Elsevier

Neuropharmacology

Volume 58, Issue 3, March 2010, Pages 676-681
Neuropharmacology

Zinc enhances ethanol modulation of the α1 glycine receptor

https://doi.org/10.1016/j.neuropharm.2009.11.001Get rights and content

Abstract

Glycine receptor function mediates most inhibitory neurotransmission in the brainstem and spinal cord and is enhanced by alcohols, volatile anesthetics, inhaled drugs of abuse, and endogenous compounds including zinc. Because zinc exists ubiquitously throughout the brain, investigations of its effects on the enhancement of GlyR function by alcohols and anesthetics are important to understanding the effects of these agents in vivo. In the present study, the effects of zinc plus ethanol, pentanol, or isoflurane were tested on homomeric α1 glycine receptors to determine if concurrent applications of physiological concentrations of zinc with each of these modulators changed the magnitude of their effects. Homomeric α1 glycine receptors were expressed in Xenopus laevis oocytes, and the two-electrode voltage-clamp technique was used to measure glycine-mediated currents in the presence of combinations of zinc with ethanol, pentanol or isoflurane. The combined effects of zinc plus ethanol were greater than the sum of the effects produced by either compound alone. However, this was not seen when zinc was combined with either pentanol or isoflurane. Chelation of zinc by tricine decreased the effects of sub-maximal, but not maximal, concentrations of glycine, and diminished the magnitude of ethanol enhancement observed. These findings suggest a zinc/ethanol interaction at the α1 GlyR that results in the enhancement of the effects of ethanol action on GlyR function.

Introduction

Glycine receptors (GlyRs) are the primary mediators of inhibitory neurotransmission in the brainstem and spinal cord (Legendre, 2001) and anion-conducting members of the niciotinic acetylcholine superfamily of ligand-gated ion channels. Two classes of GlyR subunits have been identified (α and β); four α subunits, with 80–90% sequence identity among subunits, and a single β subunit, possessing ∼50% sequence identity with the α subunits (Lynch, 2004). The α subunits can express homomerically, or heteromerically with β subunits, to produce functional channels. In addition to the spinal cord and medulla, GlyRs are found in several brain regions including the hippocampus (Fatima-Shad and Barry, 1993), nucleus accumbens (Molander and Söderpalm, 2005), cerebellum (Takahashi et al., 1992) and also in the olfactory bulb (van den Pol and Gorcs, 1988).

A number of positive modulators of GlyR function exist, including alcohols, volatile anesthetics and inhaled drugs of abuse (Mihic et al., 1997, Beckstead et al., 2000). Zinc, which is found endogenously in both free and protein-bound forms, also affects GlyR function, but in a biphasic manner. Concentrations lower than 10 μM have enhancing effects, whereas concentrations greater than 10 μM inhibit GlyR function (Harvey et al., 1999, Laube et al., 2000). Zinc concentrations in the brain exceed those present in other organs, although most zinc is protein-bound (Mathie et al., 2006). In its free or rapidly exchangeable form, zinc exists in cerebospinal fluid at basal concentrations ranging from approximately 5 to 25 nM (Frederickson et al., 2006), and is predicted to remain at concentrations below 10 μM following presynaptic release from GABAergic, glutamatergic, or glycinergic terminals (Frederickson and Bush, 2001).

Because zinc exists ubiquitously throughout the brain, investigations of its effects on the enhancement of GlyR function by alcohols and anesthetics are important to understanding the effects of these agents in vivo. In the present study, we examined whether co-applying low, physiologically-relevant, concentrations of zinc with either ethanol (EtOH), pentanol, or isoflurane would result in augmented effects of these modulators on α1 GlyR function.

Section snippets

Reagents

All reagents were purchased from Sigma–Aldrich (St. Louis, MO). Xenopus laevis were obtained from Xenopus Express (Brooksville, FL).

Oocyte isolation and cDNA injection

Partial ovariectomies were performed on sexually mature female X. laevis and ovary fragments were placed in isolation media (108 mM NaCl, 2 mM KCl, 1 mM EDTA, 10 mM HEPES). Stage IV and V oocytes were manually extracted from the thecal and epithelial membranes with forceps under a light microscope. In order to remove the follicular membrane, isolated oocytes were

Results

To examine the effects of physiological-relevant concentrations of zinc on the enhancement of GlyR function by alcohols and anesthetics, we used two approaches. First, the zinc chelator tricine was included in solutions to remove any trace levels of zinc, and then in a second approach, physiologically-relevant concentrations of zinc were added to our perfusion solutions. The effects of zinc on GlyR modulation by ethanol, pentanol and isoflurane were tested.

Tricine at a concentration of 10 mM

Discussion

Most investigations of modulators of channel function involve the sole application of the modulatory compound of interest, which may inadequately represent in vivo conditions. For example, free zinc exists in the CNS at concentrations known to affect GlyR function, which led us to speculate whether it might affect the enhancing effects of other GlyR allosteric modulators. In experiments in which zinc and ethanol were concurrently applied, low physiologically-relevant concentrations of zinc

Acknowledgements

This work was supported by NIH grants R01AA11525, R01AA06399 and P01GM47818.

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