Nirupa Chaudhari

A metabotropic glutamate receptor variant functions as a taste receptor

Sensory transduction for many taste stimuli such as sugars, some bitter compounds and amino acids is thought to be mediated via G protein-coupled receptors (GPCRs), although no such receptors that respond to taste stimuli are yet identified. Monosodium L-glutamate (l-MSG), a natural component of many foods, is an important gustatory stimulus believed to signal dietary protein. We describe a GPCR cloned from rat taste buds and functionally expressed in CHO cells. The receptor couples negatively to a cAMP cascade and shows an unusual concentration–response relationship. The similarity of its properties to MSG taste suggests that this receptor is a taste receptor for glutamate.

The role of pannexin 1 hemichannels in ATP release and cell–cell communication in mouse taste buds

ATP has been shown to be a taste bud afferent transmitter, but the cells responsible for, and the mechanism of, its release have not been identified. Using CHO cells expressing high-affinity neurotransmitter receptors as biosensors, we show that gustatory stimuli cause receptor cells to secrete ATP through pannexin 1 hemichannels in mouse taste buds. ATP further stimulates other taste cells to release a second transmitter, serotonin. These results provide a mechanism to link intracellular Ca2+ release during taste transduction to secretion of afferent transmitter, ATP, from receptor cells. They also indicate a route for cell–cell communication and signal processing within the taste bud.

The cell biology of taste

Taste buds are aggregates of 50–100 polarized neuroepithelial cells that detect nutrients and other compounds. Combined analyses of gene expression and cellular function reveal an elegant cellular organization within the taste bud. This review discusses the functional classes of taste cells, their cell biology, and current thinking on how taste information is transmitted to the brain.

Separate Populations of Receptor Cells and Presynaptic Cells in Mouse Taste Buds

Taste buds are aggregates of 50–100 cells, only a fraction of which express genes for taste receptors and intracellular signaling proteins. We combined functional calcium imaging with single-cell molecular profiling to demonstrate the existence of two distinct cell types in mouse taste buds. Calcium imaging revealed that isolated taste cells responded with a transient elevation of cytoplasmic Ca2+ to either tastants or depolarization with KCl, but never both. Using single-cell reverse transcription (RT)-PCR, we show that individual taste cells express either phospholipase C β2 (PLCβ2) (an essential taste transduction effector) or synaptosomal-associated protein 25 (SNAP25) (a key component of calcium-triggered transmitter exocytosis). The two functional classes revealed by calcium imaging mapped onto the two gene expression classes determined by single-cell RT-PCR. Specifically, cells responding to tastants expressed PLCβ2, whereas cells responding to KCl depolarization expressed SNAP25. We demonstrate this by two methods: first, through sequential calcium imaging and single-cell RT-PCR; second, by performing calcium imaging on taste buds in slices from transgenic mice in which PLCβ2-expressing taste cells are labeled with green fluorescent protein. To evaluate the significance of the SNAP25-expressing cells, we used RNA amplification from single cells, followed by RT-PCR. We show that SNAP25-positive cells also express typical presynaptic proteins, including a voltage-gated calcium channel (α1A), neural cell adhesion molecule, synapsin-II, and the neurotransmitter-synthesizing enzymes glutamic acid decarboxylase and aromatic amino acid decarboxylase. No synaptic markers were detected in PLCβ2 cells by either amplified RNA profiling or by immunocytochemistry. These data demonstrate the existence of at least two molecularly distinct functional classes of taste cells: receptor cells and synapse-forming cells.

