Shin D W, Ma J, Kim D H
Department of Life Science, Kwangju Institute of Science and Technology, Kwangju, South Korea.
FEBS Lett. 2000 Dec 8;486(2):178-82. doi: 10.1016/s0014-5793(00)02246-8.
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.
肌集钙蛋白（CSQ）是横纹肌连接肌质网中一种高容量的Ca(2+)结合蛋白，并且已证明它通过三联蛋白和连接蛋白调节兰尼碱受体（RyR）。为了确定CSQ上特定区域的功能作用，通过分子克隆和大肠杆菌表达制备了几种CSQ缺失突变体。使用天然凝胶系统的45Ca(2+)覆盖分析表明，CSQ的主要Ca(2+)结合基序位于富含天冬氨酸的区域（氨基酸354 - 367）。在使用谷胱甘肽-S-转移酶亲和柱的体外结合分析中，发现CSQ与三联蛋白之间的相互作用是Ca(2+)依赖性的，并且相互作用位点局限于CSQ的富含天冬氨酸的区域。我们的结果表明，CSQ的富含天冬氨酸的区域可以通过为腔内Ca(2+)以及三联蛋白提供直接结合位点来参与RyR介导的Ca(2+)释放过程。
