For cytosolic aequorin and mitochondrial aequorin measurements, cells growing on coverslips were incubated with 5 μM coelenterazine for 2 hours in KRB supplemented with 1% fetal calf serum and then transferred to the perfusion chamber. To reconstitute the aequorin chimeras targeted to the ER, the luminal [Ca2+] of this compartment had to be first reduced by incubating the cells for 1 hour at 4°C in KRB supplemented with 5 μM ionomycin and 600 μM EGTA in the presence of 5 μM coelenterazine, followed by extensive washing of the cells with KRB supplemented with 2% BSA and 1 mM EGTA. All aequorin measurements were performed in KRB supplemented with 1 mM CaCl2, and agonists and other drugs were added to the same medium, as specified in the figure legends. Experiments terminated by lysing the cells with 100 μM digitonin in a hypotonic, Ca2+-rich solution to discharge the remaining aequorin pool. The output of the discriminator was captured by a Thorn EMI photon counting board and stored in an IBM-compatible computer for further analyses. The aequorin luminescence data were calibrated offline into [Ca2+] values using a computer algorithm based on the Ca2+ response curve of WT and mutant aequorins.

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