Zinc transporter 10 (ZnT10)-dependent extrusion of cellular Mn² is driven by an active Ca²-coupled exchange

Manganese (Mn²) is extruded from the cell by the zinc transporter 10 (ZnT10). Loss of ZnT10 expression caused by auto-somal mutations in the ZnT10 gene leads to hypermanganesemia in multiple organs. Here, combining fluorescent monitoring of cation influx in HEK293-T cells expressing human ZnT10 with molecular modeling of ZnT10 cation selectivity, we show that ZnT10 is exploiting the transmembrane Ca² inward gradient for active cellular exchange of Mn². In analyzing ZnT10 activity we used the ability of Fura-2 to spec-trally distinguish between Mn² and Ca² fluxes. We found that (a) application of Mn²-containing Ca²-free solution to ZnT10-expressing cells triggers an influx of Mn², (b) reintroduction of Ca² leads to cellular Mn² extrusion against an inward Mn² gradient, and (c) the cellular transport of Mn² by ZnT10 is coupled to a reciprocal movement of Ca². Remarkably, replacing a single asparagine residue in ZnT10 (Asp-43) with threonine (ZnT10 N43T) converted the Mn²/Ca² exchange to an uncoupled channel mode, permeable to both Ca² and Mn². The findings in our study identify the first ion transporter that uses the Ca² gradient for active counter-ion exchange. They highlight a remarkable versatility in metal selectivity and mode of transport controlled by the tetrahedral metal transport site of ZnT proteins.