Reversal of Ethanol-induced Intoxication by a Novel Modulator of Gβγ Protein Potentiation of the Glycine Receptor

Abstract

The acute intoxicating effects of ethanol in the central nervous system result from the modulation of several molecular targets. It is widely accepted that ethanol enhances the activity of the glycine receptor (GlyR), thus enhancing inhibitory neurotransmission, leading to motor effects, sedation, and respiratory depression. We previously reported that small peptides interfered with the binding of G␤␥ to the GlyR and consequently inhibited the ethanol-induced potentiation of the receptor. Now, using virtual screening, we identified a subset of small molecules capable of interacting with the binding site of G␤␥. One of these compounds, M554, inhibited the ethanol potentiation of the GlyR in both evoked currents and synaptic transmission in vitro. When this compound was tested in vivo in mice treated with ethanol (1-3.5 g/kg), it was found to induce a faster recovery of motor incoordination in rotarod experiments and a shorter sedative effect in loss of righting reflex assays. This study describes a novel molecule that might be relevant for the design of useful therapeutic compounds in the treatment of acute alcohol intoxication.Ethanol is a brain-depressing drug possessing well recognized acute physiological effects. Within the different molecular targets for ethanol, those that best explain the acute effects of ethanol are those affecting fast neurotransmission. It is believed that ethanol effects on sensorial transmission, motor control, respiratory rhythms, and cognitive processing are caused by changes in the activity of several ligand-gated ion channels (1) and particularly through the potentiation of the glycine receptor (GlyR) 2 (2). The GlyR has been extensively studied as a molecular target for ethanol, and electrophysiological experiments have demonstrated that ethanol potentiates GlyR activity (3), as determined by increased glycine-evoked currents (4, 5), an increased decay time constant in spontaneous synaptic events (6), increased probability of channel opening in single-channel analysis (7), and increased agonist affinity (8). It was shown previously that G proteins participate in the ethanol effect on the GlyR (4). In addition, more recent studies have determined that the G protein ␤␥ dimer acts as an intermediary for the ethanol action on the GlyR (7, 10), where amino acids in the GlyR intracellular domain are essential for the interaction with G␤␥ (11, 12). Interestingly, a small peptide (RQHc7) that binds G␤␥ inhibited the potentiation effect of ethanol on both evoked and synaptic currents (6, 11). Using in silico analysis, an aspartic pocket (Asp-186, Asp-228, and Asp-246) in G␤ was identified as the binding region for RQHc7. Thus, the inhibition of the interaction between G␤␥ and the GlyR intracellular domain prevented the ethanol effect in native and recombinant systems (6). Therefore, in this study, we aimed to identify small molecules capable of binding G␤␥ and able to inhibit the potentiation of the glycine current induced by ethanol. One of these compounds was assayed in in vivo pharmaco...

Presence of ethanol‐sensitive glycine receptors in medium spiny neurons in the mouse nucleus accumbens

“…The present data, together with a recent study that showed that a small molecule can reduce the sedative effects of ethanol (San Martin et al . ) by interfering with the GlyR‐Gβγ protein/protein interaction, support the notion that the present type of study can lead to the development of therapeutics to treat toxic and addictive ethanol behaviours.…”

“…These regions should play a role on the protein–protein interaction with Gβγ and potentially for therapeutic development (San Martin et al . ).…”

The Effects of Enriched Environmental Intervention on the Parturition Outcome and Filial Health of Pregnant Mice With Chronic Pain

“…For evaluation of the righting reflex, the offspring mice were placed in the supine position. When they were able to right themselves within 2 s and repeat the movement 3 times successfully, we recorded that as achievement of the righting reflex (San Martin et al, 2016). To evaluate crawling ability (Nagao, Kawachi, Kagawa, & Komada, 2014), we observed whether the offspring could crawl for a distance longer than 3 cm without rearing.…”

Brucine N‐Oxide Reduces Ethanol Intake and Preference in Alcohol‐Preferring Male Fawn‐Hooded Rats

“…Meanwhile, the structure Loop 2 of a glycine receptor is considered a key structure and determines sensitivity to EtOH (Crawford et al, 2007; Eggers and Berger, 2004; Perkins et al, 2012). In addition, homomeric α1 or α2 GlyRs in Xenopus oocytes may be potentiated, while the activities of GlyR can be enhanced by prolonging the burst duration by EtOH (Mascia et al, 1996; San Martin et al, 2016; Welsh et al, 2009). Furthermore, BNO and brucine antagonize the α1 and α1β glycine receptors with Ki values of 1.4 and 1.7 μmol/l, respectively, in human embryonic kidney 293 cells (Jensen et al, 2006).…”