The thiol-modifying agent N-ethylmaleimide elevates the cytosolic concentration of free Zn2+ but not of Ca2+ in murine cortical neurons

The membrane permeant alkylating agent N-ethylmaleimide (NEM) regulates numerous biological processes by reacting with thiol groups. Among other actions, NEM influences the cytosolic concentration of free Ca2+ ([Ca2+]i). Depending on the cell type and the concentration used, NEM can promote the release of Ca2+, affect its extrusion, stimulate or block its entry. However, most of these findings were obtained in experiments that employed fluorescent Ca2+ probes and one major disadvantage of such experimental setting derives from the lack of specificity of the probes as all the so-called “Ca2+-sensitive” indicators also bind metals like Zn2+ or Mn2+ with higher affinities than Ca2+. In this study, we examined the effects of NEM on the [Ca2+]i homeostasis of murine cortical neurons. We performed live-cell Ca2+ and Zn2+ imaging experiments using the fluorescent probes Fluo-4, FluoZin-3 and RhodZin-3 and found that NEM does not affect the neuronal [Ca2+]i homeostasis but specifically increases the cytosolic and mitochondrial concentration of free Zn2+([Zn2+]i). In addition, NEM triggers some neuronal loss that is prevented by anti-oxidants such as N-acetylcysteine or glutathione. NEM-induced toxicity is dependent on changes in [Zn2+]i levels as chelation of the cation with the cell-permeable heavy metal chelator, N,N,N′N′-tetrakis(−)[2-pyridylmethyl]-ethylenediamine (TPEN), promotes neuroprotection of cortical neurons exposed to NEM. Our data indicate that NEM affects [Zn2+]i but not [Ca2+]i homeostasis and shed new light on the physiological actions of this alkylating agent on central nervous system neurons.