The molecular and cellular basis of bitter taste in Drosophila

The extent of diversity among bitter-sensing neurons is a fundamental issue in the field of taste. Data are limited and conflicting as to whether bitter neurons are broadly tuned and uniform, resulting in indiscriminate avoidance of bitter stimuli, or diverse, allowing a more discerning evaluation of food sources. We provide a systematic analysis of how bitter taste is encoded by the major taste organ of the Drosophila head, the labellum. Each of 16 bitter compounds is tested physiologically against all 31 taste hairs, revealing responses that are diverse in magnitude and dynamics. Four functional classes of bitter neurons are defined. Four corresponding classes are defined through expression analysis of all 68 gustatory taste receptors. A receptor-to-neuron-to-tastant map is constructed. Misexpression of one receptor confers bitter responses as predicted by the map. These results reveal a degree of complexity that greatly expands the capacity of the system to encode bitter taste.     

Two antagonistic gustatory receptor neurons responding to sweet-salty and bitter taste in Drosophila

In Drosophila, gustatory receptor neurons (GRNs) occur within hair-like structures called sensilla. Most taste sensilla house four GRNs, which have been named according to their preferred sensitivity to basic stimuli: water (W cell), sugars (S cell), salt at low concentration (L1 cell), and salt at high concentration (L2 cell). Labellar taste sensilla are classified into three types, l-, s-, and i-type, according to their length and location. Of these, l- and s-type labellar sensilla possess these four cells, but most i-type sensilla house only two GRNs. In i-type sensilla, we demonstrate here that the first GRN responds to sugar and to low concentrations of salt (10-50 mM NaCl). The second GRN detects a range of bitter compounds, among which strychnine is the most potent; and also to salt at high concentrations (over 400 mM NaCl). Neither type of GRN responds to water. The detection of feeding stimulants in i-type sensilla appears to be performed by one GRN with the combined properties of S+L1 cells, while the other GRN detects feeding inhibitors in a similar manner to bitter-sensitive L2 cells on the legs. These sensilla thus house two GRNs having an antagonistic effect on behavior, suggesting that the expression of taste receptors is segregated across them accordingly.     

Molecular basis of bitter taste

The sense of taste responds to a large variety of stimuli through specific transduction mechanisms. The molecular events in the perception of bitter taste are believed to start with the binding of specific water-soluble molecules to G-protein-coupled receptors encoded by the type 2 family of taste receptor genes and expressed at the surface of taste receptor cells. Recent advances in the identification and cloning of the complete repertoire of genes of this family in humans and rodents provide an opportunity to address unresolved questions in bitter taste. The functional characterization of some of the receptors that these genes encode suggests that it will be possible to understand more precisely their specific functions.     

Two antagonistic gustatory receptor neurons responding to sweet‐salty and bitter taste in Drosophila

In Drosophila, gustatory receptor neurons (GRNs) occur within hair‐like structures called sensilla. Most taste sensilla house four GRNs, which have been named according to their preferred sensitivity to basic stimuli: water (W cell), sugars (S cell), salt at low concentration (L1 cell), and salt at high concentration (L2 cell). Labellar taste sensilla are classified into three types, l‐, s‐, and i‐type, according to their length and location. Of these, l‐ and s‐type labellar sensilla possess these four cells, but most i‐type sensilla house only two GRNs. In i‐type sensilla, we demonstrate here that the first GRN responds to sugar and to low concentrations of salt (10–50 mM NaCl). The second GRN detects a range of bitter compounds, among which strychnine is the most potent; and also to salt at high concentrations (over 400 mM NaCl). Neither type of GRN responds to water. The detection of feeding stimulants in i‐type sensilla appears to be performed by one GRN with the combined properties of S + L1 cells, while the other GRN detects feeding inhibitors in a similar manner to bitter‐sensitive L2 cells on the legs. These sensilla thus house two GRNs having an antagonistic effect on behavior, suggesting that the expression of taste receptors is segregated across them accordingly. © 2004 Wiley Periodicals, Inc. J Neurobiol, 2004     

Receptors for bitter and sweet taste

The identification of two families of receptors, T1Rs and T2Rs, for sweet and bitter taste stimuli has opened the door to understanding some of the basic mechanisms underlying taste transduction in mammals. Studies of the functions of these receptors and their patterns of expression provide important information regarding the detection of structurally diverse taste compounds and the manner in which different taste qualities are encoded in the mouth.     

