Desformylflustrabromine (dFBr), a positive allosteric modulator of the α4β2 nicotinic receptor modulates the hypnotic response to ethanol

Abstract

Background: Binge drinking can increase an individual's risk of developing alcohol use disorder (AUD). Ethanol targets multiple neurotransmitter systems; however, not much is known about its effects on the cholinergic system. Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels, the heteromeric α₄β₂ nAChR being a commonly expressed subtype. Desformylflustrabromine (dFBr), a positive allosteric modulator (PAM), increases the efficacy of α₄β₂ nAChR in vitro and has previously been shown to have translational potential. In this study, we investigated whether dFBr modulates the hypnotic response to ethanol.

Methods: Ethanol-induced loss of righting reflex (LORR) duration was measured in the presence and absence of dFBr. The β₂ nAChR selective antagonist dihydro-β-erythroidine (DHβE) was used to study the involvement of the β₂ subunit. Additionally, we used a crosslinking-based western blot assay to estimate changes in total versus intracellular α₄ nAChR protein in thalamic tissue of rats treated with vehicle, dFBr, ethanol, or ethanol and dFBr. Lastly, using Xenopus oocyte two-electrode voltage clamp (TEVC) studies, we determined the effects of ethanol and dFBr on α₄β₂ nAChR.

Results: Pretreatment with 6 mg/kg dFBr reduced ethanol-induced LORR duration as compared to rats treated with ethanol alone. LORR studies with DHβE suggest that dFBr reduced ethanol-induced LORR duration via the β₂ nAChR subunit. Crosslinking-based western analyses revealed that ethanol caused early increases in total and presumably surface thalamic α₄ nAChR subunit protein levels. This ethanol-induced α₄ nAChR upregulation was significantly reduced in rats pretreated with 6 mg/kg dFBr. In TEVC studies, ethanol potentiated ACh-induced currents in α₄β₂ nAChR, while it slightly reduced dFBr potentiation of maximal ACh currents.

Conclusions: Our results suggest that thalamic nAChRs containing the α₄ subunit are rapidly upregulated by a single intoxicating dose of ethanol. Furthermore, dFBr, an α₄β₂ nAChR-selective PAM, significantly attenuates the hypnotic response to ethanol via actions on β₂ nAChR. Overall, these results indicate that dFBr represents an option to reverse ethanol intoxication.

Keywords: Ethanol; Intracellular protein; Positive allosteric modulator; Sedative-hypnotic; Total protein; dFBr; nAChR.