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

Research Division, Nihon Nohyaku Co., Ltd., Osaka, Japan

Received November 1, 2005; accepted February 15, 2006

	Abstract

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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.

Flubendiamide, a new phthalic diamide compound (Fig. 1), stabilizes insect RyR to an open state, evoking massive calcium release from intracellular stores (Ebbinghaus-Kintscher et al., 2006). The compound possesses distinct pharmacological characteristics, mediated by a binding site different from that of ryanodine, a widely used probe for the RyR. Interestingly, flubendiamide achieved maximum activation of the RyR in the presence of a broad range of calcium concentrations, including in low calcium conditions. This characteristic might contribute to revealing the relationships between functionally linked components by minimizing the complexity in functional modulation of the RyR. Thus, this compound is expected to provide a new experimental device for exploring the impacts of RyR activity.

View larger version (15K): [in this window] [in a new window]   Fig. 1. Flubendiamide.

The Ca²⁺ released via the RyR during excitatory states is eventually returned to intracellular stores by the Ca²⁺ pump. Functional interference with the Ca²⁺ pump by the RyR is implied in the recent controversy around studies of cADPR (Lukyanenko et al., 2001a). cADPR was first reported as an endogenous RyR activator, but subsequent studies suggested RyR activation by cADPR was a result of Ca²⁺ pump activation mediated by increased luminal calcium.

In this study, we used a unique approach to elucidate the physiological impact of RyR activity using this new probe in a distinct RyR isoform derived from an insect. In particular, we focused our attention on SR Ca²⁺ pump activity, a functionally complementary factor of the RyR. The results presented here demonstrate an intriguing relationship between the Ca²⁺ pump and RyR activation by flubendiamide, mediated by luminal Ca²⁺.

	Materials and Methods

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Chemicals and Reagents. Flubendiamide (Fig. 1) was synthesized at Nihon Nohyaku Co., Ltd. The compound was dissolved in dimethyl sulfoxide for all assays (below 0.5% final concentration). [³H]Ryanodine (56 Ci/mmol) was purchased from PerkinElmer Life and Analytical Sciences (Boston, MA). Ryanodine, ATP/Tris, thapsigargin, A23187 [GenBank] (calcimycin), and caffeine were purchased from Sigma-Aldrich (St. Louis, MO). DiBr-BAPTA and Rhod-2 were purchased from Invitrogen (Carlsbad, CA). All other chemicals were the best available grade.

Insects. The strain of Spodoptera litura (Lepidoptera: Noctuidae) used has been maintained at Nihon Nohyaku Co., Ltd. for 15 years. Larvae of S. litura were reared on an artificial diet of Insecta LFS, purchased from Nihon Nohsan Kogyo (Yokohama, Japan). The colony was maintained at 25°C under a 16-h light/8-h dark photoperiod, with constant access to the larval diet.

Preparation of Muscle Membranes. Sixth instars of S. litura were dissected under a stereoscopic microscope, and longitudinal ventral muscle tissues were collected. Collected tissues were homogenized on ice in 9 volumes (based on tissue wet weight) of the homogenization solution (250 mM sucrose, 5 mM DTT, and 10 mM Tris/HCl, pH 7.4) with a Teflon-pestle homogenizer. The homogenates obtained were centrifuged at 9000g_max (7000g_avg) for 30 min at 4°C. The resulting supernatants were filtered through a cell strainer (BD Falcon, Bedford, MA) and centrifuged at 140,000g_max, (104,000g_avg) for 1 h at 4°C. The resulting supernatants were discarded, and pellets were resuspended in four volumes (based on tissue wet weight) of the homogenization solution without DTT, followed by recentrifugation under the conditions described above. The resulting pellets were suspended in a half volume of the homogenization solution without DTT. The suspensions obtained were stored at -80°C until use.

