Odorant response assays for a heterologously expressed olfactory receptor

Odorant responsiveness of a mouse olfactory receptor, mOR-EG, was investigated in various heterologous cells using a variety of detection methods. Odorant-induced Ca(2+) response was observed in HEK293 cells that coexpressed mOR-EG and the promiscuous G protein, G alpha 15. Without G alpha 15, a robust increase in cAMP level was observed upon odorant-stimulation in various mammalian cells. A luciferase reporter gene assay using zif268 promoter was adopted to amplify the cAMP signals. In Xenopus laevis oocytes, odorant-stimulated currents were recorded when mOR-EG cRNA was co-injected with either G alpha 15 or cAMP-dependent channel. These results suggest that odorant responsiveness can be monitored via a signaling pathway mediated by endogenous G alphas or transfected G alpha 15 in heterologous cell systems. Various functional assays for a heterologously expressed olfactory receptor reported in this study, are potentially useful for high-throughput ligand screening and functional analyses of hundreds of olfactory receptors.     

Identification of specific ligands for orphan olfactory receptors. G protein-dependent agonism and antagonism of odorants

Olfactory receptors are the largest group of orphan G protein-coupled receptors with an infinitely small number of agonists identified out of thousands of odorants. The de-orphaning of olfactory receptor (OR) is complicated by its combinatorial odorant coding and thus requires large scale odorant and receptor screening and establishing receptor-specific odorant profiles. Here, we report on the stable reconstitution of OR-specific signaling in HeLa/Olf cells via G protein alphaolf and adenylyl cyclase type-III to the Ca2+ influx-mediating olfactory cyclic nucleotide-gated CNGA2 channel. We demonstrate the central role of Galphaolf in odorant-specific signaling out of OR. The employment of the non-typical G protein alpha15 dramatically altered the odorant specificities of 3 of 7 receptors that had been characterized previously by different groups. We further show for two OR that an odorant may be an agonist or antagonist, depending on the G protein used. HeLa/Olf cells proved suitable for high-throughput screening in fluorescence-imaging plate reader experiments, resulting in the de-orphaning of two new OR for the odorant (-)citronellal from an expression library of 93 receptors. To demonstrate the G protein dependence of its odorant response pattern, we screened the most sensitive (-)citronellal receptor Olfr43 versus 94 odorants simultaneously in the presence of Galpha15 or Galphaolf. We finally established an EC50-ranking odorant profile for Olfr43 in HeLa/Olf cells. In summary, we conclude that, in heterologous systems, odorants may function as agonists or antagonists, depending on the G protein used. HeLa/Olf cells provide an olfactory model system for functional expression and de-orphaning of OR.     

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.     

Origin of basal activity in mammalian olfactory receptor neurons

Mammalian odorant receptors form a large, diverse group of G protein-coupled receptors that determine the sensitivity and response profile of olfactory receptor neurons. But little is known if odorant receptors control basal and also stimulus-induced cellular properties of olfactory receptor neurons other than ligand specificity. This study demonstrates that different odorant receptors have varying degrees of basal activity, which drives concomitant receptor current fluctuations and basal action potential firing. This basal activity can be suppressed by odorants functioning as inverse agonists. Furthermore, odorant-stimulated olfactory receptor neurons expressing different odorant receptors can have strikingly different response patterns in the later phases of prolonged stimulation. Thus, the influence of odorant receptor choice on response characteristics is much more complex than previously thought, which has important consequences on odor coding and odor information transfer to the brain.     

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.     

Engineered Yeasts as Reporter Systems for Odorant Detection

Abstract

The functional expression of olfactory receptors (ORs) is a primary requirement to utilize olfactory detection systems. We have taken advantage of the functional similarities between signal transduction cascades in the budding yeast Saccharomyces cerevisiae and mammalian cells. The yeast pheromone response pathway has been adapted to allow ligand‐dependent signaling of heterologous expressed G‐protein coupled receptors (GPCRs) via mammalian or chimeric yeast/mammalian Gα proteins. Two different strategies are reported here which offer a positive screen for functional pairs. The OR and Gα protein are introduced into the modified yeast cells such that they hijack the pheromone response pathway usually resulting in cell cycle arrest. The first strategy utilizes ligand‐induced expression of a FUS1‐HIS3 reporter gene to permit growth on a selective medium lacking histidine; the second to induce ligand‐dependent expression of a FUS1‐Hph reporter gene, conferring resistance to hygromycin. Validation of the systems was performed using the rat I7 receptor response to a range of aldehyde odorants previously characterized as functional ligands. Of these only heptanal produced a positive growth response in the concentration range 5 × 10^?8 to 5 × 10^?6 M. Induction conditions appear to be critical for functional expression, and the solvents of odorants have a toxic effect for the highest odorant concentrations. The preference of rat I7 receptor for the ligand heptanal in yeast has to be compared to concurrent results obtained with mammalian expression systems.     

