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.

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.

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.

Bitter taste receptors (TAS2R proteins) allow mammals to detect and avoid ingestion of toxins in food. Thus, TAS2Rs play an important role in food choice and are subject to complex natural selection pressures. In our previous study, we examined nucleotide variation in TAS2R38, a gene expressing bitter taste receptor for phenylthiocarbamide (PTC), in 333 Japanese macaques (Macaca fuscata) from 9 local populations in Japan. We identified a PTC "non-taster" TAS2R38 allele in Japanese macaques that was caused by a loss of the start codon. This PTC non-taster allele was only found in a limited local population (the Kii area), at a frequency of 29%. In this study, we confirmed that this allele was present in only the Kii population by analyzing an additional 264 individuals from eight new populations. Using cellular and behavioral experiments, we found that this allele lost its receptor function for perceiving PTC. The nucleotide sequences of the allele including flanking regions (of about 10 kb) from 23 chromosomes were identical, suggesting that a non-taster allele arose and expanded in the Kii population during the last 13,000 years. Genetic analyses of non-coding regions in Kii individuals and neighboring populations indicated that the high allele frequency in the Kii population could not be explained by demographic history, suggesting that positive selection resulted in a rapid increase in PTC non-tasters in the Kii population. The loss-of-function that occurred at the TAS2R38 locus presumably provided a fitness advantage to Japanese macaques in the Kii population. Because TAS2R38 ligands are often found in plants, this functional change in fitness is perhaps related to feeding habit specificity. These findings should provide valuable insights for elucidating adaptive evolutionary changes with respect to various environments in wild mammals.

Signal peptide peptidase (SPP) is a multi-transmembrane aspartic protease involved in intramembrane-regulated proteolysis (RIP). RIP proteases mediate various key life events by releasing bioactive peptides from the plane of the membrane region. We have previously isolated Arabidopsis SPP (AtSPP) and found that this protein is expressed in the ER. An AtSPP-knockout plant was found to be lethal because of abnormal pollen formation; however, there is negligible information describing the physiological function of AtSPP. In this study, we have investigated the proteolytic activity of AtSPP to define the function of SPPs in plants.

Taste signals and nutrient stimuli sensed by the gastrointestinal tract are transmitted to the brain to regulate feeding behavior and energy homeostasis. This system is referred to as the gut-brain axis. Here we show that both brush cells and type II taste cells are eliminated in the gastrointestinal tract of transcription factor Skn-1 knockout (KO) mice. Despite unaltered food intake, Skn-1 KO mice have reduced body weight with lower body fat due to increased energy expenditure. In this model, 24-h urinary excretion of catecholamines was significantly elevated, accompanied by increased fatty acid β-oxidation and fuel dissipation in skeletal muscle and impaired insulin secretion driven by glucose. These results suggest the existence of brain-mediated energy homeostatic pathways originating from brush cells and type II taste cells in the gastrointestinal tract and ending in peripheral tissues, including the adrenal glands. The discovery of food-derived factors that regulate these cells may open new avenues the treatment of obesity and diabetes.

The molecular mechanisms of the mammalian gustatory system have been examined in many studies using rodents as model organisms. In this study, we examined the mRNA expression of molecules involved in taste signal transduction in the fungiform papillae (FuP) and circumvallate papillae (CvP) of the rhesus macaque, Macaca mulatta, using in situ hybridization. TAS1R1, TAS1R2, TAS2Rs, and PKD1L3 were exclusively expressed in different subsets of taste receptor cells (TRCs) in the FuP and CvP. This finding suggests that TRCs sensing different basic taste modalities are mutually segregated in macaque taste buds. Individual TAS2Rs exhibited a variety of expression patterns in terms of the apparent level of expression and the number of TRCs expressing these genes, as in the case of human TAS2Rs. GNAT3, but not GNA14, was expressed in TRCs of FuP, whereas GNA14 was expressed in a small population of TRCs of CvP, which were distinct from GNAT3- or TAS1R2-positive TRCs. These results demonstrate similarities and differences between primates and rodents in the expression profiles of genes involved in taste signal transduction.

Each of the olfactory sensory neurons (OSNs) chooses to express a single G protein-coupled olfactory receptor (OR) from a pool of hundreds. Here, we show the receptor transporting protein (RTP) family members play a dual role in both normal OR trafficking and determining OR gene choice probabilities. Rtp1 and Rtp2 double knockout mice (RTP1,2DKO) show OR trafficking defects and decreased OSN activation. Surprisingly, we discovered a small subset of the ORs are expressed in larger numbers of OSNs despite the presence of fewer total OSNs in RTP1,2DKO. Unlike typical ORs, some overrepresented ORs show robust cell surface expression in heterologous cells without the co-expression of RTPs. We present a model in which developing OSNs exhibit unstable OR expression until they choose to express an OR that exits the ER or undergo cell death. Our study sheds light on the new link between OR protein trafficking and OR transcriptional regulation.

