How Do Bugs Smell?

In general, each insect olfactory sensory neuron expresses one member of the olfactory receptor (OR) gene family along with the highly conserved Or83b co-receptor, and the two ORs form a heteromeric complex to function as a chemosensor. Here we provide evidence that heteromeric insect ORs comprise a novel class of ligand-activated nonselective cation channels. Heterologous cells expressing silk moth, fruit fly, or mosquito heteromeric OR complexes exhibited extracellular calcium influx and cation-nonselective ion conductance upon odorant or pheromone stimulation. G protein-mediated signaling was negligible in producing the current elicited by ligand activation of insect ORs. Direct evidence for ligand-gated channels was obtained by outside-out patch-clamp single-channel recording of Xenopus oocyte and HEK293T cell membranes expressing insect OR complexes. The olfactory signal transduction mechanism in insects is clearly distinct from that in vertebrates and appears to be a unique strategy that insects have acquired to respond to the olfactory environment.

Since the emergence of the first living cells, survival has hinged on the ability to detect and localize chemicals in the environment. Modern animal species ranging from insects to mammals express large odorant receptor repertoires to detect the structurally diverse array of volatile molecules important for survival. Despite the essential nature of chemical detection, there is surprising diversity in the signaling mechanisms that different species use for odorant detection. In vertebrates, odorant receptors are classical G-protein coupled, seven transmembrane receptors that activate downstream effector enzymes that, in turn, produce second messengers that open ion channels. However, recent work reveals that insects have adopted different strategies to detect volatile chemicals. In Drosophila, the odorant receptors, predicted to have seven transmembrane domains, have reversed membrane topology compared to classical G-protein coupled receptors. Furthermore, insect odorant receptors appear to form odorant-gated ion channels. Pheromone detection in insects is even more unusual, utilizing soluble, extracellular receptors that undergo conformational activation. These alternate olfactory signaling strategies are discussed in terms of receptor design principles.

The mouse receptor transporting protein RTP1S and the fly SNMP1 support the functional expression of the Drosophila odorant coreceptor Orco in mammalian culture cells

Flying insects have developed a remarkably sensitive olfactory system to detect faint and turbulent odor traces. This ability is linked to the olfactory receptors class of odorant receptors (ORs), occurring exclusively in winged insects. ORs form heteromeric complexes of an odorant specific receptor protein (OrX) and a highly conserved co-receptor protein (Orco). The ORs form ligand gated ion channels that are tuned by intracellular signaling systems. Repetitive subthreshold odor stimulation of olfactory sensory neurons sensitizes insect ORs. This OR sensitization process requires Orco activity. In the present study we first asked whether OR sensitization can be monitored with heterologously expressed OR proteins. Using electrophysiological and calcium imaging methods we demonstrate that D. melanogaster OR proteins expressed in CHO cells show sensitization upon repeated weak stimulation. This was found for OR channels formed by Orco as well as by Or22a or Or56a and Orco. Moreover, we show that inhibition of calmodulin (CaM) action on OR proteins, expressed in CHO cells, abolishes any sensitization. Finally, we investigated the sensitization phenomenon using an ex vivo preparation of olfactory sensory neurons (OSNs) expressing Or22a inside the fly's antenna. Using calcium imaging, we observed sensitization in the dendrites as well as in the soma. Inhibition of calmodulin with W7 disrupted the sensitization within the outer dendritic shaft, whereas the sensitization remained in the other OSN compartments. Taken together, our results suggest that CaM action is involved in sensitizing the OR complex and that this mechanisms accounts for the sensitization in the outer dendrites, whereas further mechanisms contribute to the sensitization observed in the other OSN compartments. The use of heterologously expressed OR proteins appears to be suitable for further investigations on the mechanistic basis of OR sensitization, while investigations on native neurons are required to study the presently unknown additional mechanisms involved in OSN sensitization.

