Biochemistry of an Olfactory Purinergic System: Dephosphorylation of Excitatory Nucleotides and Uptake of Adenosine

Abstract: The olfactory organ of the spiny lobster, Panu-lirus argus , is composed of chemosensory sensilla containing the dendrites of primary chemosensory neurons. Receptors on these dendrites are activated by the nucleotides AMP, ADP, and ATP but not by the nucleoside adenosine. It is shown here that the lobster chemosensory sensilla contain enzymes that dephosphorylate excitatory nucleotides and an uptake system that internalizes the nonexcitatory dephosphorylated product adenosine. The uptake of [³H]-adenosine is saturable with increasing concentration, linear with time for up to 3h, sodium dependent, insensitive to moderate pH changes and has a K _m of 7.1 μ M and a V_max of 5.2 fmol/sensillum/min (573 fmol/μg of protein/min). Double-label experiments show that sensilla dephosphorylate nucleotides extracellularly; ³H from adenine-labeled AMP or ATP is internalized, whereas ³²P from phosphate-labeled nucleotides is not. The dephosphorylation of AMP is very rapid; ³H from AMP is internalized at the same rate as ³H from adenosine. Sensillar 5'-ectonucleotidase activity is inhibited by ADP and the ADP analog α,β-methylene ADP. Collectively, these results indicate that the enizymes and the uptake system whereby chemosensory sensilla of the lobster inactivate excitatory nucleotides and clear adenosine from extracellular spaces are very similar to those present in the internal tissues of vertebrates, where nucleotides have many neuroactive effects.     

Bovine olfactory cilia preparation: thiol-modulated odorant-sensitive adenylyl cyclase

We have characterized the adenylyl cyclase activity in a newly developed preparation of isolated olfactory cilia from the bovine chemosensory neuroepithelium. Like its counterparts from frog and rat, the ciliary enzyme was stimulated by guanine nucleotides, by forskolin, and by a variety of odorants in the presence of GTP. The main difference between the bovine olfactory cilia preparation and the frog and rat olfactory cilia preparation is that odorant stimulation of the bovine olfactory adenylyl cyclase is strongly inhibited by submillimolar concentrations of dithiothreitol. This inhibition is a consequence of a concomitant increase in the GTP-stimulated level and the decrease of the odorant stimulation of the enzyme. Nasal respiratory cilia have a much lower level of adenylyl cyclase activity and show no odorant stimulation. Owing to the large quantities of material available, the bovine olfactory cilia preparation is advantageous for studies of the proteins involved in chemosensory transduction.     

Advantage of the Highly Restricted Odorant Receptor Expression Pattern in Chemosensory Neurons of Drosophila

A fundamental molecular feature of olfactory systems is that individual neurons express only one receptor from a large odorant receptor gene family. While numerous theories have been proposed, the functional significance and evolutionary advantage of generating a sophisticated one-receptor-per neuron expression pattern is not well understood. Using the genetically tractable Drosophila melanogaster as a model, we demonstrate that the breakdown of this highly restricted expression pattern of an odorant receptor in neurons leads to a deficit in the ability to exploit new food sources. We show that animals with ectopic co-expression of odorant receptors also have a competitive disadvantage in a complex environment with limiting food sources. At the level of the olfactory system, we find changes in both the behavioral and electrophysiological responses to odorants that are detected by endogenous receptors when an olfactory receptor is broadly misexpressed in chemosensory neurons. Taken together these results indicate that restrictive expression patterns and segregation of odorant receptors to individual neuron classes are important for sensitive odor-detection and appropriate olfactory behaviors.     