Iontophoretic application of bitter taste stimuli in hamsters

Recent electrophysiological studies on the iontophoretic applicationof taste stimuli by weak electric currents using rodents andfrogs have produced stimuli which appear to mimic the actionof salty, sour and sweet solutions. However, there has beenno report of an ionic stimulus which might serve as a bitteriontophoretic probe. Many common bitter stimuli are either uncharged(e.g. quinine, urea) or have mixed quality sensations (e.g.the bitter salts KCl, MgCl₂) and therefore are unsuitable. Thisreport investigates the use of four organic anions, all of whichare bitter to humans, which may serve as potential bitter stimulifor iontophoretic application to the tongue of the hamster whilerecording electrophysiologically from its chorda tympani nerve.These anions are m-nitrobenzene sulfonate (NBSA), picrate, cholateand m-nitrobenzoate (NBA). The electrophysiological responsesto cathodal polarization via these four anions plus saccharin,an effective cathodal stimulus in the hamster, form the sameefficacy series as chemical (i.e. normal sapid) presentationsof sodium salts of these anions, i.e. saccharin > NBSA >picrate > NBA > cholate. Behavioral evidence suggeststhat NBSA is sweet to hamsters while the latter three anions,picrate, NBA and cholate, are bitter. Electrophyiological observations,based on magnitude of response, appear to support these behavioralfindings. It was concluded that picrate, NBA and cholate mayserve as useful bitter stimulus probes for ionto-phoretic applicationin the hamster.     

Postnatal development of bitter taste avoidance behavior in mice is associated with ACTIN-dependent localization of bitter taste receptors to the microvilli of taste cells

Bitter taste avoidance behavior (BAB) plays a fundamental role in the avoidance of toxic substances with a bitter taste. However, the molecular basis underlying the development of BAB is unknown. To study critical developmental events by which taste buds turn into functional organs with BAB, we investigated the early phase development of BAB in postnatal mice in response to bitter-tasting compounds, such as quinine and thiamine. Postnatal mice started to exhibit BAB for thiamine and quinine at postnatal day 5 (PD5) and PD7, respectively. Histological analyses of taste buds revealed the formation of microvilli in the taste pores starting at PD5 and the localization of type 2 taste receptor 119 (TAS2R119) at the microvilli at PD6. Treatment of the tongue epithelium with cytochalasin D (CytD), which disturbs ACTIN polymerization in the microvilli, resulted in the loss of TAS2R119 localization at the microvilli and the loss of BAB for quinine and thiamine. The release of ATP from the circumvallate papillae tissue due to taste stimuli was also declined following CytD treatment. These results suggest that the localization of TAS2R119 at the microvilli of taste pores is critical for the initiation of BAB.     

Interstrain differences in bitter taste responses in mice

Interstrain differences in bitter taste responses were examinedusing inbred strains of mice. Taste responses were recordedfrom the glossopharyngeal and chorda tympani nerves of SWR/J,LP/J, BDP/J and DBA/2J mice. There were large differences inthe magnitude of responses to sucrose octaacetate (SOA) in boththe glossopharyngeal and chorda tympaninerves of SWR/J miceas compared with the other strains of mice. SOA thresholds ofSWR/J mice were 10^–7–^–6 M, whereas they were– 10^–4 M in LP/J mice. On the other hand, no appreciabledifferences were observed in the responses to quinine hydrochlorideand pnenyl-thio-carbamide. The results obtained in the presentexperiments fully explain the findings in behavioral studiesshowing that only SWR/J mice avoid SOA solutions whereas otherstrains do not. ^*Present address: Department of Physiology, Niigata UniversitySchool of Dentistry, Niigata 951, Japan     

Functional characterization of human bitter taste receptors

The T2Rs belong to a multi-gene family of G-protein-coupled receptors responsible for the detection of ingested bitter-tasting compounds. The T2Rs are conserved among mammals with the human and mouse gene families consisting of about 25 members. In the present study we address the signalling properties of human and mouse T2Rs using an in vitro reconstitution system in which both the ligands and G-proteins being assayed can be manipulated independently and quantitatively assessed. We confirm that the mT2R5, hT2R43 and hT2R47 receptors respond selectively to micromolar concentrations of cycloheximide, aristolochic acid and denatonium respectively. We also demonstrate that hT2R14 is a receptor for aristolochic acid and report the first characterization of the ligand specificities of hT2R7, which is a broadly tuned receptor responding to strychnine, quinacrine, chloroquine and papaverine. Using these defined ligand-receptor interactions, we assayed the ability of the ligand-activated T2Rs to catalyse GTP binding on divergent members of the G(alpha) family including three members of the G(alphai) subfamily (transducin, G(alphai1) and G(alphao)) as well as G(alphas) and G(alphaq). The T2Rs coupled with each of the three G(alphai) members tested. However, none of the T2Rs coupled to either G(alphas) or G(alphaq), suggesting the T2Rs signal primarily through G(alphai)-mediated signal transduction pathways. Furthermore, we observed different G-protein selectivities among the T2Rs with respect to both G(alphai) subunits and G(betagamma) dimers, suggesting that bitter taste is transduced by multiple G-proteins that may differ among the T2Rs.     

