electrophysiological-recording-from-drosophila-labellar-taste-sensilla

Electrophysiological Recording From <em>Drosophila</em> Labellar Taste Sensilla

The peripheral taste response of insects can be powerfully investigated with electrophysiological techniques. The method described here allows the ...

Yale University

Olfactory receptors (ORs) are seven transmembrane proteins that are responsible for the transduction of volatiles into neuronal signals. Their low sequence homology means that the prediction of ligands for ORs based on extrapolation from empirical data of other ORs is difficult, so an experimental approach must be used. Here, we report a functional assay for insect ORs using calcium-imaging in Sf9 cells. We find that the interaction of the odorant, ethyl butyrate, with the Drosophila melanogaster olfactory receptor Or22a is both dose-dependent and highly sensitive, with Or22a responding to ethyl butyrate with an EC(50) of (1.58+/-0.82)x10(-11)M. This degree of sensitivity does not require the addition of odorant binding proteins or downstream signal transduction elements. Furthermore, we demonstrate that Or22a expressed in Sf9 cells has a similar response profile to a range of odorants previously tested in vivo. This functional assay system will provide a useful tool for the de-orphaning of ORs from a wide range of insect species that are yet to have ligands assigned, and will help provide insight into OR specificity and mechanism of activation.

Functional analysis of a Drosophila melanogaster olfactory receptor expressed in Sf9 cells.

Olfaction in Drosophila is mediated by a large family of membrane-bound odorant receptor proteins (Ors). In heterologous cells, we investigated whether the structural features and signalling mechanisms of ligand-binding Drosophila Ors are consistent with them being G protein-coupled receptors (GPCRs). The detailed membrane topology of Or22a was determined by inserting epitope tags into the termini and predicted loop regions. Immunocytochemistry experiments in Drosophila S2 cells imply that Or22a has seven transmembrane domains but that its membrane topology is opposite to that of GPCRs, with a cytoplasmic N-terminus and extracellular C-terminus. To investigate Or signalling mechanisms, we expressed Or43b in Sf9 and HEK293 cells, and show that inhibitors of heterotrimeric G proteins (GDP-beta-S), adenylate cyclase (SQ22536), guanylyl cyclase (ODQ), cyclic nucleotide phosphodiesterases (IBMX) and phospholipase C (U73122) have negligible impact on Or43b responses. Whole cell patching of Or43b/Or83b-transfected HEK293 cells revealed the opening of plasma membrane cation channels on addition of ligand. The response was blocked by lanthanum and by 2-APB, but not by Ruthenium red or SKF96365. Based on these data, we conclude that Drosophila Ors comprise a novel family of seven transmembrane receptors that in HEK293 cells signal by opening cation channels, through a mechanism that is largely independent of G proteins.

Drosophila odorant receptors are novel seven transmembrane domain proteins that can signal independently of heterotrimeric G proteins.

Moths recognize a wide range of volatile compounds, which they use to locate mates, food sources, and oviposition sites. These compounds are recognized by odorant receptors (OR) located within the dendritic membrane of sensory neurons that extend into the lymph of sensilla, covering the surface of insect antennae. We have identified 3 genes encoding ORs from the tortricid moth, Epiphyas postvittana, a pest of horticulture. Like Drosophila melanogaster ORs, they contain 7 transmembrane helices with an intracellular N-terminus, an orientation in the plasma membrane opposite to that of classical GPCRs. EpOR2 is orthologous to the coreceptor Or83b from D. melanogaster. EpOR1 and EpOR3 both recognize a range of terpenoids and benzoates produced by plants. Of the compounds tested, EpOR1 shows the best sensitivity to methyl salicylate [EC(50) = 1.8 x 10(-12) M], a common constituent of floral scents and an important signaling compound produced by plants when under attack from insects and pathogens. EpOR3 best recognizes the monoterpene citral to low concentrations [EC(50) = 1.1 x 10(-13) M]. Citral produces the largest amplitude electrophysiological responses in E. postvittana antennae and elicits repellent activity against ovipositing female moths. Orthologues of EpOR3 were found across 6 families within the Lepidoptera, suggesting that the ability to recognize citral may underpin an important behavior.

Aidan Kiely

MCDB

Yale University

Electrophysiological Recording From Drosophila Labellar Taste Sensilla

Aidan Kiely

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Yale University

Electrophysiological Recording From Drosophila Labellar Taste Sensilla

MCDB