Liquan Huang - US grants

sensory receptors; taste; receptor expression; biological signal transduction; complementary DNA; cell type; RNase protection assay; laboratory mouse; genetically modified animals; in situ hybridization; immunocytochemistry; molecular cloning; electrophysiology; polymerase chain reaction;

DESCRIPTION (provided by applicant): The long-term objective is to understand the molecular mechanisms that different types of morphologically and physiologically heterogeneous taste cells utilize in receiving, processing and transmitting gustatory signals. Using a set of known genes we have successfully applied single cell RT-PCR amplification and filter hybridization methods to determine limited gene expression patterns for some taste cells, and to identify several taste cell type selective signaling elements thought to play a critical role in bitter sensation. In this application, we will use single cell RT-PCR products to probe DNA arrays to establish global gene expression profiles for many taste cells, to determine and predict taste cell types/subtypes based on their expression profiles, to identify additional taste type or subtype- selective genes or clusters of genes that define this type or subtype's physiological functions in taste perception. The specific goals of this application are: 1. To generate DNA probes from taste buds, filiform papillae and 100 individual taste cells using both an aRNA amplification method and a modified single cell RT-PCR procedure; 2. To screen genome-wide DNA arrays with both taste bud and filiform probes, to identify and re-array genes that are enriched in taste buds over filiform papillae to prepare taste DNA arrays (taste chips); 3. To screen the taste chips in pair wise fashion with a taste bud probe paired with one of the 100 single taste cell probes to be generated, and to establish the gene expression profiles of the individual taste cells by identifying the expressed genes on the taste chips in each single cell probe, to determine and discover taste cell types or subtypes using unsupervised partitional clustering method, and to identify additional gene classifiers with the neighborhood analysis method. The results of these studies will contribute to the development of new technologies in pursuing post-genomic biomedical research, provide genetic bases for taste cell classification and yield significant novel insights into the molecular mechanisms underlying taste transduction. The knowledge gained from this application should further our understanding of genetic differences in taste sensitivity, and the gustatory and metabolic disorders such as malgeusia, dysgeusia and cachexia.

[unreadable] DESCRIPTION (provided by applicant): Taste perception is initiated by the interaction of sapid molecules with proteins on the surface of taste bud cells. These gustatory signals are subsequently transduced, processed in taste buds, coded and transmitted by cranial nerves to the brain. The long-term objective of our research program is to molecularly identify and functionally characterize key proteins that are involved in taste signal transduction and coding in taste buds, and decoding in the central nervous system, and eventually to reconstruct the molecular events of taste sensation and perception. We have used molecular cloning and functional analyses to identify and characterize a number of taste transduction components, including a G-protein coupled taste receptor T1R3, G protein subunits and a transient receptor potential ion channel TRPM5. In order to understand the molecular events downstream of TRPM5 and to reveal ion channels that are responsible for sour and salty tastes, we have determined the expression of six voltage-dependent calcium channels in taste bud cells. The specific aims of this proposal are: 1) to identify and colocalize voltage-dependent calcium channel subunits with known taste signaling molecules in taste bud cells; 2) to heterologously express and functionally characterize novel isoforms of these voltage-dependent calcium channels isolated from taste buds; 3) to determine possible roles of these voltage-dependent calcium channels in taste bud synaptic transmission by monitoring the effect of pharmacological and genetic perturbation of these channels on the elevation of intracellular calcium concentrations and synaptic activity in taste bud cells in response to taste stimuli; 4) finally, to determine possible roles of these voltage-dependent calcium channels in taste sensation by gustatory nerve recording and animal behavioral tests with mutant animals. The results of these studies will yield new insights into the molecular mechanisms underling sour, salt, bitter, sweet and umami taste transduction, synaptic transmission and peripheral coding in the end organs of taste. The knowledge gained from this endeavor will further our understanding of the molecular bases of taste disorders such as malgeusia, dysgeusia, hypogeusia and ageusia and may lead to effective treatment. [unreadable] [unreadable] [unreadable]

One of the unique characteristics of the Monell Center is that researchers use multidisciplinary approaches to address scientific questions regarding taste, smell, nutrition, and their relationship to human health and well-being. The primary expertise of Monell investigators ranges from analytical chemistry and chemical ecology (Drs. Preti and Kimball) to biochemistry and biophysics (Drs. Brand and Margolskee), molecular and cellular biology (Drs. Margolskee, Huang, and Wang), genetics and neuroscience (Drs. Tordoff, Bachmanov, Beauchamp, Reed. Yamazki, Reisert, Gelperin, and Lowe), endocrinology and physiology (Drs. Margolskee, Teff, Friedman, Wysocki, and Zhao), psychophysics and behavioral science (Drs. Mennella, Dalton, Breslin, Lundstrom, Pelchat, and Wise), and clinical research (Drs. Cowart and Teff). Regardless of their primary technical expertise, a number of these laboratories have found it very useful to incorporate histology and cellular localization methods into their research programs (e.g.. Fig. 1)[1]. However, other Center laboratories are less familiar with the techniques and/or equipment involved in anatomical/histological studies. For instance, geneticists such as Drs. Reed and Bachmanov had identified candidate genes for taste receptors that detect the bitter-tasting compound phenylthiocarbamide and sweet-tasting compounds, respectively[2,3]. They needed to determine, first, whether these genes were expressed in the peripheral taste organs, and then, in which types of taste bud cells. These researchers could use reverse-transcripfion PCR with RNA extracted from taste tissue to determine if these candidate gene products are preferentially expressed in taste buds. However, to determine which type of taste bud cells express these genes, in situ hybridization and immunohistochemistry are mostly commonly used on taste fissures. This Core will provide a common resource and reservoir of experience in chemosensory anatomical analysis and state-of-the-art visualization techniques that would be inefficient to replicate in each individual laboratory. Thus, the availability of Core services would extend the capabilities of the participating laboratories beyond each laboratory's capabilities.

Project Summary Salt/water balance is crucial to mammalian physiology. Overconsumption of salt has been linked with health problems, particularly hypertension. Development of salt taste substitutes or enhancers has been ineffective partly due to the incomplete knowledge of the receptors and other signal transduction components responsible for salt taste. While an epithelial sodium channel has been shown to be involved in the amiloride-sensitive salt taste transduction, the amiloride-insensitive (AI) salt taste transduction mechanism remains to be elucidated. The goal of this proposed research is to advance our understanding of salt taste by identifying the AI salt transduction components. We will functionally characterize taste bud cells and identify and individually collect AI salt-responsive type II bitter receptor cells and AI salt type III taste bud cells as well as control cells salt-unresponsive type II and III cells, respectively (Aim 1). We will amplify and interrogate these single cells' transcriptomes using deep sequencing, and identify genes that are preferentially expressed in the salt-responsive AI type II and III cells. We will perform in-depth bioinformatic analysis on these differentially expressed genes to identify candidate AI salt receptor genes (Aim 2). The role of these candidate genes in the AI salt taste will be further investigated in our follow-up studies. Results from these studies can fill an important gap in understanding salt taste sensation and perception, thus leading to effective means to reduce salt consumption.