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CHANNELING
Taste Stimulation Is Easy To Do, But Hard To Follow
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COVER STORY
September 10, 2001

Volume 79, Number 37
CENEAR 79 37 pp. 42-46
ISSN 0009-2347
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UNLOCKING THE SECRETS OF TASTE
Newly identified taste receptors and the molecules that stimulate them provide entry point for researchers who investigate our least understood sense

A. MAUREEN ROUHI, C&EN WASHINGTON

Eating--whether a plain hot dog from a sidewalk vendor or an elaborate meal in a five-star restaurant--engages all the senses. Our eyes light up when presented with vivid colors of food and the artful creations of chefs. We inhale the aroma of wine. We hear the crispy crunch of a potato chip. We feel the tingling of soda or the smoothness of cream. We feel both heat and cold with a mouthful of apple pie á la mode. We taste.

CASCADE Binding of chemicals to receptors in taste cells bundled in a taste bud triggers a series of reactions that culminates in signals to a taste center in the brain.
LYDIA V. KUBIUK, COPYRIGHT 2001
"If you have a receptor and a meaningful readout system, you can go fishing in the universe of organic chemistry and find active molecules everywhere."
The brain synthesizes these different sensory messages, and we feel good. Satisfied diners usually remark that the food tastes good. Sometimes they might say the food looks good or smells good. Rarely would they say that the food sounds or feels good. Invariably, the pleasures of food are linked with taste.

Yet taste is the least understood of the human senses. Unlike vision, audition, or olfaction, taste is an area of neuroscience in which fundamental questions have not yet been fully answered because the biological system is not easy to study, according to Nirupa Chaudhari, an associate professor of physiology and biophysics at the University of Miami School of Medicine. Among such basic questions awaiting answers are how tastes are detected and how the brain knows what the mouth tastes.

ADDING TO THE complexity of this sense is the fact that taste cells are not static. "They are continually being born, turning over, and dying," explains Sue C. Kinnamon, an associate professor of anatomy and neurobiology at Colorado State University. "The nervous system is having to make new connections with the newly born cells and to lose connections with the cells that are dying. And somehow it has to maintain continuity so that sugar will always taste sweet. This constant turnover is fascinating, and we don't understand it yet."

Understanding how taste perception works has far-reaching implications. The food and beverage industries are seeking better artificial sweeteners. Pharmaceutical companies need compounds to neutralize the bitter taste of most drugs, whereas beer producers would welcome new bitter compounds. Salt enhancers would benefit people on low-salt diets. Even in agriculture, understanding how insects and other pests are able to taste could lead to management practices based on foul-tasting, rather than toxic, compounds.

Together with the sense of smell, taste also regulates a wide range of behaviors. The two senses "regulate caloric intake for proper diet and nutrition," says Barry J. Davis, director of the Taste & Smell Program at the National Institute on Deafness & Other Communication Disorders (NIDCD), which supports and conducts research on the chemical senses. "They play an important role in the avoidance of foul, odorous, or bitter substances, which can be life threatening."

In humans, taste sensation is launched from taste buds in the mouth. These are onionlike clusters of 30 to 100 taste cells embedded in peglike structures on the tongue, called papillae. At the tip of a taste bud is a pore, formed by the bundling of taste cells. Extending through this pore into the oral cavity are fingerlike protrusions. These are microvilli from individual taste cells, which bear the actual taste receptors.

Researchers generally agree that humans perceive five basic taste qualities: salty, sour, sweet, bitter, and umami. Compounds with sodium and hydrogen ions, respectively, are perceived as salty and sour. Carbohydrates are generally associated with sweetness, although other compounds may elicit the same sweet perception. The alkaloids caffeine and quinine are quintessential bitter compounds, but, likewise, many other compound types taste bitter. Umami is the savory taste frequently associated with protein-rich foods such as meat and cheese. It is exemplified by monosodium glutamate, a flavor enhancer commonly used in Asian cooking and a natural component of many foods humans eat and like.

Taste perception begins when a taste-eliciting molecule, or tastant, interacts with specialized receptors in the membrane of a taste cell. In general, salty and sour tastes are transduced by ion channels; bitter, sweet, and umami, by receptors coupled to internal signaling molecules called G proteins. This interaction triggers a signaling cascade that culminates with signals to the brain through a network of taste nerve fibers. If the brain recognizes the signal as pleasant, the mouth swallows; if unpleasant, the mouth spits.

