Sour Ageusia in Two Individuals Implicates Ion Channels of the ASIC and PKD Families in Human Sour Taste Perception at the Anterior Tongue

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

RT-PCR and genomic analysis of polycystic kidney disease (PKD) gene expression in sour-ageusic patients and sour-normal subjects.

<p>Sour-ageusic patients do not express detectable transcripts for either PKD1L3 or PKD2L1, even after 50 cycles of amplification, and for PKD2L1 this is not the result of a loss of the <i>PKD2L1</i> gene. Sour-normal subjects express both transcripts. Lane identifications: (A) 100 bp DNA marker with the brightest band being 500 bp; (B–D) Transcript for PKD1L3 in, respectively, sour-normal Subject 65, sour-ageusic Patient 8689 and sour-ageusic Patient 8716; (E–G) Transcript for PKD2L1 in, respectively, sour-normal subject 65, sour-ageusic Patient 8689 and sour-ageusic Patient 8716; (H) 1 kb DNA marker; (I) genomic DNA of Patient 8689 amplified with primer pair HPKD2F1 and HPKD2R1 (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007347#pone-0007347-t001" target="_blank">Table 1</a>); (J) RT-PCR for full coding sequence of PKD2L1 of sour-normal Subject 49. RT-PCR was performed with cDNA from fungiform papillae (B–G and J), but only the data for the tubes containing reverse transcriptase are presented. No products were detected in the tubes lacking reverse transcriptase. Genomic analysis (I) was performed using buccal tissue from Patient 8689. Patient 8716 did not provide buccal tissue. Individual amplification reactions were performed for each named molecular target and the data are shown in collated form. Identities of all amplification products were confirmed by sequencing.</p

RT-PCR and genomic analysis of gene expression in sour-normal subjects.

<p>Transcripts for all ASICs tested are clearly detectable, as also are those for housekeeping and taste-related genes. Lane identifications: (A) 100 bp DNA marker, with the brightest band being 500 bp; (B) Transcript for 460-bp fragment of β-actin in a pooled sample of fungiform papillae from Subjects 45 and 49; (C–G) Transcripts for, respectively, ASICs 1a, 1β, 2a, 2b (weakly-staining band at 467 bp in Lane 6), and ASIC3 in a pooled sample of fungiform papillae from Subjects 45 and 49; (H) Transcript for PLC-β2 in Subject 49; (I) 1 kb DNA marker; (J) full coding sequence of ASIC1a of Subject 49; (K) full coding sequence of bitter receptor T2R14 of Subject 49; (L) full coding sequence of δ-ENaC of Subject 45; (M) genomic DNA of Subject 65 amplified for <i>ASIC1</i> gene with primer pair ASICMCF1 and ASICMCR1 (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007347#pone-0007347-t001" target="_blank">Table 1</a>); and (N) genomic DNA of Subject 65 amplified for <i>ASIC1</i> gene with primer pair HASIC3 and HASIC4 (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007347#pone-0007347-t001" target="_blank">Table 1</a>). RT-PCR (B–H and J–L) was performed with cDNA from fungiform papillae but only the data for the tubes containing reverse transcriptase are presented. No products were detected in the tubes lacking reverse transcriptase. Genomic analysis (M, N) was performed using buccal tissue. Individual amplification reactions were performed for each named molecular target and the data are shown in collated form. Identities of all amplification products were confirmed by sequencing. All three sour normal subjects expressed all the named transcripts that were probed for, but to avoid repetition of the same pattern of data only selected transcripts are shown.</p

List of primers used in this study.

<p>List of primers used in this study.</p

RT-PCR and genomic analysis of acid sensing ion channel (ASIC) gene expression for sour-ageusic Patient 8689.

