期刊
CELL CALCIUM
卷 87, 期 -, 页码 -出版社
ELSEVIER SCI LTD
DOI: 10.1016/j.ceca.2020.102188
关键词
Acetoxymethyl ester; Ca2+ signal; Ca2+ indicator; Endoplasmic reticulum; Inositol 1,4,5-trisphosphate receptor
类别
资金
- Biotechnology and Biological Sciences Research Council UK [BB/P005330/1]
- Wellcome [101844]
- BBSRC [BB/P005330/1] Funding Source: UKRI
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 mu M 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 (K-D(Ca) similar to 22 mu M). 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) similar to 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+].
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