Functional dissection of Odorant binding protein genes in Drosophila melanogaster

Most organisms rely on olfaction for survival and reproduction. The olfactory system of Drosophila melanogaster is one of the best characterized chemosensory systems and serves as a prototype for understanding insect olfaction. Olfaction in Drosophila is mediated by multigene families of odorant receptors and odorant binding proteins (OBPs). Although molecular response profiles of odorant receptors have been well documented, the contributions of OBPs to olfactory behavior remain largely unknown. Here, we used RNAi-mediated suppression of Obp gene expression and measurements of behavioral responses to 16 ecologically relevant odorants to systematically dissect the functions of 17 OBPs. We quantified the effectiveness of RNAi-mediated suppression by quantitative real-time polymerase chain reaction and used a proteomic liquid chromatography and tandem mass spectrometry procedure to show target-specific suppression of OBPs expressed in the antennae. Flies in which expression of a specific OBP is suppressed often show altered behavioral responses to more than one, but not all, odorants, in a sex-dependent manner. Similarly, responses to a specific odorant are frequently affected by suppression of expression of multiple, but not all, OBPs. These results show that OBPs are essential for mediating olfactory behavioral responses and suggest that OBP-dependent odorant recognition is combinatorial.     

Improving the odorant sensitivity of olfactory receptor-expressing yeast with accessory proteins

Olfaction depends on the selectivity and sensitivity of olfactory receptors. Previous attempts at constructing a mammalian olfactory receptor-based artificial odorant sensing system in the budding yeast Saccharomyces cerevisiae suffered from low sensitivity and activity. This result may be at least in part due to poor functional expression of olfactory receptors and/or limited solubility of some odorants in the medium. In this study, we examined the effects of two types of accessory proteins, receptor transporting protein 1 short and odorant binding proteins, in improving odor-mediated activation of olfactory receptors expressed in yeast. We found that receptor transporting protein 1 short enhanced the membrane expression and ligand-induced responses of some olfactory receptors. Coexpression of odorant binding proteins of the silkworm moth Bombyx mori enhanced the sensitivity of a mouse olfactory receptor. Our results suggest that different classes of accessory proteins can confer sensitive and robust responses of olfactory receptors expressed in yeast. Inclusion of accessory proteins may be essential in the future development of practical olfactory receptor-based odorant sensors.     

昆虫嗅觉相关蛋白的研究进展

嗅觉是昆虫产生行为的重要物质基础,阐明昆虫嗅觉机理有助于调控昆虫行为和进行害虫治理。近年来,许多与嗅觉相关的生物活性分子和相关基因的发现和克隆,对揭示嗅觉机理具有重要作用。作者针对近年来研究较多的气味结合蛋白、化学感受蛋白、气味受体、气味降解酶以及感觉神经元膜蛋白等,就其生化特性、表达部位、分子结构、生理功能等进行了综述。     

Chemical sensing in Drosophila

Chemical sensing begins when peripheral receptor proteins recognise specific environmental stimuli and translate them into spatial and temporal patterns of sensory neuron activity. The chemosensory system of the fruit fly, Drosophila melanogaster, has become a dominant model to understand this process, through its accessibility to a powerful combination of molecular, genetic and electrophysiological analysis. Recent results have revealed many surprises in the biology of peripheral chemosensation in Drosophila, including novel structural and signalling properties of the insect odorant receptors (ORs), combinatorial mechanisms of chemical recognition by the gustatory receptors (GRs), and the implication of Transient Receptor Potential (TRP) ion channels as a novel class of chemosensory receptors.     

Ion channels from chemosensory olfactory neurons

The olfactory epithelium has the ability to respond to a large number of volatile compounds of small molecular weight. Ultimately, such a property lies on a specialized type of neuron, the olfactory receptor cell. In the presence of odorants, the olfactory receptor neuron responds with action potentials whose frequency depends on odorant concentration. The primary events in the process of olfactory transduction are thought to occur at the cilia of olfactory receptor neurons and involve the binding of odorants to receptor molecules followed by the opening of ion channels. A crucial step in understanding olfactory transduction requires identifying the mechanisms that regulate the electrical activity of olfactory cells. In the last couple of years, patch-clamp recording from isolated olfactory cells and reconstitution of olfactory membranes in planar lipid bilayers have begun to shed light on some of these mechanisms. Although the information emerging from such studies is still preliminary, there are already well-defined hypotheses on the molecular events that might underlie the primary events in olfactory transduction. Currently, attention is being focused on the notions that second messengers might be involved in the activation of ion channels in olfactory cilia, and that odorant binding to a receptor molecule might lead directly to the gating of ion channels in chemosensory olfactory membranes. The coming years promise to be exciting ones in the field of olfactory transduction. We have now the necessary tools to be able to confront hypotheses and experimental facts.     