<p>The patient expresses housekeeping and taste-related genes but ASIC transcripts are undetectable even after 50 cycles of amplification, and for ASICs 1a and 1β this is not the result of a loss of the <i>ASIC1</i> gene. Lane identifications: (A) 100 bp DNA marker with the brightest band being 500 bp; (B) Transcript of β-actin; (C–G) Transcripts of, respectively, ASICs 1a, 1β, 2a, 2b and 3; (H) Transcript of δ-ENaC; (I) 1 kb DNA marker; (J) ASIC1a, full coding sequence; (K) genomic DNA amplified with primer pair ASICMCF1 and ASICMCR1 (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007347#pone-0007347-t001" target="_blank">Table 1</a>); and (L) genomic DNA amplified with primer pair HASIC3 and HASIC4 (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007347#pone-0007347-t001" target="_blank">Table 1</a>). RT-PCR was performed with cDNA from fungiform papillae (B–H and J), but only the data for the tubes containing reverse transcriptase are presented. No products were detected in the tubes lacking reverse transcriptase. Genomic analysis (K, L) was performed using buccal tissue. Individual amplification reactions were performed for each named molecular target and the data are shown in collated form. Identities of all amplification products were confirmed by sequencing.</p

Immunohistochemical staining of the protein products of taste-related genes expressed in the fungiform papillae of sour-normal subjects.

<p>The gray image of Panel A is a fungiform section showing taste buds under Nomarski optics. The image in Panel B is of the same section as A, but showing the section immunostained with antibodies to PLC-β2. Panel C displays immunostaining with antibody to δ-ENaC, overlaid on a Nomarski picture of the same tissue section. The human taste bud is known to be invested with the δ form of ENaC at the expense of the α form <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007347#pone.0007347-Huque3" target="_blank">[36]</a>. Panel E shows immunoreactivity to antibody against ASIC1a, with the Nomarski optics of the same section displayed in Panel D. The taste bud area of the section in Panel E is magnified to a larger black and white image showing the specific labeling of the characteristic spindle-shaped cells within a taste bud. Panel F shows immunoreactivity to antibody against ASIC1β in the taste bud (outlined) and in the plexus, overlaid on a photo of the same section under Nomarski optics. The distribution of the channels PKD2Ll and PKD1L3 in the human fungiform papilla is shown in Panels G, H and I. Panels G and H display immunoreactivity toward the ion channel PKD2L1. A DAPI overlay marks each cell. The arrow in Panel G points to a taste bud. This taste bud appears in Panel H at an approximately five-fold magnification. Note the labeling of the membranes of several taste cells. Panel I shows distribution of immunoreactivity to an antibody made against PKD1L3. It shows the upper portion of a human fungiform papilla using Nomarski optics overlaid with the immunohistochemical label from an antibody against PKD1L3 and DAPI stain. A taste bud is outlined. The label for PKD1L3 appears between cells and is likely not labeling the membrane of the taste bud cells because the label does not appear in the cells, as it would were the antibody recognizing an antigen on the taste cell. The bars on each Panel show the magnification: A & B: the box width is 40 µm, blow-up in Panel B is a 2-fold magnification of the left smaller box; C: 20 µm; D & E: 25 µm with Insert E: 10 µm; F: 25 µm; G: 80 µm; H: 15 µm; I: 15 µm.</p

RT-PCR analysis of gene expression for sour-ageusic Patient 8716.

<p>The patient expresses transcripts for housekeeping genes and those involved in sweet, bitter and salty taste, but ASIC transcripts are undetectable even after 50 cycles of amplification. Lane identifications: (A) 100 bp DNA marker with the brightest band being 500 bp; (B) Transcript for 460-bp fragment of β-actin; (C–G) Transcripts for, respectively, ASICs 1a, 1β, 2a, 2b and 3; (H) Transcript for PLC-β2, which is involved in the transduction pathways of sweet and bitter; (I) 1 kb DNA marker; (J) full coding sequence of β-actin; (K) full coding sequence of bitter receptor T2R14; (L) full coding sequence of δ-ENaC; and (M) partial coding sequence of α-ENaC. RT-PCR was performed with cDNA from fungiform papillae but only the data for the tubes containing reverse transcriptase are presented. No products were detected in the tubes lacking reverse transcriptase. Individual amplification reactions were performed for each named molecular target and the data are shown in collated form. Identities of all amplification products were confirmed by sequencing.</p