Gelsemine is one of the principal alkaloids produced by the Gelsemium genus of plants belonging to the Loganiaceae family. The extracts of these plants have been used for many years, for a variety of medicinal purposes. Coincidentally, recent studies have shown that gelsemine exerts anxiolytic and analgesic effects on behavioural models. Several lines of evidence have suggested that these beneficial actions were dependent on glycine receptors, which are inhibitory neurotransmittergated ion channels of the CNS. However, it is currently unknown whether gelsemine can directly modulate the function of glycine receptors. EXPERIMENTAL APPROACHWe examined the functional effects of gelsemine on glycine receptors expressed in transfected HEK293 cells and in cultured spinal neurons by electrophysiological techniques. KEY RESULTSGelsemine directly modulated recombinant and native glycine receptors and exerted conformation-specific and subunit-selective effects. Gelsemine modulation was voltage-independent and was associated with differential changes in the apparent affinity for glycine and in the open probability of the ion channel. In addition, the alkaloid preferentially targeted glycine receptors in spinal neurons and showed only minor effects on GABA A and AMPA receptors. Furthermore, gelsemine significantly diminished the frequency of glycinergic and glutamatergic synaptic events without altering the amplitude. CONCLUSIONS AND IMPLICATIONSOur results provide a pharmacological basis to explain, at least in part, the glycine receptor-dependent, beneficial and toxic effects of gelsemine in animals and humans. In addition, the pharmacological profile of gelsemine may open new approaches to the development of subunit-selective modulators of glycine receptors. Abbreviations

Ethanol increased the frequency of miniature glycinergic currents [miniature inhibitory postsynaptic currents (mIPSCs)] in cultured spinal neurons. This effect was dependent on intracellular calcium augmentation, since preincubation with BAPTA (an intracellular calcium chelator) or thapsigargin [a sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) pump inhibitor] significantly attenuated this effect. Similarly, U73122 (a phospholipase C inhibitor) or 2-aminoethoxydiphenyl borate [2-APB, an inositol 1,4,5-trisphosphate (IP₃) receptor (IP3R) inhibitor] reduced this effect. Block of ethanol action was also achieved after preincubation with Rp-cAMPS, inhibitor of the adenylate cyclase (AC)/PKA signaling pathway. These data suggest that there is a convergence at the level of IP₃R that accounts for presynaptic ethanol effects. At the postsynaptic level, ethanol increased the decay time constant of mIPSCs in a group of neurons (30 ± 10% above control, n = 13/26 cells). On the other hand, the currents activated by exogenously applied glycine were consistently potentiated (55 ± 10% above control, n = 11/12 cells), which suggests that ethanol modulates synaptic and nonsynaptic glycine receptors (GlyRs) in a different fashion. Supporting the role of G protein modulation on ethanol responses, we found that a nonhydrolyzable GTP analog [guanosine 5'-O-(3-thiotriphosphate) (GTPγS)] increased the decay time constant in ∼50% of the neurons (28 ± 12%, n = 11/19 cells) but potentiated the glycine-activated Cl(-) current in most of the neurons examined (83 ± 29%, n = 7/9 cells). In addition, confocal microscopy showed that α1-containing GlyRs colocalized with Gβ and Piccolo (a presynaptic cytomatrix protein) in ∼40% of synaptic receptor clusters, suggesting that colocalization of Gβγ and GlyRs might account for the difference in ethanol sensitivity at the postsynaptic level.

Previous studies have shown that the effect of ethanol on glycine receptors (GlyRs) containing the a1 subunit is affected by interaction with heterotrimeric G proteins (Gbg). GlyRs containing the a3 subunit are involved in inflammatory pain sensitization and rhythmic breathing and have received much recent attention. For example, it is unknown whether ethanol affects the function of this important GlyR subtype. Electrophysiologic experiments showed that GlyR a3 subunits were not potentiated by pharmacologic concentrations of ethanol or by Gbg. Thus, we studied GlyR a1-a3 chimeras and mutants to determine the molecular properties that confer ethanol insensitivity. Mutation of corresponding glycine 254 in transmembrane domain 2 (TM2) found in a1 in the a3 A254G -a1 chimera induced a glycine-evoked current that displayed potentiation during application of ethanol (46 6 5%, 100 mM) and Gbg activation (80 6 17%). Interestingly, insertion of the intracellular a3L splice cassette into GlyR a1 abolished the enhancement of the glycine-activated current by ethanol (5 6 6%) and activation by Gbg (21 6 7%). Incorporation of the GlyR a1 C terminus into the ethanol-resistant a3S A254G mutant produced a construct that displayed potentiation of the glycine-activated current with 100 mM ethanol (40 6 6%) together with a current enhancement after G protein activation (68 6 25%). Taken together, these data demonstrate that GlyR a3 subunits are not modulated by ethanol. Residue A254 in TM2, the a3L splice cassette, and the C-terminal domain of a3 GlyRs are determinants for low ethanol sensitivity and form the molecular basis of subtype-selective modulation of GlyRs by alcohol.

Background: G␤␥ interaction with GlyR is an important determinant in ethanol potentiation of this channel. Results: A small peptide, RQH C7, can inhibit ethanol potentiation of GlyR currents. Conclusion: Results with RQH C7 indicate that ethanol mediated potentiation of GlyR is in part by G␤␥ activation. Significance: Molecular interaction between G␤␥ and GlyR could be used as a target for pharmacological modification of ethanol effects.

Background and Purpose The precise mechanism/s of action of ethanol, although studied for many years, are not well understood. Like other drugs of abuse, ethanol affects dopamine levels in the nucleus accumbens (nAc), an important region of the mesolimbic system, causing a reinforcing effect. It has been shown that glycine receptors (GlyRs) present in the nAc are potentiated by clinically relevant concentrations of ethanol, where α1 and α2 are the predominant subunits expressed. Experimental Approach Using a combination of electrophysiology and behavioural assays, we studied the involvement of GlyR α2 subunits on the effects of low and high doses of ethanol, as well as on consumption using mice lacking the GlyR α2 subunit (male Glra2−/Y and female Glra2−/−). Key Results GlyR α2 subunits exist in accumbal neurons, since the glycine‐evoked currents and glycinergic miniature inhibitory postsynaptic currents (mIPSCs) in Glra2−/Y mice were drastically decreased. In behavioural studies, differences in ethanol consumption and sedation were observed between wild‐type (WT) and Glra2 knockout (KO) mice. Using the drinking in the dark (DID) paradigm, we found that Glra2−/Y mice presented a binge‐like drinking behaviour immediately when exposed to ethanol rather than the gradual consumption seen in WT animals. Interestingly, the effect of knocking out Glra2 in female (Glra2−/−) mice was less evident, since WT female mice already showed higher DID. Conclusion and Implications The differences in ethanol consumption between WT and KO mice provide additional evidence supporting the conclusion that GlyRs are biologically relevant targets for the sedative and rewarding properties of ethanol.

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...

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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, patchclamp, and biochemical techniques. A conserved substitution (K385R) did not affect either the apparent glycine EC 50 (40 Ϯ 1 versus 41 Ϯ 0.5 M) or the ethanol-induced potentiation (53 Ϯ 5 versus 46 Ϯ 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 Ϯ 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 Ϯ 4 and 17 Ϯ 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.