Guo W, Campbell K P
Howard Hughes Medical Institute, University of Iowa College of Medicine, Iowa City 52242, USA.
J Biol Chem. 1995 Apr 21;270(16):9027-30. doi: 10.1074/jbc.270.16.9027.
Triadin is a major membrane protein that is specifically localized in the junctional sarcoplasmic reticulum of skeletal muscle and is thought to play an important role in muscle excitation-contraction coupling. In order to identify the proteins in the skeletal muscle that interact with triadin, the cytoplasmic and luminal domains of triadin were expressed as glutathione S-transferase fusion proteins and immobilized to glutathione-Sepharose to form affinity columns. Using these affinity columns, we find that triadin binds specifically to the ryanodine receptor/Ca2+ release channel and the Ca(2+)-binding protein calsequestrin from CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid)-solubilized skeletal muscle homogenates. The luminal but not the cytoplasmic domain of triadin-glutathione S-transferase fusion protein binds [3H]ryanodine receptor, whereas neither the cytoplasmic nor the luminal portion of triadin binds [3H]PN-200-100-labeled dihydropyridine receptor. In addition, the luminal domain of triadin interacts with calsequestrin in a Ca(2+)-dependent manner and is capable of inhibiting the reassociation of calsequestrin to the junctional face membrane. These results suggest that triadin is the previously unidentified transmembrane protein that anchors calsequestrin to the junctional region of the sarcoplasmic reticulum, and is involved in the functional coupling between calsequestrin and the ryanodine receptor/Ca2+ release channel.
三联蛋白是一种主要的膜蛋白，特异性定位于骨骼肌的连接肌浆网，被认为在肌肉兴奋 - 收缩偶联中起重要作用。为了鉴定骨骼肌中与三联蛋白相互作用的蛋白质，将三联蛋白的胞质结构域和腔结构域表达为谷胱甘肽S - 转移酶融合蛋白，并固定在谷胱甘肽 - 琼脂糖上以形成亲和柱。使用这些亲和柱，我们发现三联蛋白与来自CHAPS（3 - [(3 - 胆酰胺丙基)二甲基铵基]-1 - 丙烷磺酸）溶解的骨骼肌匀浆中的兰尼碱受体/Ca2+释放通道和Ca(2+)结合蛋白钙结合蛋白特异性结合。三联蛋白 - 谷胱甘肽S - 转移酶融合蛋白的腔结构域而非胞质结构域结合[3H]兰尼碱受体，而三联蛋白的胞质部分和腔部分均不结合[3H]PN - 200 - 100标记的二氢吡啶受体。此外，三联蛋白的腔结构域以Ca(2+)依赖的方式与钙结合蛋白相互作用，并且能够抑制钙结合蛋白与连接面膜的重新结合。这些结果表明，三联蛋白是先前未鉴定的跨膜蛋白，它将钙结合蛋白锚定到肌浆网的连接区域，并参与钙结合蛋白与兰尼碱受体/Ca2+释放通道之间的功能偶联。
