Kavalactones from Kava (Piper methysticum) root extract as modulators of recombinant human glycine receptors.

Roots of kava (Piper methysticum) plant are used in almost all Pacific Ocean cultures to prepare a drink with sedative, anesthetic and euphoric properties. One of the main active ingredients of the extract are kava lactones. Here, kava root CO2 extract and three kavalactones, DL-kavain, dihydrokavain and yangonin (isolated from whole extract by column chromatography) were tested for their inhibitory action on recombinant homomeric human α1 glycine receptors expressed in HEK293 cells. Kava CO2 root extract, as well as the individual components DL-kavain, dihydrokavain and yangonin inhibited glycine receptor activity in a dose-dependent manner. DL-kavain was the most potent inhibitor (IC50 = 0.077 ± 0.002 mm), followed by yangonin (IC50 = 0.31 ± 0.04 mm) and dihydrokavain (IC50 = 3.23 ± 0.10 mm) which were 4- and 40-fold less active than DL-kavain, respectively. Application of kava root extract did not reduce maximum currents, but increased EC50 of glycine. Simultaneous application of kava extract and strychnine showed additive inhibition, suggesting that binding of kavalactones and strychnine on the receptor is mutually exclusive. Overall, kavalactones exert a moderate inhibitory effect on the human α1 glycine receptor with DL-kavain being the most potent constituent.

Subunit-specific inhibition of glycine receptors by curcumol.

Emerging evidence has suggested that inhibitory glycine receptors (GlyRs) are an important molecular target in the treatment of numerous neurological disorders. Rhizoma curcumae is a medicinal plant with positive neurological effects. In this study, we showed that curcumol, a major bioactive component of R. curcumae, reversibly and concentration-dependently inhibited the glycine-activated current (IGly) in cultured rat hippocampal neurons. The inhibitory effect was neither voltage- nor agonist concentration-dependent. Moreover, curcumol selectively inhibited homomeric α2-containing, but not α1- or α3-containing, GlyRs. The addition of ß subunit conferred the curcumol sensitivity of α3-containing, but not α1-containing, GlyRs. Site-directed mutagenesis analysis revealed that a threonine at position 59 of the α2 subunit is critical for the susceptibility of GlyRs to curcumol-mediated inhibition. Furthermore, paralleling a decline of α2 subunit expression during spinal cord development, the degree of IGly inhibition by curcumol decreased with prolonged culture of rat spinal dorsal horn neurons. Taken together, our results suggest that the GlyRs are novel molecular targets of curcumol, which may underlie its pharmaceutical effects in the central nervous system.

The effect of supplemental dietary taurine on tinnitus and auditory discrimination in an animal model.

Loss of central inhibition has been hypothesized to underpin tinnitus and impact auditory acuity. Taurine, a partial agonist at inhibitory glycine and γ-amino butyric acid receptors, was added to the daily diet of rats to examine its effects on chronic tinnitus and normal auditory discrimination. Eight rats were unilaterally exposed once to a loud sound to induce tinnitus. The rats were trained and tested in an operant task shown to be sensitive to tinnitus. An equivalent unexposed control group was run in parallel. Months after exposure, 6 of the exposed rats showed significant evidence of chronic tinnitus. Two concentrations of taurine in drinking water were given over several weeks (attaining average daily doses of 67 mg/kg and 294 mg/kg). Water consumption was unaffected. Three main effects were obtained: (1) The high taurine dose significantly attenuated tinnitus, which returned to near pre-treatment levels following washout. (2) Auditory discrimination was significantly improved in unexposed control rats at both doses. (3) As indicated by lever pressing, taurine at both doses had a significant group-equivalent stimulant effect. These results are consistent with the hypothesis that taurine attenuates tinnitus and improves auditory discrimination by increasing inhibitory tone and decreasing noise in the auditory pathway.

Glycine receptors contribute to hypnosis induced by ethanol.