The signaling cascades launched by sour and salty tastants are better understood than those for other tastants. But the picture still is far from complete. In humans, for example, only 20% of salt perception can be accounted for by the known sodium channel that mediates salty taste. This sodium channel is called amiloride-sensitive, because it can be blocked by amiloride, a diuretic. "The other 80% must be mediated by another channel that is not blockable by amiloride," says Bernd Lindemann, a professor of physiology at Saarland University, Homburg, Germany. "This channel remains to be discovered. It will be sensational if somebody finds it. The clinical importance is huge because of the impact of sodium metabolism on health." 

7937coverstory1
TASTE EXPLORERS Chaudhari (left), Lindemann, and Kinnamon.
PHOTO BY MAUREEN ROUHI
Companies are being born as the biology of taste is becoming understood.
FOR A LONG TIME, the receptors for bitter, sweet, and umami stimuli eluded researchers. But in recent years, researchers have made great strides, aided by two developments. First was the cloning of genes for a family of about 300 odorant receptors by Linda B. Buck, now a Howard Hughes Medical Institute investigator at Harvard University Medical School, and Richard Axel, a professor of biochemistry and molecular biophysics at Columbia University (C&EN, Dec. 23, 1996, page 18).

"Taste and olfaction are very related. They are both concerned with detecting chemicals in the environment," Lindemann explains. "So if in olfaction a big family of receptors is discovered, that is very inspiring for people working on taste."

The work of Buck and Axel also further bolstered what had long been accepted about the chemoreceptive senses--that the receptors are seven-helix membrane-spanning proteins coupled to G proteins. Researchers base this conclusion on analysis of events occurring after responses to sweet or bitter tastants, Lindemann points out. The biochemical products formed are typical of responses mediated by G-protein-coupled receptors (GPCRs).

Sequencing of genomes has also provided a major impetus to the search for taste receptors. By scanning mouse and human genomic databases in regions associated with taste discrimination, researchers quickly found several genes encoding potential taste receptors. This year, for example, a gene that codes for a putative sweet receptor was found by this approach. Last year, a family of genes encoding putative bitter taste receptors was discovered similarly.

The successes come from several groups. Those of Buck at Harvard and of Charles S. Zuker, a Howard Hughes Medical Institute investigator and professor of biology and neurosciences at the University of California, San Diego, in collaboration with Nicholas J. P. Ryba and others at the National Institute of Dental & Craniofacial Research, reported the bitter receptors last year. And this year, the gene for a sweet receptor was discovered independently by Buck's group; a second group led by Robert F. Margolskee, an associate professor of physiology, biophysics, and pharmacology and a Howard Hughes Medical Institute associate investigator at Mount Sinai School of Medicine, New York City; a third group led by Susan L. Sullivan, a research fellow at NIDCD; and a fourth group led by Alexander A. Bachmanov, an assistant member at the Monell Chemical Senses Center, Philadelphia.

"THERE'S A COMPOUND called PROP [6-n-propyl-2-thiouracil] that is tasteless to some people but is so bitter to others that they just want to vomit," Kinnamon tells C&EN. "The difference between tasters and nontasters had been mapped to a particular locus of a human chromosome. When the human genome was sequenced, researchers went to this area of the genome and pulled out some receptors that had the right structural characteristics." Similarly, researchers found a sweet receptor by focusing on an area of the human genome that is similar to the section of the mouse genome that controls sensitivity to sweet tastants such as sucrose and saccharin.

"The field is indeed moving fast now," Lindemann says. "Three months after the candidate sweet receptor appeared in print, one group proved its definitive function as a sweet receptor. These results are marvelous," he adds. The study, from the group led by Zuker and Ryba, has just been published [Cell, 106, 381 (2001)].

A better understanding of bitter sensitivity will help treat dysgeusia, a condition characterized by the persistent perception of a bitter aftertaste. It will also help in development of bitter blockers to make medication more palatable, especially for children and the elderly. Similarly, the discovery of a gene for sweet sensitivity, Davis says, has "obvious implications for intervention and treatment" for individuals coping with diabetes or obesity.

"Sequencing of the human genome provided the big opportunity to obtain the receptors," Kinnamon says. "Cloned receptors now provide us the starting point for all the steps that lead to activation of that cell."

Before genome sequences were available, researchers relied heavily on educated guesses in searching for potential taste receptors. "If you're interested in understanding how sour taste works, you might ask, What are the receptors that are known so far that sense protons in some fashion?" Chaudhari explains. "So you look for ion channels that are known to be modulated by protons, or sense protons, or are gated by protons."

IN 1998, CHAUDHARI and Stephen D. Roper, a professor of physiology and biophysics at the University of Miami School of Medicine, began building the case for an umami receptor by using such an approach. "We looked for receptors that were known to be activated by glutamate," Chaudhari says. Then they looked for molecules that resembled these receptors in taste cells. Last year, they cloned a GPCR from rat taste buds and demonstrated that it is likely an umami receptor.