CALHM1 ion channel mediates purinergic neurotransmission of sweet, bitter and umami tastes

Recognition of sweet, bitter and umami tastes requires the non-vesicular release from taste bud cells of ATP, which acts as a neurotransmitter to activate afferent neural gustatory pathways. However, how ATP is released to fulfil this function is not fully understood. Here we show that calcium homeostasis modulator 1 (CALHM1), a voltage-gated ion channel, is indispensable for taste-stimuli-evoked ATP release from sweet-, bitter- and umami-sensing taste bud cells. Calhm1 knockout mice have severely impaired perceptions of sweet, bitter and umami compounds, whereas their recognition of sour and salty tastes remains mostly normal. Calhm1 deficiency affects taste perception without interfering with taste cell development or integrity. CALHM1 is expressed specifically in sweet/bitter/umami-sensing type II taste bud cells. Its heterologous expression induces a novel ATP permeability that releases ATP from cells in response to manipulations that activate the CALHM1 ion channel. Knockout of Calhm1 strongly reduces voltage-gated currents in type II cells and taste-evoked ATP release from taste buds without affecting the excitability of taste cells by taste stimuli. Thus, CALHM1 is a voltage-gated ATP-release channel required for sweet, bitter and umami taste perception.

Breadth of Tuning and Taste Coding in Mammalian Taste Buds

A longstanding question in taste research concerns taste coding and, in particular, how broadly are individual taste bud cells tuned to taste qualities (sweet, bitter, umami, salty, and sour). Taste bud cells express G-protein-coupled receptors for sweet, bitter, or umami tastes but not in combination. However, responses to multiple taste qualities have been recorded in individual taste cells. We and others have shown previously there are two classes of taste bud cells directly involved in gustatory signaling: “receptor” (type II) cells that detect and transduce sweet, bitter, and umami compounds, and “presynaptic” (type III) cells. We hypothesize that receptor cells transmit their signals to presynaptic cells. This communication between taste cells could represent a potential convergence of taste information in the taste bud, resulting in taste cells that would respond broadly to multiple taste stimuli. We tested this hypothesis using calcium imaging in a lingual slice preparation. Here, we show that receptor cells are indeed narrowly tuned: 82% responded to only one taste stimulus. In contrast, presynaptic cells are broadly tuned: 83% responded to two or more different taste qualities. Receptor cells responded to bitter, sweet, or umami stimuli but rarely to sour or salty stimuli. Presynaptic cells responded to all taste qualities, including sour and salty. These data further elaborate functional differences between receptor cells and presynaptic cells, provide strong evidence for communication within the taste bud, and resolve the paradox of broad taste cell tuning despite mutually exclusive receptor expression.

An Optimized Method for In Situ Hybridization with Signal Amplification That Allows the Detection of Rare mRNAs

In situ hybridization (ISH) using nonradioactive probes enables mRNAs to be detected with improved cell resolution but compromised sensitivity compared to ISH with radiolabeled probes. To detect rare mRNAs, we optimized several parameters for ISH using digoxygenin (DIG)-labeled probes, and adapted tyramide signal amplification (TSA) in combination with alkaline phosphatase (AP)-based visualization. This method, which we term TSA-AP, achieves the high sensitivity normally associated with radioactive probes but with the cell resolution of chromogenic ISH. Unlike published protocols, long RNA probes (up to 2.61 KB) readily permeated cryosections and yielded stronger hybridization signals than hydrolyzed probes of equivalent complexity. RNase digestion after hybridization was unnecessary and led to a substantial loss of signal intensity without significantly reducing nonspecific background. Probe concentration was also a key parameter for improving signal-to-noise ratio in ISH. Using these optimized methods on rat taste tissue, we detected mRNA for mGluR4, a receptor, and transducin, a G-protein, both of which are expressed at very low abundance and are believed to be involved in chemosensory transduction. Because the effect of the tested parameters was similar for ISH on sections of brain and tongue, we believe that these methodological improvements for detecting rare mRNAs may be broadly applicable to other tissues.

