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

  1. Yutaka Hirata1,
  2. Norimichi Nakahata1,2 and
  3. Yasushi Ohizumi1
  1. 1Department of Pharmaceutical Molecular Biology (Y.H., N.N., Y.O.) and 2Cellular Signaling (N.N.), Graduate School of Pharmaceutical Sciences, Tohoku University, Aoba, Aramaki, Aoba-Ku, Sendai, Japan

    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 Kdvalue of 4.0 μM and a Bmax 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.

    Footnotes

    • Send reprint requests to: Professor Norimichi Nakahata, Department of Cellular Signaling, Graduate School of Pharmaceutical Sciences, Tohoku University, Aoba, Aramaki, Aoba-Ku, Sendai 980-8578, Japan. E-mail: nakahata{at}mail.pharm.tohoku.ac.jp

    • This work was partially supported by Research Fellowships of the Japan Society for the Promotion of Science for Young Scientists (Y.H.) and Grant-in-Aid for Scientific Research from the Ministry of Education, Sciences and Culture of Japan (N.N. and Y.O.).

    • Abbreviations:
      RyR
      ryanodine receptor
      SR
      sarcoplasmic reticulum
      MP
      mastoparan
      HSR
      heavy fraction of sarcoplasmic reticulum
      SR Ca2+-pump
      sarco/endoplasmic reticulum Ca2+-ATPase
      Sulfo-SANPAH
      sulfosuccinimidyl-6-[4′-azido-2′-nitrophenylamino] hexanoate
      CHAPS
      3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid
      Fluo-3
      1-[2-amino-5-(2,7-dichloro-6-hydroxy-3-oxy-9-xanthenyl)phenoxy]-2-(2-amino-5-methylphenoxy)ethane-N,N,N′,N′-tetraacetic acid
      PAGE
      polyacrylamide gel electrophoresis
      MOPS
      3-(N-morpholino)propanesulfonic acid
      Ck
      creatinine kinase
      • Received August 30, 1999.
      • Accepted February 15, 2000.
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    1. Molecular Pharmacology June 1, 2000 vol. 57 no. 6 1235-1242
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