Understanding of taste perception rests heavily on identifying receptors, says Lindemann. "When the receptors are known, these can be generated in bacteria or yeast. The protein perhaps can be crystallized and the binding site imaged. This binding site, once you know it, will allow design of inhibitors and enhancers."

Tastant binding to a receptor is just the beginning of taste perception. The subsequent events within the taste cell are also still being elucidated. An earlier important advance was identification and cloning of gustducin, a taste-specific G protein, from taste tissue in 1992 by researchers led by Margolskee, who at the time was affiliated with the Roche Research Center, Nutley, N.J.

Gustducin appears to be crucial to sweet and bitter perception. Margolskee and coworkers have shown that mice that have been genetically altered so that they do not produce gustducin show a greatly reduced sensitivity for both sweet and bitter tastants. And last year, a team of scientists led by Zuker and Ryba demonstrated that members of a family of bitter taste receptors they identified, called T2R, are coexpressed with gustducin. They further showed that binding of a bitter tastant to the receptor activates gustducin. Elucidating the precise role of gustducin in bitter and sweet transduction is an important goal of research, Kinnamon says.

COMPANIES are being born as the biology of taste is becoming understood. For example, in 1995, Margolskee--with D. Scott Linthicum, formerly a professor of biochemistry, biophysics, veterinary pathobiology, and medical physiology at Texas A&M University, and Richard C. Lufkin, an entrepreneur--founded Linguagen Corp., based in Paramus, N.J. The company seeks to develop novel taste modifiers, like bitter blockers and sweet enhancers, based on the chemistry of gustducin.

A bigger company, Senomyx, based in La Jolla, Calif., has broader goals. The company uses identified taste, as well as odorant, receptors in a receptor-based approach to discovery of novel odor and taste compounds. "The approach is very common in pharmaceutical research but is only now being applied to the food, flavor, and fragrance industries," says Klaus M. Gubernator, vice president for chemistry at Senomyx.

Senomyx was established in 1999 by Zuker; Lubert Stryer and Denis A. Baylor, professors of neurobiology at Stanford University; Roger Y. Tsien, professor of pharmacology, chemistry, and biochemistry at UCSD; Harold McGee, an author and food chemistry expert; and Paul Grayson, a business development expert and the company's chief executive officer and chairman.

Senomyx' process of discovery is based on taste and odorant receptors and the molecules that bind to them. The company has invested heavily in "the art of developing assays for these receptors that allow Senomyx to screen rapidly hundreds of thousands of molecules," Gubernator says.

"If you have a receptor and a meaningful readout system, you can go fishing in the universe of organic chemistry and find active molecules everywhere," Gubernator tells C&EN. "We choose compound types that are readily accessible by synthesis and generate, through combinatorial methods, potential ligands and test them against the receptors. At some point, we will probably optimize molecules by synthesizing analogs in a traditional synthetic chemistry fashion."

There's much work ahead for researchers such as Chaudhari, Kinnamon, and Lindemann to understand how receptors bind tastants. Complementing their efforts are those of researchers who are trying to understand the neural circuits that process information about taste stimuli, such as David V. Smith, a professor of anatomy and neurobiology at the University of Maryland School of Medicine, in Baltimore.

"Between the receptor and the perception is a whole central nervous system," Smith tells C&EN. "I'm interested in how information about taste is represented."

Smith and his colleagues record the electrophysiological activity of taste cells and neurons of the taste system to determine their responsiveness to taste stimuli. They have confirmed that taste cells are broadly tuned. That is, individual cells respond to more than one of the taste qualities. Such multiple sensitivity is well known for neurons of the taste system and had been suggested previously for taste cells as well. However, earlier experiments with taste cells had not given definitive results.

Smith and his colleagues use whole-cell recordings of 120 receptor cells maintained in an intact epithelium from a portion of the tongue or the palate of a rat. By applying up to six taste stimuli to only the apical membrane of each cell, they have shown that individual taste receptor cells exhibit a range of chemical sensitivities. Almost 75% of the cells respond to more than one stimulus.

The breadth of tuning can be quantified through a mathematical equation based on entropy, Smith tells C&EN. A cell responding to all taste qualities has an entropy measure of one, and a cell responding to only one has an entropy measure of zero.