Human olfactory receptor families and their odorants

The human nose detects volatile chemical stimuli by at least three different receptor families: odorant receptors, trace amine-associated receptors, and vomeronasal type-1 receptors. As G protein-coupled receptors, all of the few functionally characterized olfactory receptors share major functional features: when expressed in heterologous cell systems, they 1) respond to odorants of certain chemical groups, e.g., amines, aliphatic carboxylic acids or aldehydes, floral or fruity odorants, including certain key-food odorants, and putative pheromones, and 2) transduce their signals to intracellular cAMP signaling. However, little is known yet about specific differences in the functional designation of the three olfactory receptor families. Recently, two heterologous cell systems expressing olfactory signaling molecules have been developed. Different screening strategies will shed light on the yet sparsely available odorant specificity profiles and structure-function relationships of olfactory receptors, as well as the structure-activity relationships of their odorants.     

Odorant receptor gene regulation: implications from genomic organization.

Odorant receptor genes comprise the largest known family of G-protein-coupled receptors in vertebrates. These receptor genes are tightly clustered in the genomes of every vertebrate organism investigated, including zebrafish, mice and humans, and they appear to have expanded and duplicated throughout evolution. In a mechanism that has yet to be elucidated, each olfactory neuron expresses a single receptor gene. This highly restricted expression pattern underlies the ability to distinguish between a wide variety of odorants. Here, we address the evolutionary expansion of odorant receptor genes and the role genomic organization of these genes might have in their tightly regulated expression.     

Cross-adaptation between olfactory responses induced by two subgroups of odorant molecules

It has long been believed that vertebrate olfactory signal transduction is mediated by independent multiple pathways (using cAMP and InsP3 as second messengers). However, the dual presence of parallel pathways in the olfactory receptor cell is still controversial, mainly because of the lack of information regarding the single-cell response induced by odorants that have been shown to produce InsP3 exclusively (but not cAMP) in the olfactory cilia. In this study, we recorded activities of transduction channels of single olfactory receptor cells to InsP3-producing odorants. When the membrane potential was held at -54 mV, application of InsP3-producing odorants to the ciliary region caused an inward current. The reversal potential was 0 +/- 7 mV (mean +/- SD, n = 10). Actually, InsP3-producing odorants generated responses in a smaller fraction of cells (lilial, 3.4%; lyral, 1.7%) than the cAMP-producing odorant (cineole, 26%). But, fundamental properties of responses were surprisingly homologous; namely, spatial distribution of the sensitivity, waveforms, I-V relation, and reversal potential, dose dependence, time integration of stimulus period, adaptation, and recovery. By applying both types of odorants alternatively to the same cell, furthermore, we observed cells to exhibit symmetrical cross-adaptation. It seems likely that even with odorants with different modalities adaptation occurs completely depending on the amount of current flow. The data will also provide evidence showing that olfactory response generation and adaptation are regulated by a uniform mechanism for a wide variety of odorants.     

The molecular basis of odor coding in the Drosophila antenna

We have undertaken a functional analysis of the odorant receptor repertoire in the Drosophila antenna. Each receptor was expressed in a mutant olfactory receptor neuron (ORN) used as a "decoder," and the odor response spectrum conferred by the receptor was determined in vivo by electrophysiological recordings. The spectra of these receptors were then matched to those of defined ORNs to establish a receptor-to-neuron map. In addition to the odor response spectrum, the receptors dictate the signaling mode, i.e., excitation or inhibition, and the response dynamics of the neuron. An individual receptor can mediate both excitatory and inhibitory responses to different odorants in the same cell, suggesting a model of odorant receptor transduction. Receptors vary widely in their breadth of tuning, and odorants vary widely in the number of receptors they activate. Together, these properties provide a molecular basis for odor coding by the receptor repertoire of an olfactory organ.     