Calcium Release from the Muscle Membrane Preparations. Calcium release from the membrane preparation was investigated by calcium fluorimetry. The membrane preparations (40 µg) described above were diluted with 315 µl of assay solution (100 mM potassium phosphate buffer, pH 7.4, 3 mM MgCl₂, 100 mM sucrose, 25 µM CaCl₂, and 5 µM Rhod-2). The processes of calcium influx and efflux were monitored by the fluorescence derived from the Ca²⁺/Rhod-2 complexes using a fluorescence spectrophotometer (F-4500; Hitachi, Tokyo, Japan) with excitation at 552 nm and emission at 580 nm. The assays were performed at 22°C with sustained stirring of the assay solution. In the absence of Rhod-2, no background fluorescence was detected. The free-calcium concentrations in the assay solutions were calculated according to Berman (2000).

Measurements of ATPase Activity. Membrane preparations (20 µg) were suspended in 100 µl of Ca²⁺-ATPase assay solution (100 mM KCl, 6 mM MgCl₂, and 50 mM Tris/MOPS, pH 7.4) with various concentrations of Ca²⁺ or Ca²⁺ buffer (for fixing the free calcium concentration below 2.3 µM), as detailed in the figure legends. After a 30-min preincubation at 25°C, reactions were initiated by the addition of ATP/Tris. Unless otherwise stated, the added ATP/Tris concentration was fixed at 1 mM. For determination of the basal activity, 0.8 mM EGTA was added to the assay solution. Inorganic phosphate liberated during the reaction was colorimetrically determined based on a previously described method (Marsh, 1959). In addition, ATPase activity was evaluated using the ATP-regenerating system according to Chu et al. (1988), with minor modification. In brief, 40 µg of membrane preparation was suspended in 2 ml of an assay solution containing coupling mixture (100 mM KCl, 6 mM MgCl₂, 0.05 mM CaCl₂, 50 mM Tris/HCl, pH 7.4, 0.2 mM -NADH, 2 mM phosphoenolpyruvate, 4.3 units/ml pyruvate kinase, and 6.3 units/ml L-lactate dehydrogenase). The reactions were performed at 25°C with continuous stirring of the reaction mixture. Consumption of -NADH was monitored by measuring the absorbance at 340 nm with a double-beam spectrophotometer (U-3000; Hitachi).

Equilibrium [³H]Ryanodine Binding Assay. [³H]Ryanodine was incubated at 25°C with 40 to 80 µg of the membrane preparation in 0.4 ml of binding solution containing 1 M KCl, 0.3 M sucrose, and 10 mM Tris/HCl, pH 7.4, in the absence or presence of flubendiamide. After incubation for 80 min, the assay solutions were rapidly filtered through Whatman GF/F filters (Whatman, Maidstone, UK) presoaked in the binding solution. The filters were then rinsed with 20 ml of washing solution (1 M KCl, 0.3 M sucrose, and 10 mM Tris/HCl, pH 7.4). The radioactivity remaining on the filter was counted using a liquid scintillation counter (model 1409; Perkin-Elmer Wallac, Turku, Finland). In all assays, nonspecific binding of [³H]ryanodine was determined in the presence of 10 µM unlabeled ryanodine. Under the experimental conditions, specific binding was greater than 70% of the total binding of 5 nM [³H]ryanodine. Each data point represents the mean of at least triplicate determinations.

	Results

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Calcium Release from the S. litura Membrane Preparation by Flubendiamide. It has been demonstrated that flubendiamide fixes insect RyR in its open state, which induces cytoplasmic calcium transients in cultured nerve cells (Ebbinghaus-Kintscher et al., 2006). To reveal the functional consequence of the specific interaction between insect RyRs and flubendiamide, calcium release by flubendiamide was first examined using muscle membrane preparations from S. litura. As shown in Fig. 2, flubendiamide induced remarkable calcium release only in the presence of the Ca²⁺ pump inhibitor thapsigargin. Excluding thapsigargin from the assay system concealed the observable calcium release induced by flubendiamide (Fig. 2A), suggesting that the released Ca²⁺ might be rapidly resequestered by the Ca²⁺ pump, which is tightly coupled to RyR activity as described in the next section. The calcium release induced by flubendiamide in the presence of 3 mM Mg²⁺ was suppressed by increasing the Mg²⁺ concentration to 10 mM. This inhibition by Mg²⁺, an endogenous inhibitor of RyR (Scott-Ward et al., 2001), supports the view that the RyR mediates the calcium release by flubendiamide. A calcium ionophore, A23187 [GenBank] , released a large fraction of the residual Ca²⁺ after the completion of calcium release by flubendiamide, which confirmed that P_i precipitation in the vesicle did not have a limiting effect on calcium release.