BACKGROUND: Glycine is a major inhibitory neurotransmitter in the adult central nervous system (CNS), and its receptors (GlyRs) are well known for their effects in the spinal cord and the lower brainstem. Accumulating evidence indicates that GlyRs are more widely distributed in the CNS, including many supraspinal regions. Previous in vitro studies have demonstrated that ethanol potentiates the function of these brain GlyRs, yet the behavioral role of the brain GlyRs has not been well explored. METHODS: Experiments were conducted in rats. The loss of righting reflex (LORR) was used as a marker of the hypnotic state. We compared the LORR induced by systematic administration of ethanol and of ketamine in the absence and presence of the selective glycine receptor antagonist strychnine. Ketamine is a general anesthetic that does not affect GlyRs. RESULTS: Systemically administered (by intraperitoneal injection) ethanol and ketamine dose-dependently induced LORR in rats. Furthermore, systemically administered (by subcutaneous injection) strychnine dose-dependently reduced the percentage of rats exhibiting LORR induced by ethanol, increased the onset time, and decreased the duration of LORR. Strychnine had no effect, however, on the LORR induced by ketamine. CONCLUSIONS: Given that hypnosis is caused by neuronal depression in upper brain areas, we therefore conclude that brain GlyRs contribute at least in part to the hypnosis induced by ethanol.

Behavior and cellular evidence for propofol-induced hypnosis involving brain glycine receptors.

BACKGROUND: It is well documented that several general anesthetics, including propofol, potentiate glycine receptor function. Furthermore, glycine receptors exist throughout the central nervous system, including areas of the brain thought to be involved in sleep. However, the role of glycine receptors in anesthetic-induced hypnosis has not been determined. METHODS: Experiments were conducted in rats where the loss of righting reflex (LORR) was used as a marker of the hypnotic state. Propofol-induced LORR was examined in the presence and absence of strychnine (a glycine receptor antagonist), GABAzine (a gamma-aminobutyric acid A receptor antagonist), as well as ketamine (an antagonist of N-methyl-D-aspartic acid subtype of glutamate receptors). Furthermore, the effects of propofol on the currents elicited by glycine and gamma-aminobutyric acid were analyzed in neurons isolated from the posterior hypothalamus of rats. The effects of strychnine and GABAzine on propofol-induced currents were also evaluated. RESULTS: Strychnine and GABAzine dose-dependently reduced the percentage of rats exhibiting LORR induced by propofol. Furthermore, strychnine significantly increased the onset time and reduced the duration of LORR induced by propofol. In contrast, strychnine did not affect the LORR induced by ketamine. In addition, propofol markedly increased the currents elicited by glycine and GABA of hypothalamic neurons. Conversely, strychnine and GABAzine both profoundly attenuated the current induced by propofol. CONCLUSION: Strychnine, the glycine receptor antagonist, dose-dependently reduced propofol-induced LORR in rats and propofol-induced current of rat hypothalamic neurons. These results suggest that neuronal glycine receptors partially contribute to propofol-induced hypnosis.

Subunit-specific modulation of glycine receptors by cannabinoids and N-arachidonyl-glycine.

Glycine receptors (GlyRs) mediate inhibitory neurotransmission in spinal cord motor and pain sensory neurons. Recent studies demonstrated apparently contradictory (potentiating versus inhibitory) effects of the endocannabinoid anandamide on these receptors. The present study characterised the effects of cannabinoid agonists on alpha1, alpha1beta, alpha2 and alpha3 GlyRs recombinantly expressed in HEK293 cells with the aims of reconciling effects of cannabinoids on these receptor subtypes and to establish the potential of different GlyR isoforms as novel physiological or analgesic targets for cannabinoids. The compounds investigated were anandamide, HU-210, HU-308, WIN55,212-2 and the endogenous non-cannabinoid, N-arachidonyl-glycine. The latter compound was chosen due to the structural similarity with anandamide and known analgesic actions in the spinal cord. Recombinant alpha1 and alpha1beta GlyRs were potentiated by anandamide and HU-210 at submicromolar concentrations, whereas WIN55,212-2 had no effect and HU-308 produced only weak inhibition. By contrast, N-arachidonyl-glycine exerted complex effects including both potentiation and inhibition. Anandamide had no effect at alpha2 or alpha3 GlyRs although the other cannabinoids produced potent inhibition. On alpha2 GlyRs, the inhibitory potency sequence was HU-210=WIN55,212-2>HU-308>N-arachidonyl-glycine but on alpha3 GlyRs it was HU-210=WIN55212=HU-308>N-arachidonyl-glycine. These results suggest that alpha1, alpha2 and alpha3 containing GlyRs exhibit distinct pharmacological profiles for cannabinoids. We conclude that cannabinoid agonists may be useful as pharmacological tools for selectively inhibiting alpha2 and alpha3 GlyRs. Our results also establish GlyRs as potential novel targets for endogenous and exogenous cannabinoids.