APPLYING THIS FORMULA to taste cells and neurons of the taste system, Smith and coworkers have calculated entropy measures of 0.46 for rat taste receptor cells; 0.56 for the chorda tympani, the cranial nerve branch that connects the anterior tongue to the brain; and 0.79 for the nucleus of the solitary tract, a line of cells in the medulla where the primary taste nerve fibers converge. "There's a huge convergence in the brain," Smith says. "A cell in the brain may get input from fibers that are more sensitive to salt and others that are more sensitive to sucrose. These things get more and more broadly tuned as you go into the brain."

How, then, does the brain recognize a taste quality?

"My point of view is that quality is represented in the nervous system by population responses," Smith says. What seems to be happening is that when a population of taste cells and neurons is presented with a stimulus, only a subset is activated, generating a pattern of activity in the brain. "If you stimulate the same population with a different chemical, a different ensemble of cells will be activated and will give a completely different pattern. You can think of the pattern as being the neural information. Your brain does all kinds of things all the time with ensembles of neurons, not one neuron at a time. The patterns of activity that finally emerge from the central nervous system represent the perception."

The broad tuning notwithstanding, some taste cells and taste neurons are more sensitive to certain tastants than others. Among those activated by salty stimuli, for example, some would be much more responsive than the rest. Some researchers consider these highly responsive cells the "label line" for salty taste. According to this school of thought, certain taste cells and taste neurons are dedicated to signaling specific taste qualities.

SMITH DOESN'T FIND this label-line approach convincing. "The problem with the label-line hypothesis for me is that those same cells respond to sucrose and acid," he says. "It could well be that label lines exist. If so, the signal-to-noise ratio of these label lines is very poor. One of the general principles in neural coding is that if you have alternative codes, the one that gives the best signal-to-noise ratio is probably the correct one."

If all the questions were answered, "I'd have to find another job," Smith jokingly tells C&EN, before continuing: "Sometimes it's hard to know what the impact of basic science is. But if you think about the development of medical science, people are now finding cures for diseases because others have been simply trying to understand cellular mechanisms."

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CHANNELING
Taste Stimulation Is Easy To Do, But Hard To Follow

Individual taste cells can respond to more than one type of taste stimulation. Some of the cells' response pathways are shown schematically here. Salty- and sour-tasting stimuli are transduced through ion channels. Sodium cations, and probably protons and potassium cations, can pass through the tight junctions between cells in the taste bud (far left) to enter ion channels in the basolateral (bottom) membrane. Another route for sodium cations and protons is amiloride-blockable ion channels on the apical (top) membrane. One such channel is shown at the upper left. Both routes lead directly to cell depolarization. In the absence of stimuli, taste cells have an excess of negative charge and are said to be polarized. An increase in positive charge, for example, through the entry of cations, depolarizes the cell. In mammals, protons gate--or open--some apical ion channels, allowing entry of cations.

Sweet-tasting stimuli (S1) are transduced via receptors coupled to G proteins (G). Transduction of sugars involves activation of adenylate cyclase (AC). Formation of cyclic adenosine monophosphate (cAMP) from adenosine triphosphate (ATP) leads to the closing of K+ channels on the basolateral membrane. The cell is depolarized as potassium ions accumulate. Artificial sweeteners (S2), however, appear to trigger a different transduction pathway, one that involves three membrane-bound molecules--phospholipase C (PLC), diacylglycerol (DAG), and protein kinase C (PKC)--and culminates in the release of inositol 1,4,5-triphosphate (IP3). Binding of IP3 to its receptor (IP3R) releases calcium ions stored in organelles within the cell. It's not yet absolutely clear whether stimuli that engage the IP3 pathway also cause cell depolarization.

Some bitter stimuli (B1) bind to receptors that are coupled to the G protein gustducin. The -subunit of gustducin engages the IP3 pathway, whereas the -subunit activates taste-specific phosphodiesterase (PDE). This converts to 5'-AMP a cAMP blocking a calcium ion channel. The transformation of cAMP opens the channel. Many bitter substances (B2), such as quinine, block potassium ion channels, directly depolarizing the cells. Lipophilic bitter substances (B3) pass directly through the membrane. Once inside, they can engage any of the pathways to stimulate the cell.

Amino acids (A), such as glutamate, also stimulate taste cells through G-protein-coupled receptors, although the secondary messenger molecules involved in this pathway are not known.

All of these mechanisms increase the levels of calcium ion inside the cell, which leads to release of neurotransmitters (NT) onto terminals of the taste nerve.

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ADAPTED FROM DAVID V. SMITH, STEVEN J. ST. JOHN, AND JOHN D. BOUGHTER JR., THE GUSTATORY SYSTEM, IN "NEUROSCIENCE IN MEDICINE," 2ND EDITION, EDITED BY P. M. CONN, HUMANA PRESS, TOWATA, N.J., IN PRESS

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