Odorant molecules interact with odorant receptors (ORs) lining the pores on the surface of the sensilla on an insect's antennae and maxillary palps. This interaction triggers an electrical signal that is transmitted to the insect's nervous system, thereby influencing its behavior. Orco, an OR coreceptor, is crucial for olfactory transduction, as it possesses a conserved sequence across the insect lineage. In this study, we focused on 2,4-di-tert-butylphenol (DTBP), a single substance present in acetic acid bacteria culture media. We applied DTBP to oocytes expressing various Drosophila melanogaster odor receptors and performed electrophysiology experiments. After confirming the activation of DTBP on the receptor, the binding site was confirmed through point mutations. Our findings confirmed that DTBP interacts with the insect Orco subunit. The 2-heptanone, octanol, and 2-hexanol were not activated for the Orco homomeric channel, but DTBP was activated, and the EC50 value was 13.4 ± 3.0 μM. Point mutations were performed and among them, when the W146 residue changed to alanine, the Emax value was changed from 1.0 ± 0 in the wild type to 0.0 ± 0 in the mutant type, and all activity was decreased. Specifically, DTBP interacted with the W146 residue of the Orco subunit, and the activation manner was concentration-dependent and voltage-independent. This molecular-level analysis provides the basis for novel strategies to minimize pest damage. DTBP, with its specific binding to the Orco subunit, shows promise as a potential pest controller that can exclusively target insects.
Keywords: 2,4-di-tert-butylphenol (DTBP); insect behavior; insect control; odorant receptor; odorant receptor coreceptor (Orco); two-electrode voltage clamp (TEVC).