High-Throughput Screening Using Photoluminescence Probe to Measure Intracellular Calcium Levels

Aequorin, a 22 kDa protein produced by the jellyfish Aequorea victoria, was the first probe used to measure Ca²⁺ concentrations ([Ca²⁺]) of specific intracellular organelles in intact cells. After the binding of Ca²⁺ to three high-affinity binding sites, an irreversible reaction occurs leading to the emission of photons that is proportional to [Ca²⁺]. While native aequorin is suitable for measuring cytosolic [Ca²⁺] after cell stimulation in a range from 0.5 to 10 μM, it cannot be used in organelles where [Ca²⁺] is much higher, such as in the lumen of endoplasmic/sarcoplasmic reticulum (ER/SR) and mitochondria. However, some modifications made on aequorin itself or on coelenterazine, its lipophilic prosthetic luminophore, and the addition of targeting sequences or the fusion with resident proteins allowed the specific organelle localization and the measurements of intra-organelle Ca²⁺ levels. In the last years, the development of multiwell plate readers has opened the possibility to perform aequorin-based high-throughput screenings and has overcome some limitation of the standard method. Here we present the procedure for expressing, targeting, and reconstituting aequorin in intact cells and for measuring Ca²⁺ in the bulk cytosol, mitochondria, and ER by a high-throughput screening system.