The use of synthetic pesticides carries a significant risk of pests developing resistance, leading to decreased pesticide effectiveness. ATP-binding cassette (ABC) transporters, especially the ABCC subfamily members, have been suggested to act as efflux pumps for various pesticides, thereby contributing to pesticide resistance. So far, the identification of potential pesticide substrates of insect ABC transporters is most often based on the quantification of transcript in arthropods. Here, we screened and identified the potential pesticide substrates of ABCC-9C from Tribolium castaneum based on an in vitro ATPase activity assay. Together with affinity evaluation-, cytotoxicity analysis-, and RNA interference-based bioactivity tests, we revealed that the insecticides, carbofuran, and buprofezin, are potential substrates of TcABCC-9C. Additionally, we identified an amphipathic translocation channel in the transmembrane domain of TcABCC-9C formed by 8 transmembrane helices. Molecular docking suggested that both carbofuran and buprofezin bind at the same site within the translocation channel via hydrophobic interactions. These findings indicate that TcABCC-9C might play a critical role in multi-pesticide resistance, providing a potential target for managing pesticide resistance and laying the groundwork for future pest control strategies. Given the conservations among ABCC subfamily members, the experimental model we developed in this study can be also applied to identify the potential substrates of other ABCC transporters, as well as to predict insecticide resistance mediated by ABCC transporters.
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