Douglas L. Bayley

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, 1β, 2a, 2b, and 3; and polycystic kidney disease (PKD) channels PKD1L3 and PKD2L1. Patients and sour-normals expressed the taste-related phospholipase C-β2, the δ-subunit of epithelial sodium channel (ENaC) and the bitter receptor T2R14, as well as β-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 1β, PKD2L1, phospholipase C-β2, and δ-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.

Transduction mechanisms for the taste of amino acids

Amino acids are important taste stimuli for a variety of animals. One animal model, the channel catfish, I. punctatus, possesses sensitive taste receptor systems for several amino acids. Neurophysiological and biochemical receptor binding studies suggest the presence of at least three receptor pathways: one is a relatively nonspecific site(s) responsive to short-chain neutral amino acids such as L-alanine (L-ALA); another is responsive to the basic amino acid L-arginine (L-ARG); still another is a low affinity site for L-proline (L-PRO). Several possible transduction pathways are available in the taste system of this animal model for these amino acids. One of these, formation of inositol trisphosphate (IP3) and cyclic AMP (cAMP), is mediated by GTP-binding regulatory proteins, while another involves ion channels directly activated by stimuli. L-ALA is a potent stimulus to cAMP and IP3 accumulation, while L-ARG at low concentrations is without effect. On the other hand, L-ARG and L-PRO, but not L-ALA, are able to activate stimulus-specific and cation-selective channels in taste epithelial membranes reconstituted in phospholipid bilayers at the tips of patch pipettes. Preliminary studies using mouse taste tissue demonstrate that monosodium-L-glutamate (MSG) did not enhance production of IP3 or cAMP. However, in reconstitution experiments using taste epithelium of mouse, conductance changes due to MSG are observed. The specificity of this channel(s) and its uniqueness have yet to be determined.

Cats Lack a Sweet Taste Receptor

Domestic cats (Felis silvestris catus) (herein referred to as “cats”) are neither attracted to, nor show avoidance of the taste of sweet carbohydrates and high-intensity sweeteners (1-3), yet they do show a preference for selected amino acids (4), and avoid stimuli that taste either bitter or very sour to humans (1,4). Consistent with this behavioral evidence, recordings from cat taste nerve fibers and from units of the geniculate ganglion innervating taste cells demonstrated responses to salty, sour, and bitter stimuli as well as to amino acids and nucleotides, but showed no response to sucrose and several other sugars (4-11). The sense of taste in cats appears similar to that of other mammals with the exception of an inability to taste sweet stimuli. Because only the sweet taste modality appears absent, we postulated that the defect in cats (and likely in other obligate carnivores of Felidae) lay at the receptor step, subtending this modality. The possible defects at the molecular level could range from a single to a few amino acid substitutions, such as is found between sweet “taster” and “nontaster” strains of mice (12-14), to more radical mechanisms, such as an unexpressed pseudogene. To distinguish among these possibilities, we identified the DNA sequences and examined the structures of the 2 known genes Tas1r2 and Tas1r3 that encode the sweet taste receptor heteromer T1R2/T1R3 in other mammals. We compared these with the sequence and structure of the same genes in dogs, humans, mice and rats, all species that respond to sweet stimuli.

Biochemical enrichment and biophysical characterization of a taste receptor for L-arginine from the catfish, Ictalurus puntatus

