Mery L, Mesaeli N, Michalak M, Opas M, Lew D P, Krause K H
Division of Infectious Diseases, University Hospital, Geneva, Switzerland.
J Biol Chem. 1996 Apr 19;271(16):9332-9. doi: 10.1074/jbc.271.16.9332.
The widely distributed and highly conserved Ca(2+)-binding protein calreticulin has been suggested to play a role as a Ca2+ storage protein of intracellular Ca+ stores. To test this hypothesis, we have generated a mouse L fibroblast cell line stably transfected with a calreticulin expression vector. The calreticulin content of the overexpressers was increased by 1.6 +/- 0.2-fold compared with mock-transfected cells. The total cellular Ca2+ content of calreticulin-overexpressing and control cells, as assessed by equilibrium 45Ca+2 uptake, was 141 +/- 8 and 67 +/- 6 pmol of Ca2+/10(6) cells, respectively (i.e. a 2.1 +/- 0.2-fold increase in the Ca2+ content of calreticulin-overexpressing cells). Over 80% of the increased Ca2+ content was found within thapsigargin-sensitive Ca2+ stores. The pattern of calreticulin distribution, revealed by immunofluorescence microscopy, showed an endoplasmic reticulum-like pattern and was identical in overexpressers and control cells. In overexpressers, cytosolic free [Ca2+] elevations due to Ca2+ release were enhanced when either ATP or a combination of ionomycin and thapsigargin was used as a stimulus. In contrast, thapsigargin-induced Ca2+ and Mn2+ influxes from the extracellular space were markedly diminished in calreticulin-overexpressing cells, suggesting an active involvement of calreticulin in the regulation of store-operated Ca2+ influx.
广泛分布且高度保守的钙结合蛋白钙网蛋白被认为作为细胞内钙库的钙储存蛋白发挥作用。为了验证这一假设，我们构建了一个稳定转染钙网蛋白表达载体的小鼠L成纤维细胞系。与mock转染细胞相比，过表达细胞中钙网蛋白的含量增加了1.6±0.2倍。通过平衡45Ca+2摄取评估，钙网蛋白过表达细胞和对照细胞的总细胞钙含量分别为141±8和67±6 pmol Ca2+/10(6)细胞（即钙网蛋白过表达细胞的钙含量增加了2.1±0.2倍）。超过80%增加的钙含量存在于毒胡萝卜素敏感的钙库中。免疫荧光显微镜显示的钙网蛋白分布模式呈内质网样模式，在过表达细胞和对照细胞中相同。在过表达细胞中，当使用ATP或离子霉素与毒胡萝卜素的组合作为刺激时，由于钙释放导致的胞质游离[Ca2+]升高增强。相反，在钙网蛋白过表达细胞中，毒胡萝卜素诱导的细胞外空间钙和锰内流明显减少，表明钙网蛋白积极参与调控储存操纵性钙内流。
