Reversal of Ethanol-induced Intoxication by a Novel Modulator of G?? 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.
Project description:GABA type A receptors (GABA(A)-R) are important for ethanol actions and it is of interest to link individual subunits with specific ethanol behaviors. We studied null mutant mice for six different GABA(A)-R subunits (?1, ?2, ?3, ?4, ?5 and ?). Only mice lacking the ?2 subunit showed reduction of conditioned taste aversion (CTA) to ethanol. These results are in agreement with data from knock-in mice with mutation of the ethanol-sensitive site in the ?2-subunit (Blednov et al., 2011). All together, they indicate that aversive property of ethanol is dependent on ethanol action on ?2-containing GABA(A)-R. Deletion of the ?2-subunit led to faster recovery whereas absence of the ?3-subunit slowed recovery from ethanol-induced incoordination (rotarod). Deletion of the other four subunits did not affect this behavior. Similar changes in this behavior for the ?2 and ?3 null mutants were found for flurazepam motor incoordination. However, no differences in recovery were found in motor-incoordinating effects of an ?1-selective modulator (zolpidem) or an ?4-selective agonist (gaboxadol). Therefore, recovery of rotarod incoordination is under control of two GABA(A)-R subunits: ?2 and ?3. For motor activity, ?3 null mice demonstrated higher activation by ethanol (1 g/kg) whereas both ?2 (-/-) and ?3 (-/Y) knockout mice were less sensitive to ethanol-induced reduction of motor activity (1.5 g/kg). These studies demonstrate that the effects of ethanol at GABAergic synapses containing ?2 subunit are important for specific behavioral effects of ethanol which may be relevant to the genetic linkage of the ?2 subunit with human alcoholism.
Project description:Glycine receptors (GlyRs) are broadly expressed in the central nervous system. Ethanol enhances the function of brain GlyRs, and the GlyR?1 subunit is associated with some of the behavioral actions of ethanol, such as loss of righting reflex. The in vivo role of GlyR?2 and ?3 subunits in alcohol responses has not been characterized despite high expression levels in the nucleus accumbens and amygdala, areas that are important for the rewarding properties of drugs of abuse. We used an extensive panel of behavioral tests to examine ethanol actions in mice lacking Glra2 (the gene encoding the glycine receptor alpha 2 subunit) or Glra3 (the gene encoding the glycine receptor alpha 3 subunit). Deletion of Glra2 or Glra3 alters specific ethanol-induced behaviors. Glra2 knockout mice demonstrate reduced ethanol intake and preference in the 24-hour two-bottle choice test and increased initial aversive responses to ethanol and lithium chloride. In contrast, Glra3 knockout mice show increased ethanol intake and preference in the 24-hour intermittent access test and increased development of conditioned taste aversion to ethanol. Mutants and wild-type mice consumed similar amounts of ethanol in the limited access drinking in the dark test. Other ethanol effects, such as anxiolysis, motor incoordination, loss of righting reflex, and acoustic startle response, were not altered in the mutants. The behavioral changes in mice lacking GlyR?2 or ?3 subunits were distinct from effects previously observed in mice with knock-in mutations in the ?1 subunit. We provide evidence that GlyR?2 and ?3 subunits may regulate ethanol consumption and the aversive response to ethanol.
Project description:Increased use of gene manipulation in mice (e.g., targeted or random mutagenesis) has been accompanied by increased reliance on a very few rapid and simple behavioral assays, each of which aspires to model a human behavioral domain. Yet, each assay comprises multiple traits, influenced by multiple genetic factors. Motor incoordination (ataxia), a common characteristic of many neurological disorders, may reflect disordered balance, muscle strength, proprioception, and/or patterned gait. Impaired motor performance can confound interpretation of behavioral assays of learning and memory, exploration, motivation, and sensory competence. The rotarod is one of the most commonly used tests to measure coordination in mice. We show here that exactly how the rotarod test is performed can markedly alter the apparent patterns of genetic influence both in undrugged performance and sensitivity to ethanol intoxication. However, when tested with well chosen parameters, the accelerating rotarod test showed very high inter- and intralaboratory reliability. Depending on test conditions, ethanol can either disrupt or enhance performance in some strains. Genetic contribution to performance on the accelerating versus the fixed-speed rotarod assay can be completely dissociated under some test conditions, and multiple test parameters are needed to assess the range of genetic influence adequately.