BackgroundThe channel catfish, Ictalurus punctatus, is invested with a high density of cutaneous taste receptors, particularly on the barbel appendages. Many of these receptors are sensitive to selected amino acids, one of these being a receptor for L-arginine (L-Arg). Previous neurophysiological and biophysical studies suggested that this taste receptor is coupled directly to a cation channel and behaves as a ligand-gated ion channel receptor (LGICR). Earlier studies demonstrated that two lectins, Ricinus communis agglutinin I (RCA-I) and Phaseolus vulgaris Erythroagglutinin (PHA-E), inhibited the binding of L-Arg to its presumed receptor sites, and that PHA-E inhibited the L-Arg-stimulated ion conductance of barbel membranes reconstituted into lipid bilayers.ResultsBoth PHA-E and RCA-I almost exclusively labeled an 82–84 kDa protein band of an SDS-PAGE of solubilized barbel taste epithelial membranes. Further, both rhodamine-conjugated RCA-I and polyclonal antibodies raised to the 82–84 kDa electroeluted peptides labeled the apical region of catfish taste buds. Because of the specificity shown by RCA-I, lectin affinity was chosen as the first of a three-step procedure designed to enrich the presumed LGICR for L-Arg. Purified and CHAPS-solubilized taste epithelial membrane proteins were subjected successively to (1), lectin (RCA-I) affinity; (2), gel filtration (Sephacryl S-300HR); and (3), ion exchange chromatography. All fractions from each chromatography step were evaluated for L-Arg-induced ion channel activity by reconstituting each fraction into a lipid bilayer. Active fractions demonstrated L-Arg-induced channel activity that was inhibited by D-arginine (D-Arg) with kinetics nearly identical to those reported earlier for L-Arg-stimulated ion channels of native barbel membranes reconstituted into lipid bilayers. After the final enrichment step, SDS-PAGE of the active ion channel protein fraction revealed a single band at 82–84 kDa which may be interpreted as a component of a multimeric receptor/channel complex.ConclusionsThe data are consistent with the supposition that the L-Arg receptor is a LGICR. This taste receptor remains active during biochemical enrichment procedures. This is the first report of enrichment of an active LGICR from the taste system of vertebrata.

Lipid profiles of taste and non-taste epithelial tissues from steer tongues

Some hypotheses on taste reception have implicated lipids of taste cells as major receptor constitutents. This study reports detailed lipid profiles of the taste bud-containing epidermis from circumvallate papillae and fungiform papillae as well as profiles from two non-taste bud tissues: circumvallate papillae dermis and epidermis from the lateral posterior of the tongue. Differences in levels of triglycerides and phosphatidylcholines were observed but these were not directly related to the presence of taste buds. At this level of analysis, it is evident that there are no unusual distributions of phospholipid classes in the taste bud epidermis when compared with the non-taste bud lingual epidermis.

Lipid characterization and14C-acetate metabolism in catfish taste epithelium

The catfish,Ictalurus punctatus is an important model system for the study of the biochemical mechanisms of taste reception. A detailed lipid analysis of epithelial tissue from the taste organ (barbel) of the catfish has been performed. Polar lipids account for 62±1% of the total, neutrals for 38±1%. Phosphatidyl-cholines, serines and ethanolamines are the major constitutents of the polar fraction. Plasmalogen concentration is high relative to that of non-neural tissues. [14C]-Acetate is incorporated into cell lipid fractions after incubation of barbel tissue at 37°C for 60 min. Percentage amounts of most lipids change with time during this in vitro incubation. The phospholipids are the most metabolically active fractions. This work yields information for continuing reconstitution experiments and indicates that the taste epithelium of this important model system is a metabolically active tissue capable of supporting lipid turnover/synthesis.

Comparison of fatty acid patterns of polar and neutral lipid classes and cyclo-oxygenase activity in taste and non-taste epithelium of steer tongues

Epithelial tissues and papilla from several regions of the steer tongue were isolated and the fatty acids from each lipid class in the polar and neutral fractions were assayed. The observed profiles indicated regional differences. Arachidonic acid and other fatty acids containing long carbon chains (greater than 22) were found in all tissues sampled, particularly in the phosphatidyls of the inositols, ethanolamines, cholines, and in the cholesterylesters. Production of prostaglandin E2 was measured through cyclo-oxygenase activity and the presence of plasmalogens was observed in the phosphatidylethanolamine and choline fractions. Higher rates of PGE2 synthesis and greater amounts of plasmologens were found in taste-related epithelial samples compared to lingual epithelial control samples not containing taste buds. The heterogeneity of patterns of lipids and fatty acids found in the epithelium of the tongue suggests possible zonal specialization to satisfy regional physiological needs.

