Molecular Characterization of Flubendiamide Sensitivity in the Lepidopterous Ryanodine Receptor Ca²⁺ Release Channel

Flubendiamide is a benzenedicarboxamide derivative that shows selective insecticidal activity against lepidopterous insects. The specific modulatory effects of flubendiamide on ryanodine binding in insect muscle microsomal membranes suggest that the ryanodine receptor (RyR) Ca(2+) release channel is a primary target of flubendiamide. However, the molecular mechanisms underlying the species-specific action of flubendiamide are unclear. We have cloned cDNA encoding a novel RyR from the lepidopterous silkworm RyR (sRyR) and tested the sensitivity to flubendiamide of the recombinant sRyR in HEK293 cells. Confocal localization studies and Ca(2+) imaging techniques revealed that sRyRs form Ca(2+) release channels in the endoplasmic reticulum. Importantly, flubendiamide induced release of Ca(2+) through the sRyR, but not through the rabbit RyR isoforms. Photoaffinity labeling of sRyR deletion mutants using a photoreactive derivative revealed that flubendiamide is mainly incorporated into the transmembrane domain (amino acids 4111-5084) of the sRyR. The rabbit cardiac muscle isoform RyR2 (rRyR2) and the RyR mutant carrying a replacement of the transmembrane domain (residues 4084-5084) with its counterpart sequence from rRyR2 (residues 3936-4968) were not labeled by the photoreactive compound. This replacement in the sRyR significantly impaired the responses to flubendiamide but only marginally reduced the sensitivity to caffeine, a general RyR activator. Furthermore, deletion of the N-terminal sequence (residues 183-290) abolished the responses of the sRyR to flubendiamide but not the sensitivity to caffeine. Our results suggest that the transmembrane domain plays an important role in the formation of an action site for flubendiamide, while the N-terminus is a structural requirement for flubendiamide-induced activation of the sRyR.