We learned in Chapter 4 how the ester of the fluorescent small-molecule dye fura-2 can spontaneously diffuse into the cytosol from extracellular fluids, how fura-2 fluorescence at a certain wavelength increases when Ca²⁺ is bound, and how this dye can be used to measure the concentration of free Ca²⁺ in the cytosol of live cells (see Figure 4-12). Several proteins also emit light when they have bound Ca²⁺ and can be used experimentally to determine the free Ca²⁺ concentration in other subcellular compartments.

One such protein is aequorin, a calcium-activated bioluminescent protein isolated from the hydrozoan Aequorea victoria. Aequorin consists of a protein subunit that can be expressed in cells by recombinant DNA technologies as a fusion protein with a signal sequence that targets it to a specific organelle, such as the ER lumen (see Figure 13-6) or the mitochondrial intermembrane space or matrix (see Figure 13-26). Aequorin contains three EF hands (see Chapter 3) that function as binding sites for Ca²⁺. When the small-molecule prosthetic group coelenterazine is added to the culture medium, it diffuses into the cell and binds to aequorin, and the cell emits light at a specific wavelength proportional to the concentration of free Ca²⁺ in that subcellular space.

The total calcium present in a subcellular compartment is the sum of the free Ca²⁺, which can be measured by aequorin and other calcium sensors whose fluorescence is proportional to the free Ca²⁺ concentration, and the bound Ca²⁺, the amounts of which in the ER and mitochondrion are thought to be much greater than those of free Ca²⁺. Measurements of free Ca²⁺ have indicated that, while the concentrations can vary considerably among different types of resting cells, typically Ca^2+cytosol = ~100 nM, Ca^2+ER = ~400 µM, and Ca^{2+mitochondria} = ~100 nM. The ER lumen contains several Ca²⁺-binding proteins, including the chaperones calreticulin and calnexin (page 605), that have a low affinity and large capacity for Ca²⁺ binding and that buffer Ca^2+ER.

Stimulation of different types of cells by hormones or neuronal signals can result in considerable variation in the free Ca²⁺concentration in organelles, but it invariably results in an increase in Ca^2+cytosol to about 1 µM and in Ca^{2+mitochondria} to approximately 1–10 µM, as well as a decrease in Ca^2+ER to about 100 µM. Transport of Ca²⁺ into and out of the ER lumen and the mitochondrial matrix plays an important role in controlling the nature of these changes and thus calcium signaling throughout the cell.