Flubendiamide binding

At 10 pM [Ca ], flubendiamide binding was clearly cooperative as indicated by the Hill coefficient, n = 2.1. From the sigmoidal fit an apparent Kd = 14 nM was estimated. Additionally, the B ax was drastically reduced to 1800 fmol/mg, indicating that a larger fraction of receptor sites was insensitive to the radioligand. 

Without any calcium added, specific binding increased with the radioligand concentration, but saturation was not reached and meaningful kinetics could not be calculated. None of the known RyR ligands like ryanodine, calmodulin, methylxanthines, ATP, cADP-ribose, nor dantrolene affected flubendiamide binding. 

Saturable binding of [ H]flubendiamide to Heliothis microsomes was measured at a calcium concentration of 800 pM. Interestingly, the 3.5 to 4 times higher binding site density (Bn x) compared to [ H]iyanodine may reflect the stoichiometry of one flubendiamide binding site per RyR subunit in contrast to the accepted stoichiometry of one ryanodine site per homotetrameric channel. However, this needs to be explored further. 

Flubendiamide is an example of a new chemical class of insecticides that have been termed phthalic acid diamides (Nauen 2006, Copping and Duke 2007). They are related to the alkaloid ryanodine, which is extracted from Ryania species. Ryanodine affects muscles by binding to calcium channels of the sarcoplasmic reticulum. Ca + ions act as intracellular messengers, and their flux is modulated by calcium channels of this type. The toxic action of ryanodine and synthetic insecticides related to it is due to the disturbance of calcium flux. 

Therefore it was concluded that flubendiamide acts as a selective activator of the insect ryanodine receptor, inducing ryanodine-sensitive cytosolic Ca transients. Furthermore, radioligand binding studies using microsomal membranes from Heliothis flight muscles demonstrated that flubendiamide allosterically increased the ryanodine affinity. Flubendiamide was found to bind to Heliothis microsomal membranes with an apparent of 4.7 nM. Known ryanodine receptor ligands such as cyclic ADP-ribose, caffeine, ryanodine, and dantrolene did not interfere... 

The rapid calcium efflux efficiently decreases luminal calcium concentration, which induced acceleration of Ca -pump activity due to facilitation of calcium dissociation from a low affinity calcium-binding site (luminal site). To investigate the possible involvement of luminal calcium in the Ca pump stimulation, the luminal Ca concentration was indirectly manipulated by a Ca -ionophore and a calcium chelator (Figure 5). A23187 induced a five-fold increase in Ca pump activity [3] due to an increase of calcium permeability. Under this condition, Ca pump stimulation by flubendiamide was mostly eliminated [3], suggesting the involvement of luminal Ca in the Ca pump stimulation by flubendiamide. 

The stimulatory effect of flubendiamide on the Ca pump was also diminished in the calcium buffers comprised of calcium chelators with high and low calcium affinity [3]. The low affinity calcium chelator, diBr-BAPTA (Kd = 3.7 pM), evidently accelerated the catalytic cycles of Ca pump as in the case with A23187. The result also implies importance of luminal calcium, since the low affinity of this chelator could not interrupt the calcium association with high affinity binding sites (cytoplasmic site) on Ca -ATPase. In addition, the results also demonstrate... 

Flubendiamide (54 ISO-proposed Nihon Nohyaku Co., Ltd./Bayer CropSdence) [125, 126,127] (Fig. 35.13) with a heptafluoro-isopropyl moiety in the anilide part of the molecule (Chapter 34) induces ryanodine-sensitive cytosoUc Ca + transients that were independent of extracellular Ca concentration in isolated neurons from the pest insect Heliothis virescens as well as in transfected CHO cells expressing the RyR from Drosophila mdanogaster. Binding studies on microsomal membranes from H. virescens flight muscle revealed that 54 interacts with a site distinct from the ryanodine binding site and disrupted the Ca + regulation of ryanodine binding by an allosteric mechanism. 

Figure 4. Effect of flubendiamide-sulfoxide on equilibrium saturation binding of fHJryanodine at two different calcium concentrations. Saturation binding was performed essentially as described earlier with 100 pM (A) or 800 pM (B) calcium, respectively. Control asst s (9) and assess including 1.0 pM of flubendiamide-sulfoxide (A.) were performed in quadruplicate and the data were fitted to the binding equation (Hill).

Remarkably, in the presence of 1.0 pM flubendiamide-sulfoxide, the resulting binding isotherm had a simple hyperbolic character (Hill coefficient, n = 1.0), which indicated a homogeneous population of independent sites. Ryanodine affmity was increased with an apparent dissociation constant Kd = 5.2 +/- 0.8 nM (4 experiments). However, Ae number of binding sites, Bn, . remained essentially imchanged (1072 fmol/mg) as compared to the control. 

When increasing the calcium concentration to 800 pM, specific binding of ryanodine could be fitted to the simple hyperbolic equation with n = 1.0 (Figure 4B). The Kd of ryanodine was calculated as 13.6 +/- 1.4 nM (4 experiments) d the receptor density was determined as 1086 finol/mg. Flubendiamide-sulfoxide (1.0 pM) decreased the apparent Kq slightly, but still significantly, to 7.9 +/- 0.9 nM (t-test p < 0.03). 

Flubendiamide Increased H]Ryanodine Binding in a Concentration-Dependent Fashion... 

Ryanodine binding to the Heliothis RyR was regulated by both the calcium and the flubendiamide concentrations (Figure 5). At 800 pM [Ca ],... 

Figure 5. Concentration-dependent effect of flubendiamide on high-affinity fHJryanodine binding (4 nM) at different calcium concentrations. Calcium concentrations (9) 800 pM, fW) 100 pM, (A) 50 pM, and (M) 10 pM(n = 4).

Flubendiamide Affected the Calcium Regulation of [ H]Ryanodine Binding... 

Figure 6. Ryanodine binding as a function of the calcium concentration was affected by flubendiamide (I), The concentration of [ H]ryanodine was 4.4 nM the Bmax 540 +/- 36fmol/mg protein. Flubendiamide (I) concentrations ...

A relatively low concentration of flubendiamide (30 nM) enhanced the high-affinity binding of ryanodine in the micromolar [Ca ] range so that maximum binding was achieved at 18 pM [Ca ]. Remarkably, the inhibitory effect of millimolar calcium concentrations was completely abolished. In the presence of 300 nM flubendiamide, ryanodine binding became independent of [Ca ]. 

Figure 7. Equilibrium saturation binding of H]flubendiamide to Heliothis microsomal membranes. Specific binding was measured at 800 pM (9), at 10 pM( ), and without added calcium (W), respectively.

High-Affinity Binding of ( H]Flubendiamide was Regulated by Calcium... 

As an important feature, phthalic diamides seem to be specific for insect RyR no calcium release could be measured after application of flubendiamide to the mammalian skeletal muscle subtypes RyRl (and RyR3) present in differentiated mouse C2CI2 cells (16). In addition, flubendiamide had no effect on [ Hjryanodine binding to the mammalian cardiac muscle subtype RyR2 prepared from pig heart (unpublished results). 

---