Silencing of ZnT1 reduces Zn super(2+) efflux in cultured cortical neurons

Zinc dyshomeostasis in brain might be involved in the pathogenesis of a series of brain diseases such as Alzheimer's disease and stroke. It is essential that the level of intracellular free Zn super(2+) in neurons is tightly controlled to maintain a narrow window of optimal concentration. The plasma membrane bound transporter ZnT1 is suggested to lower intracellular Zn super(2+) concentration. In this study, the function of ZnT1 in cultured cortical neurons was studied. Using vector-based shRNA interference, the expression of this protein was reduced approximately 40% in cultured rat cortical neurons when measured by immunofluorescence using a ZnT1 antibody. Changes in intracellular Zn super(2+) levels were tracked in individual neurons by microfluorometry using the Zn super(2+) selective fluorophore, FluoZin3. Unopposed Zn super(2+) efflux was measured by first loading cultured cortical neurons with Zn super(2+) then reducing extracellular Zn super(2+) to near zero by addition of EDTA. Reducing ZnT1 expression caused Zn super(2+) efflux to decrease compared with the Zn super(2+) efflux measured in nonsense transfected neurons, suggesting that ZnT1 plays a direct role in Zn super(2+) efflux. ZnT1 dependent Zn super(2+) efflux rate was higher in the first 10 min than at later time periods suggesting that ZnT1-mediated efflux was heavily dependent on the intracellular free Zn super(2+) concentration and/or required an outwardly directed Zn super(2+) gradient.