Receptive range analysis of a mouse odorant receptor subfamily

Mammals deploy a large array of odorant receptors (ORs) to detect and distinguish a vast number of odorant molecules. ORs vary widely in the type of odorant structures recognized and in the breadth of molecular receptive range (MRR), with some ORs recognizing a small group of closely related molecules and other ORs recognizing a wide range of structures. While closely related ORs have been shown to have similar MRRs, the functional relationships among less closely related ORs are unclear. We screened a small group of ORs with a diverse odorant panel to identify a new odorant‐OR pairing (unsaturated aldehydes and MOR263‐3). We then extensively screened MOR263‐3 and a series of additional MORs related to MOR263‐3 in various ways. MORs related by phylogenetic analysis (several other members of the MOR263 subfamily) had MRRs that overlapped with the MRR of MOR263‐3, even with amino acid identity as low as 48% (MOR263‐2). MOR171‐17, predicted to be functionally related to MOR263‐3 by an alternative bioinformatic analysis, but with only 39% amino acid identity, had a distinct odorant specificity. Our results support the use of phylogenetic analysis to predict functional relationships among ORs with relatively low amino acid identity.

     

Molecular receptive range variation among mouse odorant receptors for aliphatic carboxylic acids

The ability of mammals to identify and distinguish among many thousands of different odorants suggests a combinatorial use of odorant receptors, with each receptor detecting multiple odorants and each odorant interacting with multiple receptors. Numerous receptors may be devoted to the sampling of particularly important regions of odor space. In this study, we explore the similarities and differences in the molecular receptive ranges of four mouse odorant receptors (MOR23-1, MOR31-4, MOR32-11 and MOR40-4), which have previously been identified as receptors for aliphatic carboxylic acids. Each receptor was expressed in Xenopus oocytes, along with Gα_olf and the cystic fibrosis transmembrane regulator to allow electrophysiological assay of receptor responses. We find that even though these receptors are relatively unrelated, there is extensive overlap among their receptive ranges. That is, these receptors sample a similar region of odor space. However, the receptive range of each receptor is unique. Thus, these receptors contribute to the depth of coverage of this small region of odor space. Such a group of receptors with overlapping, but distinct receptive ranges, may participate in making fine distinctions among complex mixtures of closely related odorant compounds.     

Metabotropic glutamate receptor expression in olfactory receptor neurons from the channel catfish,Ictalurus punctatus

Metabotropic glutamate receptors (mGluRs) were identified in olfactory receptor neurons of the channel catfish, Ictalurus punctatus, by polymerase chain reaction. DNA sequence analysis confirmed the presence of two subtypes, mGluR1 and mGluR3, that were coexpressed with each other and with the putative odorant receptors within single olfactory receptor neurons. Immunocytochemical data showed that both mGluR subtypes were expressed in the apical dendrites and some cilia of olfactory neurons. Pharmacological analysis showed that antagonists to each mGluR subtype significantly decreased the electrophysiological response to odorant amino acids. α-Methyl-L -CCG1/(2S,3S,4S)-2-methyl-2-(carboxycyclopropyl)glycine (MCCG), a known antagonist to mGluR3, and (S)-4-carboxyphenylglycine (S-4CPG), a specific antagonist to mGluR1, each significantly reduced olfactory receptor responses to L -glutamate. S-4CPG and MCCG reduced the glutamate response to 54% and 56% of control, respectively, which was significantly greater than their effect on a neutral amino acid odorant, methionine. These significant reductions of odorant response by the antagonists, taken with the expression of these receptors throughout the dendritic and ciliated portions of some olfactory receptor neurons, suggest that these mGluRs may be involved in olfactory reception and signal transduction. © 1998 John Wiley & Sons, Inc. J Neurobiol 35: 94–104, 1998     

