Hyperpolarization-activated channels HCN1 and HCN4 mediate responses to sour stimuli.

Sour taste is initiated by protons acting at receptor proteins or channels. In vertebrates, transduction of this taste quality involves several parallel pathways. Here we examine the effects of sour stimuli on taste cells in slices of vallate papilla from rat. From a subset of cells, we identified a hyperpolarization-activated current that was enhanced by sour stimulation at the taste pore. This current resembled Ih found in neurons and cardio-myocytes, a current carried by members of the family of hyperpolarization-activated and cyclic-nucleotide-gated (HCN) channels. We show by in situ hybridization and immunohistochemistry that HCN1 and HCN4 are expressed in a subset of taste cells. By contrast, gustducin, the G-protein involved in bitter and sweet taste, is not expressed in these cells. Lowering extracellular pH causes a dose-dependent flattening of the activation curve of HCN channels and a shift in the voltage of half-maximal activation to more positive voltages. Our results indicate that HCN channels are gated by extracellular protons and may act as receptors for sour taste.     

Cyclic nucleotides may mediate taste transduction

Taste stimulus adsorption is believed to occur at the taste cell microvillous membrane. But due to technical difficulties of inserting glass electrodes into the mammalian taste cell, little is known about the mechanisms of taste transduction. Reliable intracellular recordings are necessary to determine the characteristics of taste cells. This has been accomplished previously in the mouse and is reported here. Recent experiments indicated that cyclic nucleotides can act on the inner surface of the membranes of a variety of cells to alter their ion-channel activity, and these substances might act as intracellular transmitters in taste cells. But tight junctions found at the apical membrane of mammalian taste cells do not allow stimuli to enter the taste bud, making it difficult to alter the environment of the taste cell by perfusing with chemical solutions. Here we report that cyclic AMP, cyclic GMP, EGTA or tetraethyl-ammonium electrophoretically injected into the mouse taste cell induce membrane depolarization and increased membrane resistance. These results suggest that a cyclic nucleotide enzymatic cascade, modulated by calcium ions, may mediate the potassium permeability that controls taste, in a way analogous to visual and olfactory transduction.     

The development of taste transduction and taste chip technology

Taste is one of important sensations. The primary taste sensations commonly are categorized as sweet, acid, bitter, salty and umami, of which various tastes are composed. The sensation of taste is initiated by the interaction of tas- tants with receptors and ion channels in the apical micro- villi of taste receptor cells (TRCs) when some sapid molecules (tastants) dissolve in saliva. Subsequently, through a cellular signaling pathway (TRC depolarization and Ca2+ release) gustatory signals a…     

Possible role for salivary gland protein in taste reception indicated by homology to lipophilic-ligand carrier proteins.

Sensory transduction in taste and olfaction, the principal chemical senses, seems to be mediated by membrane-associated proteins on the apical surfaces of the respective receptor cells. The recent isolation and characterization of soluble 'odorant-binding proteins' secreted from the nasal glands of rat, cow and frog, led to the hypothesis that these proteins function as necessary cofactors in olfactory transduction by concentrating and delivering odorants to the receptors. The primary reception of taste stimuli occurs in specialized neuroepithelial receptor cells bundled in taste buds that are clustered in various types of papillae in the lingual epithelium of the tongue. Small tubulo-alveolar salivary glands, the von Ebner's glands, are located beneath the circumvallate and the foliate papillae. Their ducts open exclusively into the trough at the base of the papillae. Taste buds located in the medial and lateral walls of the papillae open with their taste pores into the trough and consequently are in direct contact with the secretions of von Ebner's glands. Here we report the molecular cloning and characterization of a protein of relative molecular mass 18,000 that is highly expressed in von Ebner's glands. Like the odorant-binding proteins, this protein shows similarity to members of a protein superfamily of hydrophobic molecule transporters, indicating that pre-receptor events could also be necessary for the concentration and delivery of sapid molecules in the gustatory system, and emphasizing the close relationship of taste and olfaction.     