View larger version (22K): [in this window] [in a new window]   Fig. 2. Calcium release from the insect membrane preparation induced by flubendiamide. To load calcium into the vesicles, 1 mM ATP was added at time 0. A, extravesicular Ca2+ was monitored in the presence of 3 mM Mg2+. Flubendiamide was added after calcium sequestration was completed. Residual Ca2+ in the vesicles was released by the addition of 2 µM A23187 [GenBank] . B, extravesicular Ca2+ was monitored in the presence of 3 mM (solid line) or 10 mM (dashed line) Mg2+. After calcium sequestration had been completed, the Ca2+ pump was inhibited by addition of 0.4 µM thapsigargin; thereafter, flubendiamide was added. Residual Ca2+ in the vesicles was released by 2 µM A23187 [GenBank] .

Specific Stimulation of the Ca²⁺ Pump by Flubendiamide. The effects of flubendiamide on calcium transport by the Ca²⁺ pump were evaluated by measuring Ca²⁺-ATPase activity, indicative of the catalytic cycles of the Ca²⁺ pump, because calcium transport is stoichiometrically coupled to hydrolysis of ATP. As shown in Fig. 3, flubendiamide in the nanomolar range specifically stimulated Ca²⁺-ATPase in a concentration-dependent manner (EC₅₀ = 11 nM). The maximum velocity of Ca²⁺-ATPase was 160% of control activity at supramaximal concentrations of flubendiamide. The potency of flubendiamide was evidently pronounced in comparison with the effects of the known RyR modulators ryanodine and caffeine (Fig. 3). Contrary to the definitive stimulation of the Ca²⁺-ATPase, flubendiamide exerted no stimulation on Mg²⁺- and Na⁺/K⁺-dependent ATPase activity, indicating specificity for Ca²⁺-ATPase. Identical experiments using an ATP-regenerating system reproduced these results (data not shown). The ADP concentration is known to be a kinetic factor that can suppress the catalytic cycles of the Ca²⁺ pump by accumulation of ADP-bound phosphoenzymes (Inesi and de Meis, 1989). This can be avoided in the ATP-regenerating system, because the ADP formed is efficiently converted to ATP. Hence, the result obtained with the ATP-regenerating system proved that flubendiamide stimulated the Ca²⁺ pump by a mechanism other than enhancement of ADP dissociation from phosphoenzymes.

View larger version (14K): [in this window] [in a new window]   Fig. 3. Effect of flubendiamide (), ryanodine () and caffeine (x) on the Ca2+-ATPase activity of the membrane preparation. Assays were performed in Ca2+-ATPase assay solution containing 50 µM free Ca2+. The activity was determined by Pi production over 20 min at 25 °C. Data represents the mean ± S.D. of quadruplicate samples. The ordinate axis shows ATPase activity in the presence of flubendiamide normalized to control activity.

