Wang Ying, Li Xinghai, Duan Hongzhe, Fulton Timothy R, Eu Jerry P, Meissner Gerhard
Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, NC 27599-7260, United States.
Cell Calcium. 2009 Jan;45(1):29-37. doi: 10.1016/j.ceca.2008.05.006. Epub 2008 Jul 11.
Triadin and junctin are integral sarcoplasmic reticulum membrane proteins that form a macromolecular complex with the skeletal muscle ryanodine receptor (RyR1) but their roles in skeletal muscle calcium homeostasis remain incompletely understood. Here we report that delivery of siRNAs specific for triadin or junctin into C2C12 skeletal myoblasts reduced the expression of triadin and junctin in 8-day-old myotubes by 80 and 100%, respectively. Knocking down either triadin or junctin in these cells reduced Ca2+ release induced by depolarization (10mM KCl) by 20-25%. Unlike triadin knockdown myotubes, junctin knockdown and junctin/triadin double knockdown myotubes also had reduced Ca2+ release induced by 400 microM 4-chloro-m-cresol, 10mM caffeine, 400 microM UTP, or 1 microM thapsigargin. Thus, knocking down junctin compromised the Ca2+ stores in the sarcoplasmic reticulum of these cells. Our subsequent studies showed that in junctin knockdown myotubes at least two sarcoplasmic reticulum proteins (RyR1 and skeletal muscle calsequestrin) were down-regulated while these proteins' mRNA expression was not affected. The results suggest that triadin has a role in facilitating KCl depolarization-induced Ca2+ release in contrast to junctin which has a role in maintaining sarcoplasmic reticulum Ca2+ store size in C2C12 myotubes.
三联蛋白和连接蛋白是肌浆网的整合膜蛋白，它们与骨骼肌雷诺丁受体（RyR1）形成大分子复合物，但其在骨骼肌钙稳态中的作用仍未完全明确。在此我们报告，将针对三联蛋白或连接蛋白的小干扰RNA（siRNA）导入C2C12骨骼肌成肌细胞后，8日龄肌管中三联蛋白和连接蛋白的表达分别降低了80%和100%。在这些细胞中敲低三联蛋白或连接蛋白，可使去极化（10mM氯化钾）诱导的Ca2+释放减少20 - 25%。与敲低三联蛋白的肌管不同，敲低连接蛋白以及同时敲低连接蛋白和三联蛋白的肌管，由400μM 4 - 氯间甲酚、10mM咖啡因、400μM尿苷三磷酸（UTP）或1μM毒胡萝卜素诱导的Ca2+释放也减少。因此，敲低连接蛋白会损害这些细胞肌浆网中的Ca2+储存。我们随后的研究表明，在敲低连接蛋白的肌管中，至少两种肌浆网蛋白（RyR1和骨骼肌肌钙蛋白）表达下调，而这些蛋白的mRNA表达未受影响。结果表明，与连接蛋白不同，三联蛋白在促进氯化钾去极化诱导的Ca2+释放中起作用，而连接蛋白在维持C2C12肌管中肌浆网Ca2+储存量方面起作用。
