Localization of the strychnine binding site on the 48-kilodalton subunit of the glycine receptor

Amino acid residues that participate in antagonist binding to the strychnine-sensitive glycine receptor (GlyR) have been identified by selectively modifying functional groups with chemical reagents. Moreover, a region directly involved with strychnine binding has been localized in the 48-kDa subunit of this receptor by covalent labeling and proteolytic mapping. Modification of tyrosyl or arginyl residues promotes a marked decrease of specific [3H]strychnine binding either to rat spinal cord plasma membranes or to the purified GlyR incorporated into phospholipid vesicles. Occupancy of the receptor by strychnine, but not by glycine, completely protects from the inhibition caused by chemical reagents. Furthermore, these tyrosine- or arginine-specific reagents decrease the number of binding sites (Bmax) for [3H]strychnine binding without affecting the affinity for the ligand (Kd). These observations strongly suggest that such residues are present at, or very close to, the antagonist binding site. In order to localize the strychnine binding domain within the GlyR, purified and reconstituted receptor preparations were photoaffinity labeled with [3H]strychnine. The radiolabeled 48-kDa subunit was then digested with specific chemical proteolytic reagents, and the peptides containing the covalently bound radioligand were identified by fluorography after gel electrophoresis. N-Chlorosuccinimide treatment of [3H]strychnine-labeled 48K polypeptide yielded a single labeled peptide of Mr approximately 7300, and cyanogen bromide gave a labeled peptide of Mr 6200.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of Strychnine-sensitive Glycine Receptor in the Intact Frog Retina: Modulation by Protein Kinases

We studied ³H-glycine and ³H-strychnine specific binding to glycine receptor (GlyR) in intact isolated frog retinas. To avoid glycine binding to glycine uptake sites, experiments were performed at low ligand concentrations in a sodium-free medium. The binding of both radiolabeled ligands was saturated. Scatchard analysis of bound glycine and strychnine revealed a K_D of 2.5 and 2.0 M, respectively. Specific binding of glycine was displaced by -alanine, sarcosine, and strychnine. Strychnine binding was displaced 50% by glycine, and sarcosine. Properties of the strychnine-binding site in the GlyR were modified by sarcosine. Binding of both radioligands was considerably reduced by compounds that inhibit or activate adenylate cyclase and increased cAMP levels. A phorbol ester activator of PKC remarkably decreased glycine and strychnine binding. These results suggest modulation of GlyR in response to endogenous activation of protein kinases A and C, as well as protein phosphorylation modulating GlyR function in retina.     

Polyclonal Antibodies to the Glycine Receptor Antagonist Strychnine

Polyclonal antibodies have been raised in rabbits against the glycine receptor antagonist strychnine, coupled through a 2-amino substituent to the antigenic protein key-hole limpet haemocyanin. Strychnine binding of the predominantly immunoglobulin G (IgG) class of antibodies was measured by incubation with [3H]strychnine, followed by adsorption of IgG onto Staphylococcus aureus cells and filtration through glass-fibre filters under vacuum. Only strychnine and structurally related alkaloids or derivatives were able to inhibit [3H]strychnine binding to the IgG. A significant rank correlation was found between the potencies of these compounds to inhibit [3H]strychnine binding to the antibodies and to the glycine receptor in mouse spinal cord membranes. In contrast, preincubation of strychnine antibodies with a variety of ligands at other neurotransmitter, drug, or hormone receptors in the CNS (at 10(-4) M) failed to inhibit binding significantly. The failure of glycine to inhibit strychnine antibody binding is consistent with previous suggestions that the recognition sites for this amino acid on the CNS receptor may be conformationally distinct from those for the antagonist alkaloid. Strychnine antibodies may now help in the identification and purification of possible endogenous ligands at this alkaloid binding site in the CNS.     

Hamster sperm glycine receptor: evidence for its presence and involvement in the acrosome reaction

Recent reports have provided evidence for the presence of amino acid neurotransmitter receptor/chloride channels in human and porcine spermatozoa and their involvement in the acrosome reaction (AR). In this work we investigated whether a glycine receptor (GlyR) was present in golden hamster sperm, and whether it had a role in the hamster AR. The neuronal GlyR agonist glycine, stimulated in a dose-dependent manner, the AR of hamster spermatozoa previously capacitated for at least 3 hr. This stimulation was completely inhibited by 50 microM (+)-bicuculline and by concentrations of strychnine as low as 10-50 nM; both agents are antagonists of neuronal GlyR when used at the concentrations reported in this study. beta-Alanine, another agonist of the neuronal GlyR, also stimulated the AR. The AR-stimulatory effect of this compound was completely abolished by 50 nM strychnine. The inhibitory effect of strychnine on the glycine-induced hamster sperm AR was completely overcome by subsequent treatment with the calcium ionophore ionomycin, demonstrating that the strychnine effect was specific for GlyR. Additional binding studies with (3)[H]-strychnine, the typical radioligand used to detect GlyR in several cells, demonstrated for the first time the presence of specific binding sites for strychnine in the hamster spermatozoa. Interestingly, binding increased during in vitro capacitation, particularly in those sperm suspensions showing high percentages of AR. Taken together these results strongly suggest the presence of a GlyR in the hamster spermatozoa, with a role in the AR when activated.     

