Altered Sedative Effects of Ethanol in Mice with α1 Glycine Receptor Subunits that are Insensitive to Gβγ Modulation

Abstract

Alcohol abuse and alcoholism are major health problems and one of the leading preventable causes of death. Before achieving better treatments for alcoholism, it is necessary to understand the critical actions of alcohol on membrane proteins that regulate fundamental functions in the central nervous system. After generating a genetically modified knock-in (KI) mouse having a glycine receptor (GlyR) with phenotypical silent mutations at KK385/386AA, we studied its cellular and in vivo ethanol sensitivity. Analyses with western blotting and immunocytochemistry indicated that the expression of a1 GlyRs in nervous tissues and spinal cord neurons (SCNs) were similar between WT and KI mice. The analysis of synaptic currents recorded from KI mice showed that the glycinergic synaptic transmission had normal properties, but the sensitivity to ethanol was significantly reduced. Furthermore, the glycine-evoked current in SCNs from KI was resistant to ethanol and G-protein activation by GTP-g-S. In behavioral studies, KI mice did not display the foot-clasping behavior upon lifting by the tail and lacked an enhanced startle reflex response that are characteristic of other glycine KI mouse lines with markedly impaired glycine receptor function. The most notable characteristic of the KI mice was their significant lower sensitivity to ethanol (B40%), expressed by shorter times in loss of righting reflex (LORR) in response to a sedative dose of ethanol (3.5 g/Kg). These data provide the first evidence to link a molecular site in the GlyR with the sedative effects produced by intoxicating doses of ethanol.

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

“…Differences in the latency to LORR were not detected in any of the molecules studied (data not shown). Finally, we tested the duration of LORR in KI mice that had a reduced LORR in the presence of ethanol because of point mutations in the intracellular domain of the GlyR␣1 subunit (K385A/K386A GlyR␣ 1 ) important for the intracellular modulation of G␤␥ (16). No differences were found in the duration of LORR with ethanol alone (29 Ϯ 1 min) and ethanol plus M554 (31 Ϯ 1 min, p ϭ 0.27119) in these mice, supporting the notion that the effect of M554 is due to the interference in the intracellular signaling mechanism for the potentiation by ethanol in glycine currents (Fig.…”

Glycine Receptors Containingα2 orα3 Subunits Regulate Specific Ethanol-Mediated Behaviors

“…Mutations in GlyRa1 often result in a dramatic increase in the startle response (Harvey et al, 2008) and can affect other neurotransmitter systems such as GABA A , N-methyl-D-aspartate, or nicotinic acetylcholine (Quinlan et al, 2002;Blednov et al, 2012). Mutations in the GlyRa1 subunit that prevent G protein and ethanol modulation reduce the duration of LORR and increase the locomotor-stimulating actions without changing ethanol-induced ataxia (Aguayo et al, 2014). Thus, it appears that the GlyRa1 subunit regulates ethanol-induced motor activity and sedation.…”

“…Studies using transgenic mice expressing a mutation (S267Q) in GlyRa1 subunits (Findlay et al, 2002), as well as heterozygous knock-in mice with mutations (Q266I, M287L, or D80A) in the a1 subunit, demonstrated changes in ethanol-induced incoordination and LORR (Blednov et al, 2012;McCracken et al, 2013a). Furthermore, knock-in mice with a mutation that reduces ethanol sensitivity of the GlyRa1 subunit show reduced duration of the LORR produced by ethanol (Aguayo et al, 2014).…”

Structure and Pharmacologic Modulation of Inhibitory Glycine Receptors

“…Additional analysis found that low ethanol concentrations (#50 mM) potentiate glycinergic currents by increasing the apparent affinity of GlyR with no change in efficacy (Aguayo et al, 1996;Crawford et al, 2007;Perkins et al, 2008), and showed that alcohol effects on glycinergic currents were developmentally regulated in cultured spinal neurons (Tapia et al, 1997;Tapia and Aguayo, 1998). Other studies have demonstrated that ethanol affects GlyR expressed in hypoglossal motoneurons (Eggers and Berger, 2004;Aguayo et al, 2014), the spinal cord (Celentano et al, 1988;Mariqueo et al, 2014), and ventral tegmental area neurons (Ye et al, 2001).…”