The Journal of Membrane Biology

, Volume 142, Issue 3, pp 281–288

Endogenous, Ca2+-dependent cysteine-protease cleaves specifically the ryanodine receptor/Ca2+ release channel in skeletal muscle

  • V. Shoshan-Barmatz
  • S. Weil
  • H. Meyer
  • M. Varsanyi
  • L. M. G. Heilmeyer


The association of an endogenous, Ca2+-dependent cysteine-protease with the junctional sarcoplasmic reticulum (SR) is demonstrated. The activity of this protease is strongly stimulated by dithiothreitol (DTT), cysteine and β-mercaptoethanol, and is inhibited by iodoacetamide, mercuric chloride and leupeptin, but not by PMSF. The activity of this thiol-protease is dependent on Ca2+ with half-maximal activity obtained at 0.1 μm and maximal activity at 10 μm. Mg2+ is also an activator of this enzyme (CI50=22 μm). These observations, together with the neutral pH optima and inhibition by the calpain I inhibitor, suggest that this enzyme is of calpain I type.

This protease specifically cleaves the ryanodine receptor monomer (510 kD) at one site to produce two fragments with apparent molecular masses of 375 and 150 kD. The proteolytic fragments remain associated as shown by purification of the cleaved ryanodine receptor. The calpain binding site is identified as a PEST (proline, glutamic acid, serine, threonine-rich) region in the amino acid sequence GTPGGTPQPGVE, at positions 1356–1367 of the RyR and the cleavage site, the calmodulin binding site, at residues 1383–1400. The RyR cleavage by the Ca2+-dependent thiol-protease is prevented in the presence of ATP (1–5 mm) and by high NaCl concentrations. This cleavage of the RyR has no effect on ryanodine binding activity but stimulates Ca2+ efflux. A possible involvement of this specific cleavage of the RyR/Ca2+ release channel in the control of calpain activity is discussed.

