Changes in free calcium in the endoplasmic reticulum of living cells detected using targeted aequorin.
The Ca(2+)-activated photoprotein aequorin has been engineered with the endoplasmic reticulum (ER)-targeting sequence from calreticulin at the N-terminus and the KDEL sequence at the C-terminus so that it locates in the ER of living cells. Targeting of apoaequorin to the ER of COS7 cells was demonstrated by immunolocalization. Selective permeabilization of cells expressing the modified protein suggested that targeting was highly efficient. Functional photoprotein was reconstituted in live cells by incubating them with coelenterazine. Light emission from cells expressing ER aequorin showed that the estimated free Ca2+ within the ER of live cells at 37 degrees C was 0.3-1.0 microM, some 10 times that in the cytosol. An increase in the rate constant for aequorin light emission was demonstrated when the cells were warmed from 4 degrees C. This increase could be in part, but not wholly, explained by an increase in rate constants for aequorin at higher temperatures and a change in kinetics as a result of the ER targeting of aequorin. The increase in rate constants in the cells was inhibited by thapsigargin and occurred in the presence or absence of extracellular Ca2+. These results highlight the importance of converting aequorin light emission to rate constants and of calibrating any variants if qualitative and quantitative conclusions are to be drawn about free Ca2+ in intracellular compartments.
Kendall
J M
JM
Department of Medical Biochemistry, University of Wales College of Medicine, Cardiff, United Kingdom.
Badminton
M N
MN
Dormer
R L
RL
Campbell
A K
AK
D000331:Aequorin; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D008163:Luminescent Measurements; D008969:Molecular Sequence Data; D015202:Protein Engineering; D013696:Temperature; D013729:Terpenes / Q000494:pharmacology; D019284:Thapsigargin
D013729:Terpenes; D000331:Aequorin; D019284:Thapsigargin; D002118:Calcium