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

Area of Science:

• Neuroscience
• Cell Biology
• Biochemistry

Background:

• Intracellular zinc (Zn2+) homeostasis is crucial for neuronal function.
• Acidosis can trigger rapid changes in intracellular Zn2+ levels.
• The role of ATP in buffering intracellular Zn2+ during acidosis is not fully understood.

Purpose of the Study:

• To investigate the source and regulation of intracellular Zn2+ elevations during acidosis in primary neuronal cultures.
• To determine the contribution of ATP-dependent stores to intracellular Zn2+ buffering.
• To elucidate the role of cysteine ligands in Zn2+ binding and release.

Main Methods:

• Primary cultures of murine cortical neurons were used.
• Acid-induced intracellular Zn2+ elevations were measured using ratiometric probes (FuraZin-1 and FluoZin-3).
• ATP depletion was induced by inhibiting mitochondrial and glycolytic pathways, and confirmed with a luciferin/luciferase assay.

Main Results:

• Acidification (pH 7.2 to 6.1) caused intracellular Zn2+ elevations up to 2 µM.
• N-ethylmaleimide (NEM) treatment further increased Zn2+ to 5.6 µM, indicating release from thiol ligands.
• ATP depletion did not affect acid-induced Zn2+ elevations, suggesting negligible contribution from ATP-dependent stores.

Conclusions:

• Acid-sensitive intracellular ligands, likely cysteine-containing molecules, are the primary source of Zn2+ released during acidosis.
• ATP-dependent Zn2+ stores play a minimal role in buffering intracellular Zn2+ during neuronal acidification.
• NEM-induced Zn2+ increase highlights the importance of cysteine residues in Zn2+ chelation in vivo.