Key words

Ryanodine receptor Ca2+ release channel Calpain Junctional sarcoplasmic reticulum Ca2+ release 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Aebersold, R.H., Leavitt, J., Saavedra, R.A., Hood, L.E., Kent, S.B.H. 1987. Internal amino acid sequence analysis of proteins separated by one- or two-dimensional gel electrophoresis after in situ protease digestion on nitrocellulose. Proc. Natl. Acad. Sci. USA 84:6970–6974Google Scholar
  2. 2.
    Brandt, N.R., Caswell, A.H., Brandt, T., Brew, K., Mellgren, R.L. 1992. Mapping of the calpain proteolysis products of the junctional foot protein of the skeletal muscle triad junction. J. Membrane Biol. 127:35–47Google Scholar
  3. 3.
    Busch, W.A., Stromer, M.A., Goll, D.E., Suzuki, A. 1972. Ca2+ specific removal of Z-lines from rabbit skeletal muscle. J. Cell Biol. 52:367–381Google Scholar
  4. 4.
    Caswell, A.H., Lau, Y.H., Brunschwig, J.P. 1976. Ouabain-binding vesicles from skeletal muscle. Arch. Biochem. Biophys. 176:417–430Google Scholar
  5. 5.
    Dayton, W.R., Reville, W.J., Goll, D.E., Stromer, M.A. 1976. A Ca2+-activated protease possible involvement in myofibrillar protein turnover. Partial characterization of the purified enzyme. Biochemistry 15:2159–2167Google Scholar
  6. 6.
    Dayton, W.R., Schollmeyer, J.V., Lepley, R.A., Cortes, L.R. 1981. A calcium-activated protease possibly involved in myofibrillar protein turnover. Biochim. Biophys. Acta 659:48–61Google Scholar
  7. 7.
    DeMartino, G.N., Blumenthal, D.K. 1982. Identification and partial purification of a factor that stimulates calcium-dependent protease. Biochemistry 21:4297–4303Google Scholar
  8. 8.
    Fabiato, A. 1988. Computer programs for calculating total from specified free or free from specified total ionic concentrations in aqueous solutions containing multiple metals and ligands. Methods. Enzymol. 157:378–417Google Scholar
  9. 9.
    Gopalakrishan, R., Barsky, S.H. 1986. Hydrophobic association of calpains with subcellular organs compartmentalization of calpains and the endogenous inhibitor calpastatin in tissues. J. Biol. Chem. 261:13936–13942Google Scholar
  10. 10.
    Guroff, G. 1964. A neutral, calcium-activated protease from soluble fraction of rat brain. J. Biol. Chem. 239:149–155Google Scholar
  11. 11.
    Hewick, R.M., Hunkapiller, M.W., Hood, L.E., Dreyer, W.J. 1981. A gas-liquid solid phase peptide and protein sequenator. J. Biol. Chem. 256:7990–7997Google Scholar
  12. 12.
    Inomata, M., Hayashi, M., Nakamura, M., Imahori, K., Kawashima, S. 1983. Purification and characterization of a calciumactivated neutral protease from rabbit skeletal muscle which requires calcium ions of μm order of concentration. J. Biochem. 93:291–294Google Scholar
  13. 13.
    Kaplan, R.S., Pedersen, F.L. 1985. Determination of microgram quantities of protein in the presence of milligrams level of lipid with amido black 10B. Anal. Biochem. 150:95–104Google Scholar
  14. 14.
    Kar, N.C., Pearson, C.M. 1976. A calcium-activated neutral protease in normal and dystrophic human muscle. Clin. Chim. Acta 73:293–298Google Scholar
  15. 15.
    Kay, J. 1984. Ca2+-activated proteases, protein degradation and muscular dystrophy. In: Proteases: Potential Role in Health and Disease (W.H. Hörl, and A. Heidland), editors, pp. 519–532. Plenum, New YorkGoogle Scholar
  16. 16.
    Laemmli, U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685PubMedGoogle Scholar
  17. 17.
    Lai, F.A., Erickson, H.P., Rousseau, E., Liu, Q.Y., Meissner, G. 1988. Purification and reconstitution of the calcium release channel from skeletal muscle. Nature 331:315–319Google Scholar
  18. 18.
    Lane, R.D., Mellger, R.L., Mericle, M.T. 1985. Subcellular localization of bovine heart calcium-dependent protease inhibitor. J. Mol. Cell. Cardiol. 17:863–872Google Scholar
  19. 19.
    Lowry, O.H., Rosenbrough, N.J., Farr, A.L., Randall, R.J. 1951. Protein measurements with folin phenol reagent. J. Biol. Chem. 193:265–275PubMedGoogle Scholar
  20. 20.
    Mellgren, R.L. 1980. Canine cardiac calcium-dependent proteases: Resolution of two forms with different requirements for calcium. FEBS Lett. 109:129–133Google Scholar
  21. 21.
    Mellgren, R.L. 1987. Calcium-dependent protease: an enzyme system active at cellular membranes. FASEB J. 1:110–115Google Scholar
  22. 22.
    Mellgren, R.L., Lane, R.D., Kakar, S.S. 1987. Isolated bovine myocardial sarcolemma and sarcoplasmic reticulum vesicles contain tightly bound calcium dependent protease inhibitor. Biochem. Biophys. Res. Commun. 142:1025–1031Google Scholar
  23. 23.
    Murachi, T. 1983. Calpain and calpastatine. Trends Biochem. Sci. 8:167–169Google Scholar