Evidence Suggesting the Importance of Luminal Ca²⁺ in Ca²⁺ Pump Stimulation by Flubendiamide. Ca²⁺ within intracellular stores (luminal Ca²⁺) is regarded as an essential factor in the kinetic regulation of the Ca²⁺ pump. In the presence of sufficient extravesicular Ca²⁺, a decrease in luminal calcium induced acceleration of the Ca²⁺ pump activity because of facilitation of calcium dissociation from a low-affinity calcium binding site (luminal site). To investigate the possible involvement of luminal calcium in Ca²⁺ pump stimulation, the luminal Ca²⁺ concentration was indirectly manipulated by a Ca²⁺ ionophore and a calcium chelator (Table 1). As shown in Table 1, A23187 [GenBank] induced a 5-fold increase in Ca²⁺ pump activity. This effect of A23187 [GenBank] was attributable to a decrease in luminal calcium, because A23187 [GenBank] reduced ⁴⁵Ca²⁺ accumulation in the membrane preparations to 24.6% of control (T. Masaki, unpublished data) as a result of an increase in calcium permeability (Scarpa et al., 1972). Under this condition, Ca²⁺ pump stimulation by flubendiamide was mostly eliminated, suggesting the involvement of luminal Ca²⁺ in the Ca²⁺ pump stimulation by flubendiamide.

The stimulatory effect of flubendiamide on Ca²⁺ pump was also diminished in the calcium buffers containing calcium chelators with high and low calcium affinity (Table 1). The low-affinity calcium chelator diBr-BAPTA (K_d = 3.7 µM) evidently accelerated the catalytic cycles of Ca²⁺ pump, as in the case with A23187 [GenBank] . The result also implies importance of luminal calcium, because the low affinity of this chelator could not interrupt the calcium association with high-affinity binding sites (cytoplasmic site) on Ca²⁺-ATPase. Thus, the acceleration of Ca²⁺-ATPase suggests only calcium dissociation from luminal site was facilitated in the presence of diBr-BAPTA. Conversely, high calcium affinity of EGTA (K_d = 0.1 µM) disturbed calcium binding to both of the cytoplasmic and luminal site of the molecule, which suppressed whole catalytic cycles of the Ca²⁺ pump (Table 1). The results demonstrate that affinities for calcium are essential for apparent effect on Ca²⁺ pump activity of the chelators. Simultaneously, the elimination of effect of flubendiamide on Ca²⁺ pump with A23187 [GenBank] or the chelators inferred flubendiamide has no direct effect on the Ca²⁺ pump.

In addition, the maximum velocity of the flubendiamidestimulated Ca²⁺-ATPase activity in the presence of 50 µM Ca²⁺ was notably augmented under low calcium conditions (1 µM of free Ca²⁺, as imposed by a Ca²⁺/EGTA buffer; Fig. 4), whereas apparent flubendiamide affinity based on the EC₅₀ value for Ca²⁺-ATPase activity was unchanged (11 nM; Fig. 4). The limited calcium conditions would make for a steeper transmembrane calcium gradient, which can enhance the efflux of luminal Ca²⁺. Hence, the results further support the possibility that luminal Ca²⁺ is involved in Ca²⁺ pump stimulation by flubendiamide. It was also indicated that the extravesicular calcium concentration failed to affect the affinity of flubendiamide for the molecule.

View larger version (19K): [in this window] [in a new window]   Fig. 4. Concentration dependence of the effect of flubendiamide on Ca2+-ATPase activity in the membrane preparation. Assays were performed in Ca2+-ATPase assay solution containing 1 µM (; made up of 0.46 mM Ca2+ and 0.5 mM EGTA) or 50 µM (; replot of data from Fig. 3) free Ca2+. Activities were determined in the presence of graded concentrations of flubendiamide.

View larger version (13K): [in this window] [in a new window]   Fig. 5. Ca2+-ATPase stimulation by flubendiamide at various concentrations of Ca2+ (A) and ATP (B). A, assays were performed in Ca2+-ATPase assay solution containing 2.6 to 2300 nM free Ca2+. Activities were determined by Pi production over 20 min at 25°C in the presence () or absence () of flubendiamide (100 nM). Free Ca2+ concentrations imposed by the presence of 0.5 mM EGTA were calculated using the free software program WINMAXC (http://www.stanford.edu/~cpatton/maxc.html). B, assays were performed in Ca2+-ATPase assay solution containing 50 µM Ca2+ and 0.03 to 3 mM ATP/Tris.

