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Targeted recombinant aequorins represent to date the most
specific means of monitoring [Ca
Volume 270,
Number 17,
Issue of April 28, pp. 9896-9903, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Concentration (
Ca
)
A CRITICAL EVALUATION
] in
subcellular organelles (Rizzuto, R., Simpson, A. W. M., Brini, M., and
Pozzan, T. (1992) Nature 358, 325-328; Brini, M.,
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
-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.
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