Given the abundance of stroke patients and deaths from stroke worldwide, many studies concerning the aftermath of stroke are being carried out. To reveal the precise effect of ischemic infarction, we conducted a comprehensive gene expression analysis. Alongside a middle cerebral artery occlusion (MCAO) Sprague-Dawley rat model, we used a group undergoing sham surgery for comparison, which was the same as MCAO surgery but without blood vessel occlusion. Subsequently, infarction of the brains of MCAO-treated rats occurred, but did not occur in the sham-treated rats. Using whole blood, we carried out DNA microarray analysis, revealing the gene expression alterations caused by stroke. Downregulation of immune pathways and cluster of differentiation (CD) molecules indicated immunodepression. By conducting miRNA microarray analysis, we extracted seven miRNAs as significantly regulated: miR-107-5p, miR-383-5p, miR-24-1-5p, mir-191b, miR-196b-5p, and miR-3552 were upregulated, and mir-194-1 was downregulated. Among these seven miRNAs, three had one target mRNA each that was extracted as differentially expressed, and the expression levels of all pairs were inversely correlated. This indicates the occurrence of miRNA-mRNA regulatory systems in blood: between miR-107-5p and H2A histone family member Z (H2afz), miR-196b-5p and protein tyrosine phosphatase receptor type C (Ptprc), and miR-3552 and serine/arginine-rich splicing factor 2 (Srsf2). Moreover, six miRNAs had matching human miRNAs with similar sequences, which are potential human stroke biomarkers.

Taste is a vital sensation for vertebrates, enabling the detection of nutritionally important substances or potential toxins. A heteromeric complex of two class C GPCRs, T1R1 and T1R3, was identified as the umami (savory) taste receptor. Amino acids and 5'-ribonucleotides are well known to be natural ligands for human T1R1/T1R3. In this study, we reveal that methional, which is a familiar flavor component in foods, is an allosteric modulator of T1R1/T1R3. Receptor expression experiments showed that methional served as a positive allosteric modulator (PAM) of human T1R1/T1R3 and functioned as a negative allosteric modulator (NAM) of mouse T1R1/T1R3. Although amino acids and 5'-ribonucleotides bound to the extracellular domain of T1R1, the use of interspecies chimeric receptors demonstrated that methional interacted with the transmembrane domain of T1R1. Site-directed mutagenesis and molecular modeling showed that methional could potentially bind at two distinct sites in the transmembrane domain of T1R1 and that the amino acid residues in the bottom of the allosteric pocket engendered the switch between the PAM and NAM modes, which could contribute to switching the binding position of methional. These results may be applicable for elucidating the molecular mechanisms underlying ligand recognition by other class C GPCRs.

G-protein-coupled receptors mediate the senses of taste, smell, and vision in mammals. Humans recognize thousands of compounds as bitter, and this response is mediated by the hTAS2R family, which is one of the G-protein-coupled receptors composed of only 25 receptors. However, structural information on these receptors is limited. To address the molecular basis of bitter tastant discrimination by the hTAS2Rs, we performed ligand docking simulation and functional analysis using a series of point mutants of hTAS2R16 to identify its binding sites. The docking simulation predicted two candidate binding structures for a salicin-hTAS2R16 complex, and at least seven amino acid residues in transmembrane 3 (TM3), TM5, and TM6 were shown to be involved in ligand recognition. We also identified the probable salicin-hTAS2R16 binding mode using a mutated receptor experiment. This study characterizes the molecular interaction between hTAS2R16 and beta-D-glucopyranoside and will also facilitate rational design of bitter blockers.

The polycystic kidney disease 1-like 3 (PKD1L3)-polycystic kidney disease 2-like 1 (PKD2L1) complex functions as a Ca(2+)-permeable, non-selective cation channel that is activated by acid and its subsequent removal; this is called an off-response. In this study, we identified a single aspartic residue in PKD2L1 that is responsible for the Ca(2+) permeation of the PKD1L3/PKD2L1 complex. Calcium imaging analysis using point mutants of negatively charged amino acids present in the putative pore regions of PKD1L3 and PKD2L1 revealed that neutralization of the aspartic residue in PKD2L1 (D523N), which is conserved among PKD2 family members, abolished Ca(2+) permeation, despite robust cell surface expression. In contrast, neutralization of the other negatively charged residues of PKD1L3 (D2049N and E2072Q) and PKD2L1 (D525N and D530N) as well as substitution of Asp(523) with a glutamate residue (D523E) had little effect on Ca(2+) permeation properties. These results demonstrate that Asp(523) in PKD2L1 is a key determinant of Ca(2+) permeation into the PKD1L3/PKD2L1 complex and that PKD2L1 contributes to forming the pore of the PKD1L3/PKD2L1 channel.