Conformational changes of the sweet protein monellin as measured by fluorescence emission.

Abstract Monellin is a protein that tastes sweet. In the native state it is a dimer composed of two dissimilar noncovalently associated polypeptides. The conformation of the protein is a determinant of its sweetness, and the present investigation takes advantage of the fluorescence spectrum being a sensitive index of its conformation. The emission spectrum is used to evaluate the ability of temperature and pH to alter the conformation and the sweetness of the protein. When monellin dissolved in water is heated in discrete steps from 25 to 100°C, its sweetness decreases. The halfwidth of the fluorescence emission band increases in parallel with the loss of sweetness. The increase in halfwidth is due primarily to an increase in the intensity of tyrosine emission that may be the result of the two dissimilar polypeptides of monellin beginning to separate. Tyrosine residues are present in both chains, while the single tryptophan occurs in only one. Monellin is less susceptible to denaturation by increasing temperature when dissolved in sodium acetate buffer at pH 4 than it is at pH 3 or 7. When the pH of a solution containing monellin in 0.1 M KCl is varied from 2 to 13, there is a broad pH range (pH 4 to 9) where monellins conformation is not markedly altered. Below pH 3.5 and above pH 10.5, however, the emission spectra indicate that substantial denaturation occurs. However, monellin can be partially renatured following pH 12 treatment with only minimal loss of sweetness. The sweetness of monellin under these two types of denaturing conditions, temperature and pH, can be predicted by the fluorescence emission spectrum of the protein. In addition, this study confirms that the biological activity of monellin, its sweetness, is a function of quaternary structure of the protein.

Lipid metabolic interrelationships and phospholipase activity in gustatory epithelium ofictalurus punctatus in vitro

The catfish,Ictalurus punctatus, is an important model for studying the biochemical mechanisms of taste at the peripheral level. The type, amount and metabolic activity of the lipids within this tissue play important roles in taste transduction by forming the matrix in which the receptors for taste stimuli are imbedded and by acting as precursors to second messengers. The metabolic interconversions that occur among the lipids on the taste organ (barbels) of this animal are reported here. When sodium [32P]phosphate was incubated with minced pieces of epithelium from the taste organ ofI. punctatus, phospholipids became labeled. Maximal incorporation occurred near 20 min for lysophosphatidylcholines (LPC),phosphatidylcholines (PC) and phosphatidylinositols (PI). The phosphatidylethanolamines (PE) and phosphatidylserines (PS) became labeled more slowly. The label in LPC and PC declined from 20 min to 120 min, while that of the other fractions increased or was stable over the 20–120 min time period. Upon addition of 1,2-di-[1′-14C]palmitoyl-sn-glycero-3-phosphocholine to the medium,14C was found within minutes in all of the phospholipids assayed. The amount of label incorporated increased with time, with maximum labeling for all phospholipids occurring at 15 min. However,14C appeared predominantly first (by 5 min) in a neutral lipid fraction (fraction AG, consisting of free fatty acids, mono- and diglycerides, triglycerides and methyl esters), then declined rapidly as the phospholipids gradually incorporated more label. Within minutes of addition of 1-[1′-14C]palmitoyl-sn-glycero-3-phosphocholine (lysophosphatidylcholine) the14C-label was detected in the neutral lipid fraction AG, then in the PC fraction, and later in the other phospholipids. The PC fraction was maximally labeled by 40 min.Using the appropriate radiolabeled substrates, lysophosphatidylcholine phospholipase A1 and phosphatidylcholine phospholipase D activities were detected in this tissue. Very low activity of a phosphatidylcholine phospholipase A2 was observed. The experiments indicate that there are active and rapid exchange, degradation, synthesis and scavenger pathways of phospholipids in the taste organ of this animal, and suggest that phospholipases A1 and D-type activities are primarily responsible for the rapid breakdown of LPC and PC.