Fast non-genomic effects of progesterone-derived neurosteroids on nociceptive thresholds and pain symptoms.

Fast Inhibitory controls mediated by glycine (GlyRs) and GABAA receptors (GABAARs) play an important role to prevent the apparition of pathological pain symptoms of allodynia and hyperalgesia. The use of positive allosteric modulators of these receptors, specifically expressed in the spinal cord, may represent an interesting strategy to limit or block pain expression. In this study, we have used stereoisomers of progesterone metabolites, acting only via non-genomic effects, in order to evaluate the contribution of GlyRs and GABAARs for the reduction of mechanical and thermal heat hypernociception. We show that 3alpha neurosteroids were particularly efficient to elevate nociceptive thresholds in naive animal. It also reduced mechanical allodynia and thermal heat hyperalgesia in the carrageenan model of inflammatory pain. This effect is likely to be mediated by GABAA receptors since 3beta isomer was inefficient. More interestingly, 3alpha5beta neurosteroid was only efficient on mechanical allodynia while having no effect on thermal heat hyperalgesia. We characterized these paradoxical effects of 3alpha5beta neurosteroid using the strychnine and bicuculline models of allodynia. We clearly show that 3alpha5beta neurosteroid exerts an antinociceptive effect via a positive allosteric modulation of GABAARs but, at the same time, is pronociceptive by reducing GlyR function. This illustrates the importance of the inhibitory amino acid receptor channels and their allosteric modulators in spinal pain processing. Moreover, our results indicate that neurosteroids, which are synthesized in the dorsal horn of the spinal cord and have limited side effects, may be of significant interest in order to treat pathological pain symptoms.

Molecular pharmacology of the glycine receptor chloride channel.

The glycine receptor (GlyR) Cl(-) channel belongs to the cysteine-loop family of ligand-gated ion channel receptors. It is best known for mediating inhibitory neurotransmission in motor and sensory reflex circuits of the spinal cord, although glycinergic synapses are also present in the brain stem, cerebellum and retina. Extrasynaptic GlyRs are widely distributed throughout the central nervous system and they are also found in sperm and macrophages. A total of 5 GlyR subunits (alpha1-4 and beta) have been identified. Embryonic receptors comprise alpha2 homomers whereas adult receptors comprise predominantly alpha beta heteromers in a 2:3 stoichiometry. Notably, the alpha3 subunit is present in synaptic GlyRs that mediate inhibitory neurotransmission onto spinal nociceptive neurons. These receptors are specifically inhibited by inflammatory mediators, implying a role for alpha3-containing GlyRs in inflammatory pain sensitisation. Because molecules that increase GlyR current may have clinical potential as muscle relaxant and peripheral analgesic drugs, this review focuses on the molecular pharmacology of GlyR potentiating substances. Of all GlyR potentiating substances identified to date, we conclude that 5HT(3)R antagonists such as tropisetron offer the most promise as therapeutic lead compounds. However, one problem is that that virtually all known GlyR potentiating compounds, including tropisetron analogues, lack specificity for the GlyR. Another is that almost nothing is known about the pharmacological properties of alpha3-containing GlyRs, which is the subtype of choice for targeting by novel antinociceptive agents. These issues need to be addressed before GlyR-specific therapeutics can be developed.

Glycine receptor subunit composition alters the action of GABA antagonists.

