The asp-rich region at the carboxyl-terminus of calsequestrin binds to Ca2+ and interacts with triadin

Calsequestrin (CSQ) is a high capacity Ca2+ binding protein in the junctional sarcoplasmic reticulum of striated muscles, and has been shown to regulate the ryanodine receptor (RyR) through triadin and junctin. In order to identify the functional roles of specific regions on CSQ, several CSQ deletion mutants were prepared by molecular cloning and Escherichia coli expression. Ca-45(2+) overlay assay using a native gel system revealed that the major Ca2+ binding motif of CSQ resides in the asp-rich region (amino acids 354-367). In an in vitro binding assay using a glutathione-S-transferase affinity column, the interaction between CSQ and triadin was found to be Ca2+-dependent, and the site of interaction was confined to the asp-rich region of CSQ. Our results suggest that the asp-rich region of CSQ could participate in the RyR-mediated Ca2+ release process by offering a direct binding site to luminal Ca2+ as well as triadin. (C) 2000 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.