Transfected Aequorin in the Measurement of Cytosolic Ca2+ Concentration (Ca2+c)

Targeted recombinant aequorins represent to date the most specific means of monitoring Ca2+ 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 Ca2+ in the cytoplasm, a compartment for which effective Ca2+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 Ca2+ values. We show that although fluorescent indicators at the usual intracellular concentrations (50-100 μM) are associated with a significant buffering of the Ca2+ctransients, this problem is negligible with recombinantly expressed aequorin. The large dynamic range of the photoprotein also allows an accurate estimate of the large Ca2+cincreases that are observed in some cell types such as neurons. Finally, cytAEQ appears to be an invaluable tool for measuring Ca2+cin 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. Targeted recombinant aequorins represent to date the most specific means of monitoring Ca2+ 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 Ca2+ in the cytoplasm, a compartment for which effective Ca2+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 Ca2+ values. We show that although fluorescent indicators at the usual intracellular concentrations (50-100 μM) are associated with a significant buffering of the Ca2+ctransients, this problem is negligible with recombinantly expressed aequorin. The large dynamic range of the photoprotein also allows an accurate estimate of the large Ca2+cincreases that are observed in some cell types such as neurons. Finally, cytAEQ appears to be an invaluable tool for measuring Ca2+cin 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.