Searching for the Ligands of Odorant Receptors

Through the sense of smell mammals can detect and discriminate between a large variety of odorants present in the surrounding environment. Odorants bind to a large repertoire of odorant receptors located in the cilia of olfactory sensory neurons of the nose. Each olfactory neuron expresses one single type of odorant receptor, and neurons expressing the same type of receptor project their axons to one or a few glomeruli in the olfactory bulb, creating a map of odorant receptor inputs. The information is then passed on to other regions of the brain, leading to odorant perception. To understand how the olfactory system discriminates between odorants, it is necessary to determine the odorant specificities of individual odorant receptors. These studies are complicated by the extremely large size of the odorant receptor family and by the poor functional expression of these receptors in heterologous cells. This article provides an overview of the methods that are currently being used to investigate odorant receptor–ligand interactions.     

The molecular logic of olfaction in Drosophila

Drosophila fruit flies display robust olfactory-driven behaviors with an olfactory system far simpler than that of vertebrates. Endowed with 1300 olfactory receptor neurons, these insects are able to recognize and discriminate between a large number of distinct odorants. Candidate odorant receptor molecules were identified by complimentary approaches of differential cloning and genome analysis. The Drosophila odorant receptor (DOR) genes encode a novel family of proteins with seven predicted membrane-spanning domains, unrelated to vertebrate or nematode chemosensory receptors. There are on the order of 60 or more members of this gene family in the Drosophila genome, far fewer than the hundreds to thousands of receptors found in vertebrates or nematodes. DOR genes are selectively expressed in small subsets of olfactory neurons, in expression domains that are spatially conserved between individuals, bilaterally symmetric and not sexually dimorphic. Double in situ RNA hybridization with a number of pairwise combinations of DOR genes fails to reveal any overlap in gene expression, suggesting that each olfactory neuron expresses one or a small number of receptor genes and is therefore functionally distinct. How is activation of such a subpopulation of olfactory receptor neurons in the periphery sensed by the brain? In the mouse, all neurons expressing a given receptor project with precision to two of 1800 olfactory bulb glomeruli, creating a spatial map of odor quality in the brain. We have employed DOR promoter transgenes that recapitulate expression of endogenous receptor to visualize the projections of individual populations of receptor neurons to subsets of the 43 glomeruli in the Drosophila antennal lobe. The results suggest functional conservation in the logic of olfactory discrimination from insects to mammals.     

Amphioxus (Branchiostoma floridae) has orthologs of vertebrate odorant receptors

Background  

A common feature of chemosensory systems is the involvement of G protein-coupled receptors (GPCRs) in the detection of environmental stimuli. Several lineages of GPCRs are involved in vertebrate olfaction, including trace amine-associated receptors, type 1 and 2 vomeronasal receptors and odorant receptors (ORs). Gene duplication and gene loss in different vertebrate lineages have lead to an enormous amount of variation in OR gene repertoire among species; some fish have fewer than 100 OR genes, while some mammals possess more than 1000. Fascinating features of the vertebrate olfactory system include allelic exclusion, where each olfactory neuron expresses only a single OR gene, and axonal guidance where neurons expressing the same receptor project axons to common glomerulae. By identifying homologous ORs in vertebrate and in non-vertebrate chordates, we hope to expose ancestral features of the chordate olfactory system that will help us to better understand the evolution of the receptors themselves and of the cellular components of the olfactory system.     

