Identification, Characterization and Partial Purification of a Thiol-protease Which Cleaves Specifically the Skeletal Muscle Ryanodine Receptor/Ca²⁺ Release Channel

Abstract.

A 94 kDa large subunit thiol-protease, as identified by anti-calpain antibodies, has been isolated from skeletal muscle junctional sarcoplasmic reticulum (SR). This protease cleaves specifically the skeletal muscle ryanodine receptor (RyR)/Ca²⁺ release channel at one site resulting in the 375 kDa and 150 kDa fragments. The 94 kDa thiol-protease degrades neither other SR proteins nor the ryanodine receptor of cardiac nor brain membranes. The partially purified 94 kDa protease, like the SR associated protease, had an optimal pH of about 7.0, was absolutely dependent on the presence of thiol reducing reagents, and was completely inhibited by HgCl₂, leupeptin and the specific calpain I inhibitor. However, while the SR membrane-associated protease requires Ca²⁺ at a submicromolar concentration, the isolated thiol-protease has lost the Ca²⁺ requirement.

The 94 kDa thiol-protease had no effect on ryanodine binding but modified the channel activity of RyR reconstituted into planar lipid bilayer: in a time-dependent manner, the channel activity decreases and within several minutes the channel is converted into a subconducting state. The protease-modified channel activity is still Ca²⁺-dependent and ryanodine sensitive.

This 94 kDa thiol-protease cross react with anti-calpain antibodies thus, may represent the novel large subunit of the skeletal muscle specific calpain p94.