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1 Biological Sciences, Ohio University, 310 Irvine Hall, Athens, Ohio, 45701, United States; Neuroscience Program, Ohio University, Athens, Ohio, United States
2 The Mental Health Research Institute of Victoria, Parkville, Victoria, Australia; Pathology, University of Melbourne, Parkville, Victoria, Australia
3 Biological Sciences, Ohio University, 310 Irvine Hall, Athens, Ohio, 45701, United States
4 Materials and Life Sciences, Osaka University, Osaka, Japan
5 Biological Sciences, Ohio University, Athens, Ohio, United States; Neuroscience Program, Ohio University, Athens, Ohio, United States
* To whom correspondence should be addressed. E-mail: colvin{at}ohio.edu.
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 (ICP-MS), 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 (TPEN)). Three models were tested for their ability to match intracellular free Zn2+ transients and total Zn2+ content observed under these conditions. Only a model that incorporated an intracellular 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 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.
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