Taste function in mice with a targeted mutation of the pkd1l3 gene.
ABSTRACT: Recent studies, both in vitro and in vivo, have suggested the involvement of the polycystic kidney disease-1 and -2 like genes, Pkd1l3 and Pkd2l1, in acid taste transduction. In mice, disruption of taste cells expressing PKD2L1 eliminates gustatory neural responses to acids. However, no previous data exist on taste responses in the absence of PKD1L3 or on behavioral responses in mice lacking either of these proteins. In order to assess the function of PKD1L3, we genetically engineered mice with a targeted mutation of the Pkd1l3 gene. We then examined taste responsiveness of mutant and wild-type mice using several different approaches. In separate groups of mice, we measured preference scores in 48-h 2-bottle tests, determined NaCl or citric acid taste thresholds using a conditioned taste aversion technique, and conducted electrophysiological recordings of activity in the chorda tympani and glossopharyngeal nerves. Multiple taste compounds representing all major taste qualities were used in the preference tests and nerve-recording experiments. We found no significant reduction in taste responsiveness in Pkd1l3 mutant mice in behavioral or electrophysiological tests when compared with wild-type controls. Therefore, further studies are needed to elucidate the function of PKD1L3 in taste bud cells.
Project description: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.
Project description:Animals use their gustatory systems to evaluate the nutritious value, toxicity, sodium content, and acidity of food. Although characterization of molecular identities that receive taste chemicals is essential, molecular receptors underlying sour taste sensation remain unclear. Here, we show that two transient receptor potential (TRP) channel members, PKD1L3 and PKD2L1, are coexpressed in a subset of taste receptor cells in specific taste areas. Cells expressing these molecules are distinct from taste cells having receptors for bitter, sweet, or umami tastants. The PKD2L1 proteins are accumulated at the taste pore region, where taste chemicals are detected. PKD1L3 and PKD2L1 proteins can interact with each other, and coexpression of the PKD1L3 and PKD2L1 is necessary for their functional cell surface expression. Finally, PKD1L3 and PKD2L1 are activated by various acids when coexpressed in heterologous cells but not by other classes of tastants. These results suggest that PKD1L3 and PKD2L1 heteromers may function as sour taste receptors.
Project description: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 Ca(2+) 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 Ca(2+) 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.
Project description:Polycystic kidney disease 1-like 3 (Pkd1l3) is expressed specifically in sour-sensing type III taste cells that have synaptic contacts with afferent nerve fibers in circumvallate (CvP) and foliate papillae (FoP) located in the posterior region of the tongue, although not in fungiform papillae (FuP) or the palate. To visualize the gustatory neural pathways that originate from type III taste cells in CvP and FoP, we established transgenic mouse lines that express the transneuronal tracer wheat germ agglutinin (WGA) under the control of the mouse Pkd1l3 gene promoter/enhancer. The WGA transgene was accurately expressed in Pkd1l3-expressing type III taste cells in CvP and FoP. Punctate WGA protein signals appeared to be detected specifically in type III taste cells but not in other types of taste cells. WGA protein was transferred primarily to a subset of neurons located in close proximity to the glossopharyngeal (GL) nerve bundles in the nodose/petrosal ganglion (NPG). WGA signals were also observed in a small population of neurons in the geniculate ganglion (GG). This result demonstrates the anatomical connection between taste receptor cells (TRCs) in the FoP and the chorda tympani (CT) nerves. WGA protein was further conveyed to neurons in a rostro-central subdivision of the nucleus of the solitary tract (NST). These findings demonstrate that the approximately 10?kb 5'-flanking region of the mouse Pkd1l3 gene functions as a type III taste cell-specific promoter/enhancer. In addition, experiments using the pkd1l3-WGA transgenic mice reveal a sour gustatory pathway that originates from TRCs in the posterior region of the tongue.
Project description: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.
Project description:Five tastes have been identified, each of which is transduced by a separate set of taste cells. Of these sour, which is associated with acid stimuli, is the least understood. Genetic ablation experiments have established that sour is detected by a subset of taste cells that express the TRP channel PKD2L1 and its partner PKD1L3, however the mechanisms by which this subset of cells detects acids remain unclear. Previous efforts to understand sour taste transduction have been hindered because sour responsive cells represent only a small fraction of cells in a taste bud, and numerous ion channels with no role in sour sensing are sensitive to acidic pH. To identify acid-sensitive conductances unique to sour cells, we created genetically modified mice in which sour cells were marked by expression of YFP under the control of the PKD2L1 promoter. To measure responses to sour stimuli we developed a method in which suction electrode recording is combined with UV photolysis of NPE-caged proton. Using these methods, we report that responses to sour stimuli are not mediated by Na(+) permeable channels as previously thought, but instead are mediated by a proton conductance specific to PKD2L1-expressing taste cells. This conductance is sufficient to drive action potential firing in response to acid stimuli, is enriched in the apical membrane of PKD2L1-expressing taste cells and is not affected by targeted deletion of the PKD1L3 gene. We conclude that, during sour transduction, protons enter through an apical proton conductance to directly depolarize the taste cell membrane.