Insects are able to detect a plethora of olfactory cues using a divergent family of odorant receptors (ORs). Despite the divergent nature of this family, related species frequently express several evolutionarily conserved OR orthologues. In the largest order of insects, Coleoptera, it remains unknown whether OR orthologues have conserved or divergent functions in different species. Using HEK293 cells, we addressed this question through functional characterization of two groups of OR orthologues in three species of the Curculionidae (weevil) family, the conifer‐feeding bark beetles Ips typographus L. (“Ityp”) and Dendroctonus ponderosae Hopkins (“Dpon”) (Scolytinae), and the pine weevil Hylobius abietis L. (“Habi”; Molytinae). The ORs of H. abietis were annotated from antennal transcriptomes. The results show highly conserved response specificities, with one group of orthologues (HabiOR3/DponOR8/ItypOR6) responding exclusively to 2‐phenylethanol (2‐PE), and the other group (HabiOR4/DponOR9/ItypOR5) responding to angiosperm green leaf volatiles (GLVs). Both groups of orthologues belong to the coleopteran OR subfamily 2B, and share a common ancestor with OR5 in the cerambycid Megacyllene caryae, also tuned to 2‐PE, suggesting a shared evolutionary history of 2‐PE receptors across two beetle superfamilies. The detected compounds are ecologically relevant for conifer‐feeding curculionids, and are probably linked to fitness, with GLVs being used to avoid angiosperm nonhost plants, and 2‐PE being important for intraspecific communication and/or playing a putative role in beetle–microbe symbioses. To our knowledge, this study is the first to reveal evolutionary conservation of OR functions across several beetle species and hence sheds new light on the functional evolution of insect ORs.

Reconstitution of olfactory or pheromone receptors in heterologous expression systems greatly facilitates the functional analysis of these receptors. Xenopus laevis oocytes can be used to efficiently express insect olfactory or pheromone receptors. In this chapter, we describe how to use Xenopus laevis oocytes for functional assays of insect olfactory receptors. The procedure can be subdivided into the four following steps: (1) in vitro complementary RNA (cRNA) synthesis, (2) isolation of oocytes from female Xenopus laevis, (3) cRNA microinjection into oocytes, and (4) two-electrode voltage-clamp recording. This system can be used to identify odor or pheromone ligands and to analyze structure-function relationships involving receptor proteins of interest.

Insect has a highly sensitive and accurate discrimination capacity for sensing odorants in the environment, though its olfactory system is much simpler than mammalians. Insect olfactory receptor (OR) proteins play key roles in odor detection and discrimination. Unlike the complex olfactory signaling in mammalian, the insect ORs can be directly gated by odorants, and can function without the co-expression of downstream signaling molecules. Therefore, insect ORs hold great promise as detectors in a biosensor for detection of volatile chemicals. Several important OR genes in Drosophila, which respond to explosive vapor or related precursors such as PTEN and 2-ehtyl-hexanol, have been reported. The present work screened and selected OR genes which are sensitive to explosive vapor and related precursors by calcium imaging and electrophysiological techniques. Furthermore, a biomimetic odorant sensor which co-expresses these OR genes and a companion receptor Or83b in living cells is being constructed, in order to classify unknown volatile chemicals and detect specific types of illicit substances. This study provides an alternative way for the construction of odorant sensors, comparing to the traditional electronic noses which are not as sensitive and accurate as natural noses, and would promote the development and application of biomimetic odorant sensors in a range of applications such as security and environmental monitoring.

Insect Olfactory Receptors (ORs) are diverse family of membrane protein receptors responsible for most of the insect olfactory perception and communication, and hence they are of utmost importance for developing repellents or pesticides. Accurate gene prediction of insect ORs from newly sequenced genomes is an important but challenging task. We have developed a dedicated webserver, 'insectOR', to predict and validate insect OR genes using multiple gene prediction algorithms, accompanied by relevant validations. It is possible to employ this server nearly automatically and perform rapid prediction of the OR gene loci from thousands of OR-protein-to-genome alignments, resolve gene boundaries for tandem OR genes and refine them further to provide more complete OR gene models. InsectOR outperformed the popular genome annotation pipelines (MAKER and NCBI eukaryotic genome annotation) in terms of overall sensitivity at base, exon and locus level, when tested on two distantly related insect genomes. It displayed more than 95% nucleotide level precision in both tests. Finally, given the same input data and parameters, InsectOR missed less than 2% gene loci, in contrast to 55% loci missed by MAKER for Drosophila melanogaster. The webserver is freely available on the web at http://caps.ncbs.res.in/insectOR/ and the basic package can be downloaded from https://github.com/sdk15/insectOR for local use. This tool will allow biologists to perform quick preliminary identification of insect olfactory receptor genes from newly sequenced genomes and also assist in their further detailed annotation. Its usage can also be extended to other divergent gene families.