GABA receptor antagonists produce an unexpectedly significant inhibition of native glycine receptors in retina and in alpha1 or alpha2 homomeric glycine receptors (GlyRs) expressed in HEK 293 cells. In this study we evaluate this phenomenon in heteromeric glycine receptors, formed by mixing alpha1, alpha2, and beta subunits. Picrotoxinin, picrotin, SR95531, and bicuculline are all more effective antagonists at GlyRs containing alpha2 subunits than alpha1 subunits. Inclusion of beta subunits reduces the inhibitory potency of picrotoxinin and picrotin but increases the potency of SR95531 and bicuculline. As a result of these two factors, bicuculline is particularly poor at discriminating GABA and glycine receptors. Picrotin, which has been reported to be inactive at GABA receptors, blocks glycine currents in retina and in HEK293 cells, suggesting its utility as a selective glycine antagonist. However, picrotin is a more potent inhibitor of GABA than glycine in retinal neurons. We also tested if GABA and glycine receptor subunits can combine to form functional receptors. If GABAAR gamma2S subunits are co-expressed with GlyR alpha subunits, the mixed receptor is glycine-sensitive and GABA-insensitive. But the mixed receptor exhibits a non-competitive picrotoxinin inhibition that is not observed in the homomeric GlyRs. This suggests that glycine and GABA subunits can co-assemble to form functional glycine receptors.

Accessibility to residues in transmembrane segment four of the glycine receptor.

Glycine receptors (GlyRs) are members of the ligand-gated ion channel superfamily. Each subunit has four transmembrane segments (TM1-TM4). Several studies suggest that amino acids in all four TMs face into a water-filled, alcohol and anesthetic binding cavity in the extracellular portion of the transmembrane domain. TM4 should contribute a "wall" to this cavity, but the residues involved are unknown. Here, we determined the ability of an alcohol analog, propyl methanethiosulfonate (propyl MTS), to covalently react with twelve GlyR TM4 positions (I401-I412) after mutating the original amino acids to cysteines. Reactivity of a cysteine with propyl MTS implies that the cysteine is exposed to water. W407C, I409C, Y410C, and K411C showed altered receptor function following reaction with propyl MTS in the presence or absence of glycine. The cysteine mutations alone eliminated the effects of ethanol for I409C, Y410C, and K411C, and reduced the effects of octanol for I409C and isoflurane for K411C. The ability of propyl MTS to reduce isoflurane and chloroform potentiation was examined in the reactive mutants. Potentiation by isoflurane was significantly reduced for I409C after reaction. These data demonstrate water-accessibility of specific TM4 positions in the GlyR and suggest involvement of these residues with alcohol and anesthetic action.

A yellow fluorescent protein-based assay for high-throughput screening of glycine and GABAA receptor chloride channels.

There is a significant clinical need to identify novel ligands with high selectivity and potency for GABA(A), GABA(C) and glycine receptor Cl- channels. Two recently developed, yellow fluorescent protein variants (YFP-I152L and YFP-V163S) are highly sensitive to quench by small anions and are thus suited to reporting anionic influx into cells. The aim of this study was to establish the optimal conditions for using these constructs for high-throughput screening of GABA(A), GABA(C) and glycine receptors transiently expressed in HEK293 cells. We found that a 70% fluorescence reduction was achieved by quenching YFP-I152L with a 10 s influx of I- ions, driven by an external I- concentration of at least 50 mM. The fluorescence quench was rapid, with a mean time constant of 3 s. These responses were similar for all anion receptor types studied. We also show the assay is sufficiently sensitive to measure agonist and antagonist concentration-responses using either imaging- or photomultiplier-based detection systems. The robustness, sensitivity and low cost of this assay render it suited for high-throughput screening of transiently expressed anionic ligand-gated channels.

Pentobarbital induces thalamic oscillations in brain slices, modulated by GABA and glycine receptors.

