Neuronal acid‐induced [Zn 2+ ] 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 [Zn 2+ ] ([Zn 2+ ] i ) elevations, a low affinity ( K d  = 39 μM at pH 6.1) ratiometric Zn 2+ probe, FuraZin‐1, was used. A pH i drop from 7.2 to 6.1 caused [Zn 2+ ] i elevations reaching 2 μM; when the thiol‐reactive agent N ‐ethylmaleimide ( NEM ) was subsequently applied, [Zn 2+ ] i increased further to 5.6 μM; analogous acid‐ and NEM ‐induced [Zn 2+ ] i elevations could also be detected but not calibrated, using the high affinity Zn 2+ probe FluoZin‐3. The data indicate that NEM causes Zn 2+ release from ligands that chelate Zn 2+ at pH 6.1. ATP could also chelate Zn 2+ at pH 6.1 because its pK a is about 6.8. Therefore, it was tested whether an ATP depletion affects the acid‐induced [Zn 2+ ] 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 [Zn 2+ ] i increases. These data suggest that the total amount of Zn 2+ accumulated in intracellular ATP ‐dependent stores (Zn 2+ ‐ ATP complexes and organelles that accumulate Zn 2+ in an ATP ‐dependent manner) is negligible compared to the amount of Zn 2+ accumulated in the acid‐sensitive intracellular ligands.In vitro, upon acidification, Zn 2+ ‐cysteine complexes release Zn 2+ and ATP chelates the released Zn 2+ . However, in vivo (cultured neurons), an ATP depletion failed to enhance acid‐induced [Zn 2+ ] i elevations. These [Zn 2+ ] i elevations were calibrated using a low affinity ratiometric probe FuraZin‐1; they reached 2 µM levels and increased to 5 µM when a thiol‐reactive agent, N ‐ethylmaleimide, compromised Zn 2+ binding by cysteines.