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- Title
Neuronal acid-induced [Zn<sup>2+</sup>]<sub>i</sub> elevations calibrated using the low-affinity ratiometric probe FuraZin-1.
- Authors
Kiedrowski, Lech
- Abstract
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 ( Kd = 39 μM at pH 6.1) ratiometric Zn2+ probe, FuraZin-1, was used. A pHi drop from 7.2 to 6.1 caused [Zn2+]i elevations reaching 2 μM; when the thiol-reactive agent N-ethylmaleimide ( NEM) was subsequently applied, [Zn2+]i increased further to 5.6 μM; 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 pKa 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.
- Subjects
NEURONS; N-ethylmaleimide; LIGANDS (Biochemistry); ADENOSINE triphosphate; LUCIFERASES; INTRACELLULAR membranes
- Publication
Journal of Neurochemistry, 2015, Vol 135, Issue 4, p777
- ISSN
0022-3042
- Publication type
Article
- DOI
10.1111/jnc.13282