Sour taste responses in mice lacking PKD channels

Background

The polycystic kidney disease-like ion channel PKD2L1 and its associated partner PKD1L3 are potential candidates for sour taste receptors. PKD2L1 is expressed in type III taste cells that respond to sour stimuli and genetic elimination of cells expressing PKD2L1 substantially reduces chorda tympani nerve responses to sour taste stimuli. However, the contribution of PKD2L1 and PKD1L3 to sour taste responses remains unclear.

Methodology/Principal Findings

We made mice lacking PKD2L1 and/or PKD1L3 gene and investigated whole nerve responses to taste stimuli in the chorda tympani or the glossopharyngeal nerve and taste responses in type III taste cells. In mice lacking PKD2L1 gene, chorda tympani nerve responses to sour, but not sweet, salty, bitter, and umami tastants were reduced by 25–45% compared with those in wild type mice. In contrast, chorda tympani nerve responses in PKD1L3 knock-out mice and glossopharyngeal nerve responses in single- and double-knock-out mice were similar to those in wild type mice. Sour taste responses of type III fungiform taste cells (GAD67-expressing taste cells) were also reduced by 25–45% by elimination of PKD2L1.

Conclusions/Significance

These findings suggest that PKD2L1 partly contributes to sour taste responses in mice and that receptors other than PKDs would be involved in sour detection.     

Acetic acid activates PKD1L3-PKD2L1 channel—A candidate sour taste receptor

The polycystic kidney disease (PKD) 1L3-PKD2L1 channel is a candidate sour taste receptor expressed in mammalian taste receptor cells. Various acids are reported to activate PKD channels after the removal of the acid stimuli, but little information is available on the activation of these channels by acetic acid. It was difficult to analyze the PKD channel activation by acetic acid using Ca²⁺ imaging experiments because this acid induces a transient and nonspecific response in cultured cells. Here, we developed a novel method to evaluate PKD channel activation by acetic acid. Nonspecific responses were observed only over a short period after the application of acetic acid. In contrast, PKD channel activation evoked by acetic acid as well as citric acid was detected even at a later time point. This method revealed that PKD1L3-PKD2L1 channel activation by acetic acid was pH-dependent and occurred when the ambient pH was <3.1.     

Activation of Polycystic Kidney Disease-2-like 1 (PKD2L1)-PKD1L3 Complex by Acid in Mouse Taste Cells

Five basic tastes (bitter, sweet, umami, salty, and sour) are detected in the four taste areas where taste buds reside. Although molecular mechanisms for detecting bitter, sweet, and umami have been well clarified, those for sour and salty remain poorly understood. Several channels including acid-sensing ion channels have been proposed as candidate sour receptors, but they do not encompass all sour-sensing abilities in vivo. We recently reported a novel candidate for sour sensing, the polycystic kidney disease-2-like 1 (PKD2L1)-PKD1L3 channel complex. This channel is not a traditional ligand-gated channel and is gated open only after removal of an acid stimulus, called an off response. Here we show that off responses upon acid stimulus are clearly observed in native taste cells from circumvallate, but not fungiform papillae, of glutamate decarboxylase 67-green fluorescent protein (GAD67-GFP) knock-in mice, from which Type III taste cells can be visualized, using Ca²⁺ imaging and patch clamp methods. Off responses were detected in most cells where PKD2L1 immunoreactivity was observed. Interestingly, the pH threshold for acid-evoked intracellular Ca²⁺ increase was around 5.0, a value much higher than that observed in HEK293 cells expressing the PKD2L1-PKD1L3 complex. Thus, PKD2L1-PKD1L3-mediated acid-evoked off responses occurred both in HEK293 cells and in native taste cells, suggesting the involvement of the PKD2L1-PKD1L3 complex in acid sensing in vivo.     

Two members of the TRPP family of ion channels, Pkd1l3 and Pkd2l1, are co-expressed in a subset of taste receptor cells

Taste receptors cells are responsible for detecting a wide variety of chemical stimuli. Several molecules including both G protein coupled receptors and ion channels have been shown to be involved in the detection and transduction of tastants. We report on the expression of two members of the transient receptor potential (TRP) family of ion channels, PKD1L3 and PKD2L1, in taste receptor cells. Both of these channels belong to the larger polycystic kidney disease (PKD or TRPP) subfamily of TRP channels, members of which have been demonstrated to be non-selective cation channels and permeable to both Na(+) and Ca(2+). Pkd1l3 and Pkd2l1 are co-expressed in a select subset of taste receptor cells and therefore may, like other PKD channels, function as a heteromer. We found the taste receptor cells expressing Pkd1l3 and Pkd2l1 to be distinct from those that express components of sweet, bitter and umami signal transduction pathways. These results provide the first evidence for a role of TRPP channels in taste receptor cell function.     

