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

Accepted version
Peer-reviewed

Type

Article

Change log

Authors

Rossi, Ana M 
Taylor, Colin W 

Abstract

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 (KDCa ∼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 (KDCa ∼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+].

Description

Keywords

Acetoxymethyl ester, Ca(2+) indicator, Ca(2+) signal, Endoplasmic reticulum, Inositol 1,4,5-trisphosphate receptor, Aniline Compounds, Animals, Antibodies, Calcium, Cell Line, Chickens, Endoplasmic Reticulum, Esterification, Fluorescence, Humans, Xanthenes

Journal Title

Cell Calcium

Conference Name

Journal ISSN

0143-4160
1532-1991

Volume Title

87

Publisher

Elsevier BV
Sponsorship
Wellcome Trust (101844/Z/13/Z)
Biotechnology and Biological Sciences Research Council (BB/P005330/1)