A brief-access test for bitter taste in mice

Inbred mouse strains vary in their response to bitter-tasting compounds as assessed by 48 h preference tests. These differences are generally assumed to result from altered gustatory function, although such long-term tests could easily reflect additional factors. We developed a brief-access taste test and tested the responses of two inbred strains, as well as C3. SW congenic mice, to the bitter stimulus sucrose octaacetate (SOA). Water-deprived trained mice were tested with five concentrations of SOA (0.00018-0.18 mM) and distilled water in a Davis MS- 160 apparatus. Trials were 5 s in duration and stimuli were presented randomly within blocks; each stimulus trial was preceded by a water rinse trial. Each concentration was presented twice in a session and mice were repeatedly tested across consecutive days. SOA-taster mice, including the SWR/J (SW) inbred and C3. SW congenic taster (T) mice, avoided licking SOA at concentrations >0.003 mM. In comparison, C3HeB/FeJ (C3) and C3. SW demitaster mice (D) licked all concentrations at the same rate as water. Concentration-response functions were similar across strains for both the brief-access test and a parallel 48 h preference test run on separate groups of mice. Furthermore, concentration-response functions were similar whether or not the brief-access test was preceded by a 4 day, single concentration pretest with SOA. The brief-access test is a suitable assay for bitter taste function in mice because it minimizes possible post-ingestive influences on taste.     

Evolution of bitter taste receptors in humans and apes

Bitter taste perception is crucial for the survival of organismsbecause it enables them to avoid the ingestion of potentiallyharmful substances. Bitter taste receptors are encoded by agene family that in humans has been shown to contain 25 putativelyfunctional genes and 8 pseudogenes and in mouse 33 putativelyfunctional genes and 3 pseudogenes. Lineage-specific expansionsof bitter taste receptors have taken place in both mouse andhuman, but very little is known about the evolution of thesereceptors in primates. We report the analysis of the almostcomplete repertoires of bitter taste receptor genes in human,great apes, and two Old World monkeys. As a group, these genesseem to be under little selective constraint compared with olfactoryreceptors and other genes in the studied species. However, incontrast to the olfactory receptor gene repertoire, where humanshave a higher proportion of pseudogenes than apes, there isno evidence that the rate of loss of bitter taste receptor genesvaries among humans and apes.     

Dynamic evolution of bitter taste receptor genes in vertebrates

Background  

Sensing bitter tastes is crucial for many animals because it can prevent them from ingesting harmful foods. This process is mainly mediated by the bitter taste receptors (T2R), which are largely expressed in the taste buds. Previous studies have identified some T2R gene repertoires, and marked variation in repertoire size has been noted among species. However, the mechanisms underlying the evolution of vertebrate T2R genes remain poorly understood.     

Computational studies of ligand-receptor interactions in bitter taste receptors

Phenylthiocarbamide tastes intensely bitter to some individuals, but others find it completely tasteless. Recently, it was suggested that phenylthiocarbamide elicits bitter taste by interacting with a human G protein-coupled receptor (hTAS2R38) encoded by the PTC gene. The phenylthiocarbamide nontaster trait was linked to three single nucleotide polymorphisms occurring in the PTC gene. Using the crystal structure of bovine rhodopsin as template, we generated the 3D structure of hTAS2R38 bitter taste receptor. We were able to map on the receptor structure the amino acids affected by the genetic polymorphisms and to propose molecular functions for two of them that explained the emergence of the nontaster trait. We used molecular docking simulations to find that phenylthiocarbamide exhibited a higher affinity for the target receptor than the structurally similar molecule 6-n-propylthiouracil, in line with recent experimental studies. A 3D model was constructed for the hTAS2R16 bitter taste receptor as well, by applying the same protocol. We found that the recently published experimental ligand binding affinity data for this receptor correlated well with the binding scores obtained from our molecular docking calculations.     

Three-dimensional mapping of the bitter taste receptor site

The essential geometrical features of the receptor site of bittermolecules were found through a detailed mapping procedure basedon the use of the shapes of conformationally rigid molecules.     

The sweet and the bitter of mammalian taste

The discovery of two families of mammalian taste receptors has provided important insights into taste recognition and taste perception. Recent studies have examined the receptors and signaling pathways that mediate sweet, bitter, and amino acid taste detection in mammals. These studies demonstrate that taste cells are selectively tuned to different taste modalities and clarify the logic of taste coding in the periphery.