Reliable measurement of free Ca2+ concentrations in the ER lumen using Mag-Fluo-4

Synthetic CA²⁺ indicators are widely used to report changes in free [CA²⁺], usually in the cytosol but also within organelles. Mag-Fluo-4, loaded into the endoplasmic reticulum (ER) by incubating cells with Mag-Fluo-4 AM, has been used to measure changes in free [CA²⁺] within the ER, where the free [CA²⁺] is estimated to be between 100 μM and 1 mM. Many results are consistent with Mag-Fluo-4 reliably reporting changes in free [CA²⁺] within the ER, but the results are difficult to reconcile with the affinity of Mag-Fluo-4 for CA²⁺ measured in vitro (K_D^CA ∼22 μM). Using an antibody to quench the fluorescence of indicator that leaked from the ER, we established that the affinity of Mag-Fluo-4 within the ER is much lower (K_D^CA ∼1 mM) than that measured in vitro. We show that partially de-esterified Mag-Fluo-4 has reduced affinity for CA²⁺, suggesting that incomplete de-esterification of Mag-Fluo-4 AM within the ER provides indicators with affinities for CA²⁺ that are both appropriate for the ER lumen and capable of reporting a wide range of free [CA²⁺].