Insect odorant receptors have been reconstituted into lipid nanodiscs and tethered to carbon nanotube field-effect transistors to function as a biosensor. Here, four different insect odorant receptors (ORs) from Drosophila melanogaster (DmelOR10a, DmelOR22a, DmelOR35a, and DmelOR71a) were expressed in Sf9 cells, purified, and reconstituted into lipid nanodiscs. We have demonstrated that each of these ORs produce a selective and highly sensitive electrical response to their respective positive ligands, methyl salicylate, methyl hexanoate, trans-2-hexen-1-al, and 4-ethylguaiacol, with limits of detection in the low femtomolar range. No detection was observed for each OR against control ligands, and empty nanodiscs showed no specific sensor signal for any of the odorant molecules. Our results are the first evidence that insect ORs can be integrated into lipid nanodiscs and used as primary sensing elements for bioelectronic nose technologies.

Drosophila melanogaster vinegar flies have two olfactory organs: the antenna and maxillary palp. Olfactory neurons in these tissues respond to odorants via odorant receptors. Insect odorant receptors are heterotetramers of two proteins: an odorant binding OrX subunit and an Odorant Receptor Co-Receptor (Orco). Mutation of Orco disrupts odorant receptor formation, and abolishes olfactory responses. Some antennal olfactory neurons in Orco mutants have been previously shown to degenerate. Here, we examine if maxillary palp olfactory neurons also degenerate in Orco mutants. We find degeneration occurs both more broadly and more rapidly in Orco mutant maxillary palp olfactory neurons than reported for antennae, with ~60% of all mutant olfactory neurons absent in maxillary palps by 7 days post eclosion. Interestingly, the subset of Orco mutant olfactory neurons that express the Or42a receptor appear resistant to degeneration. These results suggest the maxillary palp might be a suitable model for examining the molecular mechanisms underlying neurodegeneration in sensory neurons.

The striatum performs essential brain functions including movement control, regulation of attention and is a critical component of reward systems. Current druggable targets have displayed limited efficacy in treating striatal diseases including addiction, schizophrenia and Parkinson's disease. G protein‐coupled receptors (GPCRs) are essential drug targets with ~35% of FDA approved medicines targeting these receptors. Of the 350 non‐sensory GPCRs, nearly 120 remain orphan GPCRs, whose endogenous ligands and G protein signaling are unknown. A previous challenge with orphan receptors has been the design of reliable drug screens, but a recent advance in the PRESTO‐Tango assay now allows for a measurement of receptor activation for over 300 GPCRs. Here we pursue the objective of identifying new druggable GPCRs, selectively expressed in the human striatum, using human tissue transcriptomics and the PRESTO‐Tango platform. We conducted a comprehensive genome‐wide survey of human tissue‐selective gene expression using 1640 high‐quality RNA‐seq samples from the Genotype Tissue Expression (GTEx) project. To identify receptor function and screen ligands, human clones of receptors were expressed in HEK293 cells and signaling was assessed using the Glo‐sensor cAMP and Tango beta‐arrestin assay. As a ligand discovery proof‐of‐concept, the Tango assay was used to screen the LOPAC library against five striatal orphan GPCRs. Human gene expression analyses identified expression of 18 striatal‐selective GPCRs. 11 of these receptors have known ligands including established therapeutic drug targets (e.g. dopamine D1, D2 receptors). The remaining 7 human striatal GPCRs, namely, GPR6, 52, 55, 88, 101, 139 and 149 were identified as class A orphan receptors. Biochemical and CRISPR/Cas9 knockout studies confirmed GPR6, GPR52 and GPR101 increased cAMP via Gs/olf G proteins, while GPR88 decreased cAMP levels via Gi/o G proteins. Screening the LOPAC library in parallel against five orphans (6, 52, 88, 101, 149) using the Tango assay revealed 60 compounds that selectively induced a 2‐fold activity above or below vehicle treated cells for one of the five receptors. Dose response assays confirmed two compounds with agonist activity for GPR88 (clozapine and carbachol) and one with agonist activity for GPR149 (PDTC). Interestingly, carbachol and clozapine are FDA approved drugs. Clozapine is a classic atypical antipsychotic medication and inverse agonist at serotonin and DA receptors; and carbachol is a cholinergic agonist currently used for the treatment of glaucoma. PDTC agonist activity for GPR149 represents the first described agonist compound for this orphan receptor. This research provides a template for identifying brain region specific targets using human tissue RNAseq transcriptomic datasets. Our findings also suggest the seven identified human orphan GPCRs are potential drug targets amenable to screening, whose pharmacological modulation may be therapeutic for treating striatum‐related neurological diseases.Support or Funding InformationRising Star Award, UT System (JAA); UTMB institutional funding (JAA); T32 DA07287 (DEF)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Enhanced sensitivity of chimeric insect olfactory co-receptors for detecting odorant molecules

