Monitoring dynamic changes in free Ca2+ concentration in the endoplasmic reticulum of intact cells.
Direct monitoring of the free Ca2+ concentration in the lumen of the endoplasmic reticulum (ER) is an important but still unsolved experimental problem. We have shown that a Ca(2+)-sensitive photoprotein, aequorin, can be addressed to defined subcellular compartments by adding the appropriate targeting sequences. By engineering a new aequorin chimera with reduced Ca2+ affinity, retained in the ER lumen via interaction of its N-terminus with the endogenous resident protein BiP, we show here that, after emptying the ER, Ca2+ is rapidly re-accumulated up to concentrations of > 100 microM, thus consuming most of the reporter photoprotein. An estimate of the steady-state Ca2+ concentration was obtained using Sr2+, a well-known Ca2+ surrogate which elicits a significantly slower rate of aequorin consumption. Under conditions in which the rate and extent of Sr2+ accumulation in the ER closely mimick those of Ca2+, the steady-state mean lumenal Sr2+ concentration ([Sr2+]er) was approximately 2 mM. Receptor stimulation causes, in a few seconds, a 3-fold decrease of the [Sr2+]er, whereas specific inhibition of the ER Ca2+ ATPase leads to an approximately 10-fold drop in a few minutes.
Montero
M
M
Department of Biomedical Sciences, University of Padova, Italy.
Brini
M
M
Marsault
R
R
Alvarez
J
J
Sitia
R
R
Pozzan
T
T
Rizzuto
R
R
D000331:Aequorin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism*; D002413:Cations, Divalent; D017931:DNA Primers; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012519:Sarcoplasmic Reticulum / Q000201:enzymology; D014407:Tumor Cells, Cultured
D002413:Cations, Divalent; D017931:DNA Primers; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D000331:Aequorin; D002118:Calcium