Stimulation of specific GTP binding and hydrolysis activities in lymphocyte membrane by interleukin-2

Interleukin-2 (IL-2) is a polypeptide growth factor which stimulates the proliferation and differentiation of T lymphocytes. The receptor for IL-2 is expressed on activated T lymphocytes, cloned IL-2 dependent cells and several other cell types. Analysis of the primary structure and of immune-precipitated receptor suggests that this molecule has no intrinsic signal transduction function, unlike other growth factors. IL-2 interaction with a high affinity receptor has been shown, however, to activate the calcium/phospholipid-dependent protein kinase C (PK-C) presumably via phosphoinositide hydrolysis. Members of a family of closely related guanine nucleotide binding proteins (G proteins) regulate a diverse group of metabolic events. Two of them, Gs and Gi, stimulate and inhibit adenylate cyclase activity respectively, and other G proteins are involved in diverse signal transduction system. Another member, Go, has no known function and activation of phospholipase C has been attributed to the action of an unidentified G protein, Gp. Since it has been observed that IL-2 inhibits the catalytic activity of adenylate cyclase and that agents such as PGE2 which stimulate adenylate cyclase activity inhibit the lymphoproliferative response to IL-2, association of GTP binding proteins with IL-2 signal transduction was investigated. In this report we describe for the first time the participation of a GTP binding protein in the action of a polypeptide growth factor, interleukin-2.     

Primary structure and functional expression of a cyclic nucleotide-activated channel from olfactory neurons.

Odorant signal transduction occurs in the specialized cilia of the olfactory sensory neurons. Considerable biochemical evidence now indicates that this process could be mediated by a G protein-coupled cascade using cyclic AMP as an intracellular second messenger. A stimulatory G protein alpha subunit is expressed at high levels in olfactory neurons and is specifically enriched in the cilia, as is a novel form of adenylyl cyclase. This implies that the olfactory transduction cascade might involve unique molecular components. Electrophysiological studies have identified a cyclic nucleotide-activated ion channel in olfactory cilia. These observations provide evidence for a model in which odorants increase intracellular cAMP concentration, which in turn activates this channel and depolarizes the sensory neuron. An analogous cascade regulating a cGMP-gated channel mediates visual transduction in photoreceptor cells. The formal similarities between olfactory and visual transduction suggest that the two systems might use homologous channels. Here we report the molecular cloning, functional expression and characterization of a channel that is likely to mediate olfactory transduction.     

The cells and logic for mammalian sour taste detection

Mammals taste many compounds yet use a sensory palette consisting of only five basic taste modalities: sweet, bitter, sour, salty and umami (the taste of monosodium glutamate). Although this repertoire may seem modest, it provides animals with critical information about the nature and quality of food. Sour taste detection functions as an important sensory input to warn against the ingestion of acidic (for example, spoiled or unripe) food sources. We have used a combination of bioinformatics, genetic and functional studies to identify PKD2L1, a polycystic-kidney-disease-like ion channel, as a candidate mammalian sour taste sensor. In the tongue, PKD2L1 is expressed in a subset of taste receptor cells distinct from those responsible for sweet, bitter and umami taste. To examine the role of PKD2L1-expressing taste cells in vivo, we engineered mice with targeted genetic ablations of selected populations of taste receptor cells. Animals lacking PKD2L1-expressing cells are completely devoid of taste responses to sour stimuli. Notably, responses to all other tastants remained unaffected, proving that the segregation of taste qualities even extends to ionic stimuli. Our results now establish independent cellular substrates for four of the five basic taste modalities, and support a comprehensive labelled-line mode of taste coding at the periphery. Notably, PKD2L1 is also expressed in specific neurons surrounding the central canal of the spinal cord. Here we demonstrate that these PKD2L1-expressing neurons send projections to the central canal, and selectively trigger action potentials in response to decreases in extracellular pH. We propose that these cells correspond to the long-sought components of the cerebrospinal fluid chemosensory system. Taken together, our results suggest a common basis for acid sensing in disparate physiological settings.     

