RECOMBINANT APOAEQUORIN ACTING AS A PSEUDO-LUCIFERASE REPORTS MICROMOLAR CHANGES IN THE ENDOPLASMIC-RETICULUM FREE CA2-CELLS( OF INTACT)

We describe a novel method to monitor the endoplasmic reticulum (ER) f ree Ca2+ in intact cells. Continuous perfusion of HeLa cells, expressi ng ER-targeted apoaequorin, with coelenterazine allowed the apoprotein to act as a pseudo-luciferase capable of reporting free Ca2+ from 0.1 -100 mu M. In intact HeLa cells, addition of ionomycin increased apoae quorin-generated light by 91%, indicating that resting ER free Ca2+ wa s approx. 2 mu M. Agonist stimulation decreased the ER apoaequorin sig nal and proportionally increased cytosolic free Ca2+ consistent with a gonist-induced release of Ca2+ from the ER.