Establishment of clonal cell lines of taste buds from a <Emphasis Type="Italic">p53</Emphasis><Superscript>−/−</Superscript> mouse tongue

A taste bud is a sensory organ and consists of 50-100 spindle-shaped cells. The cells function as taste acceptors. They have characteristics of both epithelial and neuronal cells. A taste bud contains four types of cells, type I, type II, type III cells, and basal cells. Taste buds were isolated from a tongue of a p53-deficient mouse at day 12, and 11 clonal taste bud (TBD) cell lines were established. In immunochemical analysis, all cell lines expressed cytokeratin 18, gustducin, T1R3, and neural cellular adhesion molecule, but not GLAST. In RT-PCR analysis, shh was not expressed in any of the cell lines. Further analysis with RT-PCR was conducted on four cell lines. They expressed G protein-coupled taste receptors; T1R3, T2R8 for sweet, bitter, umami. And they also expressed α-ENaC for salty taste. While, a candidate for sour receptor HCN4 was expressed in TBD-a1 and TBD-a7 lines. And another candidate for sour receptor PKD1L3 was slightly expressed in TBD-a1 and TBD-c1.     

Molecular mechanisms underlying the reception and transmission of sour taste information

Taste enables organisms to determine the properties of ingested substances by conveying information regarding the five basic taste modalities: sweet, salty, sour, bitter, and umami. The sweet, salty, and umami taste modalities convey the carbohydrate, electrolyte, and glutamate content of food, indicating its desirability and stimulating appetitive responses. The sour and bitter modalities convey the acidity of food and the presence of potential toxins, respectively, stimulating aversive responses to such tastes. In recent years, the receptors mediating sweet, bitter, and umami tastes have been identified as members of the T1R and T2R G-protein-coupled receptor families; however, the molecular mechanisms underlying sour taste detection have yet to be clearly elucidated. This review covers the molecular mechanisms proposed to mediate the detection and transmission of sour stimuli, focusing on polycystic kidney disease 1-like 3 (Pkd1l3), Pkd2l1, and carbonic anhydrase 4 (Car4).     

Molecular evolution of candidate sour taste receptor gene PKD1L3 in mammals

The PKD1L3 gene encodes an ion channel protein that can interact with the PKD2L1 protein to form a candidate sour taste receptor. In the present study, we have analyzed the evolutionary patterns of PKD1L3 genes from 10 mammalian species. The results showed that PKD1L3 genes have evolved under a dominant purifying selection force. However, for some branches and sites, PKD1L3 genes were detected to have been operated by positive selection. Moreover, some of these positive evolutionary sites are likely to participate in acid stimulus recognition. In rodents, PKD1L3 genes evolved more rapidly than other mammalian lineages. Combined with other functional research reports, our results suggest that rodents may not be the most appropriate model for functional research on the PKD1L3 gene.     

Genetic variation in taste and its influence on food selection

Abstract Taste perception plays a key role in determining individual food preferences and dietary habits. Individual differences in bitter, sweet, umami, sour, or salty taste perception may influence dietary habits, affecting nutritional status and nutrition-related chronic disease risk. In addition to these traditional taste modalities there is growing evidence that "fat taste" may represent a sixth modality. Several taste receptors have been identified within taste cell membranes on the surface of the tongue, and they include the T2R family of bitter taste receptors, the T1R receptors associated with sweet and umami taste perception, the ion channels PKD1L3 and PKD2L1 linked to sour taste, and the integral membrane protein CD36, which is a putative "fat taste" receptor. Additionally, epithelial sodium channels and a vanilloid receptor, TRPV1, may account for salty taste perception. Common polymorphisms in genes involved in taste perception may account for some of the interindividual differences in food preferences and dietary habits within and between populations. This variability could affect food choices and dietary habits, which may influence nutritional and health status and the risk of chronic disease. This review will summarize the present state of knowledge of the genetic variation in taste, and how such variation might influence food intake behaviors.     

