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.

Clioquinol and pyrithione activate TRPA1 by increasing intracellular Zn 2+

“…CQ exerts its actions in other systems by acting as a moderate affinity Cu/Zn chelator and a Zn 2ϩ ionophore (7,8,20). Therefore, we investigated whether CQ activates TRPA1 by increasing the concentration of intracellular zinc ([Zn 2ϩ ] i ).…”

Physiological factors could enhance amyloid-beta toxicity

“…5d). In addition, the Zn 2+ ionophore pyrithione (Colvin et al, 2008) caused the clearance of Zn 2+ from both young and old slices within 10 minutes (Fig. 5d), confirming that we observed uptake.…”

An optical method to evaluate both mass and functional competence of pancreatic α- and β-cells

“…2C). Extracellular application of TPEN, a small membranepermeable chemical that exhibits 10 11 times greater affinity for Zn 2+ than ZRL1 (Colvin et al, 2008), almost completely quenched ZRL1 fluorescence in non-α-cells within ∼100 s (quenching coefficient, 16±1% of the control, mean±s.e.m., n=9, Fig. 1D), but only marginally affected the fluorescence in EYFP-positive α-cells (quenching coefficient, 79±2% of the control, n=11, Fig.…”