Structural basis of odorant recognition by a human odorant receptor.

Our sense of smell enables us to navigate a vast space of chemically diverse odour molecules. This task is accomplished by the combinatorial activation of approximately 400 odorant G protein-coupled receptors encoded in the human genome1-3. How odorants are recognized by odorant receptors remains unclear. Here we provide mechanistic insight into how an odorant binds to a human odorant receptor. Using cryo-electron microscopy, we determined the structure of the active human odorant receptor OR51E2 bound to the fatty acid propionate. Propionate is bound within an occluded pocket in OR51E2 and makes specific contacts critical to receptor activation. Mutation of the odorant-binding pocket in OR51E2 alters the recognition spectrum for fatty acids of varying chain length, suggesting that odorant selectivity is controlled by tight packing interactions between an odorant and an odorant receptor. Molecular dynamics simulations demonstrate that propionate-induced conformational changes in extracellular loop 3 activate OR51E2. Together, our studies provide a high-resolution view of chemical recognition of an odorant by a vertebrate odorant receptor, providing insight into how this large family of G protein-coupled receptors enables our olfactory sense.

Structural basis of amine odorant perception by a mammal olfactory receptor.

Odorants are detected as smell in the nasal epithelium of mammals by two G-protein-coupled receptor families, the odorant receptors and the trace amine-associated receptors1,2 (TAARs). TAARs emerged following the divergence of jawed and jawless fish, and comprise a large monophyletic family of receptors that recognize volatile amine odorants to elicit both intraspecific and interspecific innate behaviours such as attraction and aversion3-5. Here we report cryo-electron microscopy structures of mouse TAAR9 (mTAAR9) and mTAAR9-Gs or mTAAR9-Golf trimers in complex with ß-phenylethylamine, N,N-dimethylcyclohexylamine or spermidine. The mTAAR9 structures contain a deep and tight ligand-binding pocket decorated with a conserved D3.32W6.48Y7.43 motif, which is essential for amine odorant recognition. In the mTAAR9 structure, a unique disulfide bond connecting the N terminus to ECL2 is required for agonist-induced receptor activation. We identify key structural motifs of TAAR family members for detecting monoamines and polyamines and the shared sequence of different TAAR members that are responsible for recognition of the same odour chemical. We elucidate the molecular basis of mTAAR9 coupling to Gs and Golf by structural characterization and mutational analysis. Collectively, our results provide a structural basis for odorant detection, receptor activation and Golf coupling of an amine olfactory receptor.

Cryo-EM structure of the insect olfactory receptor Orco.

The olfactory system must recognize and discriminate amongst an enormous variety of chemicals in the environment. To contend with such diversity, insects have evolved a family of odorant-gated ion channels comprised of a highly conserved co-receptor (Orco) and a divergent odorant receptor (OR) that confers chemical specificity. Here, we present the single-particle cryo-electron microscopy structure of an Orco homomer from the parasitic fig wasp Apocrypta bakeri at 3.5 Å resolution, providing structural insight into this receptor family. Orco possesses a novel channel architecture, with four subunits symmetrically arranged around a central pore that diverges into four lateral conduits that open to the cytosol. The Orco tetramer has few inter-subunit interactions within the membrane and is bound together by a small cytoplasmic anchor domain. The minimal sequence conservation among ORs maps largely to the pore and anchor domain, shedding light on how the architecture of this receptor family accommodates its remarkable sequence diversity and facilitates the evolution of odour tuning.

Ultrastructure of the olfactory epithelium in a flatfish, barfin flounder (Verasper moseri).

In this study, we examined the olfactory epithelium (OE) of the barfin flounder by transmission electron microscopy. As in the case of the ordinary teleost, the OE of the barfin flounder had 3 types of olfactory receptor cells (ciliated olfactory receptor cell, microvillous olfactory receptor cell and crypt cell), 3 types of supporting cells (ciliated, microvillous and crypt supporting cells) and basal cells. Each type of OE cells in the barfin flounder had similar ultrastructure to that of the ordinary teleost. Crypt cell is the third type of olfactory receptor cell unique to fish, whose function is unclear. The barfin flounder may be a suitable material to study crypt cells because it has relatively abundant crypt cells in the OE.

Characterization of perinatally born glutamatergic neurons of the mouse olfactory bulb based on NeuroD6 expression reveals their resistance to sensory deprivation.