Development of planar patch clamp technology and its application in the analysis of cellular electrophysiology

A patch clamp chip, as a novel cell-based chip for electrophysiological recordings, has many prominent advantages such as high resolution, accuracy, high throughput and automation. It can be used to perform multivariate and real-time measurements of cell networks in situ. Therefore, this technology will dramatically promote the research on ionic channels, neuronal networks and the application of this technology in drug screening. This paper reviews the development of planar patch clamp technology and its applications in detail. The latest progress in the research of taste cells electrophysiology and taste transduction is also presented. Finally, this paper analyzes the methodology of neural chips. Based on the current research of our laboratory, the prospective applications of a patch clamp chip in the research of taste sensation and transduction mechanisms at molecular and cellular levels are discussed.     

Receptors and transduction in taste

Taste is the sensory system devoted primarily to a quality check of food to be ingested. Although aided by smell and visual inspection, the final recognition and selection relies on chemoreceptive events in the mouth. Emotional states of acute pleasure or displeasure guide the selection and contribute much to our quality of life. Membrane proteins that serve as receptors for the transduction of taste have for a long time remained elusive. But screening the mass of genome sequence data that have recently become available has provided a new means to identify key receptors for bitter and sweet taste. Molecular biology has also identified receptors for salty, sour and umami taste.     

Heat activation of TRPM5 underlies thermal sensitivity of sweet taste

TRPM5, a cation channel of the TRP superfamily, is highly expressed in taste buds of the tongue, where it has a key role in the perception of sweet, umami and bitter tastes. Activation of TRPM5 occurs downstream of the activation of G-protein-coupled taste receptors and is proposed to generate a depolarizing potential in the taste receptor cells. Factors that modulate TRPM5 activity are therefore expected to influence taste. Here we show that TRPM5 is a highly temperature-sensitive, heat-activated channel: inward TRPM5 currents increase steeply at temperatures between 15 and 35 degrees C. TRPM4, a close homologue of TRPM5, shows similar temperature sensitivity. Heat activation is due to a temperature-dependent shift of the activation curve, in analogy to other thermosensitive TRP channels. Moreover, we show that increasing temperature between 15 and 35 degrees C markedly enhances the gustatory nerve response to sweet compounds in wild-type but not in Trpm5 knockout mice. The strong temperature sensitivity of TRPM5 may underlie known effects of temperature on perceived taste in humans, including enhanced sweetness perception at high temperatures and 'thermal taste', the phenomenon whereby heating or cooling of the tongue evoke sensations of taste in the absence of tastants.     

A family of candidate taste receptors in human and mouse

The gustatory system of mammals can sense four basic taste qualities, bitter, sweet, salty and sour, as well as umami, the taste of glutamate. Previous studies suggested that the detection of bitter and sweet tastants by taste receptor cells in the mouth is likely to involve G-protein-coupled receptors. Although two putative G-protein-coupled bitter/sweet taste receptors have been identified, the chemical diversity of bitter and sweet compounds leads one to expect that there is a larger number of different receptors. Here we report the identification of a family of candidate taste receptors (the TRBs) that are members of the G-protein-coupled receptor superfamily and that are specifically expressed by taste receptor cells. A cluster of genes encoding human TRBs is located adjacent to a Prp gene locus, which in mouse is tightly linked to the SOA genetic locus that is involved in detecting the bitter compound sucrose octaacetate. Another TRB gene is found on a human contig assigned to chromosome 5p15, the location of a genetic locus (PROP) that controls the detection of the bitter compound 6-n-propyl-2-thiouracil in humans.     

