Odorant Ligands Research Articles

Odr-10 Encodes a Seven Transmembrane Domain Olfactory Receptor Required for Responses to the Odorant Diacetyl

Olfactory signaling is initiated by interactions between odorants and olfactory receptors. We show that the C. elegans odr-10 gene is likely to encode a receptor for the odorant diacetyl. odr-10 mutants have a specific defect in chemotaxis to diacetyl, one of several odorants detected by the AWA olfactory neurons. odr-10 encodes a predicted seven transmembrane domain receptor; a green fluorescent protein–tagged Odr-10 protein is localized to the AWA sensory cilia. odr-10 expression is regulated by odr-7, a transcription factor implicated in AWA sensory specification. Expression of odr-10 from a heterologous promoter directs behavioral responses to diacetyl, but not to another odorant detected by the AWA neurons. These results provide functional evidence for a specific interaction between an olfactory receptor protein and its odorant ligand.

Purification and characterization of multiple forms of odorant/pheromone binding proteins in the antennae of mamestra brassicae (noctuidae)

Proteins extracted from the antennae of Mamestra brassicae (L.) (Lepidoptera: Noctuidae) adults were biochemically characterized as pheromone-binding proteins (PBP) and general odorant-binding proteins (GOBP). PBP and GOBP were purified by two successive and different HPLC (high performance liquid chromatography) systems and native polyacrylamide gel electrophoresis (native-PAGE). Their N-terminal sequence was determined by Edman microsequencing. The combined results showed evidence for three different PBPs in males, and two different PBPs in females. In addition, one GOBP was characterized in both males than in females antennae. In the males, two isoforms of PBP have the same N-terminal sequence, but different apparent mobilities and hydrophobicities: they could be separated by electrophoresis and reverse phase-HPLC (RP-HPLC). The other PBP sequence (SQEIM) showed particularly high homology (88 %) with the PBP of Heliothis virescens, another noctuid moth. The existence of several forms of PBP in the same animal strongly supports the hypothesis of the specificity of binding between the proteins and their odorant ligands, the pheromonal compounds. The observed microdiversity at the soluble proteins level could provide a good model for studying their involvement in the initial stages of odor discrimination.

Expression of an olfactory receptor in Escherichia coli: purification, reconstitution, and ligand binding.

An olfactory receptor has been expressed in bacterial cells as a fusion protein with glutathione S-transferase (GST). Overexpression of receptor protein yielding about 10% of the cell protein was achieved with mutants lacking the N-terminus and the first transmembrane region or with mutants carrying three positively charged residues in the first intracellular loop. The overexpressed fusion protein accumulated in inclusion bodies and could be solubilized in detergent. It was purified by metal chelation chromatography based on a C-terminal 6-histidine tag, and the GST portion was removed after proteolytic cleavage. The purified receptor was reconstituted into lipid vesicles and specific binding of odor ligands was shown by photoaffinity labeling and tryptophan fluorescence measurements. Thus, for the first time, an odorant receptor/ligand pair becomes available in large amounts for biophysical and screening studies.

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.

Spatial segregation of odorant receptor expression in the mammalian olfactory epithelium

The signal elicited by the interaction of odorous ligands with receptors on olfactory sensory neurons must be decoded by the brain to determine which of the numerous receptors have been activated. We have examined the patterns of odorant receptor expression in the rat olfactory epithelium to determine whether the mammalian olfactory system employs spatial segregation of sensory input to encode the identity of an odorant stimulus. In situ hybridization experiments with probes for 11 different odorant receptors demonstrate that sensory neurons expressing distinct receptors are topologically segregated into a small number of broad, yet circumscribed, zones within the olfactory epithelium. Within a given zone, however, olfactory neurons expressing a specific receptor appear to be randomly distributed, rather than spatially localized. The complex mammalian olfactory system may therefore compartmentalize the epithelium into anatomically and functionally discrete units, such that each zone expresses only a subset of the entire receptor repertoire.

Coding of odor molecules by mitral/tufted cells in rabbit olfactory bulb. I. Aliphatic compounds