Ontogeny of odorant receptor gene expression in zebrafish,Danio rerio

We cloned three putative odorant receptor (OR) genes from the zebrafish to use as in situ hybridization probes to follow the temporal patterns of neurons expressing OR genes through a developmental progression from embryo (12 h postfertilization) to adult. The identification of these genes is supported by sequence homology to previously reported ORs and by the morphology and location of labeled cells in in situ hybridization experiments. Cells expressing OR mRNA were first observed in the olfactory placodes between 31 and 38 h after fertilization (fish reared at 26°C). Initially, only single cells were observed to hybridize the probe; the number of labeled cells increased throughout the remainder of embryogenesis and through postembryonic growth and morphogenesis of the olfactory organ. At all ages, the positively hybridizing cells were scattered throughout the olfactory epithelium but not in the nonsensory epithelium of the olfactory organ. © 1996 John Wiley & Sons, Inc.     

Developing and maintaining a nose-to-brain map of odorant identity

Olfactory sensory neurons (OSNs) in the olfactory epithelium of the nose transduce chemical odorant stimuli into electrical signals. These signals are then sent to the OSNs'' target structure in the brain, the main olfactory bulb (OB), which performs the initial stages of sensory processing in olfaction. The projection of OSNs to the OB is highly organized in a chemospatial map, whereby axon terminals from OSNs expressing the same odorant receptor (OR) coalesce into individual spherical structures known as glomeruli. This nose-to-brain map of odorant identity is built from late embryonic development to early postnatal life, through a complex combination of genetically encoded, OR-dependent and activity-dependent mechanisms. It must then be actively maintained throughout adulthood as OSNs experience turnover due to external insult and ongoing neurogenesis. Our review describes and discusses these two distinct and crucial processes in olfaction, focusing on the known mechanisms that first establish and then maintain chemospatial order in the mammalian OSN-to-OB projection.     

Functional evolution of mammalian odorant receptors

The mammalian odorant receptor (OR) repertoire is an attractive model to study evolution, because ORs have been subjected to rapid evolution between species, presumably caused by changes of the olfactory system to adapt to the environment. However, functional assessment of ORs in related species remains largely untested. Here we investigated the functional properties of primate and rodent ORs to determine how well evolutionary distance predicts functional characteristics. Using human and mouse ORs with previously identified ligands, we cloned 18 OR orthologs from chimpanzee and rhesus macaque and 17 mouse-rat orthologous pairs that are broadly representative of the OR repertoire. We functionally characterized the in vitro responses of ORs to a wide panel of odors and found similar ligand selectivity but dramatic differences in response magnitude. 87% of human-primate orthologs and 94% of mouse-rat orthologs showed differences in receptor potency (EC50) and/or efficacy (dynamic range) to an individual ligand. Notably dN/dS ratio, an indication of selective pressure during evolution, does not predict functional similarities between orthologs. Additionally, we found that orthologs responded to a common ligand 82% of the time, while human OR paralogs of the same subfamily responded to the common ligand only 33% of the time. Our results suggest that, while OR orthologs tend to show conserved ligand selectivity, their potency and/or efficacy dynamically change during evolution, even in closely related species. These functional changes in orthologs provide a platform for examining how the evolution of ORs can meet species-specific demands.     

Differential development of odorant receptor expression patterns in the olfactory epithelium: a quantitative analysis in the mouse septal organ

The rodent olfactory epithelium expresses more than 1000 odorant receptors (ORs) with distinct patterns, yet it is unclear how such patterns are established during development. In the current study, we investigated development of the expression patterns of different ORs in the septal organ, a small patch of olfactory epithelium predominantly expressing nine identified ORs. The presumptive septal organ first appears at about embryonic day 16 (E16) and it completely separates from the main olfactory epithelium (MOE) at about postnatal day 7 (P7). Using in situ hybridization, we quantified the densities of the septal organ neurons labeled by specific RNA probes of the nine abundant OR genes from E16 to postnatal 3 months. The results indicate that olfactory sensory neurons (OSNs) expressing different ORs have asynchronous temporal onsets. For instance, MOR256-17 and MOR236-1 cells are present in the septal organ at E16; however, MOR0-2 cells do not appear until P0. In addition, OSNs expressing different ORs show distinct developmental courses and reach their maximum densities at different stages ranging from E16 (e.g. MOR256-17) to 1 month (e.g. MOR256-3 and MOR235-1). Furthermore, early onset does not correlate with high abundance in adult. This study reveals a dynamic composition of the OSNs expressing different ORs in the developing olfactory epithelium.     

