Kendall J M, Badminton M N, Sala-Newby G B, Campbell A K, Rembold C M
Department of Medical Biochemistry, University of Wales College of Medicine, Health Park, Cardiff, U.K.
Biochem J. 1996 Sep 1;318 ( Pt 2)(Pt 2):383-7. doi: 10.1042/bj3180383.
We describe a novel method to monitor the endoplasmic reticulum (ER) free Ca2+ in intact cells. Continuous perfusion of HeLa cells, expressing ER-targeted apoaequorin, with coelenterazine allowed the apoprotein to act as a pseudo-luciferase capable of reporting free Ca2+ from 0.1-100 microM. In intact HeLa cells, addition of ionomycin increased apoaequorin-generated light by 91%, indicating that resting ER free Ca2+ was approx. 2 microM. Agonist stimulation decreased the ER apoaequorin signal and proportionally increased cytosolic free Ca2+ consistent with agonist-induced release of Ca2+ from the ER.
我们描述了一种监测完整细胞内质网(ER)游离Ca2+的新方法。用腔肠素持续灌注表达内质网靶向脱辅基水母发光蛋白的HeLa细胞,使脱辅基蛋白充当能够报告0.1 - 100微摩尔游离Ca2+的假荧光素酶。在完整的HeLa细胞中,添加离子霉素使脱辅基水母发光蛋白产生的光增加了91%,表明内质网静息游离Ca2+约为2微摩尔。激动剂刺激降低了内质网脱辅基水母发光蛋白信号,并相应增加了胞质游离Ca2+,这与激动剂诱导的Ca2+从内质网释放一致。