Genetic and pharmacological inhibition of the Kir2A channel in diamondback moth exhibits ovicidal activity

Inward rectifier potassium (Kir) channels play crucial roles in regulating physiological processes in insects; however, the physiological and pharmacological characteristics of Kir2A channels in Lepidoptera remain unclear. This study employs CRISPR/Cas9-mediated gene knockout, patch-clamp electrophysiology, and toxicological assays to investigate the PxKir2A channel in diamondback moth, Plutella xylostella (L.). The segregation ratio of self-crossed offspring from PxKir2A heterozygous knockouts implies lethality of homozygous mutants during the egg stage. The channel current of PxKir2A was successfully recorded using the Sf9 expression system. Single-cell patch-clamp recordings revealed that the small-molecule inhibitors VU625, VU590, and chlorfenapyr significantly suppressed PxKir2A channel activity, with half-maximal inhibitory concentrations (IC₅₀) of 0.2673 µmol/L, 0.6647 µmol/L, and 6.056 µmol/L, respectively. Confocal laser microscopy observations revealed that the PxKir2A-GFP fusion protein was predominantly localized on the cell membrane in Sf9 cells. In contrast, HEK293T cells did not show clear membrane localization of the protein, which explains the absence of detectable channel currents in HEK293T cells expressing PxKir2A. Toxicological evaluations indicate that these PxKir2A inhibitors exert dose-dependent ovicidal effects during the late stage of eggs, with mortality rates at 2 mg/mL of 87.87% ± 5.51%, 73.85% ± 4.11%, and 89.76% ± 8.51% for VU625, VU590, and chlorfenapyr, respectively. This study highlights the crucial role of PxKir2A channels in the egg development and provides important electrophysiological and pharmacological data on PxKir2A channels, offering valuable insights for future research on pest control strategies targeting PxKir2A.