ZnT1 is the main transporter mediating Zn²⁺ efflux from the cytoplasm to the extracellular space. Paradoxically, due to relatively low Zn²⁺ affinity of ZnT1, physiological levels of cytoplasmic free Zn²⁺ are seemingly insufficient to support ZnT1 function. However, physiological modulation of NMDA receptor responses by synaptically released Zn²⁺ requires a Zn²⁺ rise in the postsynaptic neurons, accompanied by ZnT1 activity. We hypothesized that Zn²⁺ import from the extracellular space could generate intracellular Zn²⁺ microdomains in the vicinity of ZnT1 to enable its activity, thus forming a localized "Zn²⁺-cycle." To identify the functional machinery that may drive this process, we investigated the expression and function of ZIP proteins that import Zn²⁺ into the neuronal cytoplasm. We focused on the dorsal cochlear nucleus (DCN) and hippocampus, regions where synaptic Zn²⁺ modulates postsynaptic NMDA responses and synaptic plasticity. We demonstrate that ZIP3 is expressed on DCN cartwheel cells and required for postsynaptic Zn²⁺ influx. Importantly, ZIP3 physically interacts with ZnT1, suggesting that it is an integral component of the synaptic Zn²⁺-cycle machinery. In the hippocampus, ZIP1, but not ZIP3, mediates Zn²⁺ import into postsynaptic CA3 cells and we find that ZIP1 interacts with ZnT1 in this brain region. Importantly, Zn²⁺ efflux rates are enhanced in SH-SY5Y cells co-expressing ZnT1 and either ZIP3 or ZIP1, compared to rates in cells expressing ZnT1 alone. Our findings indicate that ZnT1-dependent Zn²⁺ efflux is facilitated by ZIP-ZnT1 complexes, which potentially induce local intracellular Zn²⁺ microdomains supporting a Zn²⁺-cycle capable of modulating synaptic signaling.

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