Abstract

Mastoparan (MP) and radiolabeled [Tyr³]MP caused a transient Ca²⁺ release from the heavy fraction of sarcoplasmic reticulum, which was inhibited by ryanodine. MP enhanced [³H]ryanodine binding in a concentration-dependent manner with an EC₅₀ value of approximately 0.3 μM. The⁴⁵Ca²⁺ release was accelerated by MP, [Tyr³]MP, or caffeine in a concentration-dependent manner. The EC₅₀ values for MP, [Tyr³]MP, and caffeine were approximately 2.0 μM, 7.7 μM, and 1.8 mM, respectively. MP, like caffeine, shifted the stimulatory limb of a bell-shaped curve of Ca²⁺ dependence to the left.⁴⁵Ca²⁺ release induced by caffeine was completely inhibited by typical blockers of Ca²⁺-induced Ca²⁺ release, such as Mg²⁺, ruthenium red, or procaine. However, ⁴⁵Ca²⁺ release induced by MP was completely inhibited by Mg²⁺, but it was only partially inhibited by ruthenium red or procaine. The rate of⁴⁵Ca²⁺ release induced by MP was further increased in the presence of caffeine, showing that the MP binding site is different from that of caffeine on Ca²⁺ release channels. We succeeded in the synthesis of¹²⁵I-[Tyr³]MP with a high specific activity.¹²⁵I-[Tyr³]MP bound specifically to heavy fraction of sarcoplasmic reticulum with a K_dvalue of 4.0 μM and a B_max value of 3.0 nmol/mg. Furthermore, ¹²⁵I-[Tyr³]MP specifically cross-linked to the 97-kDa protein without direct binding to ryanodine receptor. The protein was not triadin or Ca²⁺-pump, because antitriadin antibody and anti-Ca²⁺-pump antibody did not immunoprecipitate the protein. These results suggest that the 97-kDa MP-binding protein may have an important role in the excitation-contraction coupling of skeletal muscle.