During postnatal olfactory bulb (OB) neurogenesis, predetermined stem cells residing in the ventricular-subventricular zone continuously generate progenitors that migrate in the rostral migratory stream and integrate into the OB. Although the vast majority of these postnatally generated interneurons are inhibitory, a sub-fraction represents glutamatergic neurons that integrate into the superficial glomerular layer. In the present work, we demonstrate that the bHLH transcription factor NeuroD6 is specifically and transitorily expressed in the dorsal neurogenic lineage that generates glutamatergic juxtaglomerular cells (JGCs) for the OB. Using lineage tracing combined with whole brain clearing, we provide new insight into timing of generation, morphology, and connectivity of glutamatergic JGCs. Specifically, we show that all glutamatergic JGCs send complex axons with varying projection patterns into different layers of the OB. Moreover, we find that, contrary to GABAergic OB interneurons, glutamatergic JGCs survive under sensory deprivation, indicating that inhibitory and excitatory populations are differentially susceptible to environmental stimulation.

Insect-like olfactory adaptations in the terrestrial giant robber crab.

The robber crab (Birgus latro), also known as the coconut crab, is the world's largest land-living arthropod, with a weight reaching 4 kg and a length of over half a meter. Apart from the marine larval stage, this crab is fully terrestrial, and will actually drown if submerged in water. A transition from sea to land raises dramatically new demands on the sensory equipment of an animal. In olfaction, the stimulus changes from hydrophilic molecules in aqueous solution to mainly hydrophobic in the gaseous phase. The olfactory system of land crabs thus represents an excellent opportunity for investigating the effects of the transition from sea to land. Have land crabs come to the same solutions as other terrestrial animals, or is their olfactory sense characterized by unique innovations? Here, we show that the robber crab has evolved an olfactory sense with a high degree of resemblance to the insect system. The similarities extend to physiological, behavioral, and morphological characters. The insect nose of the robber crab is a striking example of convergent evolution and nicely illustrates how similar selection pressures result in similar adaptation.

Histochemical and ultrastructural analyses of the lubrication systems in the olfactory organs of soft-shelled turtle.

In general, the nasal cavity of turtles is divided into two chambers: the upper chamber, lined with the olfactory epithelium containing ciliated olfactory receptor cells, and the lower chamber, lined with the vomeronasal epithelium containing microvillous receptor cells. In the nasal cavity of soft-shelled turtles, however, differences between the upper and lower chamber epithelia are unclear due to the presence of ciliated receptor cells in both epithelia. In the olfactory organ of vertebrates, the surface of sensory epithelium is covered with secretory products of associated glands and supporting cells, playing important roles in the olfaction by dissolving odorants and transporting them to the olfactory receptors. Here, the associated glands and supporting cells in the olfactory organ of soft-shelled turtles were analyzed histochemically and ultrastructurally. The upper chamber epithelium possessed associated glands, constituted by cells containing serous secretory granules; whereas, the lower chamber epithelium did not. In the upper chamber epithelium, secretory granules filled the supranuclear region of supporting cells, while most of the granules were distributed near the free border of supporting cells in the lower chamber epithelium. The secretory granules in the supporting cells of both epithelia were seromucous, but alcian blue stained them differently from each other. In addition, distinct expression of carbohydrates was suggested by the differences in lectin binding. These data indicate the quantitative and qualitative differences in the secretory properties between the upper and lower chamber epithelia, suggesting their distinct roles in the olfaction.

Immunohistochemical analysis for G protein in the olfactory organs of soft-shelled turtle, Pelodiscus sinensis.

In turtles, the epithelia lining the upper and lower chambers of the nasal cavity project axons to the ventral and dorsal parts of the olfactory bulbs, respectively. In a semi-aquatic soft-shelled turtle, Pelodiscus sinensis, more than 1,000 odorant receptor genes have been found, but it is not known where they are expressed. In this study, we aimed to clarify the distribution of cells expressing these genes in the olfactory organs of soft-shelled turtles. Immunoreactions for the Gαolf, the α subunit of G protein coupled to the odorant receptors, were detected on the surface of epithelia lining both the upper and lower chambers of the nasal cavity. The receptor cells in the epithelium of both chambers possessed cilia on the tip of their dendrites, whereas microvillous, non-ciliated, receptor cells were not found. These data suggest that the odorant receptor genes are expressed by the ciliated receptor cells in the upper and lower chamber epithelia. Precise location of the vomeronasal epithelium is not known at present.

Odorant receptors activated by amino acids in sensory neurons of the channel catfish Ictalurus punctatus.