动物感光器中的G蛋白及其偶联的光信号转导

G蛋白偶联的信号转导系统是细胞跨膜信号转导的重要途径，在动物的光感觉生理中，G蛋白在信号的转导和放大过程中起了重要的作用，有关这方面的研究已是动物光感觉研究中的一个热点，作者介绍了国内外这方面的研究进展，以及研究的方法，并对动物感光器中G蛋白的类型、性质和功能作了简要的概括。     

Stimulation of the phosphatidylinositol pathway can induce T-cell activation

The T-cell antigen receptor (TCR) regulates two signal transduction pathways: the phosphatidylinositol (PtdIns) and tyrosine kinase pathways. Stimulation of T cells with antigen or anti-TCR monoclonal antibodies induces an increase in inositol phosphates and diacylglycerol, the second messengers responsible for the mobilization of cytoplasmic free calcium and activation of protein kinase C-4. The TCR also activates a tyrosine kinase that is not intrinsic to the TCR. The relationship between these two signal transduction pathways and their contribution to later T-cell responses is unclear. Studies using variants of a murine hybridoma suggested that the PtdIns pathway might not be necessary for or be involved in regulating interleukin-2 (IL-2) production. To address the relationship between later T-cell responses and the early biochemical signals, we investigated the ability of a heterologous receptor with defined signal transduction function to induce T-cell activation. The human muscarinic subtype-1 receptor (HM1), which elicits PtdIns metabolism in neuronal cells through a G protein-coupled mechanism, also functionally activates this pathway when expressed in the T-cell line Jurkat-derived host, J-HM1-2.2 (ref.8). We show here that stimulation of HM1 alone induced IL-2 production and IL-2 receptor alpha chain expression. HM1 does not induce the tyrosine kinase pathway, suggesting that this pathway does not directly influence later T cell-activation responses. Instead, our studies indicate that activation of the PtdIns pathway is probably sufficient to induce later T-cell responses.     

The receptors and coding logic for bitter taste

The sense of taste provides animals with valuable information about the nature and quality of food. Bitter taste detection functions as an important sensory input to warn against the ingestion of toxic and noxious substances. T2Rs are a family of approximately 30 highly divergent G-protein-coupled receptors (GPCRs) that are selectively expressed in the tongue and palate epithelium and are implicated in bitter taste sensing. Here we demonstrate, using a combination of genetic, behavioural and physiological studies, that T2R receptors are necessary and sufficient for the detection and perception of bitter compounds, and show that differences in T2Rs between species (human and mouse) can determine the selectivity of bitter taste responses. In addition, we show that mice engineered to express a bitter taste receptor in 'sweet cells' become strongly attracted to its cognate bitter tastants, whereas expression of the same receptor (or even a novel GPCR) in T2R-expressing cells resulted in mice that are averse to the respective compounds. Together these results illustrate the fundamental principle of bitter taste coding at the periphery: dedicated cells act as broadly tuned bitter sensors that are wired to mediate behavioural aversion.     

Drosophila odorant receptors are both ligand-gated and cyclic-nucleotide-activated cation channels

From worm to man, many odorant signals are perceived by the binding of volatile ligands to odorant receptors that belong to the G-protein-coupled receptor (GPCR) family. They couple to heterotrimeric G-proteins, most of which induce cAMP production. This second messenger then activates cyclic-nucleotide-gated ion channels to depolarize the olfactory receptor neuron, thus providing a signal for further neuronal processing. Recent findings, however, have challenged this concept of odorant signal transduction in insects, because their odorant receptors, which lack any sequence similarity to other GPCRs, are composed of conventional odorant receptors (for example, Or22a), dimerized with a ubiquitously expressed chaperone protein, such as Or83b in Drosophila. Or83b has a structure akin to GPCRs, but has an inverted orientation in the plasma membrane. However, G proteins are expressed in insect olfactory receptor neurons, and olfactory perception is modified by mutations affecting the cAMP transduction pathway. Here we show that application of odorants to mammalian cells co-expressing Or22a and Or83b results in non-selective cation currents activated by means of an ionotropic and a metabotropic pathway, and a subsequent increase in the intracellular Ca(2+) concentration. Expression of Or83b alone leads to functional ion channels not directly responding to odorants, but being directly activated by intracellular cAMP or cGMP. Insect odorant receptors thus form ligand-gated channels as well as complexes of odorant-sensing units and cyclic-nucleotide-activated non-selective cation channels. Thereby, they provide rapid and transient as well as sensitive and prolonged odorant signalling.     

