Zinc-dependent modulation of α2- and α3-glycine receptor subunits by ethanol.

Although ethanol (EtOH) is a prevalently used drug, the sites and mechanisms of action by which it produces its intoxicating effects are not thoroughly understood. One widely accepted idea is that EtOH acts at protein targets in the body. Among the strongly supported protein targets of EtOH are strychnine-sensitive glycine receptors (GlyRs) (Harris et al., 2008), which belong to the Cys-loop superfamily of ligand-gated ion channels. There are 4 GlyR alpha subunits (α1, α2, α3, and α4) and 1 GlyR beta subunit (Lynch, 2004). GlyRs can assemble to form α-homomeric or αβ-heteromeric chloride channels that mediate inhibition in the central nervous system (CNS). They are localized most abundantly in the spinal cord and brainstem (Legendre, 2001), but are also expressed throughout the brain (Baer et al., 2009; Jonsson et al., 2009, 2012). GlyRs are not only modulated by alcohols, but also by volatile anesthetics, and inhaled drugs of abuse (Beckstead et al., 2002; Mihic et al., 1997). In addition to these exogenous agents, endogenous cations such as zinc also allosterically modulate GlyR function. Unlike the exogenous sedative hypnotics that strictly enhance GlyR function, zinc produces biphasic effects on GlyR function such that nanomloar and low micromolar concentrations enhance glycine-activated currents, whereas higher micromolar concentrations of zinc inhibit GlyR function (Bloomenthal et al., 1994; Laube et al., 2000; Miller et al., 2005a). In addition, recent findings in the GlyR α1-subunit show that zinc at physiological concentrations enhances the magnitude of EtOH’s effects on GlyR function (McCracken et al., 2010b, 2013). Although the effects of EtOH on the α1-GlyR subunit have been relatively well studied, less work has focused on the GlyR α2-subunit, and presently, there are no published reports of EtOH modulation of the GlyR α3-subunit despite that both α2- and α3-GlyR subunits are expressed in a number of brain regions involved in EtOH-related behaviors (Jonsson et al., 2009, 2012). More specifically, in limbic and motivation centers of the brain, such as the amygdala and nucleus accumbens, studies of gene and receptor membrane expression demonstrate that there is as much or greater abundance of α2- and α3-subunits than α1-subunits in these regions (Delaney et al., 2010; Jonsson et al., 2009, 2012). Unlike GlyRs expressed in the spinal cord, which are predominantly α1β-heteromers, recent studies of GlyRs in brain suggest that they are likely to include populations of α2- or α3-homomers (Adermark et al., 2011; Chen et al., 2011; Eichler et al., 2009; Muller et al., 2008; Weltzien et al., 2012). Therefore, studies investigating these GlyRs are of physiological relevance. In addition to differences in brain region expression among GlyR alpha subunits, there is emergent evidence suggesting functional differences. For example, subpopulations, particularly of α3-subunits, undergo RNA editing that results in α3P185L-receptors (Meier et al., 2005). These edited subunits confer high sensitivity to agonists such as glycine (Legendre et al., 2009), which may be important for tonic inhibition (Eichler et al., 2009). Recent studies of RNA editing focus on α3-GlyRs, but there is evidence for a similar posttranscriptional modification in α2-GlyRs (Eichler et al., 2008). Due to their localization in limbic, motivation, and reward-related brain areas, thorough studies of the effects of EtOH at homomeric α2- and α3-GlyRs are necessary and relevant for better understanding the mechanisms by which EtOH exerts its physiological effects. In addition, because zinc exists in the CNS at tonic baseline nanomolar concentrations (Frederickson et al., 2006), which are sufficient for enhancement of GlyR function and critical for determining the magnitude of EtOH’s effects on α1-GlyRs (McCracken et al., 2010b), it is important to investigate the role of zinc in EtOH modulation of α2- and α3-GlyR subunits. Furthermore, studies of RNA-edited GlyRs in CNS inhibition (Eichler et al., 2009; Legendre et al., 2009; Meier et al., 2005) suggest that comparisons of EtOH sensitivity between edited and unedited receptors may be important for a thorough understanding of EtOH action. In this study, we tested the hypothesis that the magnitude of EtOH’s effects on α2- and α3-GlyR subunits like α1-subunits would be enhanced in the presence of physiologically relevant (nanomolar) concentrations of zinc. Because the effects of EtOH on α3-GlyRs have not been previously studied, sensitivity to EtOH was first confirmed in this subunit. A secondary goal of this study was to determine whether or not there are differences in EtOH sensitivity between edited and unedited α3-GlyRs.