Umami Responses in Mouse Taste Cells Indicate More than One Receptor

A number of gustatory receptors have been proposed to underlie umami, the taste of l-glutamate, and certain other amino acids and nucleotides. However, the response profiles of these cloned receptors have not been validated against responses recorded from taste receptor cells that are the native detectors of umami taste. We investigated umami taste responses in mouse circumvallate taste buds in an intact slice preparation, using confocal calcium imaging. Approximately 5% of taste cells selectively responded to l-glutamate when it was focally applied to the apical chemosensitive tips of receptor cells. The concentration–response range for l-glutamate fell approximately within the physiologically relevant range for taste behavior in mice, namely 10 mm and above. Inosine monophosphate enhanced taste cell responses to l-glutamate, a characteristic feature of umami taste. Using pharmacological agents, ion substitution, and immunostaining, we showed that intracellular pathways downstream of receptor activation involve phospholipase C β2. Each of the above features matches those predicted by studies of cloned and expressed receptors. However, the ligand specificity of each of the proposed umami receptors [taste metabotropic glutamate receptor 4, truncated metabotropic glutamate receptor 1, or taste receptor 1 (T1R1) and T1R3 dimers], taken alone, did not appear to explain the taste responses observed in mouse taste cells. Furthermore, umami responses were still observed in mutant mice lacking T1R3. A full explanation of umami taste transduction may involve novel combinations of the proposed receptors and/or as-yet-undiscovered taste receptors.

Biogenic amine synthesis and uptake in rodent taste buds

Although adenosine triphosphate (ATP) is known to be an afferent transmitter in the peripheral taste system, serotonin (5‐HT) and norepinephrine (NE) have also been proposed as candidate neurotransmitters and have been detected immunocytochemically in mammalian taste cells. To understand the significance of biogenic amines in taste, we evaluated the ability of taste cells to synthesize, transport, and package 5‐HT and NE. We show by reverse transcriptase‐polymerase chain reaction and immunofluorescence microscopy that the enzymes for 5‐HT synthesis, tryptophan hydroxylase (TPH) and aromatic amino acid decarboxylase (AADC) are expressed in taste cells. In contrast, enzymes necessary for NE synthesis, tyrosine hydroxylase (TH) and dopamine β‐hydroxylase (DBH) are absent. Both TH and DBH are expressed in nerve fibers that penetrate taste buds. Taste buds also robustly express plasma membrane transporters for 5‐HT and NE. Within the taste bud NET, a specific NE transporter, is expressed in some presynaptic (type III) and some glial‐like (type I) cells but not in receptor (type II) cells. By using enzyme immunoassay, we show uptake of NE, probably through NET in taste epithelium. Proteins involved in inactivating and packaging NE, including catechol‐O‐methyltransferase (COMT), monoamine oxidase‐A (MAO‐A), vesicular monoamine transporter (VMAT1,2) and chromogranin A (ChrgA), are also expressed in taste buds. Within the taste bud, ChrgA is found only in presynaptic cells and may account for dense‐cored vesicles previously seen in some taste cells. In summary, we postulate that aminergic presynaptic taste cells synthesize only 5‐HT, whereas NE (perhaps secreted by sympathetic fibers) may be concentrated and repackaged for secretion. J. Comp. Neurol. 505:302–313, 2007.

Acid-Sensing Ion Channel-2 Is Not Necessary for Sour Taste in Mice

The acid-sensitive cation channel acid-sensing ion channel-2 (ASIC2) is widely believed to be a receptor for acid (sour) taste in mammals on the basis of its physiological properties and expression in rat taste bud cells. Using reverse transcriptase-PCR, we detected expression of ASIC1 and ASIC3, but not ASIC4, in mouse and rat taste buds and nonsensory lingual epithelium. Surprisingly, we did not detect mRNA for ASIC2 in mouse taste buds, although we readily observed its expression in rat taste buds. Furthermore, in Ca2+ imaging experiments, ASIC2 knock-out mice exhibited normal physiological responses (increases in intracellular Ca2+ concentrations) to acid taste stimuli. Our results indicate that ASIC2 is not required for acid taste in mice, and that if a universal mammalian acid taste transduction mechanism exists, it likely uses other acid-sensitive receptors or ion channels.