Ethanol-oxytocin Interactions at Homomeric Glycine Receptors

Abstract

Alcohol is one of the most widely used drugs, yet its targets in the brain have not been reliably established, and effective treatments for alcohol addiction are lacking. Various ligand-gated ion channels (LGICs) are established targets of ethanol in the central nervous system, including NMDA receptors and the Cys-loop γ-aminobutyric acid type A (GABAA), 5-HT3, nicotinic acetylcholine (nACh), and glycine (Gly) receptors (Harris et al 2008, Spanagel 2009). Historically, the role of GABAA receptors in alcohol’s sedative, anxiolytic, and depressant effects has been supposed. More recently, extrasynaptic δ subunit-containing GABAA receptors have come to light as targets of behaviourally relevant concentrations of alcohol in the brain (Olsen et al 2007). Glycine receptors (GlyRs) have also been implicated in ethanol’s (EtOH) effects on motor coordination, reward pathways, sensory processing and perception. Furthermore, glycine receptors may be involved in regulating alcohol consumption (Perkins et al 2010, Soderpalm et al 2017). Ethanol may interact with the receptor via residues in transmembrane M2 and M3 regions, in addition to residues in Loop 2 of the extracellular domain (Burgos et al 2015, Crawford et al 2008, Horani et al 2015). Loop 2 residues of the extracellular domain may mediate signal transduction in the channel of both GABAA and glycine receptors (Cederholm et al 2010), and may be important in ethanol’s actions at these receptors (Crawford et al 2007). Furthermore, Loop 2 of the GABAA receptor δ subunit may be necessary for its high sensitivity to ethanol, and an ethanol ultra-sensitive mutant GlyR may be created through insertion of this sequence into the α subunit (Perkins et al 2009). Recent behavioural and electrophysiological experiments have demonstrated that the neuropeptide oxytocin (OT) attenuates ethanol-induced motor impairment in rats, and prevents ethanol-induced potentiation of GABA-gated currents via selective and specific interaction at δ-GABAA receptors (Bowen et al 2015). Although oxytocin’s binding site remains unclear, its interactions at these receptors presents a useful tool for investigation of the receptor conformations necessary for both alcohol and oxytocin’s actions. The actions of oxytocin at glycine receptors have not yet been characterised, although the functional and structural similarities between GABAA and Gly receptors - particularly as inhibitory LGIC targets of ethanol - suggested that a similar oxytocin-ethanol interaction could be possible at glycine receptors. Overall, this project aimed to investigate the interactive effects of ethanol and oxytocin at recombinant human homomeric α1 and α2 GlyRs. The role of Loop 2 in oxytocin’s effects and interactions with ethanol at ethanol-sensitive mutant α1δL2 GlyRs was also a focus of this investigation. To achieve this, human wild type α1, α2, and mutant α1δL2 homomers were expressed recombinantly in Xenopus laevis oocytes. Two-electrode voltage clamp electrophysiology was then used to evaluate the effects of EtOH and OT at these receptors. Presently, ethanol was found to potentiate α1 and α2 GlyRs at pharmacologically relevant concentrations, with the threshold concentration for potentiation greatly reduced (from 30 mM EtOH to as low as 1 mM) in the α1δL2 mutant receptor. At all of these receptors, oxytocin significantly attenuated ethanol-induced potentiation of glycine-gated current when co-applied at low micromolar concentrations. Importantly, neither ethanol nor oxytocin activated the receptors independently, and oxytocin did not modulate glycine currents in the absence of ethanol. The structurally similar neuropeptide arginine vasopressin (AVP) did not modulate ethanol potentiation when tested in place of OT at α2 receptors, suggesting structural moieties unique to the oxytocin molecule may allow modulation of ethanol’s actions at these receptors. In addition to their significantly increased sensitivity to ethanol, Loop 2-mutated GlyRs were more sensitive to glycine, although the general function of the receptor appeared unchanged. Importantly, the modulatory effects of oxytocin upon ethanol was preserved across wild type α1 and mutant α1δL2 receptors, indicating that Loop 2 is unlikely to be involved in mediating oxytocin’s interactions with the glycine receptor. However, the binding site for oxytocin at glycine and GABAA receptors has not been identified, necessitating further investigation into this novel action of oxytocin in the brain.