Project description:G?? interaction with GlyR is an important determinant in ethanol potentiation of this channel.A small peptide, RQH(C7), can inhibit ethanol potentiation of GlyR currents.Results with RQH(C7) indicate that ethanol mediated potentiation of GlyR is in part by G?? activation.Molecular interaction between G?? and GlyR could be used as a target for pharmacological modification of ethanol effects. Previous studies indicate that ethanol can modulate glycine receptors (GlyR), in part, through G?? interaction with basic residues in the intracellular loop. In this study, we show that a seven-amino acid peptide (RQH(C7)), which has the primary structure of a motif in the large intracellular loop of GlyR (GlyR-IL), was able to inhibit the ethanol-elicited potentiation of this channel from 47 ± 2 to 16 ± 4%, without interfering with the effect of G?? on GIRK (G protein activated inwardly rectifying potassium channel) activation. RQH(C7) displayed a concentration-dependent effect on ethanol action in evoked and synaptic currents. A fragment of GlyR-IL without the basic amino acids did not interact with G?? or inhibit ethanol potentiation of GlyR. In silico analysis using docking and molecular dynamics allowed to identify a region of ~350Å(2) involving aspartic acids 186, 228, and 246 in G?? where we propose that RQH(C7) binds and exerts its blocking action on the effect of ethanol in GlyR.
Project description:Ethanol is a widely used drug, yet an understanding of its sites and mechanisms of action remains incomplete. Among the protein targets of ethanol are glycine receptors (GlyRs), which are potentiated by millimolar concentrations of ethanol. In addition, zinc ions also modulate GlyR function, and recent evidence suggests that physiologic concentrations of zinc enhance ethanol potentiation of GlyRs. Here, we first built a homology model of a zinc-bound GlyR using the D80 position as a coordination site for a zinc ion. Next, we investigated in vitro the effects of zinc on ethanol action at recombinant wild-type (WT) and mutant ?1 GlyRs containing the D80A substitution, which eliminates zinc potentiation. At D80A GlyRs, the effects of 50 and 200 mM ethanol were reduced as compared with WT receptors. Also, in contrast to what was seen with WT GlyRs, neither adding nor chelating zinc changed the magnitude of ethanol enhancement of mutant D80A receptors. Next, we evaluated the in vivo effects of the D80A substitution by using heterozygous Glra1(D80A) knock-in (KI) mice. The KI mice showed decreased ethanol consumption and preference, and they displayed increased startle responses compared with their WT littermates. Other behavioral tests, including ethanol-induced motor incoordination and strychnine-induced convulsions, revealed no differences between the KI and WT mice. Together, our findings indicate that zinc is critical in determining the effects of ethanol at GlyRs and suggest that zinc binding at the D80 position may be important for mediating some of the behavioral effects of ethanol action at GlyRs.
Project description:Ethanol alters the function of several members of the Cys-loop ligand-gated ion channel superfamily. Recent studies have shown that the sensitivity of the ?1 glycine receptor (GlyR) to ethanol can be affected by the state of G protein activation mediated by the interaction of G?? with intracellular amino acids in the GlyR. Here, we evaluated the physicochemical property of Lys385 that contributes to ethanol modulation by using mutagenesis, patch-clamp, and biochemical techniques. A conserved substitution (K385R) did not affect either the apparent glycine EC?? (40 ± 1 versus 41 ± 0.5 ?M) or the ethanol-induced potentiation (53 ± 5 versus 46 ± 5%) of the human ?1 GlyR. On the other hand, replacement of this residue with glutamic acid (K385E), an acidic amino acid, reduced the potentiation of the GlyR to 10 ± 1%. Furthermore, mutations with a hydrophobic leucine (K385L), a hydrogen bond donor glutamine (K385Q), or a neutral residue (K385A) also reduced ethanol modulation. Finally, substitution by a large and hydrophobic residue (K385F) and deletion of 385 (Lys385_) reduced ethanol modulation to 10 ± 4 and 17 ± 0.4%, respectively. Experiments using dynamic cysteine substitution with a methanethiosulfonate reagent and homology modeling indicate that the basic property and the position of Lys385, probably because of its interaction with G??, is critical for ethanol potentiation of the receptor.
Project description: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 ?1 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-?-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 (?40%), 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.
