Yoshiro Ishimaru

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.

Oral and extra-oral taste perception

Of the five basic taste qualities, the molecular mechanisms underlying sweet, bitter, and umami (savory) taste perception have been extensively elucidated, including the taste receptors and downstream signal transduction molecules. Recent studies have revealed that these taste-related molecules play important roles not only in the oral cavity but also in a variety of tissues including the respiratory tract, stomach, intestines, pancreas, liver, kidney, testes, and brain. This review covers the current knowledge regarding the physiological roles of taste-related molecules in the oral and extra-oral tissues.

Interaction between PKD1L3 and PKD2L1 through their transmembrane domains is required for localization of PKD2L1 at taste pores in taste cells of circumvallate and foliate papillae

The polycystic kidney disease 1‐like 3 (PKD1L3) and polycystic kidney disease 2‐like 1 (PKD2L1) proteins have been proposed to form heteromers that function as sour taste receptors in mammals. Here, we show that PKD1L3 and PKD2L1 interact through their transmembrane domains, and not through the coiled‐coil domain, by coimmunoprecipitation experiments using a series of deletion mutants. Deletion mutants lacking the critical interaction region were not transported to the cell surface and remained in the cytoplasm, whereas PKD1L3 and PKD2L1 proteins were expressed at the cell surface when both are transfected. Calcium imaging analysis revealed that neither the coiled‐coil domain nor the EF‐hand domain located in the C‐terminal cytoplasmic tail of PKD2L1 was required for response on stimulation with an acidic solution. Finally, PKD2L1 did not localize to the taste pore but was distributed throughout the cytoplasm in taste cells of circumvallate and foliate papillae in PKD1L3−/− mice, whereas it localized to the taste pore in wild‐type mice. Collectively, these results suggest that the interaction between PKD1L3 and PKD2L1 through their transmembrane domains is essential for proper trafficking of the channels to the cell surface in taste cells of circumvallate and foliate papillae and in cultured cells.—Ishimaru, Y., Katano, Y., Yamamoto, K., Akiba, M., Misaka, T., Roberts, R. W., Asakura, T., Matsunami, H., Abe, K. Interaction between PKD1L3 and PKD2L1 through their transmembrane domains is required for localization of PKD2L1 at taste pores in taste cells of circumvallate and foliate papillae. FASEB J. 24, 4058–4067 (2010). www.fasebj.org

Global gene expression profiles in developing soybean seeds

The gene expression profiles in soybean (Glycine max L.) seeds at 4 stages of development, namely, pod, 2-mm bean, 5-mm bean, and full-size bean, were examined by DNA microarray analysis. The total genes of each sample were classified into 4 clusters based on stage of development. Gene expression was strictly controlled by seed size, which coincides with the development stage. First, stage specific gene expression was examined. Many transcription factors were expressed in pod, 2-mm bean and 5-mm bean. In contrast, storage proteins were mainly expressed in full-size bean. Next, we extracted the genes that are differentially expressed genes (DEGs) that were extracted using the Rank products method of the Bioconductor software package. These DEGs were sorted into 8 groups using the hclust function according to gene expression patterns. Three of the groups across which the expression levels progressively increased included 100 genes, while 3 groups across which the levels decreased contained 47 genes. Storage proteins, seed-maturation proteins, some protease inhibitors, and the allergen Gly m Bd 28K were classified into the former groups. Lipoxygenase (LOX) family members were present in both the groups, indicating the multi-functionality with different expression patterns.

Functional diversity of bitter taste receptor TAS2R16 in primates

In mammals, bitter taste is mediated by TAS2R genes, which belong to the large family of seven transmembrane G protein-coupled receptors. Because TAS2Rs are directly involved in the interaction between mammals and their dietary sources, it is likely that these genes evolved to reflect species-specific diets during mammalian evolution. Here, we investigated the sensitivities of TAS2R16s of various primates by using a cultured cell expression system, and found that the sensitivity of each primate species varied according to the ligand. Especially, the sensitivity of TAS2R16 of Japanese macaques to salicin was much lower than that of human TAS2R16, which was supported by behavioural tests. These results suggest the possibility that bitter-taste sensitivities evolved independently by replacing specific amino acid residues of TAS2Rs in different primate species to adapt to food items they use.

The human bitter taste receptor, hTAS2R16, discriminates slight differences in the configuration of disaccharides.

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.

Genetic tracing of the gustatory neural pathway originating from Pkd1l3-expressing type III taste cells in circumvallate and foliate papillae

J. Neurochem. (2011) 119, 497–506.

Surface plasmon resonance analysis on interactions of food components with a taste epithelial cell model.

A new device for evaluating the continuity of taste was developed with the use of surface plasmon resonance (SPR). The model of lingual cells was constructed with liposomes immobilized onto an L1 sensor chip for SPR. Using this device, we classified food components into three categories according to the sensorgram pattern and residual ratio on lipid bilayer. Samples in group A strongly interacted with lipid bilayer, those in group B poorly interacted, and those in group C belong to neither group A nor group B. Sweet proteins and gymnemic acids that prolonged sweet perception were categorized in group A. Almost all the carbohydrates investigated and aspartame, of which the taste perception does not continue, belonged to group B. This device made it possible to detect the interaction with lipid bilayer and dissected the mechanism of taste continuity.

The response of PKD1L3/PKD2L1 to acid stimuli is inhibited by capsaicin and its pungent analogs.

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 IC50 of 32.5u2003μm. Capsaicin and its analogs are thus useful tools for physiological analysis of PKD1L3/PKD2L1 function.

The single pore residue Asp523 in PKD2L1 determines Ca2+ permeation of the PKD1L3/PKD2L1 complex.

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.