Flubendiamide specific binding

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). 

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.

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. 

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had no effect on [ Hjryanodine binding to the mammalian cardiac muscle subtype RyR2 prepared from pig heart (unpublished results). 

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