Effects of Flubendiamide on [³H]Ryanodine Binding to the Membrane Preparations. Our previous report indicates that the RyR is the primary target of flubendiamide; therefore, the stimulation of the Ca²⁺ pump observed in this study should be triggered by activation of insect RyR. To confirm the involvement of RyR, conformational changes of the RyR in the membrane preparation was examined by [³H]ryanodine binding. As shown in Fig. 6, the binding isotherm indicates a single binding site for [³H]ryanodine in the membrane preparation. The K_d and B_max values for ryanodine binding were 15.4 ± 5.5 nM and 0.54 ± 0.06 pmol/mg, respectively, in optimal binding solution containing 1 mM Ca²⁺. In the presence of a supramaximal concentration (1 µM) of flubendiamide, the binding affinity of the [³H]ryanodine was significantly increased without a evident effect on the B_max value. This result indicates that flubendiamide shifts the RyR conformation to the open state in this membrane preparation from S. litura.

View larger version (20K): [in this window] [in a new window]   Fig. 6. Effect of flubendiamide on [3H]ryanodine binding to the insect membrane preparation. Saturation kinetics (A) and Scatchard transformation (B) of [3H]ryanodine binding in the presence (; Kd = 3.1 ± 0.1 nM; Bmax = 0.54 ± 0.057 pmol/mg of protein) and absence (; Kd = 15 ± 0.06 nM; Bmax = 0.47 ± 0.06 pmol/mg of protein) of flubendiamide (1 µM). Binding was equilibrated in binding solution containing 1 mM calcium. Results are expressed as the means of three preparations.

Numerous endogenous modulators such as Ca²⁺, Mg²⁺, or ATP affect the activity of RyR. The effect of those modulators on RyR was evaluated by comparing the EC₅₀ values of flubendiamide for potentiation of [³H]ryanodine binding (Table 2). The EC₅₀ value of flubendiamide in the presence of 1 mM Ca²⁺ was 0.12 nM, which was invariable even after the addition of 1 mM ATP or 6 mM Mg²⁺, whereas addition of 1 mM EGTA, which excludes free Ca²⁺ from the assay solution, drastically increased the EC₅₀ value to 26 nM. It is therefore indicated that the efficacy of flubendiamide binding is altered due to a change of the Ca²⁺ concentration. The effective concentration of flubendiamide for the Ca²⁺ pump (EC₅₀ = 11 nM) was in a similar range to the EC₅₀ values for RyR, determined in the various solutions listed in Table 2. Indeed, the EC₅₀ for RyR obtained in the Ca²⁺-ATPase assay solution (2.5 nM) was rather close to the EC₅₀ value for the Ca²⁺ pump. The consistency of the effects of the EC₅₀ values for both two components suggests that the Ca²⁺ pump stimulation by flubendiamide is accompanied by RyR activation. This evidence further supports coordination between the RyR and the Ca²⁺ pump.

	Discussion

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Flubendiamide and its analogous compounds modify intracellular calcium kinetics in cultured insect neurons. This effect is mediated by a specific interaction with the insect RyR (Ebbinghaus-Kintscher et al., 2006). The results of this study revealed Ca²⁺ release from muscle membrane preparations from an insect, S. litura, as a consequence of the functional modulation of the RyR by flubendiamide. Interestingly, Ca²⁺ pump inhibition was necessary for flubendiamide to produce an observable increase in extravesicular calcium (Fig. 2), despite the robust calcium release induced by flubendiamide. This obscuring of the observable calcium release should be achieved by rapid calcium sequestration by the Ca²⁺ pump, which was concurrently stimulated by flubendiamide (Fig. 3). The stimulation of the Ca²⁺ pump by flubendiamide was clearly pronounced compared with the effects of the classical RyR modulators ryanodine and caffeine, which are known to induce calcium release even in the absence of the Ca²⁺ pump inhibitor (Palade, 1987). Furthermore, Ca²⁺ pump stimulation by flubendiamide seemed to be linked with RyR activation, because the EC₅₀ values of flubendiamide for these two components were comparable (Fig. 3; Table 2). This evidence strongly suggests tight functional coupling between the Ca²⁺ pump and the RyR.

