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 junction al sarcoplasmic reticulum of striated muscles, and has been shown to regula te the ryanodine receptor (RyR) through triadin and junctin. In order to id entify the functional roles of specific regions on CSQ, several CSQ deletio n mutants were prepared by molecular cloning and Escherichia coli expressio n. Ca-45(2+) overlay assay using a native gel system revealed that the majo r 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 affin ity column, the interaction between CSQ and triadin was found to be Ca2+-de pendent, 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 Bioche mical Societies. Published by Elsevier Science B.V. All rights reserved.