Vol. 57, Issue 6, 1235-1242, June 2000

Identification of a 97-kDa Mastoparan-Binding Protein Involving in Ca²⁺ Release from Skeletal Muscle Sarcoplasmic Reticulum

Yutaka Hirata, Norimichi Nakahata, and Yasushi Ohizumi

Department of Pharmaceutical Molecular Biology (Y.H., N.N., Y.O.) and Cellular Signaling (N.N.), Graduate School of Pharmaceutical Sciences, Tohoku University, Aoba, Aramaki, Aoba-Ku, Sendai, Japan

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. The ⁴⁵Ca²⁺ 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 of ⁴⁵Ca²⁺ 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 of ¹²⁵I-[Tyr³]MP with a high specific activity. ¹²⁵I-[Tyr³]MP bound specifically to heavy fraction of sarcoplasmic reticulum with a K_d value 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.