Simultaneous Monitoring of Cytosolic and Reticular Ca2+ Indicates Heterogeneity of Ca2+ Store

Transduction of many agonists involves mobilization of intracellular Ca²⁺. The dynamics and shape of intracellular Ca²⁺ signals are determined by Ca²⁺ fluxes across the plasma membrane and membranes of intracellular organelles, primarily the endoplasmic reticulum (ER). Although traditionally, intracellular Ca²⁺ signaling has been studied using chemical fluorescent Ca²⁺ probes, the advent of genetically encoded Ca²⁺ sensors with different intracellular localizations has significantly expanded the instrumental capabilities. In the present work, synchronous monitoring of cytosolic Ca²⁺ in HEK-293 cells using Fluo-8 and of reticular Ca²⁺ using the genetically encoded ER-localized Ca²⁺ sensor R-CEPIA1er was performed. Upon stepwise stimulation of cells with acetylcholine (ACh), a coordinated increase in cytosolic Ca²⁺ and a decrease in ER Ca²⁺ were observed in the front phase of the Ca²⁺ response, the relaxation of which was often accompanied by superimposed oscillations. A greater detailing of the correlated behavior of the cytosolic Ca²⁺ (C) and reticular Ca²⁺ (C_s) concentrations could be provided by representing cellular responses in the phase plane (C_s, C). Since C_s and C are not measurable directly but evaluated through Ca²⁺-dependent fluorescence of Ca²⁺ probes, the experimental data were presented in the (F_s, F_c) plane, where F_s and F_c are ΔF/F₀ for R-CEPIA1er and Fluo-8, respectively. Qualitatively, this is equivalent to the presentation of data in the (C_s, C) plane. The phase trajectories indicated that Ca²⁺ responses to ACh could not be generated solely by Ca²⁺ exchange between the homogeneous Ca²⁺ store and the cytosol, since in approximately 30% of cases the phase trajectory contained a loop, wherein a simultaneous increase in C_s and C took place. The loop was observed in a calcium-free medium with negligible Ca²⁺ influx into the cell, indicating the involvement of an intracellular Ca²⁺ source that was not accessible to monitoring by R-CEPIA1er or did not significantly contribute to its fluorescence. The inhibitory analysis showed that the suggested source was not acidic endosomes and lysosomes containing two-pore channels, the Golgi apparatus, and vesicles loaded with Ca²⁺ by SPCA-type ATPase, organelles releasing Ca²⁺ through ryanodine receptors, and/or mitochondria releasing Ca²⁺ into the cytosol through the Na⁺/Ca²⁺ exchanger. The results obtained pointed out that a system mediating agonist-induced Ca²⁺ signals is more complex than one in the paradigm with one homogeneous pool of stored Ca²⁺.

intracellular Са²⁺ signaling, Fluo-8, genetically encoded Cа²⁺ sensor R-CEPIA1er, phase plane