We studied the effects of pentobarbital and antagonists of glutamate, gamma-aminobutyrate (GABA), and glycine receptors on extracellular activity in ventrobasal thalamic slices. Pentobarbital at sedative-hypnotic concentration (20 microM) reversibly induced 1-15 Hz oscillations. Sustained oscillations required electrical stimulation of internal capsule, but not elevated temperature or low [Mg2+]. Anesthetic concentration (200 microM) of pentobarbital evoked only transient oscillations. Kynurenate-sensitive glutamate receptors were essential for oscillations. GABA(A) antagonism (bicuculline, 50 microM or gabazine, 20 microM) suppressed oscillations at 5-15 Hz. GABA(B) antagonism (CGP 35348, 100 nM), or antagonism of glycine receptors (strychnine, 1 microM) suppressed oscillations at 1-4 and 11-15 Hz. GABA and glycine receptors modulated oscillation frequency. For elimination, oscillations required GABA antagonists and strychnine. Receptors for glutamate and glycine mediated oscillations during GABA receptor blockade in ventrobasal nuclei, or on disconnection from nRT. Glycine receptors were critical for oscillations in dorsal thalamic network, divested of GABAergic inhibition. Glutamate and GABA receptors mediated pentobarbital-induced oscillations in nRT, disconnected from ventrobasal nuclei. Hence, pentobarbital oscillogenesis occurred in isolated networks of the ventrobasal and reticularis nuclei mediated by glutamate receptors, with frequency modulation by GABA(A), GABA(B), and glycine receptors. These stationary oscillations represent a model of sedation-hypnosis, amenable to pharmacological analysis.

About the toxicity of some Strychnos species and their alkaloids.

Poisons are widespread in plants and animals and humankind has often tried to turn them to its own advantage. Owing to their poisonous properties, some species of Strychnos genus have been employed mainly in hunting and fishing, as an adjunct to weapons used not only in the search of food and clothes, but also for preventing depredation by wild animals. They have been employed for martial and criminal purposes and also as a means of determining guilt or innocence. By their nature, poisons such as strychnine and curare affect the functioning of the victim's body; this also means that they have been, and are, an important source of pharmacological tools and medicines all over the world. With such potentially dangerous substances, care in medication is essential to avoid complications by overdose. All these points are approached in the present review.

Glycine receptor knock-in mice and hyperekplexia-like phenotypes: comparisons with the null mutant.

Strychnine-sensitive glycine receptors (GlyRs) inhibit neurotransmission in the spinal cord and brainstem. To better define the function of this receptor in vivo, we constructed a point mutation that impairs receptor function in the alpha1-subunit and compared these knock-in mice to oscillator (spdot) mice lacking functional GlyR alpha1-subunits. Mutation of the serine residue at amino acid 267 to glutamine (alpha1S267Q) results in a GlyR with normal glycine potency but decreased maximal currents, as shown by electrophysiological recordings using Xenopus oocytes. In addition, single-channel recordings using human embryonic kidney 293 cells indicated profoundly altered properties of the mutated GlyR. We produced knock-in mice bearing the GlyR alpha1 S267Q mutation to assess the in vivo consequences of selectively decreasing GlyR efficacy. Chloride uptake into brain synaptoneurosomes from knock-in mice revealed decreased responses to maximally effective glycine concentrations, although wild-type levels of GlyR expression were observed using 3H-strychnine binding and immunoblotting. A profound increase in the acoustic startle response was observed in knock-in mice as well as a "limb clenching" phenotype. In contrast, no changes in coordination or pain perception were observed using the rotarod or hot-plate tests, and there was no change in GABA(A)-receptor-mediated chloride uptake. Homozygous S267Q knock-in mice, like homozygous spdot mice, exhibited seizures and died within 3 weeks of birth. In heterozygous spdot mice, both decreased 3H-strychnine binding and chloride flux were observed; however, neither enhanced acoustic startle responses nor limb clenching were seen. These data demonstrate that a dominant-negative point mutation in GlyR disrupting normal function can produce a more dramatic phenotype than the corresponding recessive null mutation, and provides a new animal model to evaluate GlyR function in vivo.

Effects of ginsenosides on glycine receptor alpha1 channels expressed in Xenopus oocytes.

