RESUMEN
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.
Asunto(s)
Benzamidas/farmacología , Bombyx/metabolismo , Insecticidas/farmacología , Proteínas Recombinantes/efectos de los fármacos , Proteínas Recombinantes/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/efectos de los fármacos , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Sulfonas/farmacología , Animales , Western Blotting , Bombyx/genética , Calcio/metabolismo , Línea Celular , Retículo Endoplásmico/metabolismo , Humanos , Microscopía Confocal , Modelos Biológicos , Conejos , Proteínas Recombinantes/genética , Canal Liberador de Calcio Receptor de Rianodina/genéticaRESUMEN
Flubendiamide, developed by Nihon Nohyaku Co., Ltd. (Tokyo, Japan), is a novel activator of ryanodine-sensitive calcium release channels (ryanodine receptors; RyRs), and is known to stabilize insect RyRs in an open state in a species-specific manner and to desensitize the calcium dependence of channel activity. In this study, using flubendiamide as an experimental tool, we examined an impact of functional modulation of RyR on Ca2+ pump. Strikingly, flubendiamide induced a 4-fold stimulation of the Ca2+ pump activity (EC50=11 nM) of an insect that resequesters Ca2+ to intracellular stores, a greater increase than with the classical RyR modulators ryanodine and caffeine. This prominent stimulation, which implies tight functional coupling of Ca2+ release with Ca2+ pump, resulted in a marginal net increase in the extravesicular calcium concentration despite robust Ca2+ release from the intracellular stores by flubendiamide. Further analysis suggested that luminal Ca2+ is an important mediator for the functional coordination of RyRs and Ca2+ pumps. However, kinetic factors for Ca2+ pumps, including ATP and cytoplasmic Ca2+, failed to affect the Ca2+ pump stimulation by flubendiamide. We therefore conclude that the stimulation of Ca2+ pump by flubendiamide is mediated by the decrease in luminal calcium, which may induce calcium dissociation from the luminal Ca2+ binding site on the Ca2+ pump. This mechanism should play an essential role in precise control of intracellular Ca2+ homeostasis.
Asunto(s)
Canales de Calcio/fisiología , Señalización del Calcio/fisiología , Animales , Benzamidas/farmacología , Calcio/metabolismo , Canales de Calcio/efectos de los fármacos , Señalización del Calcio/efectos de los fármacos , Membrana Celular/efectos de los fármacos , Membrana Celular/fisiología , Relación Dosis-Respuesta a Droga , Cinética , Rianodina/farmacocinética , Spodoptera , Sulfonas/farmacologíaRESUMEN
Flubendiamide represents a novel chemical family of substituted phthalic acid diamides with potent insecticidal activity. So far, the molecular target and the mechanism of action were not known. Here we present for the first time evidence that phthalic acid diamides activate ryanodine-sensitive intracellular calcium release channels (ryanodine receptors, RyR) in insects. With Ca(2+) measurements, we showed that flubendiamide and related compounds induced ryanodine-sensitive cytosolic calcium transients that were independent of the extracellular calcium concentration in isolated neurons from the pest insect Heliothis virescens as well as in transfected CHO cells expressing the ryanodine receptor from Drosophila melanogaster. Binding studies on microsomal membranes from Heliothis flight muscles revealed that flubendiamide and related compounds interacted with a site distinct from the ryanodine binding site and disrupted the calcium regulation of ryanodine binding by an allosteric mechanism. This novel insecticide mode of action seems to be restricted to specific RyR subtypes because the phthalic acid diamides reported here had almost no effect on mammalian type 1 ryanodine receptors.