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

Abstract

To understand the mechanisms of neuronal Zn2+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,65Zn2+uptake and release, and intracellular free Zn2+levels using ZnAF-2F were determined before and after neurons were exposed to increased Zn2+, either with or without the addition of a Zn2+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 Zn2+transients and total Zn2+content observed under these conditions. Only a model that incorporated a muffler with high affinity for Zn2+, trafficking Zn2+to intracellular storage sites, was able to reproduce the experimental results, both qualitatively and quantitatively. This “muffler model” estimated the resting intracellular free Zn2+concentration to be 1.07 nM. If metallothionein were to function as the exclusive cytosolic Zn2+muffler, the muffler model predicts that the cellular concentration required to match experimental data is greater than the measured resting concentration of metallothionein. Thus Zn2+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 Zn2+influx. Simulations reproduced these data by modeling CQ as an ionophore. We conclude that maintenance of neuronal Zn2+homeostasis, when challenged with Zn2+loads, relies heavily on the function of a high-affinity muffler, the characteristics of which can be effectively studied with computational models.