title: Silencing of ZnT1 reduces Zn2+ efflux in cultured cortical neurons.
abstract: 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 Zn2+ in neurons is tightly controlled to maintain a narrow window of optimal concentration. The plasma membrane bound transporter ZnT1 is suggested to lower intracellular Zn2+ 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 Zn2+ levels were tracked in individual neurons by microfluorometry using the Zn2+ selective fluorophore, FluoZin3. Unopposed Zn2+ efflux was measured by first loading cultured cortical neurons with Zn2+ then reducing extracellular Zn2+ to near zero by addition of EDTA. Reducing ZnT1 expression caused Zn2+ efflux to decrease compared with the Zn2+ efflux measured in nonsense transfected neurons, suggesting that ZnT1 plays a direct role in Zn2+ efflux. ZnT1 dependent Zn2+ 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 Zn2+ concentration and/or required an outwardly directed Zn2+ gradient.
authors.affiliation: Department of Biological Sciences, Ohio University, Athens, OH 45701, USA.
authors.forename: Yan
authors.initials: Y
authors.lastname: Qin
authors.forename: Dustin
authors.initials: D
authors.lastname: Thomas
authors.forename: Charles P
authors.initials: CP
authors.lastname: Fontaine
authors.forename: Robert A
authors.initials: RA
authors.lastname: Colvin
mesh_terms: D000818:Animals; D027682:Cation Transport Proteins; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology*; D004622:Embryo, Mammalian; D007211:Indoles; D016014:Linear Models; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000494:pharmacology; D051381:Rats; D015032:Zinc / Q000378:metabolism*
chemical_list: D027682:Cation Transport Proteins; D007211:Indoles; D008565:Membrane Proteins; D034741:RNA, Small Interfering; C092392:Slc30a1 protein, rat; C007293:DAPI; D015032:Zinc