Project description:Toluene, a widely used and commonly abused organic solvent, produces various behavioral disturbances, including motor incoordination and cognitive impairment. Toluene alters the function of a large number of receptors and ion channels. Blockade of N-methyl-d-aspartate (NMDA) receptors has been suggested to play a critical role in toluene-induced behavioral manifestations. The present study determined the effects of various toluene doses on motor coordination, recognition memory, body temperature, and intracranial self-stimulation (ICSS) thresholds in mice. Additionally, the effects of sarcosine on the behavioral and physiological effects induced by toluene were evaluated. Sarcosine may reverse toluene-induced behavioral manifestations by acting as an NMDA receptor co-agonist and by inhibiting the effects of the type I glycine transporter (GlyT1). Mice were treated with toluene alone or combined with sarcosine pretreatment and assessed for rotarod performance, object recognition memory, rectal temperature, and ICSS thresholds. Toluene dose-dependently induced motor incoordination, recognition memory impairment, and hypothermia and lowered ICSS thresholds. Sarcosine pretreatment reversed toluene-induced changes in rotarod performance, novel object recognition, and rectal temperature but not ICSS thresholds. These findings suggest that the sarcosine-induced potentiation of NMDA receptors may reverse motor incoordination, memory impairment, and hypothermia but not the enhancement of brain stimulation reward function associated with toluene exposure. Sarcosine may be a promising compound to prevent acute toluene intoxications by occupational or intentional exposure.
Project description:GABAA receptors consisting of ?1, ?2, or ?3 subunits in homo- or hetero-pentamers have been studied mainly in retina but are detected in many brain regions. Receptors formed from ?1 are inhibited by low ethanol concentrations, and family-based association analyses have linked ? subunit genes with alcohol dependence. We determined if genetic deletion of ?1 in mice altered in vivo ethanol effects. Null mutant male mice showed reduced ethanol consumption and preference in a two-bottle choice test with no differences in preference for saccharin or quinine. Null mutant mice of both sexes demonstrated longer duration of ethanol-induced loss of righting reflex (LORR), and males were more sensitive to ethanol-induced motor sedation. In contrast, ?1 null mice showed faster recovery from acute motor incoordination produced by ethanol. Null mutant females were less sensitive to ethanol-induced development of conditioned taste aversion. Measurement of mRNA levels in cerebellum showed that deletion of ?1 did not change expression of ?2, ?2, or ?6 GABAA receptor subunits. (S)-4-amino-cyclopent-1-enyl butylphosphinic acid ("?1" antagonist), when administered to wild type mice, mimicked the changes that ethanol induced in ?1 null mice (LORR and rotarod tests), but the ?1 antagonist did not produce these effects in ?1 null mice. In contrast, (R)-4-amino-cyclopent-1-enyl butylphosphinic acid ("?2" antagonist) did not change ethanol actions in wild type but produced effects in mice lacking ?1 that were opposite of the effects of deleting (or inhibiting) ?1. These results suggest that ?1 has a predominant role in two in vivo effects of ethanol, and a role for ?2 may be revealed when ?1 is deleted. We also found that ethanol produces similar inhibition of function of recombinant ?1 and ?2 receptors. These data indicate that ethanol action on GABAA receptors containing ?1/?2 subunits may be important for specific effects of ethanol in vivo.
Project description:Glycine receptors (GlyRs) are inhibitory ligand-gated ion channels. Ethanol potentiates glycine activation of the GlyR, and putative binding sites for alcohol are located in the transmembrane (TM) domains between and within subunits. To alter alcohol sensitivity of GlyR, we introduced two mutations in the GlyR ?1 subunit, M287L (TM3) and Q266I (TM2). After expression in Xenopus laevis oocytes, both mutants showed a reduction in glycine sensitivity and glycine-induced maximal currents. Activation by taurine, another endogenous agonist, was almost abolished in the M287L GlyR. The ethanol potentiation of glycine currents was reduced in the M287L GlyR and eliminated in Q266I. Physiological levels of zinc (100 nM) potentiate glycine responses in wild-type GlyR and also enhance the ethanol potentiation of glycine responses. Although zinc potentiation of glycine responses was unchanged in both mutants, zinc enhancement of ethanol potentiation of glycine responses was absent in M287L GlyRs. The Q266I mutation decreased conductance but increased mean open time (effects not seen in M287L). Two lines of knockin mice bearing these mutations were developed. Survival of homozygous knockin mice was impaired, probably as a consequence of impaired glycinergic transmission. Glycine showed a decreased capacity for displacing strychnine binding in heterozygous knockin mice. Electrophysiology in isolated neurons of brain stem showed decreased glycine-mediated currents and decreased ethanol potentiation in homozygous knockin mice. Molecular models of the wild-type and mutant GlyRs show a smaller water-filled cavity within the TM domains of the Q266I ?1 subunit. The behavioral characterization of these knockin mice is presented in a companion article (J Pharmacol Exp Ther 340:317-329, 2012).