Calcium and the Ca-ATPase SPCA1 modulate plasma membrane abundance of ZIP8 and ZIP14 to regulate Mn(II) uptake in brain microvascular endothelial cells

Manganese (II) accumulation in human brain microvascular endothelial cells is mediated by the metal-ion transporters ZRT IRT-like protein 8 (ZIP8) and ZRT IRT-like protein 14 (ZIP14). The plasma membrane occupancy of ZIP14, in particular, is increased in cells treated with Mn2+, lipopolysaccharide, or IL -6, but the mechanism of this regulation has not been eluci-dated. The calcium-transporting type 2C member 1 ATPase, SPCA1, is a Golgi-localized Ca2+-uptake transporter thought to support Golgi uptake of Mn2+ also. Here, we show using surface protein biotinylation, indirect immunofluorescence, and GFP-tagged proteins that cytoplasmic Ca2+ regulates ZIP8-and ZIP14-mediated manganese accumulation in human brain microvascular endothelial cells by increasing the plasma membrane localization of these transporters. We demonstrate that RNAi knockdown of SPCA1 expression results in an in-crease in cytoplasmic Ca2+ levels. In turn, we found increased cytoplasmic Ca2+ enhances membrane-localized ZIP8 and ZIP14 and a subsequent increase in 54Mn2+ uptake. Further-more, overexpression of WT SPCA1 or a gain-of-function mutant resulted in a decrease in cytoplasmic Ca2+ and Mn-54(2+) accumulation. While addition of Ca2+ positively regu-lated ZIP-mediated Mn-54(2+) uptake, we show chelation of Ca2+ diminished manganese transport. In conclusion, the modula-tion of ZIP8 and ZIP14 membrane cycling by cytoplasmic calcium is a novel finding and provides new insight into the regulation of the uptake of Mn2+ and other divalent metal ions-mediated ZIP metal transporters.

Automated summary

Manganese (II) accumulation in human brain microvascular endothelial cells is mediated by the metal-ion transporters ZIP8 and ZIP14. The membrane localization of ZIP8 and ZIP14 is regulated by cytoplasmic Ca2+, which affects their manganese uptake.