β-Catenin Signaling Biases Multipotent Lingual Epithelial Progenitors to Differentiate and Acquire Specific Taste Cell Fates

Continuous taste bud cell renewal is essential to maintain taste function in adults; however, the molecular mechanisms that regulate taste cell turnover are unknown. Using inducible Cre-lox technology, we show that activation of β-catenin signaling in multipotent lingual epithelial progenitors outside of taste buds diverts daughter cells from a general epithelial to a taste bud fate. Moreover, while taste buds comprise 3 morphological types, β-catenin activation drives overproduction of primarily glial-like Type I taste cells in both anterior fungiform (FF) and posterior circumvallate (CV) taste buds, with a small increase in Type II receptor cells for sweet, bitter and umami, but does not alter Type III sour detector cells. Beta-catenin activation in post-mitotic taste bud precursors likewise regulates cell differentiation; forced activation of β-catenin in these Shh⁺ cells promotes Type I cell fate in both FF and CV taste buds, but likely does so non-cell autonomously. Our data are consistent with a model where β-catenin signaling levels within lingual epithelial progenitors dictate cell fate prior to or during entry of new cells into taste buds; high signaling induces Type I cells, intermediate levels drive Type II cell differentiation, while low levels may drive differentiation of Type III cells.     

Expression of the synaptic exocytosis‐regulating molecule complexin 2 in taste buds and its participation in peripheral taste transduction

Taste information from type III taste cells to gustatory neurons is thought to be transmitted via synapses. However, the molecular mechanisms underlying taste transduction through this pathway have not been fully elucidated. In this study, to identify molecules that participate in synaptic taste transduction, we investigated whether complexins (Cplxs), which play roles in regulating membrane fusion in synaptic vesicle exocytosis, were expressed in taste bud cells. Among four Cplx isoforms, strong expression of Cplx2 mRNA was detected in type III taste cells. To investigate the function of CPLX2 in taste transduction, we observed taste responses in CPLX2‐knockout mice. When assessed with electrophysiological and behavioral assays, taste responses to some sour stimuli in CPLX2‐knockout mice were significantly lower than those in wild‐type mice. These results suggested that CPLX2 participated in synaptic taste transduction from type III taste cells to gustatory neurons.

     

A serotonin-sensitive sensor for investigation of taste cell-to-cell communication

Taste receptor cells are the taste sensation elements for sour, salty, sweet, bitter and umami sensations. It was demonstrated that there are cell-to-cell communications between type II (sour) and type III (sweet, bitter and umami) taste cells. Serotonin (5-HT) is released from type III cells, which is the only type of taste cells that has synaptic process with sensory afferent fibers. Then, taste information is transmitted via fibers to the brain. During this process, 5-HT plays important roles in taste information transmission. In order to explore a sensor to detect 5-HT released from taste cell or taste cell networks, we develop a 5-HT sensitive sensor based on LAPS chip. This sensor performs with a detection limit of 3.3 × 10(-13)M and a sensitivity of 19.1 mV per concentration decade. Upon the stimuli of sour and mix (bitter, sweet and umami) tastants, 5-HT released from taste cells could be detected flexibly, benefit from the addressability of LAPS chip. The experimental results show that the local concentration of 5-HT is around several nM, which is consistent with those from other methods. In addition, immunofluorescent imaging technique is utilized to confirm the functional existence of both type II and III cells in a cluster of isolated taste cells. Different types of taste cells are labeled with corresponding specific antibody. This 5-HT sensitive LAPS chip provides a potential and promising way to detect 5-HT and to investigate the taste coding and information communication mechanisms.     

Glutamate may be an efferent transmitter that elicits inhibition in mouse taste buds

Recent studies suggest that l-glutamate may be an efferent transmitter released from axons innervating taste buds. In this report, we determined the types of ionotropic synaptic glutamate receptors present on taste cells and that underlie this postulated efferent transmission. We also studied what effect glutamate exerts on taste bud function. We isolated mouse taste buds and taste cells, conducted functional imaging using Fura 2, and used cellular biosensors to monitor taste-evoked transmitter release. The findings show that a large fraction of Presynaptic (Type III) taste bud cells (～50%) respond to 100 μM glutamate, NMDA, or kainic acid (KA) with an increase in intracellular Ca(2+). In contrast, Receptor (Type II) taste cells rarely (4%) responded to 100 μM glutamate. At this concentration and with these compounds, these agonists activate glutamatergic synaptic receptors, not glutamate taste (umami) receptors. Moreover, applying glutamate, NMDA, or KA caused taste buds to secrete 5-HT, a Presynaptic taste cell transmitter, but not ATP, a Receptor cell transmitter. Indeed, glutamate-evoked 5-HT release inhibited taste-evoked ATP secretion. The findings are consistent with a role for glutamate in taste buds as an inhibitory efferent transmitter that acts via ionotropic synaptic glutamate receptors.     

