Identification of a 97-kDa mastoparan-binding protein involving in Ca2+ release from skeletal muscle sarcoplasmic reticulum

Mastoparan (MP) and radiolabeled [Tyr(3)]MP caused a transient Ca2+ release from the heavy fraction of sarcoplasmic reticulum, which was inhibited by ryanodine. MP enhanced [H-3]ryanodine binding in a concentration-dependent manner with an EC50 value of approximately 0.3 mu M. The Ca-45(2+) release was accelerated by MP, [Tyr(3)]MP, or caffeine in a concentration-dependent manner. The EC50 values for MP, [Tyr(3)]MP, and caffeine were approximatel y 2.0 mu M, 7.7 mu M, and 1.8 mM, respectively. MP, like caffeine, shifted the stimulatory limb of a bell-shaped curve of Ca2+ dependence to the left. Ca-45(2+) release induced by caffeine was completely inhibited by typical blockers of Ca2+-induced Ca2+ release, such as Mg2+, ruthenium red, or proc aine. However, Ca-45(2+) release induced by MP was completely inhibited by Mg2+, but it was only partially inhibited by ruthenium red or procaine. The rate of Ca-45(2+) release induced by MP was further increased in the prese nce of caffeine, showing that the MP binding site is different from that of caffeine on Ca2+ release channels. We succeeded in the synthesis of I-125[ Tyr(3)]MP with a high specific activity. I-125-[Tyr(3)]MP bound specificall y to heavy fraction of sarcoplasmic reticulum with a K-d value of 4.0 mu M and a B-max value of 3.0 nmol/mg. Furthermore, I-125-[Tyr(3)]MP specificall y cross-linked to the 97-kDa protein without direct binding to ryanodine re ceptor. The protein was not triadin or Ca2+-pump, because antitriadin antib ody and anti-Ca2+-pump antibody did not immunoprecipitate the protein. Thes e results suggest that the 97-kDa MP-binding protein may have an important role in the excitation-contraction coupling of skeletal muscle.