Neuronal acid‐induced [Zn2+]i elevations calibrated using the low‐affinity ratiometric probe FuraZin‐1

The experiments were carried out on primary cultures of murine cortical neurons from cryopreserved preparations obtained from embryonic-day-16 fetuses. To calibrate acid-induced intracelluar [Zn2+] ([Zn2+](i)) elevations, a low affinity (K-d=39M at pH 6.1) ratiometric Zn2+ probe, FuraZin-1, was used. A pH(i) drop from 7.2 to 6.1 caused [Zn2+](i) elevations reaching 2M; when the thiol-reactive agent N-ethylmaleimide (NEM) was subsequently applied, [Zn2+](i) increased further to 5.6M; analogous acid- and NEM-induced [Zn2+](i) elevations could also be detected but not calibrated, using the high affinity Zn2+ probe FluoZin-3. The data indicate that NEM causes Zn2+ release from ligands that chelate Zn2+ at pH 6.1. ATP could also chelate Zn2+ at pH 6.1 because its pK(a) is about 6.8. Therefore, it was tested whether an ATP depletion affects the acid-induced [Zn2+](i) elevations. The ATP depletion was induced by inhibiting mitochondrial and glycolytic ATP production. Interestingly, an almost complete ATP depletion (confirmed using a luciferin/luciferase assay) failed to affect the acid-induced [Zn2+](i) increases. These data suggest that the total amount of Zn2+ accumulated in intracellular ATP-dependent stores (Zn2+-ATP complexes and organelles that accumulate Zn2+ in an ATP-dependent manner) is negligible compared to the amount of Zn2+ accumulated in the acid-sensitive intracellular ligands.