Project description:Mutations in polycystin proteins PKD1 and TRPP2 lead to autosomal dominant polycystic kidney disease. These two proteins form a receptor-ion channel complex on primary cilia. PKD1 undergoes an autoproteolysis at the N terminal G-protein-coupled receptor proteolytic site (GPS), which is essential for the function of PKD1. Whether GPS cleavage happens in other PKD proteins and its functional consequence has remained elusive. Here we studied the GPS cleavage of PKD1L3, a protein that associates with TRPP3 in taste cells and may play a role in sour taste. Our results show that PKD1L3 also undergoes GPS cleavage. Mutation at the GPS abolishes the cleavage, and the non-cleavable mutant does not traffic to the plasma membrane when associated with TRPP3. We also found that a splice variant of PKD1L3, which was originally identified in taste buds, is not cleaved. Amino acids L708 and S709, which are missing in this splice variant, are crucial for the GPS cleavage of PKD1L3 and the trafficking of the PKD1L3/TRPP3 complex. Our results gain insight into the molecular mechanism of the GPS cleavage of PKD1L3. The presence of the non-cleavable variant suggests the potential in vivo function of uncleaved PKD proteins.
Project description:BACKGROUND:The perception of sour taste in humans is incompletely understood at the receptor cell level. We report here on two patients with an acquired sour ageusia. Each patient was unresponsive to sour stimuli, but both showed normal responses to bitter, sweet, and salty stimuli. METHODS AND FINDINGS:Lingual fungiform papillae, containing taste cells, were obtained by biopsy from the two patients, and from three sour-normal individuals, and analyzed by RT-PCR. The following transcripts were undetectable in the patients, even after 50 cycles of amplification, but readily detectable in the sour-normal subjects: acid sensing ion channels (ASICs) 1a, 1beta, 2a, 2b, and 3; and polycystic kidney disease (PKD) channels PKD1L3 and PKD2L1. Patients and sour-normals expressed the taste-related phospholipase C-beta2, the delta-subunit of epithelial sodium channel (ENaC) and the bitter receptor T2R14, as well as beta-actin. Genomic analysis of one patient, using buccal tissue, did not show absence of the genes for ASIC1a and PKD2L1. Immunohistochemistry of fungiform papillae from sour-normal subjects revealed labeling of taste bud cells by antibodies to ASICs 1a and 1beta, PKD2L1, phospholipase C-beta2, and delta-ENaC. An antibody to PKD1L3 labeled tissue outside taste bud cells. CONCLUSIONS:These data suggest a role for ASICs and PKDs in human sour perception. This is the first report of sour ageusia in humans, and the very existence of such individuals ("natural knockouts") suggests a cell lineage for sour that is independent of the other taste modalities.
Project description:Polycystic kidney disease (PKD) protein 2 Like 1 (PKD2L1), also called transient receptor potential polycystin-3 (TRPP3), regulates Ca(2+)-dependent hedgehog signalling in primary cilia, intestinal development and sour tasting but with an unclear mechanism. PKD2L1 is a Ca(2+)-permeable cation channel that is activated by extracellular Ca(2+) (on-response) in Xenopus oocytes. PKD2L1 co-expressed with PKD protein 1 Like 3 (PKD1L3) exhibits extracellular acid-induced activation (off-response, i.e., activation following acid removal) but whether PKD1L3 participates in acid sensing remains unclear. Here we used the two-microelectrode voltage-clamp, site directed mutagenesis, Western blotting, reverse transcriptase-polymerase chain reaction (RT-PCR) and immunofluorescence, and showed that PKD2L1 expressed in oocytes exhibits sustained off-response currents in the absence of PKD1L3. PKD1L3 co-expression augmented the PKD2L1 plasma membrane localization but did not alter the observed properties of the off-response. PKD2L1 off-response was inhibited by an increase in intracellular Ca(2+). We also identified two intra-membrane residues aspartic acid 349 (D349) and glutamic acid 356 (E356) in the third transmembrane domain that are critical for PKD2L1 channel function. Our study suggests that PKD2L1 may itself sense acids and defines off-response properties in the absence of PKD1L3.
Project description:Sensory impairments are critical for diagnosing and characterizing neurodevelopmental disorders. Taste is a sensory modality often not well characterized. Engrailed-2 (En2) is a transcription factor critical for neural development, and mice lacking En2 (En2-/-) display signs of impaired social interaction, cognitive processes (e.g., learning and memory, conditioned fear), and neurodevelopmental alterations. As such, En2-/- mice display the behavioral deficits and neural impairments characteristic of the core symptoms associated with autism spectrum disorder (ASD). The objective of this study was to characterize the taste function in En2-/- compared with En2+/+ in adult male mice. Measuring taste responsiveness by an automated gustometer, En2 null mice had decreased lick responses for 1.6?M fructose, whereas they demonstrated an increased taste responsivity (i.e., relative to water) at 0.3?M sodium chloride and 1?M monosodium glutamate. In a separate cohort of mice, En2-/- mice had an increased preference for sodium chloride over a range of concentrations (0.032-0.3?M) compared with En2+/+ mice. Regional gene expression of the tongue epithelium demonstrated an increase in Scnn1a, T2R140, T1R3, and Trpm5 and a decrease in Pkd1l3 in En2 null mice. Taken together, such data indicate that deficits in En2 can produce sensory impairments that can have a measurable impact on taste, particularly salt taste.