Umami taste perception in mammals is mediated by a heteromeric complex of two G-protein-coupled receptors, T1R1 and T1R3. T1R1/T1R3 exhibits species-dependent differences in ligand specificity; human T1R1/T1R3 specifically responds to L-Glu, whereas mouse T1R1/T1R3 responds more strongly to other L-amino acids than to L-Glu. The mechanism underlying this species difference remains unknown. In this study we analyzed chimeric human-mouse receptors and point mutants of T1R1/T1R3 and identified 12 key residues that modulate amino acid recognition in the human- and mouse-type responses in the extracellular Venus flytrap domain of T1R1. Molecular modeling revealed that the residues critical for human-type acidic amino acid recognition were located at the orthosteric ligand binding site. In contrast, all of the key residues for the mouse-type broad response were located at regions outside of both the orthosteric ligand binding site and the allosteric binding site for inosine-5'-monophosphate (IMP), a known natural umami taste enhancer. Site-directed mutagenesis demonstrated that the newly identified key residues for the mouse-type responses modulated receptor activity in a manner distinct from that of the allosteric modulation via IMP. Analyses of multiple point mutants suggested that the combination of two distinct determinants, amino acid selectivity at the orthosteric site and receptor activity modulation at the non-orthosteric sites, may mediate the ligand specificity of T1R1/T1R3. This hypothesis was supported by the results of studies using nonhuman primate T1R1 receptors. A complex molecular mechanism involving changes in the properties of both the orthosteric and non-orthosteric sites of T1R1 underlies the determination of ligand specificity in mammalian T1R1/T1R3.

Polycystic kidney disease (PKD) 2L1 protein is a member of the transient receptor potential (TRP) ion channel family. In circumvallate and foliate papillae, PKD2L1 is coexpressed with PKD1L3. PKD2L1 and PKD1L3 interact through their transmembrane domain and the resulting heteromer PKD1L3/PKD2L1 owns a unique channel property called 'off-responses' to acid stimulation, although PKD2L1 does not own this property by itself. To define the pharmacological properties of the PKD1L3/PKD2L1 channel, we developed a new method to effectively evaluate channel activity using human embryonic kidney 293T cells in which the channel was heterologously expressed. This method was applied to screen substances that potentially regulate it. We found that capsaicin and its analogs, which are TRPV1 agonists, inhibited the response to acid stimuli and that the capsaicin inhibition was reversible with an IC(50) of 32.5 μm. Capsaicin and its analogs are thus useful tools for physiological analysis of PKD1L3/PKD2L1 function.

Vertebrates receive tastants, such as sugars, amino acids, and nucleotides, via taste bud cells in epithelial tissues. In mammals, two families of G protein-coupled receptors for tastants are expressed in taste bud cells-T1Rs for sweet tastants and umami tastants (l-amino acids) and T2Rs for bitter tastants. Here, we report two families of candidate taste receptors in fish species, fish T1Rs and T2Rs, which show significant identity to mammalian T1Rs and T2Rs, respectively. Fish T1Rs consist of three types: fish T1R1 and T1R3 that show the highest degrees of identity to mammalian T1R1 and T1R3, respectively, and fish T1R2 that shows almost equivalent identity to both mammalian T1R1 and T1R2. Unlike mammalian T1R2, fish T1R2 consists of two or three members in each species. We also identified two fish T2Rs that show low degrees of identity to mammalian T2Rs. In situ hybridization experiments revealed that fish T1R and T2R genes were expressed specifically in taste bud cells, but not in olfactory receptor cells. Fish T1R1 and T1R2 genes were expressed in different subsets of taste bud cells, and fish T1R3 gene was co-expressed with either fish T1R1 or T1R2 gene as in the case of mammals. There were also a significant number of cells expressing fish T1R2 genes only. Fish T2R genes were expressed in different cells from those expressing fish T1R genes. These results suggest that vertebrates commonly have two kinds of taste signaling pathways that are defined by the types of taste receptors expressed in taste receptor cells.

New World monkeys (NWMs) are unique in that they exhibit remarkable interspecific variation in color vision and feeding behavior, making them an excellent model for studying sensory ecology. However, it is largely unknown whether non-visual senses co-vary with feeding ecology, especially gustation, which is expected to be indispensable in food selection. Bitter taste, which is mediated by bitter taste receptors (TAS2Rs) in the tongue, helps organisms avoid ingesting potentially toxic substances in food. In this study, we compared the ligand sensitivities of the TAS2Rs of five species of NWMs by heterologous expression in HEK293T cells and calcium imaging.

