![]() The problem is negligible with medium free ( m) designed to mimic an unstimulated cell ( < 200 nM) because such a small fraction (<1%) of the released indicator binds Ca 2+, but the leakage becomes more problematic as m approaches or exceeds the K D Ca. An additional concern is that after Mag-Fluo-4 AM is de-esterified within the ER, some indicator leaks into the medium, possibly through organic anion transporters. Although Mag-Fluo-4 has been used to measure luminal in the ER with evident reliability, , ], it is puzzling that an indicator with a reported K D Ca of 22 μM should not be saturated at the free thought to occur within the ER. Mag-Fluo-4 is almost non fluorescent in the absence of divalent cations, but its fluorescence increases after binding Ca 2+ or Mg 2+. Mag-Fluo-4, a Mg 2+ indicator, for which the K D Ca measured in vitro is ∼22 μM, has been widely used as an ER Ca 2+-indicator, , ]. The affinities of Mag-Fura-2 (K D Ca = 53 μM) and Fluo-5 N (K D Ca ∼90 μM) measured in vitro, suggest they would be compatible with measurements of ER, but Fluo-5 N can be effectively loaded into the ER only with the aid of ER-targeted esterases. Among commercially available Ca 2+ indicators, most have affinities best suited to measuring cytosolic (K D Ca < 1 μM). Since the free in the ER lumen is thought to be between 100 μM and 1 mM, , ], relatively low-affinity indicators are required. Typically, cells are incubated with an esterified indicator under conditions that favour accumulation within the ER, followed by selective permeabilization of the plasma membrane (with digitonin or saponin) to release cytosolic indicator. Several methods, including targeted expression of esterases, have been developed to optimize accumulation of synthetic indicators within the ER. While this serendipitous compartmentalization of indicator can compromise measurements of cytosolic, it can also be exploited to allow measurement of within organelles, like the ER. Esterified indicators can also cross intracellular membranes ER-resident carboxylesterases are then assumed to cleave the indicator, trapping it within the organelle. The AM ester is then cleaved in the cytosol by endogenous esterases that both restore the Ca 2+-binding site of the indicator and trap the indicator within the cell. Synthetic indicators, which are readily available and usually brighter than GECIs, are often loaded into cells as acetoxymethyl (AM) esters. Addition of targeting sequences to GECIs allows selective expression in the ER lumen. Both genetically-encoded Ca 2+ indicators (GECI), derived from endogenous Ca 2+ sensors and synthetic Ca 2+ indicators, derived from carboxylate-based Ca 2+ buffers, have been developed to meet these needs. There is, therefore, a need for fluorescent Ca 2+ indicators that can reliably measure free within the cytosol and lumina of intracellular organelles. Many of these signals are initiated by release of Ca 2+ from the ER through inositol 1,4,5-trisphosphate receptors. ![]() Changes in the free within organelles, notably the endoplasmic reticulum (ER), mitochondria and lysosomes, also regulate cellular activities. Spatially organized increases in cytosolic free regulate many cellular processes. We show that partially de-esterified Mag-Fluo-4 has reduced affinity for Ca 2+, suggesting that incomplete de-esterification of Mag-Fluo-4 AM within the ER provides indicators with affinities for Ca 2+ that are both appropriate for the ER lumen and capable of reporting a wide range of free. 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 ∼1 mM) than that measured in vitro. Many results are consistent with Mag-Fluo-4 reliably reporting changes in free within the ER, but the results are difficult to reconcile with the affinity of Mag-Fluo-4 for Ca 2+ measured in vitro (K D Ca ∼22 μM). 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 within the ER, where the free is estimated to be between 100 μM and 1 mM. Synthetic Ca 2+ indicators are widely used to report changes in free, usually in the cytosol but also within organelles.
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