With respect to the underlying mechanism of the functional coordination between the RyR and the Ca²⁺ pump, we focused on the possibility of luminal calcium as the mediator, an important regulator for both components (Ikemoto and Yamamoto, 2000; Lukyanenko et al., 2001b). In membrane preparations forming sealed vesicles, millimolar concentrations of luminal Ca²⁺ are quickly achieved, and thereby the catalytic cycles of the Ca²⁺ pump and calcium transport are impeded (Inesi and de Meis, 1989). This high luminal Ca²⁺ concentration is efficiently reduced in the presence of the calcium ionophore A23187 [GenBank] , which results in acceleration of Ca²⁺ pump activity (Scarpa et al., 1972). In the presence of A23187 [GenBank] , the stimulatory effect of flubendiamide on the Ca²⁺ pump activity was evidently eliminated, which was considered to be due to reduction of the luminal calcium concentration, because the effect of A23187 [GenBank] was accompanied by acceleration of the Ca²⁺ pump (Table 1).

Furthermore, the importance of luminal Ca²⁺ was also indicated by the amplification of flubendiamide-stimulated Ca²⁺ pump activity with an increased transmembrane Ca²⁺ gradient (Fig. 4). At low extravesicular calcium concentrations, Ca²⁺ pump stimulation by flubendiamide was enhanced. This might be due to an increased flow rate of Ca²⁺ through the activated RyR with the steeper transmembrane calcium gradient.

The catalytic cycles of the Ca²⁺ pump are also regulated by cytoplasmic Ca²⁺, which accelerates the catalytic cycles of the Ca²⁺ pump via cytoplasmic calcium binding site on the molecule. Flubendiamide failed to increase the apparent calcium affinity for cytoplasmic binding site (Fig. 5A). In addition, flubendiamide stimulated the Ca²⁺ pump even under buffered calcium conditions in which released calcium was rapidly clamped by the calcium chelator (Fig. 4); therefore, released Ca²⁺ could not participate in the stimulation of the Ca²⁺ pump by flubendiamide. Hence, we conclude that luminal Ca²⁺ is the pivotal mediator of the coordination between the Ca²⁺ pump and the RyR.

The luminal Ca²⁺ affects the catalytic cycles of the Ca²⁺ pump via the luminal Ca²⁺ binding site on the molecule, which has a low calcium affinity with a high dissociation constant of around 1 mM (de Meis and Vianna, 1979; Ikemoto, 1982; MacLennan et al., 1997). The decrease in luminal Ca²⁺ is considered to accelerate calcium dissociation rates from luminal binding sites and subsequently enhances the Ca²⁺ pump activity based on the unifying model of calcium transport by the Ca²⁺ pump. Similar to the case of A23187 [GenBank] , a Ca²⁺ buffer composed of diBr-BAPTA accelerated the catalytic cycles of Ca²⁺ pump activity (Table 1). This result is attributable to enhancement of the calcium dissociation rate from the luminal binding sites, because the chelator has a higher affinity for Ca²⁺ (K_d = 3.6 µM; Tsien, 1980) compared with the reported affinity of the luminal calcium binding sites (K_d = 1 mM; de Meis and Vianna, 1979). Conversely, the affinity of diBr-BAPTA for Ca²⁺ is lower compared with the cytoplasmic binding sites on the Ca²⁺ pump (K_m = 0.3 µM; Fig. 5A), which indicates that the calcium association to cytoplasmic binding site cannot be interrupted by the chelator. Therefore, enhancement of the calcium dissociation rate from the luminal binding sites by diBr-BAPTA can be directly reflected in acceleration of the Ca²⁺ pump. Hence, the elimination of Ca²⁺ pump stimulation by flubendiamide in the presence of diBr-BAPTA further supports the mediation by luminal Ca²⁺ from the view of a subsequent increase of calcium dissociation rate from luminal binding site. However, the precise mechanism for the acceleration of the Ca²⁺ pump in the presence of diBr-BAPTA remains to be clarified.