Odorant receptors activated by amino acids were investigated with patch-clamp techniques in olfactory receptor neurons of the channel catfish, Ictalurus punctatus. The L-isomers of alanine, norvaline, arginine, and glutamate, known to act predominantly on different olfactory receptor sites, activated nondesensitizing inward currents with amplitudes of -2.5 to -280 pA in olfactory neurons voltage-clamped at membrane potentials of -72 or -82 mV. Different amino acids were shown to induce responses in the same sensory neurons; however, the amplitude and the kinetics of the observed whole cell currents differed among the stimuli and may therefore reflect activation of different amino acid receptor types or combinations of receptor types in these cells. Amino acid-induced currents appeared to have diverse voltage dependence and could also be classified according to the amplitude of the spontaneous channel fluctuations underlying the macroscopic currents. A mean single-channel conductance (gamma) of 360 fS was estimated from small noise whole-cell currents evoked by arginine within the same olfactory neuron in which a mean gamma value of 23.6 pS was estimated from 'large noise' response to norvaline. Quiescent olfactory neurons fired bursts of action potentials in response to either amino acid stimulation or application of 8-Br-cyclic GMP (100 microM), and voltage-gated channels underlying generation of action potentials were similar in these neurons. However, in whole-cell voltage-clamp, 8-Br-cyclic GMP evoked large rectangular current pulses, and single-channel conductances of 275, 220, and 110 pS were obtained from the discrete current levels. These results suggest that in addition to the cyclic nucleotide-gated transduction channels, olfactory neurons of the channel catfish possess a variety of odor receptors coupled to different types of transduction channels.

Differential distribution of olfactory receptor neurons in goldfish: structural and molecular correlates.

The olfactory system of many terrestrial vertebrates comprises a main olfactory organ and a vomeronasal organ each containing a morphologically distinct type of olfactory receptor neuron (ORN). The two cell types also differ in the expression of G-proteins and odorant receptor molecules. Fish do not have a vomeronasal organ, and their olfactory neurons-three different morphological types-are contained in one epithelium. The olfactory organ of goldfish appears as a rosette, with the sensory epithelium lying along the proximal portion of each lamella, where it attaches to the midline raphe. Using immunocytochemistry, in situ hybridization, and electron microscopy, we tested whether a correlation exists between receptor cell morphology, distribution of cell type within the sensory epithelium, and expression of odorant receptors and G-proteins. A strong correlation exists between ORN morphology, type of odorant receptor and G-protein expressed and the distribution of sensory cells within the olfactory epithelium. The Buck and Axel type of odorant receptor and Galpha(olf) are expressed in tall ciliated ORNs distributed homogenously across the entire sensory epithelium. In contrast, microvillous ORNs expressing V2R-like receptors, and Galpha(o), Galpha(q), or Galpha(i-3), and crypt type ORNs expressing Galpha(o) and Galpha(q), are preferentially located along the dorsal margin of the epithelium and near the midline raphe. V2R- and OR-type receptor molecules do not colocalize in one cell, and only crypt-type ORNs express more than one G-protein.

Non-conventional role of lysosomal acid phosphatase in olfactory receptor axons: co-localization with growth-associated phosphoprotein-43.

Olfactory receptor neurons undergo a continuous turnover in adult mammals. It is largely unknown how their axons invade the olfactory bulb and induce synaptic re-organization in glomeruli. Here, the cytochemical localization of lysosomal acid phosphatase has been studied in olfactory bulbs of adult rats and mice. The enzyme has been identified by specific substrate, inhibitors and absence in lysosomal acid phosphatase-knockout mice. Lysosomal acid phosphatase is located in primary and secondary lysosomes, which are unevenly distributed in the olfactory nerve layer and among olfactory glomeruli. In consecutive sections of glomeruli, the intensity of lysosomal acid phosphatase immunoreactivity co-varied with that of growth-associated phosphoprotein. Electron microscopically, differential lysosomal acid phosphatase staining in glomeruli corresponded to different proportions of labelled and unlabelled axons. Quantification revealed that lysosomal acid phosphatase labelling was strongest in non-synaptic profiles of terminal axons, while it was weak in or even missing from most synaptic profiles. Hence, growing olfactory axons apparently carry more lysosomal acid phosphatase than those which have established synaptic contacts. Following olfactory deafferentation both lysosomal acid phosphatase activity and growth-associated phosphoprotein-43 are lost from glomeruli, suggesting that both proteins are expressed in olfactory sensory axons during growth, while lysosomal acid phosphatase is apparently not a marker of anterograde terminal degeneration.

A piezoelectric biosensor as an olfactory receptor for odour detection: electronic nose.

