Transfected Aequorin in the Measurement of Cytosolic Ca
Concentration (
Ca
)
A CRITICAL EVALUATION (*)
- Marisa Brini(§),
- Robert Marsault(§)(¶),
- Carlo Bastianutto,
- Javier Alvarez(**),
- Tullio Pozzan and
- Rosario Rizzuto(§§)
- From the (1) Department of Biomedical Sciences and Consiglio Nazionale delle Ricerche Center for the Study of Mitochondrial Physiology, University of Padova, Padova 35121, Italy
- §§ To whom correspondence should be addressed: Dept. Biomedical Sciences, University of Padova, Via Trieste 75, 35121 Padova, Italy. Tel.: 39-49-828-6569; Fax: 39-49-828-6576; E-mail: rizzuto{at}cribi1.bio.unipd.it.
Abstract
Targeted recombinant aequorins represent to date the most specific means of monitoring [Ca
] in subcellular organelles (Rizzuto, R., Simpson, A. W. M., Brini, M., and Pozzan, T. (1992) Nature 358, 325-328; Brini, M., [Medline] Murgia, M., Pasti, L., Picard, D., Pozzan, T., and Rizzuto, R. (1993) EMBO J. 12, 4813-4819; Kendall, J. M., Dormer, R. L., and Campbell, A. K. (1992) Biochem. Biophys. Res. Commun. 189, 1008-1016). Up until now, however, only limited attention has been paid to the use of recombinant photoproteins for
measuring, in mammalian cells, the [Ca
] in the cytoplasm, a compartment for which effective Ca
probes are already available. Here we describe this approach in detail, highlighting the advantages, under various experimental
conditions, of using recombinant cytosolic aequorin (cytAEQ) instead of classical fluorescent indicators. We demonstrate that
cytAEQ is expressed recombinantly at high levels in transiently transfected cell lines and primary cultures as well as in
stably transfected clones, and we describe a simple algorithm for converting aequorin luminescence data into [Ca
] values. We show that although fluorescent indicators at the usual intracellular concentrations (50-100 μM) are associated with a significant buffering of the [Ca
]
transients, this problem is negligible with recombinantly expressed aequorin. The large dynamic range of the photoprotein
also allows an accurate estimate of the large [Ca
]
increases that are observed in some cell types such as neurons. Finally, cytAEQ appears to be an invaluable tool for measuring
[Ca
]
in cotransfection experiments. In particular, we show that when cotransfected with an α1-adrenergic receptor (coupled to inositol 1,4,5-trisphosphate generation), cytAEQ faithfully monitors the subpopulation of
cells expressing the receptor, whereas the signal of fura-2, at the population level, is dominated largely by that of the
untransfected cells.
Footnotes
-
↵§ The first two authors equally contributed to this work.
-
↵¶ Recipient of a European Union Human Capital and Mobility fellowship.
-
↵** Supported by a fellowship of the Spanish Direccion General de Investigation Cientifica y Tecnica.
-
↵* This work was supported in part by grants from the Italian Research Council (CNR), Biotechnology and Oncology; Telethon; the Italian Association for Cancer Research (AIRC); the AIDS project of the Italian Health Ministry; the Italian Ministry of University and Scientific Research; and the British Research Council (to T. P. and R. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
-
↵1 The abbreviations used are:
- [Ca
] -
cytosolic calcium concentration
- PCR
-
polymerase chain reaction
- cytAEQ
-
cytosolic aequorin
- PBS
-
phosphate-buffered saline
- HEDTA
-
N-hydroxyethylethylenediaminetriacetic acid.
- [Ca
-
↵2R. Marsault, M. Brini, T. Pozzan, and R. Rizzuto, in preparation.
-
↵3L. Jaffe and G. Rutter, personal communication.
-
↵4L. Jaffe, personal communication.
-
↵5M. Brini, R. Rizzuto, and T. Pozzan, unpublished data.
- © 1995 by The American Society for Biochemistry and Molecular Biology, Inc.