The sensing of vapor odorants is highly demanded in the field of life and medical sciences. Although olfactory receptors (ORs) have potentials to recognize volatile organic compounds, the interaction of ORs, chemical vapors, and peptide components in olfactory mucus has yet to be analyzed to develop OR‐based sensors. A bioinspired electrophysiology technique is shown to record the response of reconstituted insect ORs to chemical vapors. To mimic the interface between ORs and olfactory mucus, OR expressing spheroids were loaded into a hydrogel microchamber array. A negative extracellular field potential shift of spheroids was successfully observed by the stimulation of their vapor cognate ligand. Importantly, the ligand repertoire of the OR of malaria vector mosquito examined by this method differed from that of in vivo studies. Our method is useful to develop protein‐based gas sensing techniques and to examine the binding of ORs and chemical vapors.

Insect odorant receptors are a large family of seven transmembrane proteins believed to be G-protein coupled receptors. The peptide sequences of two odorant receptors within a given species may share as little as 17% identity, and there is limited similarity between receptors of divergent species. One exception is DmOr83b, which is found in Drosophila melanogaster and is highly conserved in at least ten other insect species. DmOr83b is broadly expressed in most of the olfactory sensory neurons of D. melanogaster at most developmental stages, while other odorant receptors tend to have more restricted and specific expression patterns. DmOr83b is critical for D. melanogaster olfaction, and it is involved in properly localizing other odorant receptors possibly by forming heterodimers with these receptors. The C-terminal region has been implicated as sites for such heterodimer formation. Multiple em for motif elicitation (MEME), a hidden markov model based program, was used to uncover three conserved motifs in the C-termini of a vast majority of the odorant receptor peptides from Anopheles gambiae, D. melanogaster, and Apis mellifera. These motifs are also found in DmOr83b and its orthologs and the order of these motifs is conserved as well. The conservation of these motifs among divergent odorant receptors in divergent species suggests functional importance. We propose that these motifs are involved in receptor- receptor protein interactions, contributing to the heterodimer formation between DmOr83b (or its orthologs) and other odorant receptors.

Olfactory receptors (ORs) comprise more than half of the large class I G protein-coupled receptor (GPCR) superfamily. Although cloned over a decade ago, little is known about their properties because wild-type ORs do not efficiently reach the cell surface following heterologous expression. Receptor-receptor interactions strongly influence surface trafficking of other GPCRs, and we examined whether a similar mechanism might be involved in OR surface expression. Olfactory neurons are known to express beta-adrenergic receptors (ARs), and we found that coexpression with beta(2)-ARs, but not any other AR subtypes, dramatically increased mouse 71 (M71) OR surface expression in human embryonic kidney 293 cells. A persistent physical interaction between M71 ORs and beta(2)-ARs was shown by coimmunoprecipitation and by cointernalization of the two receptors in response to their specific ligands. Also, coexpression of wild-type M71 ORs with beta(2)-ARs resulted in cAMP responses to the M71 ligand acetophenone. Finally, in situ hybridization studies showed extensive colocalization of M71 OR and beta(2)-AR expression in mouse olfactory epithelium. These data demonstrate the successful heterologous surface expression of a functional wild-type OR and reveal that persistent physical association with other GPCRs can control OR surface expression.