1. Recordings of extracellular spike responses were made from single mitral/tufted cells in the main olfactory bulb of urethan-anesthetized rabbits. Olfactory epithelium ipsilateral to the recorded olfactory bulb was stimulated with homologous series of aliphatic compounds using periodic artificial inhalations. 2. In the dorsomedial part of the main olfactory bulb, single mitral/tufted cells were activated by subsets of n-fatty acids with similar hydrocarbon chain lengths. Response selectivities of single mitral/tufted cells were examined in detail using a series of n-fatty acids at five different concentrations. The results indicate that although the range of effective fatty acids is broader at the higher concentrations, the best response at higher concentrations was similar to that determined at lower concentrations. 3. Analysis of single-unit responses to the panel of fatty acids, including those with branched hydrocarbon chains, suggested that the determinants for the response specificities of individual mitral/tufted cells in the dorsomedial region include the overall size of hydrocarbon chains of the odor ligand molecules. 4. Single mitral/tufted cells in the dorsomedial region tended to be activated not only by fatty acids but also by n-aliphatic aldehydes. For a panel of a homologous series of n-aldehydes at five different concentrations, individual mitral/tufted cells showed response selectivity to subsets of aldehydes with similar hydrocarbon chain lengths. 5. In most cases, normal aliphatic alcohols and alkanes were ineffective in activating mitral/tufted cells in the dorsomedial region. This suggests that carbonyl group (--C = O) in the odor molecules plays an important role in determining response specificity of these neurons. 6. Examination with an expanded panel of stimulus odor molecules that included ketones and esters indicated that single mitral/tufted cells sensitive to subsets of fatty acids and n-aliphatic aldehydes were also responsive to subsets of ketones and/or esters having hydrocarbon chain lengths similar to those of the effective fatty acids and aldehydes. 7. The present results show a clear correlation between the tuning specificity of individual mitral/tufted cells and the stereochemical structure of the odor molecules, with respect to 1) length and/or structure of hydrocarbon chain, 2) difference in functional group, and 3) position of the functional group within the molecule. 8. A hypothetical diagram suggesting functional convergence of olfactory nerve input to individual glomeruli is proposed to explain the mechanism for selective activation of individual mitral/tufted cells by a range of odor molecules with similar stereochemical structures.

Expression of olfactory receptors in Xenopus oocytes.

The rat olfactory epithelium and the amino acid-sensitive catfish olfactory system have been used as models to study the molecular mechanisms of olfactory transduction. Here we report the functional expression of rat and catfish olfactory receptors in Xenopus oocytes injected with mRNA isolated from the respective tissues. Application of odor ligands to injected oocytes, monitored by two-electrode voltage clamp, activates stimulus-dependent transmembrane currents that reverse direction at about the chloride equilibrium potential. The currents show characteristic secondary oscillations that are presumed to reflect underlying Ca2+ oscillations. Similar ligand-activated membrane currents induced in oocytes after injection of other mRNAs have been shown to be due to activation of endogenous Ca(2+)-activated chloride channels. In summary, our results demonstrate the usefulness of the Xenopus oocyte expression system for cloning and characterization of olfactory receptors in both fish and mammalian species.

Toward a pharmacology of odor receptors and the processing of odor images

Odor molecules may be considered as molecular ligands which bind to receptors in the olfactory sensory neurons to give rise to the sensory response. Binding studies in whole sensory epithelia suggest that the receptors also bind muscarinic cholinergic antagonists. Preliminary electrophysiological evidence indicates that muscarinic and beta adrenergic antagonists block odor-elicited membrane currents in single isolated salamander sensory neurons. These results support the idea that models developed for analyzing ligand binding by members of the 7 transmembrane domain family of membrane receptors may apply rather closely to olfactory transduction. We suggest that sensory neurons express single receptor types with differeing degrees of affinity for different ligands. We further suggest that glomeruli in the olfactory bulb function as labeled lines for particular sets of odor ligand determinants, and that interglomerular circuits bind together similar glomeruli and enhance contrast between dissimilar glomeruli. The odor image laid down in the sensory neuron population is thus subjected to abstracting and enhancement at the glomerular stage, prior to being transmitted for further processing in the deeper layers of the olfactory bulb and in the olfactory cortex.

A novel multigene family may encode odorant receptors: A molecular basis for odor recognition

The mammalian olfactory system can recognize and discriminate a large number of different odorant molecules. The detection of chemically distinct odorants presumably results from the association of odorous ligands with specific receptors on olfactory sensory neurons. To address the problem of olfactory perception at a molecular level, we have cloned and characterized 18 different members of an extremely large multigene family that encodes seven transmembrane domain proteins whose expression is restricted to the olfactory epithelium. The members of this novel gene family are likely to encode a diverse family of odorant receptors.

Odorant-binding protein. Characterization of ligand binding.

We have characterized the odorant binding properties of purified bovine odorant-binding protein (OBP) using as a ligand [3H]3,7-dimethyloctan-1-ol ([3H]DMO). A broad variety of odorants, including terpenes, aldehydes, esters, and musks, bind to OBP with affinities of 0.2 to 100 microM. Odorant affinities for OBP correlate most closely with their stimulation of an odorant-sensitive adenylyl cyclase as well as hydrophobicity. We also measured the kinetics of binding for the ligands, [3H]DMO and 2-isobutyl-3-[3H]methoxypyrazine. Dissociation of both is markedly accelerated in the presence of excess unlabeled ligand. Competition curves of displacers for [3H]DMO binding are shallow, and saturation binding isotherms for 3H-odorants are curvilinear. These kinetic and equilibrium binding properties suggest that OBP interactions with odorant ligands are negatively cooperative.