Journal
AMERICAN JOURNAL OF PHYSIOLOGY-CELL PHYSIOLOGY
Volume 294, Issue 3, Pages C726-C742Publisher
AMER PHYSIOLOGICAL SOC
DOI: 10.1152/ajpcell.00541.2007
Keywords
muffler; zinc buffering; computational model; metal ion transport and homeostasis; metallothionein
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Funding
- NIA NIH HHS [AG20536] Funding Source: Medline
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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, Zn-65(2+) 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 mu M, 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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