ZnT1 is a neuronal Zn²⁺/Ca²⁺ exchanger

Zinc transporter 1 (ZnT1; SLC30A1) is present in the neuronal plasma membrane, critically modulating NMDA receptor function and Zn²⁺ neurotoxicity. The mechanism mediating Zn²⁺ transport by ZnT1, however, has remained elusive. Here, we investigated ZnT1-dependent Zn²⁺ transport by measuring intracellular changes of this ion using the fluorescent indicator FluoZin-3. In primary mouse cortical neurons, which express ZnT1, transient addition of extracellular Zn²⁺ triggered a rise in cytosolic Zn²⁺, followed by its removal. Knockdown of ZnT1 by adeno associated viral (AAV)-short hairpin RNA (shZnT1) markedly increased rates of Zn²⁺ rise, and decreased rates of its removal, suggesting that ZnT1 is a primary route for Zn²⁺ efflux in neurons. Although Zn²⁺ transport by other members of the SLC30A family is dependent on pH gradients across cellular membranes, altered H⁺ gradients were not coupled to ZnT1-dependent transport. Removal of cytoplasmic Zn²⁺, against a large inward gradient during the initial loading phase, suggests that Zn²⁺ efflux requires a large driving force. We therefore asked if Ca²⁺ gradients across the membrane can facilitate Zn²⁺ efflux. Elimination of extracellular Ca²⁺ abolished Zn²⁺ efflux, while increased extracellular Ca²⁺ levels enhanced Zn²⁺ efflux. Intracellular Ca²⁺ rises, measured in GCaMP6 expressing neurons, closely paralleled cytoplasmic Zn²⁺ removal. Taken together, these results strongly suggest that ZnT1 functions as a Zn²⁺/Ca²⁺ exchanger, thereby regulating the transport of two ions of fundamental importance in neuronal signaling.