The solute carriers ZIP8 and ZIP14 regulate manganese accumulation in brain microvascular endothelial cells and control brain manganese levels

Manganese (Mn) supports numerous neuronal functions but in excess is neurotoxic. Consequently, regulation of Mn flux at the blood-brain barrier (BBB)(1) is critical to brain homeostasis. However, the molecular pathways supporting the transcellular trafficking of divalent Mn ions within the microvascular capillary endothelial cells (BMVECs) that constitute the BBB have not been examined. In this study, we have determined that Zrt- and Irt-like protein 8 (ZIP8) and ZIP14 support Mn2+-uptake by BMVECs and that neither DMT1 nor an endocytosis-dependent pathway play any significant role in Mn2+-uptake. Specifically, siRNA-mediated knockdown of ZIP8 and ZIP14 coincided with a decrease in Mn uptake, and kinetic analyses revealed that Mn uptake depends on pH and bicarbonate and is up-regulated by lipopolysaccharide (LPS), all biochemical markers of ZIP8 or ZIP14 activity. Mn2+-uptake also was associated with cell-surface membrane presentation of ZIP8 and ZIP14, as indicated by membrane protein biotinylation. Importantly, surface ZIP8 and ZIP14 biotinylation, and Mn2+-uptake experiments together revealed that these transporters support Mn-uptake at both the apical, blood and basal, brain sides of BMVECs. This indicated that at in the BMVECs of the BBB, these two transporters support a bi-directional Mn2+ flux. We conclude that BMVECs play a critical role in controlling manganese homeostasis in the brain.