The importance of luminal calcium in the coordination between calcium release and resequestration in the mammalian SR has been previously reported by Ikemoto and Yamamoto (2000). Their experiments, however, used polylysine as an inducer of calcium release, which could interact with components of the membrane (de Kruijff and Cullis, 1980). Therefore, this study first elucidated the coordination between calcium release and resequestration using a specific probe for the RyR. Luminal Ca²⁺ is increasingly recognized as a modulator of RyRs, especially cardiac RyRs (Lukyanenko et al., 2001b), which can also modulate the sensitivity for exogenous modulators (Dettbarn and Palade, 1997). As for the Ca²⁺ pump, the luminal calcium binding site contributes to preventing overloading by suppressing calcium uptake. Based on the increasing background information described above, several researchers have indicated the importance of luminal Ca²⁺ homeostasis from the view of optimization of cytoplasmic calcium regulation (Fabio, 1992; Ikemoto and Yamamoto, 2000; Lukyanenko et al., 2001b). The mediation of luminal calcium with cooperation between the Ca²⁺ pump and the RyR in the insect suggests that the physiological importance of luminal calcium is ubiquitous over long evolutionary distances.

It seems that flubendiamide has no direct effect on Ca²⁺ pump, although flubendiamide binding to the isolated Ca²⁺-ATPase has not been investigated, because Ca²⁺-ATPase activity in light SR from rabbit skeletal muscles was not affected by the compound (T. Masaki, unpublished data). The elimination of effect of flubendiamide on Ca²⁺ pump with A23187 [GenBank] or the chelators (Table 1) should provide additional evidence for this speculation. It should also be noted that intracellular calcium in Chinese hamster ovary cells expressing native Ca²⁺-ATPase exhibited no fluctuations by the treatment of flubendiamide (Ebbinghaus-Kintscher et al., 2006). Hence, the stimulation of the Ca²⁺ pump observed in this study should be triggered by activation of insect RyR.

In this study, we demonstrated that the selective and straightforward RyR activation by flubendiamide enables us to elucidate sequential effects on the Ca²⁺ pump. In addition, insect RyR isoforms have distinct characteristics, such as lower calcium sensitivity than mammalian isoforms (Scott-Ward et al., 2001), which can be exactly discriminated using flubendiamide. Thus, this compound should provide a promising probe for understanding the diversity of functional regulations of the RyR. Moreover, precise investigations using the compound can provide pertinent information for discovering unexplored target site for insecticides. The practical flubendiamide binding domain on RyR and its functional role, including interactions with accessory factors, remain to be determined for further clarification of the mode of action of flubendiamide.

	Acknowledgements

 
We are indebted to Drs. Ulrich Ebbinghaus-Kintscher, Peter Lümmen, and Thomas Schulte (Bayer CropScience AG, Monheim am Rhein, Germany) for excellent contributions to these findings. We also extend our appreciation to all colleagues who participated in scientific discussions and provided useful suggestions.

	Footnotes

 
ABBREVIATIONS: RyR, ryanodine receptor; cADPR, cyclic ADP ribose; SR, sarcoplasmic reticulum; diBr-BAPTA, 1,2-bis(2-amino-5-bromophenoxy)ethane-N,N, N', N'-tetraacetic acid; DTT, dithiothreitol; MOPS, 3-(N-morpholino)propanesulfonic acid; A23187 [GenBank] , calcimycin.

Address correspondence to: Takao Masaki, Research Division, Nihon Nohyaku Co., Ltd., 345 Oyamada-cho, Kawachi-Nagano, Osaka, Japan. E-mail: masaki-takao{at}nichino.co.jp

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