Functional identification of a goldfish odorant receptor.

The vertebrate olfactory system utilizes odorant receptors to receive and discriminate thousands of different chemical stimuli. An understanding of how these receptors encode information about an odorant's molecular structure requires a characterization of their ligand specificities. We employed an expression cloning strategy to identify a goldfish odorant receptor that is activated by amino acids-potent odorants for fish. Structure-activity analysis indicates that the receptor is preferentially tuned to recognize basic amino acids. The receptor is a member of a multigene family of G protein-coupled receptors, sharing sequence similarities with the calcium sensing, metabotropic glutamate, and V2R class of vomeronasal receptors. The ligand tuning properties of the goldfish amino acid odorant receptor provide information for unraveling the molecular mechanisms underlying olfactory coding.     

The sense of smell: molecular basis of odorant recognition

Most animal species rely on odorant compounds to locate food, predators, or toxins. The sense of smell is also involved in animal communication, and revealing the underlying mechanisms will therefore facilitate a deeper understanding of animal behaviour. Since the 1940s different theories have speculated on the fundamental basis of olfaction. It was assumed that odorant molecules were recognized by selective protein receptors in the nose, triggering a nervous signal processed by the brain. The discovery of these receptors in the early 1990s allowed great progress in understanding the physiological and biochemical principles of olfaction. An overview of the different mechanisms involved in the coding of odour character as well as odour intensity is presented here, focusing on the biochemical basis of odorant recognition. Despite the enormous progress achieved in recent years, details of odorant-receptor interaction at the molecular level and the mechanisms of olfactory receptor activation are poorly understood. The likely role of metal ions in odorant recognition is discussed, and also the perireceptor events involved in odorant transport and biotransformation, with a view to providing a comprehensive overview of mammalian olfaction to guide future computational structural models and the design of functional experiments. Recent studies have analysed the olfactory genome of several species, providing information about the evolution of olfaction. The role of the olfactory system in animal communication is also described.     

Allosteric modulation of a human odorant receptor

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NMDA receptor subunit-dependent modulation by conantokin-G and Ala7-conantokin-G

The modulation of recombinant NMDA receptors by conantokin-G (con-G) and Ala7-conantokin-G (Ala7-Con-G) was investigated in Xenopus oocytes injected with capped RNA coding for NR1 splice variants and NR2 subunits using the two-electrode voltage clamp technique. Glutamate exhibited a marginally higher apparent affinity for NR2A-containing receptors than NR2B-containing receptors, regardless of the NR1 subunit present. Conantokins were bath applied to give cumulative concentration responses in the presence of 3 and 30 mum glutamate. Both contantokins exhibited biphasic concentration-response relationships at NR2A-containing NMDA receptors, producing potentiation at low conantokin concentrations and inhibition at high concentrations. These effects were stronger with glutamate concentrations near its EC50, and less marked at saturating concentrations. In contrast, the conantokin concentration-response relation was monophasic and inhibitory at NR2B-containing receptors. We conclude that the combinations of subunits that comprise the NMDA receptor complex influence conantokin and glutamate affinities and the nature of the responses to conantokins.     

