Reliable measurement of free Ca(2+) concentrations in the ER lumen using Mag-Fluo-4

Synthetic Ca(2+) indicators are widely used to report changes in free [Ca(2+)], 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 [Ca(2+)] within the ER, where the free [Ca(2+)] is estimated to be between 100 μM and 1 mM. Many results are consistent with Mag-Fluo-4 reliably reporting changes in free [Ca(2+)] within the ER, but the results are difficult to reconcile with the affinity of Mag-Fluo-4 for Ca(2+) 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 Ca(2+), suggesting that incomplete de-esterification of Mag-Fluo-4 AM within the ER provides indicators with affinities for Ca(2+) that are both appropriate for the ER lumen and capable of reporting a wide range of free [Ca(2+)].