Measurement of Intracellular Free Zinc in Living Cortical Neurons: Routes of Entry

Abstract

We used the ratioable fluorescent dye mag-fura-5 to measure intracellular free Zn 2+([Zn 2+]i) in cultured neocortical neurons exposed to neurotoxic concentrations of Zn 2+in concert with depolarization or glutamate receptor activation and identified four routes of Zn 2+entry. Neurons exposed to extracellular Zn 2+plus high K+responded with a peak cell body signal corresponding to a [Zn 2+]i of 35–45 n m. This increase in [Zn 2+]i was attenuated by concurrent addition of Gd 3+, verapamil, ω-conotoxin GVIA, or nimodipine, consistent with Zn 2+entry through voltage-gated Ca 2+channels. Furthermore, under conditions favoring reverse operation of the Na+–Ca 2+exchanger, Zn 2+application induced a slow increase in [Zn 2+]i and outward whole-cell current sensitive to benzamil–amiloride. Thus, a second route of Zn 2+entry into neurons may be via transporter-mediated exchange with intracellular Na+. Both NMDA and kainate also induced rapid increases in neuronal [Zn 2+]i. The NMDA-induced increase was only partly sensitive to Gd 3+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 Gd 3+or Na+removal in most neurons but insensitive in a minority subpopulation (“cobalt-positive cells”), suggesting that a fourth route of neuronal Zn 2+entry is through the Ca 2+-permeable channels gated by certain subtypes of AMPA or kainate receptors.