Studies of Zinc Transport and Its Contribution to Zinc Homeostasis in Cultured Cortical Neurons

Zn²⁺ dyshomeostasis in brain might be involved in the pathogenesis of brain diseases such as Alzheimer’s disease and stroke. Thus, neurons tightly control the level of intracellular free Zn²⁺ within a narrow window of optimal concentration. In this study, the mechanisms of transporter-mediated Zn²⁺ extrusion and uptake across the plasma membrane of cultured cortical neurons were studied. Changes in intracellular Zn²⁺ levels were tracked in individual neurons by microfluorometry using a Zn²⁺ selective fluorophore, FluoZin3. Zn²⁺ uptake and efflux was measured by first loading cultured cortical neurons with Zn²⁺ then reducing extracellular Zn²⁺ to near zero by addition of EDTA. Studies revealed that the primary means of Zn²⁺ efflux in cortical neurons required both extracellular Na⁺ and Ca²⁺. A Na⁺, Ca²⁺/Zn²⁺ exchanger mechanism is proposed to extrude Zn²⁺ at the expense of electrochemical sodium and calcium gradients. ZnT1 (SLC30A1) protein levels were reduced around 40% in cultured cortical neurons (*p<0.001) by vector-based shRNA interference (shRNAi). Reducing ZnT1 expression caused the Zn²⁺ efflux to decrease compared with the control neurons (*p<0.01), which are consistent with ZnT1’s role as a Zn²⁺ efflux transporter or at least regulating Zn²⁺ efflux. In case of Zn²⁺ uptake, acidosis or alkalosis both inhibited Zn²⁺ uptake at resting condition. Depolarization induced large Zn²⁺ uptake in neurons. ZIP1 (SLC39A1) protein levels were reduced around 22% by shRNAi (*p<0.001) and resulted in less Zn²⁺ uptake (*p<0.05). In addition, effects of intracellular zinc levels on the expression of zinc transporter proteins were studied. Right after hZIP1 mRNA was injected into oocytes, oocytes were incubated with different solutions with different levels zinc. The expression of hZIP1 protein on the membrane of oocytes treated with TPEN was increased compare with control (regular medium plus DMSO). When the oocytes were incubated with 10 µM ZnCl₂, the hZIP1 expression was highly reduced observed by immunostaining.

The findings of my studies can be summarized into three aspects: firstly, a Na⁺, Ca²⁺/Zn²⁺ exchanger and ZnT1 appear to be separate routes acting to reduce intracellular Zn²⁺ levels in cultured cortical neurons. Second, a Zn²⁺, HCO₃^- symporter mechanism and ZIP1 could uptake Zn²⁺ into neurons at resting condition. Third, hZIP1 protein expression can be regulated by zinc levels at translational levels.