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Neuronal acid‐induced [Zn 2+ ] i elevations calibrated using the low‐affinity ratiometric probe FuraZin‐1
Author(s) -
Kiedrowski Lech
Publication year - 2015
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/jnc.13282
Subject(s) - chemistry , intracellular , chelation , cysteine , zinc , intracellular ph , biophysics , thiol , biochemistry , inorganic chemistry , biology , enzyme , organic chemistry
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.