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. 1996 Sep 1;318(Pt 2):383–387. doi: 10.1042/bj3180383

Recombinant apoaequorin acting as a pseudo-luciferase reports micromolar changes in the endoplasmic reticulum free Ca2+ of intact cells.

J M Kendall 1, M N Badminton 1, G B Sala-Newby 1, A K Campbell 1, C M Rembold 1
PMCID: PMC1217633  PMID: 8809023

Abstract

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.

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Selected References

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  1. Badminton M. N., Sala-Newby G. B., Kendall J. M., Campbell A. K. Differences in stability of recombinant apoaequorin within subcellular compartments. Biochem Biophys Res Commun. 1995 Dec 26;217(3):950–957. doi: 10.1006/bbrc.1995.2862. [DOI] [PubMed] [Google Scholar]
  2. Berridge M. J. Capacitative calcium entry. Biochem J. 1995 Nov 15;312(Pt 1):1–11. doi: 10.1042/bj3120001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Berridge M. J. Inositol trisphosphate and calcium signalling. Nature. 1993 Jan 28;361(6410):315–325. doi: 10.1038/361315a0. [DOI] [PubMed] [Google Scholar]
  4. Button D., Eidsath A. Aequorin targeted to the endoplasmic reticulum reveals heterogeneity in luminal Ca++ concentration and reports agonist- or IP3-induced release of Ca++. Mol Biol Cell. 1996 Mar;7(3):419–434. doi: 10.1091/mbc.7.3.419. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Campbell A. K., Trewavas A. J., Knight M. R. Calcium imaging shows differential sensitivity to cooling and communication in luminous transgenic plants. Cell Calcium. 1996 Mar;19(3):211–218. doi: 10.1016/s0143-4160(96)90022-6. [DOI] [PubMed] [Google Scholar]
  6. Chatton J. Y., Liu H., Stucki J. W. Simultaneous measurements of Ca2+ in the intracellular stores and the cytosol of hepatocytes during hormone-induced Ca2+ oscillations. FEBS Lett. 1995 Jul 10;368(1):165–168. doi: 10.1016/0014-5793(95)00632-j. [DOI] [PubMed] [Google Scholar]
  7. Clapham D. E. Calcium signaling. Cell. 1995 Jan 27;80(2):259–268. doi: 10.1016/0092-8674(95)90408-5. [DOI] [PubMed] [Google Scholar]
  8. Combettes L., Cheek T. R., Taylor C. W. Regulation of inositol trisphosphate receptors by luminal Ca2+ contributes to quantal Ca2+ mobilization. EMBO J. 1996 May 1;15(9):2086–2093. [PMC free article] [PubMed] [Google Scholar]
  9. Davies E. V., Campbell A. K., Hallett M. B. Ca2+ oscillations in neutrophils triggered by immune complexes result from Ca2+ influx. Immunology. 1994 May;82(1):57–62. [PMC free article] [PubMed] [Google Scholar]
  10. Dawson A. P., Rich G. T., Loomis-Husselbee J. W. Estimation of the free [Ca2+] gradient across endoplasmic reticulum membranes by a null-point method. Biochem J. 1995 Sep 1;310(Pt 2):371–374. doi: 10.1042/bj3100371. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hirose K., Iino M. Heterogeneity of channel density in inositol-1,4,5-trisphosphate-sensitive Ca2+ stores. Nature. 1994 Dec 22;372(6508):791–794. doi: 10.1038/372791a0. [DOI] [PubMed] [Google Scholar]
  12. Hofer A. M., Machen T. E. Technique for in situ measurement of calcium in intracellular inositol 1,4,5-trisphosphate-sensitive stores using the fluorescent indicator mag-fura-2. Proc Natl Acad Sci U S A. 1993 Apr 1;90(7):2598–2602. doi: 10.1073/pnas.90.7.2598. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Kendall J. M., Badminton M. N., Dormer R. L., Campbell A. K. Changes in free calcium in the endoplasmic reticulum of living cells detected using targeted aequorin. Anal Biochem. 1994 Aug 15;221(1):173–181. doi: 10.1006/abio.1994.1394. [DOI] [PubMed] [Google Scholar]
  14. Kendall J. M., Badminton M. N., Sala-Newby G. B., Wilkinson G. W., Campbell A. K. Agonist-stimulated free calcium in subcellular compartments. Delivery of recombinant aequorin to organelles using a replication deficient adenovirus vector. Cell Calcium. 1996 Feb;19(2):133–142. doi: 10.1016/s0143-4160(96)90082-2. [DOI] [PubMed] [Google Scholar]
  15. Kendall J. M., Dormer R. L., Campbell A. K. Targeting aequorin to the endoplasmic reticulum of living cells. Biochem Biophys Res Commun. 1992 Dec 15;189(2):1008–1016. doi: 10.1016/0006-291x(92)92304-g. [DOI] [PubMed] [Google Scholar]
  16. Montero M., Brini M., Marsault R., Alvarez J., Sitia R., Pozzan T., Rizzuto R. Monitoring dynamic changes in free Ca2+ concentration in the endoplasmic reticulum of intact cells. EMBO J. 1995 Nov 15;14(22):5467–5475. doi: 10.1002/j.1460-2075.1995.tb00233.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Nomura M., Inouye S., Ohmiya Y., Tsuji F. I. A C-terminal proline is required for bioluminescence of the Ca(2+)-binding photoprotein, aequorin. FEBS Lett. 1991 Dec 16;295(1-3):63–66. doi: 10.1016/0014-5793(91)81385-l. [DOI] [PubMed] [Google Scholar]
  18. Putney J. W., Jr Capacitative calcium entry revisited. Cell Calcium. 1990 Nov-Dec;11(10):611–624. doi: 10.1016/0143-4160(90)90016-n. [DOI] [PubMed] [Google Scholar]
  19. Raju B., Murphy E., Levy L. A., Hall R. D., London R. E. A fluorescent indicator for measuring cytosolic free magnesium. Am J Physiol. 1989 Mar;256(3 Pt 1):C540–C548. doi: 10.1152/ajpcell.1989.256.3.C540. [DOI] [PubMed] [Google Scholar]
  20. Shimomura O. Cause of spectral variation in the luminescence of semisynthetic aequorins. Biochem J. 1995 Mar 1;306(Pt 2):537–543. doi: 10.1042/bj3060537. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Tse F. W., Tse A., Hille B. Cyclic Ca2+ changes in intracellular stores of gonadotropes during gonadotropin-releasing hormone-stimulated Ca2+ oscillations. Proc Natl Acad Sci U S A. 1994 Oct 11;91(21):9750–9754. doi: 10.1073/pnas.91.21.9750. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Woods N. M., Cuthbertson K. S., Cobbold P. H. Repetitive transient rises in cytoplasmic free calcium in hormone-stimulated hepatocytes. Nature. 1986 Feb 13;319(6054):600–602. doi: 10.1038/319600a0. [DOI] [PubMed] [Google Scholar]
  23. van de Put F. H., Elliott A. C. Imaging of intracellular calcium stores in individual permeabilized pancreatic acinar cells. Apparent homogeneous cellular distribution of inositol 1,4,5-trisphosphate-sensitive stores in permeabilized pancreatic acinar cells. J Biol Chem. 1996 Mar 1;271(9):4999–5006. doi: 10.1074/jbc.271.9.4999. [DOI] [PubMed] [Google Scholar]

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