, Volume 142, Issue 3, pp 281-288

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

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Abstract

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.

The work was supported by grants from the fund for basic research administered by the Israeli Academy of Science and Humanities (to V.SB.) and by Funds from the Minister für Wissenschaft und Forschüng des Landes NRW, from the fonds der Chemischen Indüstrie, from the Deütsche Forschungsgemeinschaft and the Minister für Arbeit Gesündheit ünd Soziales des landes NRW via Herzzentrüm Bad Oeynhaüsen (to the German group). We thank Prof. J. Abramson, Portland State University, for reading the manuscript and offering helpful discussions and valuable suggestions. We also thank Mr. B. Koppitz for technical assistance and Mr. G. Raziel for the photographs.