Measurement of intracellular free zinc in living cortical neurons: Routes of entry

Stefano L. Sensi, Lorella M.T. Canzoniero, Shan Ping Yu, Howard S. Ying, Jae Young Koh, Geoffrey A. Kerchner, Dennis W. Choi

Research output: Contribution to journalArticlepeer-review

Abstract

We used the ratioable fluorescent dye mag-fura-5 to measure intracellular free Zn2+ ([Zn2+](i)) in cultured neocortical neurons exposed to neurotoxic concentrations of Zn2+ in concert with depolarization or glutamate receptor activation and identified four routes of Zn2+ entry. Neurons exposed to extracellular Zn2+ plus high K+ responded with a peak cell body signal corresponding to a [Zn2+](i) of 35-45 nM. This increase in [Zn2+](i) was attenuated by concurrent addition of Gd3+, verapamil, ω- conotoxin GVIA, or nimodipine, consistent with Zn2+ entry through voltage- gated Ca2+channels. Furthermore, under conditions favoring reverse operation of the Na+-Ca2+ exchanger, Zn2+ application induced a slow increase in [Zn2+](i) and outward whole-cell current sensitive to benzamil- amiloride. Thus, a second route of Zn2+ entry into neurons may be via transporter-mediated exchange with intracellular Na+. Both NMDA and kainate also induced rapid increases in neuronal [Zn2+](i). The NMDA-induced increase was only partly sensitive to Gd3+ or to removal of extracellular Na+, consistent with a third route of entry directly through NMDA receptor- gated channels. The kainate-induced increase was highly sensitive to Gd3+ or Na+ removal in most neurons but insensitive in a minority subpopulation ('cobalt-positive cells'), suggesting that a fourth route of neuronal Zn2+ entry is through the Ca2+-permeable channels gated by certain subtypes of AMPA or kainate receptors.

Original languageEnglish (US)
Pages (from-to)9554-9564
Number of pages11
JournalJournal of Neuroscience
Volume17
Issue number24
DOIs
StatePublished - 1997

Keywords

  • AMPA
  • Calcium
  • Global ischemia
  • Glutamate
  • Hypoxia
  • Mag-fura-5
  • NMDA
  • Neurotoxicity
  • Sodium
  • Sodium-calcium exchanger
  • Voltage-gated calcium channels

ASJC Scopus subject areas

  • Neuroscience(all)

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