Thermal stimulation of taste

The first electrophysiological recordings from animal and human taste nerves gave clear evidence of thermal sensitivity, and studies have shown that as many as half of the neurons in mammalian taste pathways respond to temperature. Because temperature has never been shown to induce sensations of taste, it has been assumed that thermal stimulation in the gustatory system is somehow nulled. Here we show that heating or cooling small areas of the tongue can in fact cause sensations of taste: warming the anterior edge of the tongue (chorda tympani nerve) from a cold temperature can evoke sweetness, whereas cooling can evoke sourness and/or saltiness. Thermal taste also occurs on the rear of the tongue (glossopharyngeal nerve), but the relationship between temperature and taste is different there than on the front of the tongue. These observations indicate the human gustatory system contains several different types of thermally sensitive neurons that normally contribute to the sensory code for taste.     

TRP channels as cellular sensors

TRP channels are the vanguard of our sensory systems, responding to temperature, touch, pain, osmolarity, pheromones, taste and other stimuli. But their role is much broader than classical sensory transduction. They are an ancient sensory apparatus for the cell, not just the multicellular organism, and they have been adapted to respond to all manner of stimuli, from both within and outside the cell.     

Multiple D2 dopamine receptors produced by alternative RNA splicing

Dopamine receptor belong to a large class of neurotransmitter and hormone receptors that are linked to their signal transduction pathways through guanine nucleotide binding regulatory proteins (G proteins). Pharmacological, biochemical and physiological criteria have been used to define two subcategories of dopamine receptors referred to as D1 and D2. D1 receptors activate adenylyl cyclase and are coupled with the Gs regulatory protein. By contrast, activation of D2 receptors results in various responses including inhibition of adenylyl cyclase, inhibition of phosphatidylinositol turnover, increase in K+ channel activity and inhibition of Ca2+ mobilization. The G protein(s) linking the D2 receptors to these responses have not been identified, although D2 receptors have been shown to both copurify and functionally reconstitute with both Gi and Go related proteins. The diversity of responses elicited by D2-receptor activation could reflect the existence of multiple D2 receptor subtypes, the identification of which is facilitated by the recent cloning of a complementary DNA encoding a rat D2 receptor. This receptor exhibits considerable amino-acid homology with other members of the G protein-coupled receptor superfamily. Here we report the identification and cloning of a cDNA encoding an RNA splice variant of the rat D2 receptor cDNA. This cDNA codes for a receptor isoform which is predominantly expressed in the brain and contains an additional 29 amino acids in the third cytoplasmic loop, a region believed to be involved in G protein coupling.     

人神经节苷脂诱导分化相关蛋白cDNA的克隆、染色体定位和初步功能研究

通过构建,筛选人18周胎脑cDNA文库,克隆到一条与神经节苷脂诱导分化相关蛋白高度同源的新基因,经HUGO／GDB人类基因命名委员会的同意命名为GDAP1L1,进行新基因的全序列测定,RH定位分析,Blast分析及生物学信息分析,Northern杂交提示GDAP1L1基因在胎脑中高度表达,但在成人脑组织中低表达,新的神经节甙脂诱导分化相关蛋白的表达和功能研究初步提示：全长新神经节苷脂诱导分化相关基因核苷酸序列长1163bp,RH定位分析新基因定位在染色体20q12区,BLASTN,BLASTP,TBLASTN分析新基因的蛋白质序列与人和鼠“神经节甘脂诱导分化相关蛋白1”有58％的同源性,而与人的另一条“类似神经节苷脂诱导分化相关蛋白1”的部分蛋白序列（47－253aa)的同源性达100％,新基因蛋白在3,5端分别多出46aa和114aa的长度,生物学住处分析证实神经节苷脂诱导分化相关基因与神经营养与细胞凋亡有密切关系,全长新神经节苷脂诱导分化相关基因是一个神经营养与发育及细胞周期调控,信号传导有关的基因,可能在肿瘤的发生中具有重要作用,其功能的进一步研究将为肿瘤机理的阐明提供思路。