Reliable measurement of free Ca2+ concentrations in the ER lumen using Mag-Fluo-4.
Cell Calcium
; 87: 102188, 2020 05.
Article
in En
| MEDLINE
| ID: mdl-32179239
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 â¼22 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 â¼1 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+].
Key words
Full text:
1
Database:
MEDLINE
Main subject:
Xanthenes
/
Calcium
/
Endoplasmic Reticulum
/
Aniline Compounds
Limits:
Animals
/
Humans
Language:
En
Year:
2020
Type:
Article