Zinc release from thapsigargin/IP3-sensitive stores in cultured cortical neurons

Background

Changes in ionic concentration have a fundamental effect on numerous physiological processes. For example, IP₃-gated thapsigargin sensitive intracellular calcium (Ca²⁺) storage provides a source of the ion for many cellular signaling events. Less is known about the dynamics of other intracellular ions. The present study investigated the intracellular source of zinc (Zn²⁺) that has been reported to play a role in cell signaling.

Results

In primary cultured cortical cells (neurons) labeled with intracellular fluorescent Zn²⁺ indicators, we showed that intracellular regions of Zn²⁺ staining co-localized with the endoplasmic reticulum (ER). The latter was identified with ER-tracker Red, a marker for ER. The colocalization was abolished upon exposure to the Zn²⁺ chelator TPEN, indicating that the local Zn²⁺ fluorescence represented free Zn²⁺ localized to the ER in the basal condition. Blockade of the ER Ca²⁺ pump by thapsigargin produced a steady increase of intracellular Zn²⁺. Furthermore, we determined that the thapsigargin-induced Zn²⁺ increase was not dependent on extracellular Ca²⁺ or extracellular Zn²⁺, suggesting that it was of intracellular origin. The applications of caged IP₃ or IP₃-3Kinase inhibitor (to increase available IP₃) produced a significant increase in intracellular Zn²⁺.

Conclusions

Taken together, these results suggest that Zn²⁺ is sequestered into thapsigargin/IP₃-sensitive stores and is released upon agonist stimulation.