor cells may shed light on interactions between taste cells within the taste bud and taste cell-to nerve interactions. It is unclear for example, if activation of type II cells leads to direct activation of the gustatory nerves, or whether the information is relayed through type III cells. To determine how WGA produced in T1r3-expressing taste cells is transported into gustatory neurons, transgenic mice expressing WGA-IRES-GFP driven by the T1r3 promoter were generated.ResultsImmunohistochemistry showed co-expression of WGA, GFP and endogenous T1r3 in the taste bud cells of transgenic mice: the only taste cells immunoreactive for WGA were the T1r3-expressing cells. The WGA antibody also stained intragemmal nerves. WGA, but not GFP immunoreactivity was found in the geniculate and petrosal ganglia of transgenic mice, indicating that WGA was transported across synapses. WGA immunoreactivity was also found in the trigeminal ganglion, suggesting that T1r3-expressing cells make synapses with trigeminal neurons. In the medulla, WGA was detected in the nucleus of the solitary tract but also in the nucleus ambiguus, the vestibular nucleus, the trigeminal nucleus and in the gigantocellular reticular nucleus. WGA was not detected in the parabrachial nucleus, or the gustatory cortex.ConclusionThese results show the usefulness of genetically encoded WGA as a tracer for the first and second order neurons that innervate a subset of taste cells, but not for higher order neurons, and demonstrate that the main route of output from type II taste cells is the gustatory neuron, not the type III cells.

Claudin-based permeability barriers in taste buds

Tight junctions operate as semipermeable barriers in epithelial tissue, separating the apical from the basolateral sides of the cells. Membrane proteins of the claudin family represent the major tight junction constituents, and some reinforce permeability barriers, whereas others create pores based on solute size and ion selectivity. To outline paracellular permeability pathways in gustatory tissue, all claudins expressed in mouse taste buds and in human fungiform papillae have been characterized. Twelve claudins are expressed in murine taste‐papillae‐enriched tissue, and five of those are expressed in human fungiform papillae. A subset of the claudins expressed in mouse papillae is uniquely found in taste buds. By immunohistochemistry, claudin 4 has been found in mouse taste epithelium, with high abundance around the taste pore. Claudin 6 is explicitly detected inside the pore, claudin 7 was found at the basolateral side of taste cells, and claudin 8 was found around the pore. With the ion permeability features of the different claudins, a highly specific permeability pattern for paracellular diffusion is apparent, which indicates a peripheral mechanism for taste coding. J. Comp. Neurol. 502:1003–1011, 2007.

Sensory Attributes of Complex Tasting Divalent Salts Are Mediated by TRPM5 and TRPV1 Channels

Complex tasting divalent salts (CTDS) are present in our daily diet, contributing to multiple poorly understood taste sensations. CTDS evoking metallic, bitter, salty, and astringent sensations include the divalent salts of iron, zinc, copper, and magnesium. To identify pathways involved with the complex perception of the above salts, taste preference tests (two bottles, brief access) were performed in wild-type (WT) mice and in mice lacking (1) the T1R3 receptor, (2) TRPV1, the capsaicin receptor, or (3) the TRPM5 channel, the latter being necessary for the perception of sweet, bitter, and umami tasting stimuli. At low concentrations, FeSO4 and ZnSO4 were perceived as pleasant stimuli by WT mice, and this effect was fully reversed in TRPM5 knock-out mice. In contrast, MgSO4 and CuSO4 were aversive to WT mice, but for MgSO4 the aversion was abolished in TRPM5 knock-out animals, and for CuSO4, aversion decreased in both TRPV1- and TRPM5-deficient animals. Behavioral tests revealed that the T1R3 subunit of the sweet and umami receptors is implicated in the hedonically positive perception of FeSO4 and ZnSO4. For high concentrations of CTDS, the omission of TRPV1 reduced aversion. Imaging studies on heterologously expressed TRPM5 and TRPV1 channels are consistent with the behavioral experiments. Together, these results rationalize the complexity of metallic taste by showing that at low concentrations, compounds such as FeSO4 and ZnSO4 stimulate the gustatory system through the hedonically positive T1R3–TRPM5 pathway, and at higher concentrations, their aversion is mediated, in part, by the activation of TRPV1.