Structural Domains Critical for the Selectivity of Diamide Activators to Lepidopterous Ryanodine Receptors

Diamide insecticides, such as flubendiamide and chlorantraniliprole, show selective insecticidal activity against lepidopterous insects such as the diamondback moth. The diamide insecticides act to prolong ryanodine receptor (RyR) channel opening, resulting in uncoordinated muscle contractions in intoxicated pest insects. However, the molecular mechanisms underlying the species-specific action of diamide insecticides are unclear. Here, we characterized protein domains important for the selective flubendiamide action in lepidopterous RyRs. In silkworm RyR (sRyR), replacement of the divergent region 1 (DR1), poorly conserved among species, with that from rabbit RyR2 significantly impaired the Ca2+ release response to flubendiamide, but spared receptor sensitivity to caffeine, a universal RyR activator. Flubendiamide-acyl imidazol (flubendiamide-AI), the labeling probe with the reactive AI attached to the aliphatic amide moiety, was predominantly incorporated into the peptide sequence TLQLGISILR (3,906-3,915) of the cytoplasmic central domain adjacent to DR1 in sRyR. Importantly, replacement of T3906 with alanine abolished flubendiamide-AI incorporation but retained intact flubendiamide sensitivity in sRyR. Furthermore, the mutation G4866E, corresponding to the diamide-resistant mutation identified at the S4 transmembrane segment in RyR of the diamondback moth, abolished the flubendiamide response but retained flubendiamide-AI labeling or the caffeine response in sRyR. Our findings suggested that flubendiamide directly binds to DR1 with its aliphatic amide moiety oriented toward T3906 of the central domain, which serves as the reaction site for the AI moiety of flubendiamide-AI in sRyR, to impinge the S4 segment in opening the Ca2+-permeable channel pore of sRyR. Thus, diamides differ from non-selective modulators such as caffeine in binding to DR1, which may provide important basis for designing a class of compounds that selectively activate RyRs of specific insect taxa or mammalian RyR subtypes.