昆虫气味受体研究进展

嗅觉在昆虫的多种行为中发挥关键作用。气味分子与嗅觉神经元树突上气味受体的结合,参与了昆虫嗅觉识别的初始过程。昆虫的嗅觉神经元表达两类气味受体： 一是传统气味受体,该类受体同源性较低,在少部分嗅觉神经元中表达; 二是Or83b家族受体,该类受体不感受气味,在不同昆虫间较为保守且在大多数嗅觉神经元中表达。目前,对于单个传统气味受体的气味分子配体特异性所知甚少; 对于Or83b家族受体,一般认为其可能具有将传统气味受体运送至嗅觉神经元树突膜上的功能。此外,有一些实验证据不支持昆虫气味受体为G蛋白偶联受体的观点。     

Functional analysis of LjSUT4, a vacuolar sucrose transporter from Lotus japonicus

Sucrose transporters in the SUT family are important for phloem loading and sucrose uptake into sink tissues. The recent localization of type III SUTs AtSUT4 and HvSUT2 to the vacuole membrane suggests that SUTs also function in vacuolar sucrose transport. The transport mechanism of type III SUTs has not been analyzed in detail. LjSUT4, a type III sucrose transporter homolog from Lotus japonicus, is expressed in nodules and its transport activity has not been previously investigated. In this report, LjSUT4 was expressed in Xenopus oocytes and its transport activity assayed by two-electrode voltage clamping. LjSUT4 transported a range of glucosides including sucrose, salicin, helicin, maltose, sucralose and both alpha- and beta-linked synthetic phenyl glucosides. In contrast to other sucrose transporters, LjSUT4 did not transport the plant glucosides arbutin, fraxin and esculin. LjSUT4 showed a low affinity for sucrose (K (0.5) = 16 mM at pH 5.3). In addition to inward currents induced by sucrose, other evidence also indicated that LjSUT4 is a proton-coupled symporter: (14)C-sucrose uptake into LjSUT4-expressing oocytes was inhibited by CCCP and sucrose induced membrane depolarization in LjSUT4-expressing oocytes. A GFP-fusion of LjSUT4 localized to the vacuole membrane in Arabidopsis thaliana and in the roots and nodules of Medicago truncatula. Based on these results we propose that LjSUT4 functions in the proton-coupled uptake of sucrose and possibly other glucosides into the cytoplasm from the vacuole.     

Cholinergic modulation of progesterone-induced maturation of Xenopus oocytes in vitro

Mixed and muscarinic cholinergic agonists (acetylcholine, carbamylcholine, methacholine, oxotremorine, and pilocarpine) accelerated in a dose-dependent manner the progesterone-induced maturation of Xenopus laevis oocytes. None of these agonists induced oocyte maturation in the absence of progesterone. The accelerating effect of cholinergic agonists was blocked in a dose-dependent manner by specific muscarinic antagonists (atropine and scopolamine) but not by specific nicotinic antagonists (d-tubocurarine and hexamethonium). The specific nicotinic agonist, dimethylphenylpiperazine, alone induced maturation in the absence of progesterone. The optimal promoting effect of acetylcholine was observed when oocytes were exposed to acetylcholine for 30 min, 5 min after the addition of progesterone, and was markedly better than when oocytes were exposed to acetylcholine throughout their incubation with progesterone. The effect of acetylcholine was observed in both follicle-enclosed and in defolliculated oocytes, indicating that follicular cells were not the target of the cholinergic drugs.     

Phosphinic,phosphonic and seleninic acid bioisosteres of isonipecotic acid as novel and selective GABA(C) receptor antagonists

A number of amino acids bioisosterically derived from the specific GABA_A agonist, isonipecotic acid, were electrophysiologically characterized as antagonists at GABA_C ρ₁ receptors expressed in Xenopus oocytes. The phosphinic acid analogue of isonipecotic acid, piperidin-4-ylphosphinic acid (2), was comparable with the standard GABA_C antagonist, (1,2,5,6-tetrahydropyridin-4-yl)methylphosphinic acid (TPMPA), in terms of potency and GABA_C versus GABA_A receptor selectivity. Whereas the phosphonic acid analogue, piperidin-4-ylphosphonic acid (4), was at least an order of magnitude weaker than piperidin-4-ylphosphinic acid as a GABA_C antagonist, the seleninic acid analogue, piperidin-4-ylseleninic acid (SEPI, 6), was the most potent and selective GABA_C antagonist within the group of isonipecotic acid derived amino acids studied.     

