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

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Reliable measurement of free Ca2+ concentrations in the ER lumen using Mag-Fluo-4.
Cell Calcium ( IF 4.3 ) Pub Date : 2020-03-06 , DOI: 10.1016/j.ceca.2020.102188
Ana M Rossi ₁ , Colin W Taylor ₁

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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+].

中文翻译：

使用 Mag-Fluo-4 可靠测量 ER 腔中的游离 Ca2+ 浓度。

合成 Ca2+ 指示剂广泛用于报告游离 [Ca2+] 的变化，通常在细胞溶质中，但也在细胞器中。Mag-Fluo-4，通过用 Mag-Fluo-4 AM 孵育细胞装载到内质网 (ER) 中，已用于测量 ER 内游离 [Ca2+] 的变化，其中游离 [Ca2+] 估计为在 100 μM 和 1 mM 之间。许多结果与 Mag-Fluo-4 可靠地报告 ER 内游离 [Ca2+] 的变化一致，但结果难以与 Mag-Fluo-4 对体外测量的 Ca2+ 的亲和力相一致（KDCa ∼22 μM）。使用抗体淬灭从 ER 泄漏的指示剂的荧光，我们确定 Mag-Fluo-4 在 ER 内的亲和力比体外测量的低得多（KDCa ∼1 mM）。我们表明，部分去酯化的 Mag-Fluo-4 对 Ca2+ 的亲和力降低，

更新日期：2020-03-06

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