Acid stimulation (sour taste) elicits GABA and serotonin release from mouse taste cells

Several transmitter candidates including serotonin (5-HT), ATP, and norepinephrine (NE) have been identified in taste buds. Recently, γ-aminobutyric acid (GABA) as well as the associated synthetic enzymes and receptors have also been identified in taste cells. GABA reduces taste-evoked ATP secretion from Receptor cells and is considered to be an inhibitory transmitter in taste buds. However, to date, the identity of GABAergic taste cells and the specific stimulus for GABA release are not well understood. In the present study, we used genetically-engineered Chinese hamster ovary (CHO) cells stably co-expressing GABA(B) receptors and Gαqo5 proteins to measure GABA release from isolated taste buds. We recorded robust responses from GABA biosensors when they were positioned against taste buds isolated from mouse circumvallate papillae and the buds were depolarized with KCl or a stimulated with an acid (sour) taste. In contrast, a mixture of sweet and bitter taste stimuli did not trigger GABA release. KCl- or acid-evoked GABA secretion from taste buds was Ca(2+)-dependent; removing Ca(2+) from the bathing medium eliminated GABA secretion. Finally, we isolated individual taste cells to identify the origin of GABA secretion. GABA was released only from Presynaptic (Type III) cells and not from Receptor (Type II) cells. Previously, we reported that 5-HT released from Presynaptic cells inhibits taste-evoked ATP secretion. Combined with the recent findings that GABA depresses taste-evoked ATP secretion, the present results indicate that GABA and 5-HT are inhibitory transmitters in mouse taste buds and both likely play an important role in modulating taste responses.     

Taste receptor cell-based biosensor for taste specific recognition based on temporal firing

Taste receptor cells are the taste sensation elements expressing sour, salty, sweet, bitter and umami receptors, respectively. There are cell-to-cell communications between different types of cells. Nevertheless, the mechanism of taste sensation and taste information coded by taste receptor cell is not well understood at present and it is a long-standing issue. In order to explore taste sensation and analyze taste-firing responses from another point of view, we present a promising biomimetic taste receptor cell-based biosensor. The temporal firing responses to different tastants are recorded. Meanwhile, we investigate the firing rate and temporal firing of taste receptor cells. The experimental results are consistent with that from patch clamp and molecular biology experiment. Firing rate is dependent on the concentration of stimulus. PCA analysis (principal component analysis) of the temporal firing responses shows that the responses from different types of taste receptor cells can be distinguished. Furthermore, exogenous ATP is applied to mimic the effects of transmitter ATP (adenosine triphosphate) released from type II cells onto type III cells. Both enhanced and inhibitory effects on spontaneous firing are observed. This novel biomimetic hybrid biosensor provides a potential solution to investigate the taste sensation and coding mechanisms in a non-invasive way.     

Off-response property of an acid-activated cation channel complex PKD1L3-PKD2L1

Ligand-gated ion channels are important in sensory and synaptic transduction. The PKD1L3-PKD2L1 channel complex is a sour taste receptor candidate that is activated by acids. Here, we report that the proton-activated PKD1L3-PKD2L1 ion channels have the unique ability to be activated after the removal of an acid stimulus. We refer to this property as the off-response (previously described as a delayed response). Electrophysiological analyses show that acid-induced responses are observed only after the removal of an acid solution at less than pH 3.0. A small increase in pH is sufficient for PKD1L3-PKD2L1 channel activation, after exposure to an acid at pH 2.5. These results indicate that this channel is a new type of ion channel-designated as an 'off-channel'-which is activated during stimulus application but not gated open until the removal of the stimulus. The off-response property of PKD1L3-PKD2L1 channels might explain the physiological phenomena occurring during sour taste sensation.     

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.