Molecular characterization of flubendiamide sensitivity in the lepidopterous ryanodine receptor Ca(2+) 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.
Kato
Kenta
K
Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Kyoto 615-8510, Japan.
Kiyonaka
Shigeki
S
Sawaguchi
Yuichi
Y
Tohnishi
Masanori
M
Masaki
Takao
T
Yasokawa
Noriaki
N
Mizuno
Yusuke
Y
Mori
Emiko
E
Inoue
Keisuke
K
Hamachi
Itaru
I
Takeshima
Hiroshi
H
Mori
Yasuo
Y
D000818:Animals; D001549:Benzamides / Q000494:pharmacology*; D015153:Blotting, Western; D012831:Bombyx / Q000235:genetics / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D004721:Endoplasmic Reticulum / Q000378:metabolism; D006801:Humans; D007306:Insecticides / Q000494:pharmacology*; D018613:Microscopy, Confocal; D008954:Models, Biological; D011817:Rabbits; D011994:Recombinant Proteins / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism*; D019837:Ryanodine Receptor Calcium Release Channel / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism*; D013450:Sulfones / Q000494:pharmacology*; D005466:Fluorocarbons; D010797:Phthalimides
D001549:Benzamides; D002118:Calcium; D007306:Insecticides; D011994:Recombinant Proteins; D019837:Ryanodine Receptor Calcium Release Channel; D013450:Sulfones; C508762:flubendiamide; D005466:Fluorocarbons; D010797:Phthalimides