Zinc enhances ethanol modulation of the α1 glycine receptor
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.
References (35)
- et al.
Metals in our minds: therapeutic implications for neurodegenerative disorders
Lancet Neurology
(2004) - et al.
Concentrations of extracellular free zinc in the central nervous system during simple anesthetization, ischemia and reperfusion
Experimental Neurology
(2006) - et al.
Hyperekplexia phenotype of glycine receptor alpha1 subunit mutant mice identifies Zn2+ as an essential endogenous modulator of glycinergic neurotransmission
Neuron
(2006) - et al.
Channel gating of the glycine receptor changes accessibility to residues implicated in receptor potentiation by alcohols and anesthetics
Journal of Biological Chemistry
(2004) - et al.
Accessibility to residues in transmembrane segment four of the glycine receptor
Neuropharmacology
(2006) - et al.
Zinc and copper: pharmacological probes and endogenous modulators of neuronal excitability
Pharmacology and Therapeutics
(2006) - et al.
Molecular basis for zinc potentiation at strychnine-sensitive glycine receptors
Journal of Biological Chemistry
(2005) - et al.
Zinc at glutamatergic synapses
Neuroscience
(2009) - et al.
Functional correlation of fetal and adult forms of glycine receptors with developmental changes in inhibitory synaptic receptor channels
Neuron
(1992) - et al.
Inhibitory zinc- enriched terminals in the mouse cerebellum: double-immunohistochemistry for zinc transporter 3 and glutamate decarboxylase
Neuroscience Letters
(2002)
Glycine and gamma-aminobutyric acidA receptor function is enhanced by inhaled drugs of abuse
Molecular Pharmacology
Zinc and brain injury
Annual Review of Neuroscience
Evidence that ethanol acts on a target in Loop 2 of the extracellular domain of alpha1 glycine receptors
Journal of Neurochemistry
Multiple sites of ethanol action in alpha1 and alpha2 glycine receptors suggested by sensitivity to pressure antagonism
Journal of Neurochemistry
Anion permeation in GABA- and glycine-gated channels of mammalian cultured hippocampal neurons
Proceedings of the Royal Society of London Series B Biological Sciences
Synaptically released zinc: physiological functions and pathological effects
Biometals
Identification of an inhibitory Zn2+ binding site on the human glycine receptor alpha1 subunit
Journal of Physiology
Cited by (25)
Identification of N-acyl amino acids that are positive allosteric modulators of glycine receptors
2020, Biochemical PharmacologyKetone body modulation of ligand-gated ion channels
2019, NeuropharmacologyCitation Excerpt :To test if the modulatory effect of acetone on glycine receptors that we observed might be the result of contaminating zinc, or if acetone acted on the glycine receptor in a zinc-dependent manner, we tested the effects of 1M acetone on wildtype glycine receptors in the presence of a zinc chelator, tricine, and on mutant W170S α1 glycine receptors in which amino acid tryptophan 170 was changed to serine (Fig. 6A). The presence of tricine did not alter acetone enhancement of the glycine receptor, suggesting that there was little to no zinc contamination present in our acetone stock, and that acetone actions on the glycine receptor are not zinc dependent, similar to volatile anesthetics (McCracken et al., 2010). This contrasts with other modulators of the glycine receptor, such as alcohol and inhalants such as trichloroethane, which are influenced by zinc (McCracken et al., 2010; Cornelison et al., 2017).
Glycine Receptor Drug Discovery
2017, Advances in PharmacologyCitation Excerpt :It is possible, for example, that a potentially valuable drug may require a low concentration of Zn2+ as a coagonist. Indeed, evidence has been presented that this is the case: peptides, isoflurane, and ethanol all exert much weaker effects on GlyRs when Zn2+ is removed from the extracellular solution (Cornelison, Pflanz, Tipps, & Mihic, 2016; Kirson, Cornelison, Philpo, Todorovic, & Mihic, 2013; McCracken, Trudell, Goldstein, Harris, & Mihic, 2010). Rather than buffer Zn2+ to nonphysiologically low levels, we recommend quantifying the Zn2+ concentration in drug samples of interest via inductively coupled plasma mass spectroscopy.
Identification and characterization of heptapeptide modulators of the glycine receptor
2016, European Journal of PharmacologyCitation Excerpt :Interestingly, the actions of these peptides appear to be zinc-dependent. This zinc dependence was also previously shown to affect alcohol modulation of the glycine receptor (McCracken et al., 2010), suggesting that the presence of zinc may be necessary for efficient modulation of glycine receptor activity by allosteric modulators. Unless stated otherwise, all chemicals were obtained from Sigma-Aldrich (St. Louis, MO).
Ethanol effects on glycinergic transmission: From molecular pharmacology to behavior responses
2015, Pharmacological Research