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. 1996 Mar;7(3):419-34.
doi: 10.1091/mbc.7.3.419.

Aequorin _targeted to the endoplasmic reticulum reveals heterogeneity in luminal Ca++ concentration and reports agonist- or IP3-induced release of Ca++

D Button  1 , A Eidsath
Affiliations
Free PMC article

Aequorin _targeted to the endoplasmic reticulum reveals heterogeneity in luminal Ca++ concentration and reports agonist- or IP3-induced release of Ca++

D Button et al. Mol Biol Cell. 1996 Mar.
Free PMC article

Abstract

A chimeric protein (ERaeq) comprised of the invariant chain (Ii) of class II major histocompatability complex (MHC-II) and aequorin was localized in the endoplasmic reticulum (ER) of transfected human embryonal kidney 293 cells. The _targeted aequorin resided in the lumen of the ER membrane system, including the nuclear cistern, and following addition of the chromophore coelenterazine underwent Ca(++)-activated chemiluminescence. The majority of chemiluminescence produced by coelenterazine treatment of ERaeq-expressing 293 cells was consumed rapidly (within 2-4 min) upon re-addition of Ca++ to coelenterazine-loaded cells, a finding consistent with very high Ca++ concentrations (approximately 10(-5)-10(-3) M Ca++ ion) inside the ER. However, following the initial rapid consumption of ERaeq chemiluminescence, the activity that remained (10-30% of total sample luminescence of permeabilized cells or 50-70% of total sample luminescence of intact cells) was found to produce a stable baseline corresponding to a Ca++ ion concentration < or = 1-2 microM. The stable baseline of luminescence observed following rapid consumption of the majority of the sample's activity was not derived from re-binding of fresh chromophore to spent photoprotein, suggesting that a minority fraction of the ER membrane system within which the ERaeq chimera was distributed contained a relatively low Ca++ concentration. Addition of IP3 to digitonin-permeabilized cells, or agonist treatment of intact cells decreased this residual signal. Luminescence recordings from cells expressing an ER-_targeted aequorin with relatively high affinity for Ca++ thus reveal heterogeneity in luminal ER Ca++ concentration and permit observation of receptor- and IP3-activated release of Ca++ from the ER membrane system.

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References

    1. Annu Rev Physiol. 1993;55:427-54 - PubMed
    1. Proc Natl Acad Sci U S A. 1992 May 15;89(10):4319-23 - PubMed
    1. Cell Calcium. 1993 Mar;14(3):185-200 - PubMed
    1. Biochem J. 1993 Jul 1;293 ( Pt 1):181-5 - PubMed
    1. EMBO J. 1993 Dec;12(12):4813-9 - PubMed
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