Journal of the American Chemical Society · 2002 · 409 citations · 25 references
Life SciencesBiochemistryCell BiologyBiosensorsExocytosisMolecular ImagingFluorescence ImagingFluorescence MicroscopyFluorescence SpectroscopyIntracellular Calcium SignalingPancreatic HormonesSecretory PathwayPancreatic Islet CellsZinc Binding ProteinsZinc Biochemistry
The probe’s chelating core is based on EGTA‑derived Ca²⁺ fluoroionophores, modified by deleting one N‑acetyl group. A novel Zn²⁺‑selective visible‑wavelength fluoroionophore, FluoZin‑3, was synthesized and used to monitor Zn²⁺ co‑secreted with insulin from pancreatic β‑cells upon glucose‑stimulated exocytosis. FluoZin‑3 is non‑fluorescent without Zn²⁺ but shows a several‑hundred‑fold increase at ~100 nM Zn²⁺ (Kd ≈ 15 nM) with 1:1 stoichiometry and pH‑independent fluorescence above pH 6; it revealed ~600 nM Zn²⁺ near β‑cells, detectable up to 15 µm away, and heterogeneous secretion indicating active membrane zones.
A novel Zn2+-selective visible wavelength fluoroionophore (FluoZin-3, 9) was synthesized. The chelating portion of the molecule resembles known EGTA-based Ca2+-selective fluoroionophores, except that one of the N-acetic acid moieties has been deleted in 9. FluoZin-3 is virtually non-fluorescent in the absence of Zn2+, and exhibits a several hundred-fold fluorescence increase upon saturation with Zn2+(∼100 nM), with a Kd = 15 ± 2 nM. A 1:1 binding stoichiometry of 9:Zn2+ was determined, and the fluorescence of the complex is pH-independent at pH > 6. FluoZin-3 was used to monitor Zn2+ that was co-secreted with insulin from pancreatic β-cells by exocytosis following stimulation with glucose. The total Zn2+ concentration near the cells reached 600 nM, and Zn2+ was detectable at least 15 μm away from secreting cells. Heterogeneity in secretion among cells was indicated in that some cells in a cluster did not release Zn2+. Also, within secreting cells some regions of the cell membrane gave rise to secretion while others did not, suggesting active zones of secretion on the cell surface.
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Chemical and physiological characterization of fluo-4 Ca2+-indicator dyes
Katrina Gee, K A Brown, W-N.U. Chen et al. · Cell Calcium · 2000 · 646 citations