ENDOGENOUS, CA2-DEPENDENT CYSTEINE-PROTEASE CLEAVES SPECIFICALLY THE RYANODINE RECEPTOR CA2+ RELEASE CHANNEL IN SKELETAL-MUSCLE()

The association of an endogenous, Ca2+-dependent cysteine-protease wit h the junctional sarcoplasmic reticulum (SR) is demonstrated. The acti vity of this protease is strongly stimulated by dithiothreitol (DTT), cysteine and beta-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 obtain ed at 0.1 mu M and maximal activity at 10 mu M. Mg2+ is also an activa tor of this enzyme (CI50 = 22 mu M). These observations, together with the neutral pH optima and inhibition by the calpain I inhibitor, sugg est 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 p roteolytic 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 a nd the cleavage site, the calmodulin binding site, at residues 1383-14 00. 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 s timulates Ca2+ efflux. A possible involvement of this specific cleavag e of the RyR/Ca2+ release channel in the control of calpain activity i s discussed.