  24. 24.
    Murachi, T., Tanaka, K., Hatanaka, M., Murakami, T. 1981. Intracellular Ca2+-dependent protease (calpain) and its highmolecular weight endogenous inhibitor (calpastatin). Adv. Enzyme Regul. 19:407–424Google Scholar
  25. 25.
    Nelson, W.J., Traub, P. 1982. Purification and further characterization of the Ca2+-activated proteinase specific for the intermediate filament proteins vimentin and desmim. J. Biol. Chem. 257:5544–5553Google Scholar
  26. 26.
    Pontremoli, S., Melloni, E. 1986. Extralysosomal protein degradation. Annu. Rev. Biochem. 55:455–481Google Scholar
  27. 27.
    Puca, G.A., Nola, E., Sica, V., Bresciani, F. 1977. Estragon binding proteins of calf uterus; molecular and functional characterization of the receptor transforming factor: A calcium-activated protease. J. Biol. Chem. 252:1358–1370Google Scholar
  28. 28.
    Rardon, D.P., Cefali, D.C., Mitchell, R.D., Seiler, S.M., Hathaway, D.R., Jones, L.R. 1990. Digestion of cardiac and skeletal muscle junctional sarcoplasmic reticulum vesicles with calpain II: Effect on the Ca2+ release channel. Circ. Res. 67:84–96Google Scholar
  29. 29.
    Reddy, M.K., Etlinger, J.D., Rabinowitz, M., Fishman, D., Zak, R. 1975. Removal of Z-lines and α-actinin from isolated myofibrils by a calcium-activated neutral protease. J. Biol. Chem. 250:4278–4284Google Scholar
  30. 30.
    Rogers, S., Wells, R., Rechsteiner, M. 1986. Amino acid sequences common to rapidly degraded proteins: the pest hypothesis. Science 234:364–368Google Scholar
  31. 31.
    Saito, A., Seiler, S., Chu, A., Fleischer, S. 1984. Preparation and morphology of sarcoplasmic reticulum terminal cisternae from rabbit skeletal muscle. J. Cell Biol. 99:875–885Google Scholar
  32. 32.
    Seiler, S., Wegener, A.D., Whang, D.D., Hathaway, D.R., Jones, L.R. 1984. High molecular weight proteins in cardiac and skeletal muscle junctional sarcoplasmic reticulum vesicles bind calmodulin, are phosphorylated, and are degraded by Ca2+-activated protease. J. Biol. Chem. 259:8550–8557Google Scholar
  33. 33.
    Shoshan-Barmatz, V., Zarka, A. 1992. A simple, fast, one-step method for purification of the skeletal muscle ryanodine receptor. Biochem. J. 285:61–64Google Scholar
  34. 34.
    Suzuki, K. 1987. Calcium activated neutral protease: domain structure and activity regulation. Trends Biochem. Sci. 12:103–105Google Scholar
  35. 35.
    Takeshima, H., Nishimura, S., Matsumoto, T., Ishida, H.T., Kangawa, K., Minamino, N., Matsuo, H., Ueda, M., Hanaoka, M., Hirose, T., Numa, S. 1989. Primary structure and expression from complementary DNA of skeletal muscle ryanodine receptor. Nature 339:439–445Google Scholar
  36. 36.
    Towbin, H., Staehelin, T., Gordon, J. 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc. Natl. Acad. Sci. USA 76:4350–4354Google Scholar
  37. 37.
    Vedeckis, W.V., Freeman, M.R., Schrader, W.T., O'Malley, B.W. 1980. Progesterone-binding components of chick oviduct: Partial purification and characterization of a calcium-activated protease which hydrolyzes the progesterone receptor. Biochemistry 19:335–343Google Scholar
  38. 38.
    Wang, K.K.W., Villalobo, A., Roufogalis, B.D. 1989. Calmodulinbinding proteins as calpain substrates. Biochem. J. 262:693–706Google Scholar
  39. 39.
    Yang, H.-C., Reedy, M.M., Burke, C.L., Strasburg, G.M. 1994. Calmodulin interaction with the skeletal muscle sarcoplasmic reticulum calcium channel protein. Biochemistry 33:518–525Google Scholar
  40. 40.
    Yoshimura, N., Kikuchi, T., Sasaki, T., Kitahara, A., Hatanaka, M., Murachi, T. 1983. Two distinct Ca2+ proteases (calpain I and calpain II) purified concurrently by the same method from rat kidney. J. Biol. Chem. 258:8883–8889Google Scholar
  41. 41.
    Zarka, A., Shoshan-Barmatz, V. 1993. Characterization and photoaffinity labeling of the ATP binding site of the ryanodine receptor from skeletal muscle. Eur. J. Biochem. 213:147–154Google Scholar
  42. 42.
    Zarzato, F., Fuyjii, J., Otsu, K., Phillips, M., Green, N.M., Lai, F.A., Meissner, G., MacLennan, D.H. 1990. Molecular cloning of cDNA encoding human and rabbit forms of Ca2+ release channel (Ry-Rec) of skeletal muscle sarcoplasmic reticulum. J. Biol. Chem. 265:2244–2256Google Scholar

Copyright information

© Springer-Verlag New York Inc. 1994

Authors and Affiliations

  • V. Shoshan-Barmatz
    • 1
  • S. Weil
    • 1
  • H. Meyer
    • 2
  • M. Varsanyi
    • 2
  • L. M. G. Heilmeyer
    • 2
  1. 1.Department of Life SciencesBen Gurion University of the NegevBeer ShevaIsrael
  2. 2.Institute for Physiological ChemistryRuhr UniversityBochumGermany

Personalised recommendations