Insights into Zn²⁺homeostasis in neurons from experimental and modeling studies

To understand the mechanisms of neuronal Zn 2+ homeostasis better, experimental data obtained from cultured cortical neurons were used to inform a series of increasingly complex computational models. Total metals (inductively coupled plasma-mass spectrometry), resting metallothionein, 65 Zn 2+ uptake and release, and intracellular free Zn 2+ levels using ZnAF-2F were determined before and after neurons were exposed to increased Zn 2+ , either with or without the addition of a Zn 2+ ionophore (pyrithione) or metal chelators [EDTA, clioquinol (CQ), and N, N, N′, N′-tetrakis(2-pyridylmethyl)ethylenediamine]. Three models were tested for the ability to match intracellular free Zn 2+ transients and total Zn 2+ content observed under these conditions. Only a model that incorporated a muffler with high affinity for Zn 2+ , trafficking Zn 2+ to intracellular storage sites, was able to reproduce the experimental results, both qualitatively and quantitatively. This “muffler model” estimated the resting intracellular free Zn 2+ concentration to be 1.07 nM. If metallothionein were to function as the exclusive cytosolic Zn 2+ muffler, the muffler model predicts that the cellular concentration required to match experimental data is greater than the measured resting concentration of metallothionein. Thus Zn 2+ buffering in resting cultured neurons requires additional high-affinity cytosolic metal binding moieties. Added CQ, as low as 1 μM, was shown to selectively increase Zn 2+ influx. Simulations reproduced these data by modeling CQ as an ionophore. We conclude that maintenance of neuronal Zn 2+ homeostasis, when challenged with Zn 2+ loads, relies heavily on the function of a high-affinity muffler, the characteristics of which can be effectively studied with computational models.