Heterologous investigation of metabotropic and ionotropic odorant receptors in ab3A neurons of Drosophila melanogaster

Front Mol Biosci. 2024 Jan 25:10:1275901. doi: 10.3389/fmolb.2023.1275901. eCollection 2023.

Abstract

In insects, antennal ionotropic receptors (IRs) and odorant receptors (ORs) are among the main sensors of olfactory cues. To functionally characterize the subunits from these receptors, the use of ab3A neurons from transgenic Drosophila melanogaster represented one of the most powerful tools, allowing the identification of ligands (deorphanization) and decrypting their pharmacological properties. However, further investigation is needed to shed light on possible metabotropic functionalities behind insect olfactory receptors and test potentials from the up-to-now-used empty neuronal systems to express subunits belonging to variegate receptor classes. In this project, we adopted the most updated system of Drosophila ab3A empty neurons to test various olfactory receptors, ranging from human ORs working as metabotropic G-protein coupled receptors to insect ionotropic IRs and ORs. Testing transgenic Drosophila expressing human ORs into ab3A neurons by single sensillum recording did not result in an OR response to ligands, but it rather re-established neuronal spiking from the empty neurons. When transgenic D. melanogaster expressed ionotropic IRs and ORs, both heterologous and cis-expressed IRs were non-functional, but the Drosophila suzukii OR19A1 subunit responded to a wide asset of ligands, distinguishing phasic or tonic compound-dependent effects. Despite the use of Drosophila ab3A neurons to test the activation of some metabotropic and ionotropic receptor subunits resulted non-functional, this study deorphanized a key OR of D. suzukii demonstrating its binding to alcohols, ketones, terpenes, and esters.

Keywords: deorphanization; empty ab3A neurons; functional characterization of chemoreceptors; heterologous expression; ionotropic receptors; metabotropic receptors (mGluRs); single sensillum recording; transgenic Drosophila melanogaster.

Grants and funding

The authors declare financial support was received for the research, authorship, and/or publication of this article. Cloning of human ORs into pUAS expression vectors was supported by the Chemical Ecology-Horticulture group of SLU in the frame of the Crafoordska Stiftelsen project number 20180954, title: “Control of apple pests with fruit and yeast odorants.” Costs for part of the ligands were covered by the Martha och Dagny Larssons fond, Swedish University of Agricultural Sciences (Protokoll 172-174, Sammanträdesdatum 2018-04-24). Costs for HEK-heterologous experiments were covered by the Ache lab of the Whitney Laboratory for Marine Bioscience, University of Florida (United States). Costs for FISH, immunostaining, the cloning of IR genes, and the generation of transgenic Drosophila expressing IRs were covered by the Benton Lab, Center for Integrative Genomics, University of Lausanne (Switzerland). Costs for the generation of transgenic Drosophila expressing insect and human ORs and costs to purchase part of the synthetic ligands that were tested by SSR were covered by the Lars Hiertas Minne project number FO2022-0248; title “Wide targeting of insect chemoreceptors to the control of pests of Swedish horticulture.” This research was conducted and supported in the frame of the FORMAS Swedish research council project number 2018-00891; title “Control of fruit pests by targeting larval chemical sensing.” Costs for publication have been covered by the Lars Hiertas Minne project number FO2022-0248 and by the Chemical Ecology-Horticulture group of SLU.