Olfactory receptor antagonism between odorants

The detection of thousands of volatile odorants is mediated by several hundreds of different G protein-coupled olfactory receptors (ORs). The main strategy in encoding odorant identities is a combinatorial receptor code scheme in that different odorants are recognized by different sets of ORs. Despite increasing information on agonist-OR combinations, little is known about the antagonism of ORs in the mammalian olfactory system. Here we show that odorants inhibit odorant responses of OR(s), evidence of antagonism between odorants at the receptor level. The antagonism was demonstrated in a heterologous OR-expression system and in single olfactory neurons that expressed a given OR, and was also visualized at the level of the olfactory epithelium. Dual functions of odorants as an agonist and an antagonist to ORs indicate a new aspect in the receptor code determination for odorant mixtures that often give rise to novel perceptual qualities that are not present in each component. The current study also provides insight into strategies to modulate perceived odorant quality.     

Physical Variables in the Olfactory Stimulation Process

Electrical recording from small twigs of nerve in a tortoise showed that olfactory, vomeronasal, and trigeminal receptors in the nose are responsive to various odorants. No one kind of receptor was most sensitive to all odorants. For controlled stimulation, odorant was caused to appear in a stream of gas already flowing through the nose. Of the parameters definable at the naris, temperature, relative humidity, and nature of inert gas had little effect on olfactory responses to amyl acetate, whereas odorant species, odorant concentration, and volume flow rate effectively determined the responses of all nasal chemoreceptors. An intrinsic variable of accessibility to the receptors, particularly olfactory, was demonstrated. Flow dependence of chemoreceptor responses is thought to reflect the necessity for delivery of odorant molecules to receptor sites. Since the olfactory receptors are relatively exposed, plateauing of the response with flow rate for slightly soluble odorants suggests an approach to concentration equilibrium in the overlying mucus with that in the air entering the naris. Accordingly, data for responses to amyl acetate were fitted with Beidler's (1954) taste equation for two kinds of sites being active. The requirement for finite aqueous solubility, if true, suggests substitution of aqueous solutions for gaseous solutions. A suitable medium was found and results conformed to expectations. Olfactory receptors were insensitive to variation of ionic strength, pH, and osmotic pressure.     

Spatial representation of odorant valence in an insect brain

Brains have to decide whether and how to respond to detected stimuli based on complex sensory input. The vinegar fly Drosophila melanogaster evaluates food sources based on olfactory cues. Here, we performed a behavioral screen using the vinegar fly and established the innate valence of 110 odorants. Our analysis of neuronal activation patterns evoked by attractive and aversive odorants suggests that even though the identity of odorants is coded by the set of activated receptors, the main representation of odorant valence is formed at the output level of the antennal lobe. The topographic clustering within the antennal lobe of valence-specific output neurons resembles a corresponding domain in the olfactory bulb of mice. The basal anatomical structure of the olfactory circuit between insects and vertebrates is known to be similar; our study suggests that the representation of odorant valence is as well.     

Odor recognition and second messenger signaling in olfactory receptor neurons

The detection of volatile odorants is supposed to begin with their interaction with soluble binding proteins which shuttle the hydrophobic ligands through the aqueous mucus layer towards specific odorant receptors in the ciliary membrane of olfactory neurons. A large family of receptors for odorants has been identified recently; individual receptor types are expressed in subsets of cells distributed in distinct zones of the olfactory epithelium. Ligand-receptor interaction triggers a rapid multistep reaction cascade, ultimately leading to an electrical response of the receptor neuron. Olfactory signaling is terminated by phosphorylation of receptors via a negative feedback reaction catalyzed by two types of kinases.     

Signal recognition and chemoelectrical transduction in olfaction

The mimicking of olfaction is considered to be a promising approach for the construction of artificial odour-sensing systems. In the nose, the detection of volatile odorants begins when the odorant ligands interact with specific odorant receptors in the ciliary membrane of the olfactory neurons. A large family of genes encoding putative odorant receptors has been identified recently. Individual receptor types are expressed in subsets of cells distributed in distinct zones of the olfactory epithelium. Ligand-receptor interaction triggers a rapid multistep reaction cascade, resulting in a “pulse” of second messengers that initiates an electrical response from the receptor neuron. Olfactory signalling is terminated by phosphorylation of receptors via a negative feedback reaction, catalyzed by specific kinases.     