Functional Changes in Rat Nigral GABAA Receptors Induced by Degeneration of the Striatonigral GABAergic Pathway: An Electrophysiological Study of Receptors Incorporated into Xenopus Oocytes

Abstract: Expression of rat brain γ-aminobutyric acid type A (GABA_A) receptors in Xenopus laevis oocytes can be achieved by injection of the oocytes with synaptosomes. This approach has now been applied to evaluate changes in the function of nigral GABA_A receptors after degeneration of the striatonigral GABAergic pathway induced by the unilateral infusion of kainic acid into the rat striatum. Ten days after striatal injection, synaptosomal membranes were prepared from the substantia nigra and introduced into oocytes. Nigral GABA_A receptors incorporated into the oocyte cell membrane were then characterized electrophysiologically under voltage-clamp conditions. The maximal amplitude of GABA-induced Cl^? currents in oocytes injected with synaptosomes from denervated substantia nigra was twice that observed in oocytes injected with synaptosomes from control substantia nigra. The concentration of GABA required for the half-maximal response did not differ between the two groups of oocytes. In addition, the potentiation of GABA-induced currents by the benzodiazepine diazepam (1 µM) and the steroid derivative allopregnanolone (3 µM) was increased by ～65 and 60%, respectively, in oocytes injected with synaptosomes from denervated substantia nigra compared with those injected with control synaptosomes. The concentrations of diazepam and allopregnanolone giving half-maximal responses were not affected by denervation. In contrast, the inhibitory effects of the benzodiazepine receptor inverse agonists FG 7142 (10 µM) and 6,7-dimethoxy-4-ethyl-β-carboline-3-carboxylic acid ethyl ester (1 µM) were reduced by 48 and 38%, respectively, after denervation. These results indicate that the up-regulation of nigral GABA_A receptors induced by degeneration of the striatonigral GABAergic pathway is associated with an increased efficacy of positive allosteric modulators, such as benzodiazepines and steroids, and with a reduced efficacy of negative allosteric modulators such as β-carbolines.     

Functional characterization of a putative aquaporin from Encephalitozoon cuniculi, a microsporidia pathogenic to humans

The microsporidia are a group of obligate intracellular parasitic protists that have been implicated as both human and veterinary pathogens. The infectious process of these organisms is believed to be dependent upon the rapid influx of water into spores, presumably via aquaporins (AQPs), transmembrane channels that facilitate osmosis. An AQP-like sequence of the microsporidium Encephalitozoon cuniculi (EcAQP), when cloned and expressed in oocytes of Xenopus laevis, rendered these oocytes highly permeable to water. No permeability to the solutes glycerol or urea was observed. Pre-treatment of EcAQP-expressing oocytes with HgCl(2) failed to inhibit their osmotic permeability, as predicted from EcAQP's lack of mercury-sensitive cysteine residues near the NPA motifs which line the AQP aqueous pore. EcAQP exhibits sequence identity to AQP A of Dictyostelium discoideum (26%) and human AQP 2 (24%). Further study of AQPs in microsporidia and their potential inhibitors may yield novel therapeutic agents for microsporidian infections.     

An odorant derivative as an antagonist for an olfactory receptor

Different odorants are recognized by different combinations of G protein-coupled olfactory receptors, and thereby, odor identity is determined by a combinatorial receptor code for each odorant. We recently demonstrated that odorants appeared to compete for receptor sites to act as an agonist or an antagonist. Therefore, in natural circumstances where we always perceive a mixture of various odorants, olfactory receptor antagonism between odorants may result in a receptor code for the mixture that cannot be predicted from the codes for its individual components. Here we show that stored isoeugenol has an antagonistic effect on a mouse olfactory receptor, mOR-EG. However, freshly purified isoeugenol did not have an inhibitory effect. Instead, an isoeugenol derivative produced during storage turned out to be a potent competitive antagonist of mOR-EG. Structural analysis revealed that this derivative is an oxidatively dimerized isoeugenol that naturally occurs by oxidative reaction. The current study indicates that as odorants age, they decompose or react with other odorants, which in turn affects responsiveness of an olfactory receptor(s).