The asp-rich region at the carboxyl-terminus of calsequestrin binds to Ca(2+) and interacts with triadin.
Calsequestrin (CSQ) is a high capacity Ca(2+) 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. 45Ca(2+) overlay assay using a native gel system revealed that the major Ca(2+) 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 Ca(2+)-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 Ca(2+) release process by offering a direct binding site to luminal Ca(2+) as well as triadin.
Shin
D W
DW
Department of Life Science, Kwangju Institute of Science and Technology, Kwangju, South Korea.
Ma
J
J
Kim
D H
DH
D000818:Animals; D001224:Aspartic Acid / Q000378:metabolism*; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins; D002155:Calsequestrin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism; D002413:Cations, Divalent; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins; D006899:Mixed Function Oxygenases; D009124:Muscle Proteins / Q000378:metabolism*; D018482:Muscle, Skeletal / Q000378:metabolism; D016296:Mutagenesis; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D019837:Ryanodine Receptor Calcium Release Channel / Q000378:metabolism
D002135:Calcium-Binding Proteins; D002155:Calsequestrin; D002352:Carrier Proteins; D002413:Cations, Divalent; D008565:Membrane Proteins; D009124:Muscle Proteins; D011993:Recombinant Fusion Proteins; D019837:Ryanodine Receptor Calcium Release Channel; C498269:TRDN protein, human; C070048:triadin; D001224:Aspartic Acid; C498842:Junctin protein, Oryctolagus cuniculus; D006899:Mixed Function Oxygenases; D005982:Glutathione Transferase; D002118:Calcium