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

Abstract

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., 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 microM) are associated with a significant buffering of the [Ca2+]c transients, this problem is negligible with recombinantly expressed aequorin. The large dynamic range of the photoprotein also allows an accurate estimate of the large [Ca2+]c increases that are observed in some cell types such as neurons. Finally, cytAEQ appears to be an invaluable tool for measuring [Ca2+]c in cotransfection experiments. In particular, we show that when cotransfected with an alpha 1-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.

Intracellular ATP-sensitive K+ channels in mouse pancreatic beta cells: against a role in organelle cation homeostasis

“…Cells were infected [42] with adenoviruses encoding untargeted aequorin (AdCMVcytoAq) [43], aequorin targeted to the mitochondria (AdCMVmAq) [42], or mutant aequorin targeted to secretory vesicles (AdCMVVampAq) [44]. Aequorin was reconstituted with 5 μmol/l coelenterazine (LUX Biotechnology, Edinburgh, UK) by incubating for 2 h at 37°C in modified Krebs-Ringer bicarbonate buffer (KRB) (140 mmol/l NaCl, 3.5 mmol/l KCl, 0.5 mmol/l NaH 2 PO 4 , 0.5 mmol/l MgSO 4 , 3 mmol/l glucose, 10 mmol/l HEPES, 2 mmol/l NaHCO 3 , pH 7.4) supplemented with 1.5 mmol/l CaCl 2 .…”

“…Recombinant aequorins targeted to the mitochondrial matrix or cytosol [43,48,49] were used. As previously demonstrated [42,48], depolarisation of intact cells with 30 mmol/l KCl caused increases in [Ca 2+ ] in both compartments, with a ∼50% greater increase in the mitochondrial matrix, as expected (Fig.…”

Measuring [Ca2+] in the endoplasmic reticulum with aequorin

“…L is the luminescence value at every point (minus the background) and L max is the integral of luminescence (minus the background) from that point to the end of the experiment. L/L max values are then transformed into [Ca 2+ ] values using the following mathematical algorithm [19].…”

CALHM1 and its polymorphism P86L differentially control Ca2+ homeostasis, mitogen‐activated protein kinase signaling, and cell vulnerability upon exposure to amyloid β

“…Experiments to measure changes in [Ca 2+ ] n or [Ca 2+ ] c were performed 36 to 48 h after transfection. The two recombinant proteins were expressed in the same subset of cells (AEQ and the channel) (Brini et al ., 1995). …”

“…To ensure the total translocation of nu_AEQ to the nucleus, cells were incubated with dexamethasone 10 μ m for 2 h immediately before the experiment was carried out (Brini et al ., 1995). The cell monolayer was continuously superfused at room temperature (24 ± 2 °C) with Krebs–Hepes buffer (KHB) of the following composition: 125 m m NaCl, 5 m m KCl, 1 m m Na 3 PO 4 , 1 m m MgSO 4 , 5.5 m m glucose, and 20 m m HEPES (pH 7.4); the zero Ca 2+ solution contained 0.5 m m ethylene glycol tetraacetic acid.…”