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NERVOUS SYSTEM CELL BIOLOGY

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

¹Department of Biological Sciences, ²Neuroscience Program, and ³Quantitative Biology Institute, Ohio University, Athens, Ohio; ⁴Graduate School of Engineering, Department of Materials and Life Sciences, Osaka University, Osaka, Japan; and ⁵The Mental Health Research Institute of Victoria and ⁶Department of Pathology, The University of Melbourne, Parkville, Victoria, Australia

Submitted 14 November 2007 ; accepted in final form 29 December 2007

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.

muffler; zinc buffering; computational model; metal ion transport and homeostasis; metallothionein

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