Measurement of Free [Ca2+] Changes in Agonist-Sensitive Internal Stores Using Compartmentalized Fluo

The fact that acetoxymethyl (AM)-ester derivatives of fluorescent Ca2+ indicators accumulate not only in the cytoplasm but also in organelles was recognized long ago as a potential source of artifacts during measurements of cytoplasmic [Ca2+ ] (1 ,2 ). Later it was observed that high-affinity dyes such as fluo-3 and fura-2, normally saturated in the high-[Ca2+ ] environment of the agonist-sensitive Ca2+ store, could register [Ca2+ ] changes in this compartment under special conditions (e.g., when pools were already partially empty) (3 –6 ). The propensity of indicators to become compartmentalized was fully exploited, however, when investigators began to use lower affinity probes such as magfura-2 (7 ), to monitor [Ca2+ ] changes in the inositol(1,4,5)trisphosphate [Ins(1,4,5)P3 ]-sensitive store (a compartment largely accepted to be the endo-plasmic reticulum [ER]). Thus, the release and reloading of this organelle with Ca2+ , as reported by ER-trapped dye, could be directly visualized in single permeabilized cells with high spatiotemporal resolution (8 ,9 ). This basic approach has been adopted by a number of laboratories to investigate phenomena ranging from Ca2+ oscillations (10 –12 ) to quantal release (13 –15 ), and the subcellular localization of Ca2+ storage sites (16 –18 ).