Mutational Analysis of Cysteine Residues of the Insect Odorant Co-receptor (Orco) from Drosophila melanogaster Reveals Differential Effects on Agonist- and Odorant-tuning Receptor-dependent Activation

Olfaction plays a dominant role in the mate-finding and host selection behaviours of the codling moth (Cydia pomonella), an important pest of apple, pear and walnut orchards worldwide. Antennal transcriptome analysis revealed a number of abundantly expressed genes related to the moth olfactory system, including those encoding the olfactory receptors (ORs) CpomOR1, CpomOR3 and CpomOR6a, which belong to the pheromone receptor (PR) lineage, and the co-receptor (CpomOrco). Using heterologous expression, in both Drosophila olfactory sensory neurones and in human embryonic kidney cells, together with electrophysiological recordings and calcium imaging, we characterize the basic physiological and pharmacological properties of these receptors and demonstrate that they form functional ionotropic receptor channels. Both the homomeric CpomOrco and heteromeric CpomOrco + OR complexes can be activated by the common Orco agonists VUAA1 and VUAA3, as well as inhibited by the common Orco antagonists amiloride derivatives. CpomOR3 responds to the plant volatile compound pear ester ethyl-(E,Z)-2,4-decadienoate, while CpomOR6a responds to the strong pheromone antagonist codlemone acetate (E,E)-8,10-dodecadien-1-yl acetate. These findings represent important breakthroughs in the deorphanization of codling moth pheromone receptors, as well as more broadly into insect ecology and evolution and, consequently, for the development of sustainable pest control strategies based on manipulating chemosensory communication.

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“…The sequence is consistent with the selectivity sequences previously reported for Orco-based channels from other insects3132.…”

Section: Resultssupporting

confidence: 90%

Candidate pheromone receptors of codling moth Cydia pomonella respond to pheromones and kairomones

Cattaneo

Gonzalez

Bengtsson

et al. 2017

Sci Rep

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“…We are currently unable to distinguish ECs that mediate intramolecular versus intersubunit interactions in these receptors. Nevertheless, a systematic mutational analysis of cysteine residues in D. melanogaster ORCO revealed a particularly important contribution of those cysteines in IL3 to forming functional homomeric and/or heteromeric ion channels 35 ( Supplementary Table 2 ). In summary, although these observations do not validate the ECs and structural models per se , the striking coincidence of known functionally-important sites with highly coupled residues gives basic confidence in our results.…”

Section: Resultsmentioning

confidence: 99%

Amino acid coevolution reveals three-dimensional structure and functional domains of insect odorant receptors

et al. 2015

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Insect Odorant Receptors (ORs) comprise an enormous protein family that translates environmental chemical signals into neuronal electrical activity. These heptahelical receptors are proposed to function as ligand-gated ion channels and/or to act metabotropically as G protein-coupled receptors (GPCRs). Resolving their signalling mechanism has been hampered by the lack of tertiary structural information and primary sequence similarity to other proteins. We use amino acid evolutionary covariation across these ORs to define restraints on structural proximity of residue pairs, which permit de novo generation of three-dimensional models. The validity of our analysis is supported by the location of functionally important residues in highly constrained regions of the protein. Importantly, insect OR models exhibit a distinct transmembrane domain packing arrangement to that of canonical GPCRs, establishing the structural unrelatedness of these receptor families. The evolutionary couplings and models predict odour binding and ion conduction domains, and provide a template for rationale structure-activity dissection.

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“…AalOR10 and AalOR88 do not have the putative CaM binding site and channel gate sequences. Recent studies on structural features and function of Orco show that ICL3 is important for Orco channel activation [ 43 ], and CaM (in ICL2) activity affects the function of Orco channels [ 44 ]. Extracellular loop 2 (ECL-2) and TM4 are essential for the odorant response-specificity of AgOR15 [ 45 ].…”

Section: Resultsmentioning

confidence: 99%

Functional analysis of Orco and odorant receptors in odor recognition in Aedes albopictus

Liu

Xie

et al. 2016

Parasites Vectors

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BackgroundAedes albopictus is a globally invasive mosquito and a major vector of arboviruses, including dengue, Zika and Chikungunya. Olfactory-related behaviors, particularly host-seeking, offer opportunities to disrupt the disease-transmission process. A better understanding of odorant receptors (ORs) may assist in explaining host selection and location, and contribute to novel strategy of vector control.MethodsBased on previous prediction of 158 putative odorant receptors by Ae. albopictus genome analysis, 29 AalORs were selected for tissue-specific expression profiles in the present study. AalOrco (AalOR7), AalOR10 and AalOR88, highly expressed in female olfactory tissues, were chosen for further structure predictions as well as functional validation including calcium imaging assay in human embryonic kidney (HEK293) cells and RNA interference assay in Ae. albopictus. We also conducted electrophysiological and behavioral assays in mosquitoes after RNA interference of the three genes to determine their roles in host-seeking.ResultsThe results support previous conclusions that individual conventional (ORXs) and Orco can form heteromeric complexes to recognize odorants and respond to components of human volatiles in HEK293 cells. The reduction of AalOrco transcript levels led to a significant decrease in host-seeking and confusion in host preference. In contrast, AalOR10 and AalOR88 knockdown mosquitoes showed no significant behavioral differences compared with controls. The functions of conventional ORs at least AalOR10 and AalOR88 are abolished with inhibited expression of the Orco gene orthologs, along with the concomitant relevant olfactory behavior.ConclusionsCombining structural and functional data, we conclude that the product of the Orco gene in this mosquito is crucial for transmitting olfactory signaling and conventional ORs contribute directly to odorant recognition. Our results provide insight into the linkage between odorant receptors and host-seeking in this important vector species.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-016-1644-9) contains supplementary material, which is available to authorized users.

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Mutational Analysis of Cysteine Residues of the Insect Odorant Co-receptor (Orco) from Drosophila melanogaster Reveals Differential Effects on Agonist- and Odorant-tuning Receptor-dependent Activation

Cited by 28 publications

References 33 publications

Candidate pheromone receptors of codling moth Cydia pomonella respond to pheromones and kairomones

Candidate pheromone receptors of codling moth Cydia pomonella respond to pheromones and kairomones

Amino acid coevolution reveals three-dimensional structure and functional domains of insect odorant receptors

Functional analysis of Orco and odorant receptors in odor recognition in Aedes albopictus

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