pH dependence and compartmentalization of zinc transported across plasma membrane of rat cortical neurons

Abstract

In this study, Zn2+ transport in rat cortical neurons was characterized by successfully combining radioactive tracer experiments with spectrofluorometry and fluorescence microscopy. Cortical neurons showed a time-dependent and saturable transport of65Zn2+ with an apparent affinity of 15–20 μM. 65Zn2+ transport was pH dependent and was decreased by extracellular acidification and increased by intracellular acidification. Compartmentalization of newly transported Zn2+ was assessed with the Zn2+-selective fluorescent dye zinquin. Resting cortical neurons showed uniform punctate labeling that was found in cell processes and the soma, suggesting extrasynaptic compartmentalization of Zn2+. Depletion of intracellular Zn2+ with the membrane-permeant chelator N, N, N′, N′-tetrakis(2-pyridylmethyl)-ethylenediamine (TPEN) resulted in the complete loss of punctate zinquin labeling. After Zn2+ depletion, punctate zinquin labeling was rapidly restored when cells were placed in 30 μM Zn2+, pH 7.4. However, rapid restoration of punctate zinquin labeling was not observed when cells were placed in 30 μM Zn2+, pH 6.0. These data were confirmed in parallel 65Zn2+transport experiments.