Zip6 (LIV-1) regulates zinc uptake in neuroblastoma cells under resting but not depolarizing conditions

Impaired zinc homeostasis is implicated in many cases of brain injury and pathogenesis. While several routes of zinc influx have been identified in neurons under depolarizing conditions, zinc uptake mechanisms during resting conditions are unknown. We have previously detected Zip6 at the plasma membrane of rat neurons, suggesting a role for Zip6 in neuronal zinc uptake. Zinc uptake under resting and depolarizing membrane potentials was measured in SH-SY5Y neuroblastoma cells using ⁶⁵Zn. Zinc uptake was higher under depolarizing conditions, compared with resting conditions, and could be reduced by high extracellular calcium, gadolinium, or nimodipine, which suggests that L-type calcium channels are significant routes of zinc uptake under depolarizing membrane potential. In contrast, zinc uptake under resting conditions was not affected by calcium or calcium channel antagonists. Zip6 was localized to the plasma membrane in SH-SY5Y cells, and siRNA-mediated down-regulation of Zip6 expression reduced zinc uptake during resting, but not depolarizing conditions. Zinc treatment (100 μM Zn) reduced zinc uptake under resting, but not depolarizing conditions, which was associated with lower plasma membrane-associated and total Zip6 protein abundance. These results demonstrate that Zip6 functions as a zinc import protein in neuroblastoma cells, that zinc influx during resting and depolarizing conditions occurs via distinctly different processes in these cells, and suggest that neuronal zinc uptake may be down-regulated by excess zinc levels, but only under resting conditions.