The balance between carbon and nitrogen is a key determinant of seed storage components, and thus, is of great importance to rice and other seed-based food crops. To clarify the influence of the rhizosphere carbon/nitrogen balance during the maturation stage of several seed components, transcriptome analysis was performed on the seeds from rice plants that were provided additional nitrogen fertilization at heading time. As a result, it was assessed that genes associated with molecular processes such as photosynthesis, trehalose metabolism, carbon fixation, amino acid metabolism, and cell wall metabolism were differentially expressed. Moreover, cellulose and sucrose synthases, which are involved in cellulose synthesis, were down-regulated. Therefore, we compared cellulose content of mature seeds that were treated with additional nitrogen fertilization with those from control plants using calcofluor staining. In these experiments, cellulose content in endosperm from plants receiving additional nitrogen fertilization was less than that in control endosperm. Other starch synthesis-related genes such as starch synthase 1, starch phosphorylase 2, and branching enzyme 3 were also down-regulated, whereas some α-amylase and β-amylase genes were up-regulated. On the other hand, mRNA expression of amino acid biosynthesis-related molecules was up-regulated. Moreover, additional nitrogen fertilization caused accumulation of storage proteins and up-regulated Cys-poor prolamin mRNA expression. These data suggest that additional nitrogen fertilization at heading time changes the expression of some storage substance-related genes and reduces cellulose levels in endosperm.

"Salty taste" sensation is evoked when sodium and chloride ions are present together in the oral cavity. The presence of an epithelial cation channel that receives Na+ has previously been reported. However, no molecular entity involving Cl- receptors has been elucidated. We report the strong expression of transmembrane channel-like 4 (TMC4) in the circumvallate and foliate papillae projected to the glossopharyngeal nerve, mediating a high-concentration of NaCl. Electrophysiological analysis using HEK293T cells revealed that TMC4 was a voltage-dependent Cl- channel and the consequent currents were completely inhibited by NPPB, an anion channel blocker. TMC4 allowed permeation of organic anions including gluconate, but their current amplitudes at positive potentials were less than that of Cl-. Tmc4-deficient mice showed significantly weaker glossopharyngeal nerve response to high-concentration of NaCl than the wild-type littermates. These results indicated that TMC4 is a novel chloride channel that responds to high-concentration of NaCl.

Sweetness and bitterness are key determinants of food acceptance and rejection, respectively. Sugars, such as sucrose and fructose, are generally recognized as sweet. However, not all sugars are sweet, and even anomers may have quite different tastes. For example, gentiobiose is bitter, whereas its anomer, isomaltose, is sweet. Despite this unique sensory character, the molecular basis of the bitterness of gentiobiose remains to be clarified. In this study, we used calcium imaging analysis of human embryonic kidney 293T cells that heterologously expressed human taste receptors to demonstrate that gentiobiose activated hTAS2R16, a bitter taste receptor, but not hT1R2/hT1R3, a sweet taste receptor. In contrast, isomaltose activated hT1R2/hT1R3. As a result, these anomers elicit different taste sensations. Mutational analysis of hTAS2R16 also indicated that gentiobiose and β-D-glucopyranosides, such as salicin share a common binding site of hTAS2R16.

Taste perception plays an essential role in food selection. Umami (savory) tastes are sensed by a taste receptor complex, T1R1/T1R3, that detects proteinogenic amino acids.1 High sensitivity to l-glutamate (l-Glu) is a characteristic of human T1R1/T1R3, but the T1R1/T1R3 of other vertebrates does not consistently show this l-Glu response.1,2 Here, we demonstrate that the l-Glu sensitivity of T1R1/T1R3 is a derived state that has evolved repeatedly in large primates that rely on leaves as protein sources, after their divergence from insectivorous ancestors. Receptor expression experiments show that common amino acid substitutions at ligand binding sites that render T1R1/T1R3 sensitive to l-Glu occur independently at least three times in primate evolution. Meanwhile T1R1/T1R3 senses 5'-ribonucleotides as opposed to l-Glu in several mammalian species, including insectivorous primates. Our chemical analysis reveal that l-Glu is one of the major free amino acids in primate diets and that insects, but not leaves, contain large amounts of free 5'-ribonucleotides. Altering the ligand-binding preference of T1R1/T1R3 from 5'-ribonucleotides to l-Glu might promote leaf consumption, overcoming bitter and aversive tastes. Altogether, our results provide insight into the foraging ecology of a diverse mammalian radiation and help reveal how evolution of sensory genes facilitates invasion of new ecological niches.