Received for publication November 2, 2005.
Revised February 14, 2006.
Accepted for publication February 15, 2006.

Flubendiamide, a Novel Ca²⁺ Channel Modulator, Reveals Evidence for Functional Cooperation between Ca²⁺ Pumps and Ca²⁺ Release

Abstract

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 fourfold 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 outervesicular 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 co-ordination 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.

Key words: Ion channel regulation, Ion transporters (SERCA, Na/K ATPase, CFTR)