Ginsenosides, major active ingredients of Panax ginseng, are known to regulate the excitatory ligand-gated ion channel activity. Recent reports showed that ginsenosides attenuate nicotinic acetylcholine and NMDA receptor channel activity. However, it is not known whether ginsenosides also affect the inhibitory ligand-gated ion channel activity. We investigated the effect of ginsenosides on human glycine alpha1 receptor channel activity expressed in Xenopus oocytes using a two-electrode voltage clamp technique. Treatment of ginsenoside Rf enhances glycine-induced inward peak current (IGly) with dose dependent and reversible manner but ginsenoside Rf itself did not elicit membrane currents. The half-stimulatory concentrations (EC50) of ginsenoside Rf was 49.8 +/- 8.9 microM. Glycine receptor antagonist strychnine completely blocked the inward current elicited by glycine plus ginsenoside Rf. Cl- channel blocker 4,4'-disothiocyanostilbene-2,2'-disulfonic acid (DIDS) also blocked the inward current elicited by glycine plus ginsenoside Rf. We also tested the effect of eight individual ginsenosides (i.e., Rb1, Rb2, Rc, Rd, Re, Rg1, Rg2, and Ro) in addition to ginsenoside Rf. We found that five of them significantly enhanced the inward current induced by glycine with the following order of potency: Rb1 > Rb2 > Rg2 > or = Rc > Rf > Rg1 > Re. These results indicate that ginsenosides might regulate gylcine receptor expressed in Xenopus oocytes and this regulation might be one of the pharmacological actions of Panax ginseng.

Ivermectin, an unconventional agonist of the glycine receptor chloride channel.

The effects of the antihelmintic, ivermectin, were investigated in recombinantly expressed human alpha(1) homomeric and alpha(1)beta heteromeric glycine receptors (GlyRs). At low (0.03 microm) concentrations ivermectin potentiated the response to sub-saturating glycine concentrations, and at higher (> or =0.03 microm) concentrations it irreversibly activated both alpha(1) homomeric and alpha(1)beta heteromeric GlyRs. Relative to glycine-gated currents, ivermectin-gated currents exhibited a dramatically reduced sensitivity to inhibition by strychnine, picrotoxin, and zinc. The insensitivity to strychnine could not be explained by ivermectin preventing the access of strychnine to its binding site. Furthermore, the elimination of a known glycine- and strychnine-binding site by site-directed mutagenesis had little effect on ivermectin sensitivity, demonstrating that the ivermectin- and glycine-binding sites were not identical. Ivermectin strongly and irreversibly activated a fast-desensitizing mutant GlyR after it had been completely desensitized by a saturating concentration of glycine. Finally, a mutation known to impair dramatically the glycine signal transduction mechanism had little effect on the apparent affinity or efficacy of ivermectin. Together, these findings indicate that ivermectin activates the GlyR by a novel mechanism.

Direct inhibition of glycine receptors by genistein, a tyrosine kinase inhibitor.

Genistein, a tyrosine kinase inhibitor, has been widely used to examine potential effects of protein tyrosine kinase (PTK)-mediated regulation of receptor/channel function. Alteration of ion channel function in the presence of genistein has typically led to the conclusion that PTK regulates the activity of the channel under investigation. In the present report, we have assessed the possibility that genistein directly inhibits the glycine receptor, independent of effects on protein tyrosine kinase. Coapplication of genistein with glycine reversibly inhibited the strychnine-sensitive, glycine-activated current recorded from hypothalamic neurons. The time course of genistein action was rapid (within ms). Equilibration of genistein in the intracellular solution did not affect the ability of extracellularly applied genistein to inhibit the glycine response. Glycine concentration-response profiles generated in the absence and presence of genistein indicated the block was due to non-competitive antagonism. The genistein effect also displayed voltage-dependence. Daidzein, an analog of genistein that does not block protein kinases, also inhibited glycine-activated current. Coapplication of lavendustin A, a specific inhibitor of PTK, had no effect on the glycine response. Our results demonstrate that the tyrosine kinase inhibitor genistein has a direct inhibitory effect on glycine receptors that is not mediated via inhibition of PTK.

IV glycine and oral D-cycloserine effects on plasma and CSF amino acids in healthy humans.