Humans can detect and differentiate the presence of different odours even at trace levels of these odorous compounds. The odour quantification of any particular samples is normally based on conventional panel decisions. Other analytical instruments could be used to detect trace levels of odorous molecules. This study presents the results of a biological sensor system subject to different odorants. The system consists of a sensor in which the isolated olfactory receptor proteins (ORPs) from bullfrogs (Rana spp.) were coated onto the surface of a piezoelectric (PZ) electrode, similar to the mechanism of human olfaction. The PZ crystal served as a signal transducer. The results indicate rapid (about 400 s), reversible, and longterm (up to 3 months) stable responses to different volatile compounds such as n-caproic acid, isoamyl acetate, n-decyl alcohol, beta-ionone, linalool, and ethyl caporate. The sensitivity of the sensor ranges from 10(-6)-10(-7) g, fully correlated with the olfactory threshold values of human noses. An array of six sensors consisting of five fractionated ORPs and one referenced phospholipid probe is able to respond to different odorants and form a typical fingerprint for each odorant.

Hormonal control of phase-related changes in the number of antennal sensilla in the desert locust, Schistocerca gregaria: possible involvement of [His7]-corazonin.

The effect of [His(7)]-corazonin on the abundance of antennal sensilla in the desert locust, Schistocerca gregaria, was investigated to test the hypothesis that injection of this neuropeptide would mimic a crowding effect. Solitarious locusts (reared in isolation) were injected with [His(7)]-corazonin at the 3rd nymphal instar and the numbers of sensilla on the 2nd, 8th and 14th antennal segments in the adult stage were compared with those for oil-injected solitarious controls or un-injected gregarious locusts (reared in group). The numbers of sensilla on these antennal segments were all reduced significantly after [His(7)]-corazonin injection compared with those for oil-injected controls, but similar to the values for gregarious individuals. Among the four major types of olfactory sensilla, coeloconic, trichoid, basiconic type A and basiconic type B, [His(7)]-corazonin injection influenced the abundance of all but the last type. The effect of [His(7)]-corazonin injection varied with the time of injection; the earlier the injection the larger the effects on the abundance of total antennal sensilla on the 8th segment, although the way in which the injection affected the abundance varied with the sensillum type. A hypothesis explaining how crowding affects the abundance of antennal sensilla and other phase-related characteristics through changes in [His(7)]-corazonin concentrations was proposed.

Role of topology in electrical properties of bacterio-rhodopsin and rat olfactory receptor I7.

We report on electrical properties of the two sensing proteins: bacteriorhodopsin and rat olfactory receptor OR-I7. As relevant transport parameters we consider the small-signal impedance spectrum and the static current-voltage characteristics. Calculations are compared with available experimental results and the model predictability is tested for future perspectives.

Analysis of penetrance and expressivity during ontogenesis supports a stochastic choice of zebrafish odorant receptors from predetermined groups of receptor genes.

Olfactory receptor neurons select a single odourant receptor gene for expression out of a large gene family. The mechanisms of this extreme selectivity are largely unknown. We have determined in detail the developmental expression dynamics of a representative subset of the zebrafish odourant receptor repertoire, using in situ hybridization analysis. We have thus generated a dataset, which allows us to test hypotheses of odourant receptor gene regulation. The receptors chosen belong to four different groups with respect to ontogenetic onset of expression (onset groups). Statistical analysis of the data supports a model in which the final choice of an individual odourant receptor gene occurs stochastically from within a group of genes sharing a deterministically defined onset of expression. Genomic mapping revealed a pronounced correlation of onset of expression with genomic neighbourhood. During a protracted juvenile developmental period individual regulatory influences seem to modify the expression of odourant receptor genes, a notable example being a transient decrease in expressivity of two odourant receptor genes.

Phenol and lactone receptors in the distal sensilla of the Haller's organ in Ixodes ricinus ticks and their possible role in host perception.

Extract of steer wool odor was found to excite olfactory receptor(s) in a wall-pore olfactory sensillum on the distal knoll of the Haller's organ. Three active volatile compounds were revealed in this odor by gas chromatography. Electrophysiological experiments revealed two types of receptors (sensory neurons) within the sensilla examined. One type of receptor responded only to phenolic derivatives, such as o-chlorophenol, o-bromophenol, o-methylphenol, 2,6-dichlorophenol, 2,6-dibromophenol, 2,4,6-trichlorophenol, but not to o-nithrophenol, p-methylphenol, 2,5-dichlorophenol, 3,5-dichlorophenol, 2,6-dinithrophenol, 2,6-dimethylphenol, and pentachlorophenol. The other type of receptor responded only to gamma-valerolactone. It is assumed that these cells play an important role in perception of a host from long distances (10-15 m), which is typical of Ixodes ricinus ticks.

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.