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. 1995 Nov 15;14(22):5467–5475. doi: 10.1002/j.1460-2075.1995.tb00233.x

Monitoring dynamic changes in free Ca2+ concentration in the endoplasmic reticulum of intact cells.

M Montero 1, M Brini 1, R Marsault 1, J Alvarez 1, R Sitia 1, T Pozzan 1, R Rizzuto 1
PMCID: PMC394660  PMID: 8521803

Abstract

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.

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Selected References

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