Insights into Zn2+ homeostasis in neurons from experimental and modeling studies.
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, (65)Zn2+ 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 microM, 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.
Colvin
Robert A
RA
Dept. of Biological Sciences, Ohio Univ, Athens, OH 45701, USA. colvin@ohio.edu
Bush
Ashley I
AI
Volitakis
Irene
I
Fontaine
Charles P
CP
Thomas
Dustin
D
Kikuchi
Kazuya
K
Holmes
William R
WR
D000818:Animals; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology / Q000187:drug effects / Q000196:embryology / Q000378:metabolism*; D002614:Chelating Agents / Q000494:pharmacology; D007464:Clioquinol / Q000494:pharmacology; D003198:Computer Simulation; D004492:Edetic Acid / Q000494:pharmacology; D005029:Ethylenediamines / Q000494:pharmacology; D006706:Homeostasis; D007700:Kinetics; D013058:Mass Spectrometry / Q000379:methods; D008668:Metallothionein / Q000378:metabolism; D008959:Models, Neurological; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D011485:Protein Binding; D011725:Pyridines / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D015203:Reproducibility of Results; D013050:Spectrometry, Fluorescence; D013871:Thiones / Q000494:pharmacology; D015032:Zinc / Q000378:metabolism*
D002614:Chelating Agents; D005029:Ethylenediamines; D011725:Pyridines; D013871:Thiones; C008704:pyrithione; D007464:Clioquinol; D008668:Metallothionein; D004492:Edetic Acid; D015032:Zinc; C044387:N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine