In vitro and in vivo calibration of low affinity genetic Ca²⁺ indicators

Calcium is a universal intracellular messenger and proper Ca²⁺concentrations ([Ca²⁺]) both in the cytosol and in the lumen of cytoplasmic organelles are essential for cell functions. Ca²⁺ homeostasis is achieved by a delicate pump/leak balance both at the plasma membrane and at the endomembranes, and improper Ca²⁺ levels result in malfunction and disease. Selective intraorganellar Ca²⁺measurements are best achieved by using targeted genetically encoded Ca²⁺ indicators (GECIs) but to calibrate the luminal fluorescent signals into accurate [Ca²⁺] is challenging, especially in vivo, due to the difficulty to normalize and calibrate the fluorescent signal in various tissues or conditions. We report here a procedure to calibrate the ratiometric signal of GAP (GFP-Aequorin Protein) targeted to the endo-sarcoplasmic reticulum (ER/SR) into [Ca²⁺]_ER/SR based on imaging of fluorescence after heating the tissue at 50-52 °C, since this value coincides with that obtained in the absence of Ca²⁺ (R_min). Knowledge of the dynamic range (R_max/R_min) and the Ca²⁺-affinity (K_D) of the indicator permits calculation of [Ca²⁺] by applying a simple algorithm. We have validated this procedure in vitro using several cell types (HeLa, HEK 293T and mouse astrocytes), as well as in vivo in Drosophila. Moreover, this methodology is applicable to other low Ca²⁺ affinity green and red GECIs.