Molecular and cellular basis of human olfaction

The human olfactory systems recognize and discriminate a large number of different odorant molecules. The detection of chemically distinct odorants begins with the binding of an odorant ligand to a specific receptor protein in the ciliary membrane of olfactory neurons. To address the problem of olfactory perception at a molecular level, we have cloned, functionally expressed, and characterized some of the human olfactory receptors from chromosome 17. Our results show that a receptor protein is capable of recognizing the particular chemical substructure of an odor molecule and, therefore, is able to respond only to odorants that have a defined molecular structure. These findings represent the beginning of the molecular understanding of odorant recognition in humans. In the future, this knowledge could be used for the design of synthetic ideal receptors for specific odors (biosensors), or the perfect odor molecule for a given receptor.     

An artificial neural network approach for glomerular activity pattern prediction using the graph kernel method and the gaussian mixture functions

This paper proposes a neural network model for prediction of olfactory glomerular activity aimed at future application to the evaluation of odor qualities. The model's input is the structure of an odorant molecule expressed as a labeled graph, and it employs the graph kernel method to quantify structural similarities between odorants and the function of olfactory receptor neurons. An artificial neural network then converts odorant molecules into glomerular activity expressed in Gaussian mixture functions. The authors also propose a learning algorithm that allows adjustment of the parameters included in the model using a learning data set composed of pairs of odorants and measured glomerular activity patterns. We observed that the defined similarity between odorant structure has correlation of 0.3-0.9 with that of glomerular activity. Glomerular activity prediction simulation showed a certain level of prediction ability where the predicted glomerular activity patterns also correlate the measured ones with middle to high correlation in average for data sets containing 363 odorants.     

Anatomical contributions to odorant sampling and representation in rodents: zoning in on sniffing behavior

Odorant sampling behaviors such as sniffing bring odorant molecules into contact with olfactory receptor neurons (ORNs) to initiate the sensory mechanisms of olfaction. In rodents, inspiratory airflow through the nose is structured and laminar; consequently, the spatial distribution of adsorbed odorant molecules during inspiration is predictable. Physicochemical properties such as water solubility and volatility, collectively called sorptiveness, interact with behaviorally regulable variables such as inspiratory flow rate to determine the pattern of odorant deposition along the inspiratory path. Populations of ORNs expressing the same odorant receptor are distributed in strictly delimited regions along this inspiratory path, enabling different deposition patterns of the same odorant to evoke different patterns of neuronal activation across the olfactory epithelium and in the olfactory bulb. We propose that both odorant sorptive properties and the regulation of sniffing behavior may contribute to rodents' olfactory capacities by this mechanism. In particular, we suggest that the motor regulation of sniffing behavior is substantially utilized for purposes of "zonation" or the direction of odorant molecules to defined intranasal regions and hence toward distinct populations of receptor neurons, pursuant to animals' sensory goals.     

Functional expression of olfactory receptors in yeast and development of a bioassay for odorant screening

The functional expression of olfactory receptors (ORs) is a primary requirement to examine the molecular mechanisms of odorant perception and coding. Functional expression of the rat I7 OR and its trafficking to the plasma membrane was achieved under optimized experimental conditions in the budding yeast Saccharomyces cerevisiae. The membrane expression of the receptor was shown by Western blotting and immunolocalization methods. Moreover, we took advantage of the functional similarities between signal transduction cascades of G protein-coupled receptor in mammalian cells and the pheromone response pathway in yeast to develop a novel biosensor for odorant screening using luciferase as a functional reporter. Yeasts were engineered to coexpress I7 OR and mammalian G(alpha) subunit, to compensate for the lack of endogenous Gpa1 subunit, so that stimulation of the receptor by its ligands activates a MAP kinase signaling pathway and induces luciferase synthesis. The sensitivity of the bioassay was significantly enhanced using mammalian G(olf) compared to the G(alpha15) subunit, resulting in dose-dependent responses of the system. The biosensor was probed with an array of odorants to demonstrate that the yeast-borne I7 OR retains its specificity and selectivity towards ligands. The results are confirmed by functional expression and bioluminescence response of human OR17-40 to its specific ligand, helional. Based on these findings, the bioassay using the luciferase reporter should be amenable to simple, rapid and inexpensive odorant screening of hundreds of ORs to provide insight into olfactory coding mechanisms.