Chemosensory signaling in C. elegans

The nematode Caenorhabditis elegans can sense and respond to hundreds of different chemicals with a simple nervous system, making it an excellent model for studies of chemosensation. The chemosensory neurons that mediate responses to different chemicals have been identified through laser ablation studies, providing a cellular context for chemosensory signaling. Genetic and molecular analyses indicate that chemosensation in nematodes involves G protein signaling pathways, as it does in vertebrates, but the receptors and G proteins involved belong to nematode-specific gene families. It is likely that about 500 different chemosensory receptors are used to detect the large spectrum of chemicals to which C. elegans responds, and one of these receptors has been matched with its odorant ligand. C. elegans olfactory responses are also subject to regulation based on experience, allowing the nematode to respond to a complex and changing chemical environment.     

Olfactory receptors and signalling elements in the Grueneberg ganglion

The Grueneberg ganglion (GG) is a cluster of neurones present in the vestibule of the anterior nasal cavity. Although its function is still elusive, recent studies have shown that cells of the GG transcribe the gene encoding the olfactory marker protein (OMP) and project their axons to glomeruli of the olfactory bulb, suggesting that they may have a chemosensory function. Chemosensory responsiveness of olfactory neurones in the main olfactory epithelium (MOE) and the vomeronasal organ (VNO) is based on the expression of either odorant receptors or vomeronasal putative pheromone receptors. To scrutinize its presumptive olfactory nature, the GG was assessed for receptor expression by extensive RT-PCR analyses, leading to the identification of a distinct vomeronasal receptor which was expressed in the majority of OMP-positive GG neurones. Along with this receptor, these cells expressed the G proteins Go and Gi, both of which are also present in sensory neurones of the vomeronasal organ. Odorant receptors were expressed by very few cells during prenatal and perinatal stages; a similar number of cells expressed adenylyl cyclase type III and G(olf/s), characteristic signalling elements of the main olfactory system. The findings of the study support the notion that the GG is in fact a subunit of the complex olfactory system, comprising cells with either a VNO-like or a MOE-like phenotype. Moreover, expression of a vomeronasal receptor indicates that the GG might serve to detect pheromones.     

Subunit contributions to insect olfactory receptor function: channel block and odorant recognition

Insect olfactory receptors are heteromeric ligand-gated ion channels composed of at least one common subunit (Orco) and at least one subunit that confers odorant specificity. Little is known about how individual subunits contribute to the structure and function of the olfactory receptor complex. We expressed insect olfactory receptors in Xenopus oocytes to investigate 2 functional features, ion channel block and odorant recognition. The sensitivity of Drosophila olfactory receptors to inhibition by ruthenium red, a cation channel blocker, varied widely when different specificity subunits were present, suggesting that the specificity subunits contribute to the structure of the ion pore. Olfactory receptors formed by Dmel\Or35a and Orco subunits from several different species displayed highly similar odorant response profiles, suggesting that the Orco subunit does not contribute to the structure of the odorant-binding site. We further explored odorant recognition by conducting a detailed examination of the odorant specificity Dmel\Or67a + Dmel\Orco, a receptor that responds to aromatic structures. This screen identified agonists, partial agonists, and an antagonist of Dmel\Or67a + Dmel\Orco. Our findings favor specific subunit arrangements within the olfactory receptor complex and provide a preliminary odorophore for an olfactory receptor, offering a useful foundation for future exploration of insect olfactory receptor structure.     

The human olfactory receptor repertoire

Background  

The mammalian olfactory apparatus is able to recognize and distinguish thousands of structurally diverse volatile chemicals. This chemosensory function is mediated by a very large family of seven-transmembrane olfactory (odorant) receptors encoded by approximately 1,000 genes, the majority of which are believed to be pseudogenes in humans.     

A chemosensory gene family encoding candidate gustatory and olfactory receptors in Drosophila

A novel family of candidate gustatory receptors (GRs) was recently identified in searches of the Drosophila genome. We have performed in situ hybridization and transgene experiments that reveal expression of these genes in both gustatory and olfactory neurons in adult flies and larvae. This gene family is likely to encode both odorant and taste receptors. We have visualized the projections of chemosensory neurons in the larval brain and observe that neurons expressing different GRs project to discrete loci in the antennal lobe and subesophageal ganglion. These data provide insight into the diversity of chemosensory recognition and an initial view of the representation of gustatory information in the fly brain.