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⁺].