Recombinant apoaequorin acting as a pseudo-luciferase reports micromolar changes in the endoplasmic reticulum free Ca2+ of intact cells.
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.
Kendall
J M
JM
Department of Medical Biochemistry, University of Wales College of Medicine, Health Park, Cardiff, U.K.
Badminton
M N
MN
Sala-Newby
G B
GB
Campbell
A K
AK
Rembold
C M
CM
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000331:Aequorin / Q000031:analogs & derivatives / Q000096:biosynthesis / Q000494:pharmacology; D001059:Apoproteins / Q000096:biosynthesis; D002118:Calcium / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D007093:Imidazoles; D015759:Ionomycin / Q000494:pharmacology; D007700:Kinetics; D008156:Luciferases; D008163:Luminescent Measurements; D011719:Pyrazines; D011994:Recombinant Proteins / Q000096:biosynthesis; D012680:Sensitivity and Specificity; D013997:Time Factors
D001059:Apoproteins; D007093:Imidazoles; D011719:Pyrazines; D011994:Recombinant Proteins; C060014:apoaequorin; C017144:coelenterazine; D000331:Aequorin; D015759:Ionomycin; D000255:Adenosine Triphosphate; D008156:Luciferases; D002118:Calcium