Elsevier

Neuroscience

Volume 139, Issue 4, 2006, Pages 1315-1327
Neuroscience

Cellular neuroscience
Modulation of inhibitory glycine receptors in cultured embryonic mouse hippocampal neurons by zinc, thiol containing redox agents and carnosine

https://doi.org/10.1016/j.neuroscience.2006.01.013Get rights and content

Abstract

Modulation of inhibitory glycine receptors by zinc (Zn2+) and endogenous redox agents such as glutathione may alter inhibition in the mammalian brain. Despite the abundance of Zn2+ in the hippocampus and its ability to modulate glycine receptors, few studies have examined Zn2+ modulation of hippocampal glycine receptors. Whether redox agents modulate hippocampal glycine receptors also remains unknown. This study examined Zn2+ and redox modulation of glycine receptor-mediated currents in cultured embryonic mouse hippocampal neurons using whole-cell recordings. Zn2+ concentrations below 10 μM potentiated currents elicited by low glycine, β-alanine, and taurine concentrations by 300–400%. Zn2+ concentrations above 300 μM produced nearly complete inhibition. Potentiating Zn2+ concentrations shifted the dose-response curves for the three agonists to the left and decreased the Hill coefficient for glycine and β-alanine but not taurine. Inhibiting Zn2+ concentrations shifted the dose-response curves for glycine and β-alanine to the right but reduced the maximum taurine response. Histidine residues may participate in potentiation because diethyl pyrocarbonate and pH 5.4 diminished Zn2+ enhancement of glycine currents. pH 5.4 diminished Zn2+ block of glycine currents, but diethyl pyrocarbonate did not. These findings indicate that separate sites mediate Zn2+ potentiation and inhibition. The redox agents glutathione, dithiothreitol, tris(2-carboxyethyl)phosphine, and 5,5′-dithiobis(2-nitrobenzoic acid) did not alter glycine currents by a redox mechanism. However, glutathione and dithiothreitol interfered with the effects of Zn2+ on glycine currents by chelating it. Carnosine had similar effects. Thus, Zn2+ and thiol containing redox agents that chelate Zn2+ modulate hippocampal glycine receptors with the mechanism of Zn2+ modulation being agonist dependent.

Section snippets

Embryonic mouse hippocampal cultures

Animal care and experimental protocols complied with all applicable federal laws and regulations of the United States, and they were approved by the Animal Studies Committee of Washington University. All efforts were made to minimize the number of animals used and their suffering. Hippocampal neurons were cultured from Swiss Webster mouse embryos at day 16 of gestation as described previously (Thio et al 2003, Karkar et al 2004). Briefly, cells were obtained by enzymatically digesting

Zn2+ biphasically modulates glycine currents mediated by α2-containing GlyRs

Glycine, β-alanine, and taurine evoke dose-dependent, strychnine sensitive, chloride currents mediated by α2-containing GlyRs in cultured embryonic mouse hippocampal neurons (Thio et al 2003, Karkar et al 2004). We and others showed that GlyRs in this preparation lack α1 subunits but contain α2 subunits with α2β heteromers predominating over α2 homomers (Thio et al 2003, Mangin et al 2005).

Zn2+ biphasically modulated glycine currents mediated by α2-containing GlyRs. Zn2+ reversibly potentiated

Discussion

This study examined modulation of hippocampal GlyRs by Zn2+ and redox agents as well as the interaction between them. The findings demonstrate that Zn2+ is a potent and effective biphasic modulator of α2-containing GlyRs, though the mechanism of modulation is agonist dependent. While these receptors are relatively resistant to redox modulation, thiol containing redox agents and carnosine can prevent Zn2+ from modulating GlyRs by chelation.

Acknowledgments

This work was supported by NIH grant K02NS043278 and the Department of Neurology, Washington University School of Medicine. We thank Nicholas Rensing for preparing and maintaining the neuronal cultures, Kelvin Yamada for helpful discussions, and Edwin Trevathan for statistical advice.

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