BACKGROUND: The amino acid glycine, modulates neurotransmission via actions at GLY-A receptor and GLY-B receptor. The latter are coagonist sites associated with N-Methyl-D-Aspartate (NMDA) glutamate receptors. The central bioavailability of peripherally administered glycine has not been adequately characterized in humans. METHODS: Healthy human subjects were administered either oral D-cycloserine (50 mg or placebo) and intravenous glycine (saline, 100 mg/kg or 200 mg/kg) in random order over 4 test days under double-blind conditions. Cerebrospinal fluid was collected by lumbar puncture performed on the first test day was analyzed to determine amino acid levels. The acoustic startle response was measured on the second test day. RESULTS: Intravenous glycine dose-dependently increased both serum and CSF glycine and serine levels. Neither glycine nor DCS produced any significant effects on behavior, cognition or the acoustic startle response. Neither IV glycine nor DCS were associated with any toxicity. CONCLUSIONS: Thus, peripheral glycine administration raised CSF glycine levels without producing any clear central nervous system effects. Glycine and D-cycloserine did not worsen cognitive test performance and did not induce behavioral symptoms on their own. The possibility that glycine and D-cycloserine enhanced cognitive test performance cannot be excluded given the psychometric limitations of the test battery.

Kinetic and mutational analysis of Zn2+ modulation of recombinant human inhibitory glycine receptors.

1. The effects of Zn2+ on glycine receptor (GlyR) currents were analysed in Xenopus oocytes and human embryonic kidney cells expressing homomeric human wild-type and mutant alpha1 subunit GlyRs. 2. Low concentrations (10 microM) of extracellular Zn2+ converted the partial agonist taurine into a high-efficacy agonist. Concentration-response analysis showed that the EC50 for taurine decreased whereas the Hill coefficient increased under these conditions. In contrast, 50-500 microM Zn2+ showed an increased EC50 value and reduced maximal inducible taurine currents. The potency of competitive antagonists was not affected in the presence of Zn2+. 3. Single-channel recording from outside-out patches revealed different kinetics of glycine- and taurine-gated currents. With both agonists, Zn2+ altered the open probability of the alpha1 GlyR without changing its unitary conductance. Low Zn2+ concentrations (5 microM) increased both the opening frequency and mean burst duration, whereas higher Zn2+ concentrations (> 50 microM) reduced GlyR open probability mainly by decreasing the open frequency and the relative contribution of the longest burst of the single-channel events. 4. Site-directed mutagenesis of the GlyR alpha1 subunit identified aspartate 80 and threonine 112 as important determinants of Zn2+ potentiation and inhibition, respectively, without affecting potentiation by ethanol. 5. Our data support the view that Zn2+ modulates different steps of the receptor binding and gating cycle via specific allosteric high- and low-affinity binding sites in the extracellular N-terminal region of the GlyR alpha1 subunit.

Anxiolytic-like effects of N-methyl-D-aspartate-associated glycine receptor ligands in the rat potentiated startle test.

Competitive and non-competitive NMDA receptor complex antagonists have been shown to be active in various models of anxiolytic activity. This study examined the effects of ligands at the NMDA receptor-associated glycine site and two competitive NMDA receptor antagonists on the fear-potentiated startle response model for anxiolytic activity. The results show that the NMDA receptor antagonists, 2-amino-7-phosphonoheptanoate (30 mg/kg) and 3-(2-carboxypiperazin-4-yl)propyl-1-phosphonate (3 mg/kg), the glycine receptor antagonist 7-chlorokynurenate (100 mg/kg), and the glycine receptor partial agonists 3-amino-1-hydroxy-2-pyrrolidinone ((+)-HA-966) (30 mg/kg), 1-aminocyclopropane carboxylate (200-500 mg/kg) and D-cycloserine (30-300 mg/kg) blocked the potentiated startle effect. These results extend the findings of earlier studies showing anxiolytic-like effects of NMDA antagonists and glycine receptor ligands by demonstrating their effectiveness in the rat potentiated startle paradigm. These results also demonstrate the anxiolytic potential of D-cycloserine.