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.
Pharmacological probes have gained widespread acceptance as reagents to reveal the functional consequences of intracellular calcium regulation. Many exogenous effectors modulate intracellular calcium homeostasis through effects on ryanodine-sensitive calcium release channels (ryanodine receptors; RyRs). However, their actions on RyRs are often complex, not completely clarified, or both (Zucchi and RoncaTestoni, 1997). This complexity is thought to be attributable to calcium dependence of the functional modulation, leading to apparently antithetical effects on the RyR. Such complexity is a serious obstacle to understanding the sequential effects of RyR activity in fluctuating calcium conditions.
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.
The action of flubendiamide is highly specific to insect RyRs and results in selective toxicity in restricted taxa, including Lepidoptera (Tohnishi et al., 2005). The characteristic symptoms caused by continuous muscle contractions through straightforward activation of the insect RyR suggest a pivotal role for the RyR in insect muscle contraction. The physiological importance of RyR in insect muscle implies that basic understanding of excitation-contraction coupling, which has been postulated from evidence accumulated in comprehensive investigations (Lockyer et al., 1998; Quinn et al., 1998; Takekura and Franzini-Armstrong, 2002; Vázquez-Martínez et al., 2003), is broadly deducible in the animal kingdom, including the Insecta. However, distinct pharmacological characteristics are seen in the functional properties of the insect RyR, despite its essential similarity to mammalian isoforms (Lehmberg and Casida, 1994; Puente et al., 2000; Scott-Ward et al., 2001). We therefore believe that the insect muscle is a unique model for understanding the physiological impact of altered RyR activity.
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
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 (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 9000gmax (7000gavg) for 30 min at 4°C. The resulting supernatants were filtered through a cell strainer (BD Falcon, Bedford, MA) and centrifuged at 140,000gmax, (104,000gavg) 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 β-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 β-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.
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, released a large fraction of the residual Ca2+ after the completion of calcium release by flubendiamide, which confirmed that Pi precipitation in the vesicle did not have a limiting effect on calcium release.
The specific Ca2+ pump inhibitor thapsigargin itself induced moderate calcium release, which was not sensitive to Mg2+. Berman (2000) reported that thapsigargin could release Ca2+ bound in ATPase molecules in calcium-limited states. According to the Mg2+ sensitivity, calcium release induced by flubendiamide was characteristically distinguishable from that caused by thapsigargin.
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.
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 induced a 5-fold increase in Ca2+ pump activity. This effect of A23187 was attributable to a decrease in luminal calcium, because A23187 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 (Kd = 3.7 μM) evidently accelerated the catalytic cycles of Ca2+ pump, as in the case with A23187. 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 (Kd = 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 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.
Effect of ATP and Ca2+ Concentrations on Ca2+ Pump Stimulation by Flubendiamide. The catalytic cycles of the Ca2+ pump are also regulated by cytoplasmic Ca2+ concentrations through cytoplasmic calcium binding site on the molecule. Flubendiamide failed to affect the apparent calcium affinity of the cytoplasmic calcium binding site, because the Km values of Ca2+ (0.3 μM) for Ca2+-ATPase activity were not altered by the presence of a supramaximal concentration (100 nM) of the compound (Fig. 5A). The Km values of Ca2+ were determined at calcium concentrations around the reported Kd value for the cytoplasmic calcium binding site (1 μM; de Meis and Vianna, 1979). In this calcium concentration range, Ca2+ pump stimulation by flubendiamide was characterized by increases in the Vmax of the activity.
ATP is another kinetic factor for the catalytic cycles of Ca2+ pump. Ca2+ pump stimulation by flubendiamide was also unaffected in the presence of graded concentrations of ATP (Fig. 5B). ATP concentrations in the millimolar range enhance Ca2+-ATPase activity, which is thought to be mediated by a regulatory binding site on Ca2+-ATPase (Ogawa, 1972; Nakamura et al., 2002). Therefore, these results infer that flubendiamide could not enhance the Ca2+-ATPase activity by affecting the cytoplasmic calcium binding site or the low-affinity ATP binding site.
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 Kd and Bmax 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 Bmax value. This result indicates that flubendiamide shifts the RyR conformation to the open state in this membrane preparation from S. litura.
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.
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, which results in acceleration of Ca2+ pump activity (Scarpa et al., 1972). In the presence of A23187, 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 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, 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+ (Kd = 3.6 μM; Tsien, 1980) compared with the reported affinity of the luminal calcium binding sites (Kd = 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 (Km = 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 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.
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.
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, calcimycin.
- Received November 1, 2005.
- Accepted February 15, 2006.
- The American Society for Pharmacology and Experimental Therapeutics