For mt-AEQ measurements, coverslips with control and mutated fibroblasts overexpressing MCU were incubated for 2 hours in KRB (Krebs-Ringer modified buffer) (135 mM NaCl, 5 mM KCl, 0.4 mM KH2PO4, 1 mM MgSO4, 5.5 mM glucose, and 20 mM Hepes (pH 7.4)] at 37°C supplemented with wild-type coelenterazine (5 M) and then transferred to the perfusion chamber. In the experiments with permeabilized cells, cells were perfused in intracellular buffer [140 mM KCl, 10 mM NaCl, 1 mM KH2PO4, 5.5 mM glucose, 2 mM MgSO4, 1 mM ATP, 2 mM Na+ succinate, and 20 mM Hepes (pH 7.05)] at 37°C. Cells were permeabilized through a 1-min perfusion with 100 μM digitonin during luminescence measurements. After a 2-min washing (to remove digitonin), [Ca2+]m uptake was estimated by mt-AEQ after cell perfusion with the same intracellular solution without EGTA and containing Ca2+ at different concentrations (1 and 4 μM). Calcium uptake speed was calculated as the first derivative by using the first derivative function smoothed for three time points.

To reconstitute erAEQ with high efficiency, luminal [Ca2+] of the ER was first reduced by incubating the cells for 45 min at 4°C in KRB supplemented with 5 μM coelenterazine, the Ca2+ ionophore ionomycin, and 600 μM EGTA. After incubation, the cells were extensively washed with KRB supplemented with 2% BSA and 2 mM EGTA before the luminescence measurement was initiated. Aequorin signals were measured in KRB supplemented with either 1 mM CaCl2 or 100 μM EGTA, using a purpose-built luminometer. The agonist (bradykinin at 100 μM) was added to the same medium.