Phenethyl Acetate as an Agonist of Insect Odorant Receptor Co-Receptor (Orco): Molecular Mechanisms and Functional Insights

The insect olfactory system is vital for survival, enabling the recognition and discrimination of a wide range of odorants present in the environment. This process is mediated by odorant receptors (Ors) and the highly conserved co-receptor Orco. Insect Ors are structurally distinct from mammalian olfactory receptors, a divergence that presents unique advantages for developing insect-specific pest control strategies. In this study, we explored the molecular-level interactions between insect Ors and volatile organic compounds. Specifically, we investigated the response of Ors/Orco to phenethyl acetate (PA), a volatile compound found in the culture media of acetic acid bacteria. PA elicited activation in a concentration-dependent, reversible, and voltage-independent manner in Or1a, Or24a, and Or35a when combined with Orco, as well as in Orco homomers. Through molecular docking studies, we determined that the PA-binding site is localized to the Orco subunit, a highly conserved protein across diverse insect taxa. To further elucidate the role of key residues in the Orco homotetramer receptor, we performed site-directed mutagenesis. A mutational analysis identified W146 and E153 as critical residues for PA binding and activation. A double-mutant Orco receptor (W146A + E153A) exhibited a significant reduction in PA-induced activation compared to the wild-type receptor. These findings indicate that PA functions as an agonist for the <i>Drosophila melanogaster</i> Orco receptor and highlight its potential applications in chemosensory research and insect pest management strategies.