Glycine receptor activation regulates short-term plasticity in CA1 area of hippocampal slices of rats

Functional glycine receptors (GlyRs) are enriched in the hippocampus, but their roles in synaptic transmission are unclear. In this study, we examined the effect of GlyR activation on paired-pulse stimulation of the whole-cell postsynaptic currents (PSCs) in the Schaffer-CA1 synapses in rat hippocampal slices. Bath application of glycine reduced the amplitude of PSCs, accompanied by an increase in holding current and resting conductance. Moreover, glycine application increased the paired-pulse ratio (PPR) of PSCs significantly, an effect largely abolished by the GlyR specific antagonist strychnine. Interestingly, glycine application had no significant effect on either the amplitude or the PPR of excitatory postsynaptic currents (EPSCs). Our findings suggest that GlyR activation regulates hippocampal short-term plasticity by altering GABAergic neurotransmission.     

Thermodynamics of Agonist and Antagonist Interaction with the Strychnine-Sensitive Glycine Receptor

The thermodynamic parameters associated with the interactions of agonists and antagonists with glycine receptors in rat spinal cord membranes were determined. The binding of the antagonist [3H]strychnine and the inhibition of strychnine binding by 11 different glycinergic ligands were examined at temperatures between 0.5 and 37 degrees C. The density of receptors was not affected by the temperature at which the incubation was performed, but the ability of glycine receptor agonists and antagonists to compete with [3H]strychnine binding varied markedly. The affinity of the receptor for the antagonists strychnine, 2-aminostrychnine, RU-5135, 5,6,7,8-tetrahydro-4H-isoxazolo[5,4-c]azepin-3-ol, and the ligands bicuculline, norharmane, and PK-8165 decreased at higher temperatures. The binding of these ligands was enthalpy-driven. In contrast, the affinity of the agonists glycine, beta-alanine, and taurine and of the antihelmintic ivermectin increased at higher temperatures, and their binding was characterized by substantial increases in entropy. In addition, temperature affected the allosteric interaction between the glycine and strychnine sites of the receptor, as indicated by changes in the Hill number of the competition curves for glycine. Our results clearly indicate that the binding of agonists and antagonists to the glycine receptor is differentially affected by temperature, probably as a consequence of the different changes induced in the receptor conformation.     

Glycine inhibits angiogenic signaling in human hepatocellular carcinoma cells

Hepatocellular carcinoma (HCC) is a highly vascularized tumor with limited susceptibility to chemotherapy. Modern targeted therapies are aimed at specific properties of this neoplasm. Glycine is a simple non-essential amino acid with potential antiangiogenic effects. In this study, the amino acid’s effect on angiogenic signaling in an in vitro model of HCC was evaluated. HepG2 and Huh7 cells were treated with glycine-free DMEM supplemented with 0, 0.01, 0.1, 1.0, 2.0, 5.0 and 10 mM glycine. The direct effects of glycine on the viability of HCC cells were monitored using MTT assay. To detect angiogenic signaling, mRNA and protein levels of vascular endothelial growth factor (VEGF-A) were measured using RT-PCR and Western Blot assays. To determine whether or not glycine receptors (GlyR) played a significant role, the specific antagonist, strychnine, was used as a direct inhibitor. Western Blotting was performed to show the presence of GlyR. While there was no direct pro- or antiproliferative effect of either glycine or strychnine in both cell lines, glycine was shown to significantly decrease VEGF-A expression on mRNA and protein level up to 63 % in both cell lines. This effect was blunted by the presence of strychnine. GlyR was also identified in both cell lines. Glycine decreases GlyR-dependent, VEGF-A-mediated, angiogenic signaling in human HCC and thus might be a promising additive to chemotherapy treatment strategies for highly vascularized tumors.     

Zinc Potentiation of the Glycine Receptor Chloride Channel Is Mediated by Allosteric Pathways

Abstract: Molecular mechanisms of zinc potentiation were investigated in recombinant human α1 glycine receptors (GlyRs) by whole-cell patch-clamp recording and [³H]strychnine binding assays. In the wild-type (WT) GlyR, 1 µ M zinc enhanced the apparent binding affinity of the agonists glycine and taurine and reduced their concentrations required for half-maximal activation. Thus, in the WT GlyR, zinc potentiation apparently occurs by enhancing agonist binding. However, analysis of GlyRs incorporating mutations in the membrane-spanning domain M1–M2 and M2–M3 loops, which are both components of the agonist gating mechanism, indicates that most mutations uncoupled zinc potentiation from glycine-gated currents but preserved zinc potentiation of taurine-gated currents. One such mutation in the M2–M3 loop, L274A, abolished the ability of zinc to potentiate taurine binding but did not inhibit zinc potentiation of taurine-gated currents. In this same mutant where taurine acts as a partial agonist, zinc potentiated taurine-gated currents but did not potentiate taurine antagonism of glycine-gated currents, suggesting that zinc interacts selectively with the agonist transduction pathway. The intracellular M246A mutation, which is unlikely to bind zinc, also disrupted zinc potentiation of glycine currents. Thus, zinc potentiation of the GlyR is mediated via allosteric mechanisms that are independent of its effects on agonist binding.     

Evidence for an essential sulfhydryl group at the substrate binding site of the A-system transporter of Ehrlich cell plasma membranes

Plasma membrane suspensions of Ehrlich ascites cells solubilized with cholic acid were used to study the effects of sulfhydryl reagents on Na(+)-dependent amino acid transport. These suspensions were treated with the sulfhydryl binding agents p-chloromercuribenzenesulfonic acid or N-ethylmaleimide prior to reconstitution for the assay of transport activity. The proteoliposomes formed from dissolved membranes treated with p-chloromercuribenzenesulfonic acid showed no Na(+)-dependent alpha-aminoisobutyric acid transport, while N-ethylmaleimide pretreated membranes retained approximately 90% of the original activity. To avoid interference by the N-ethylmaleimide component, further studies were carried out with membranes pretreated with 200 microM N-ethylmaleimide prior to p-chloromercuribenzenesulfonic acid treatment. A concentration of 25 microM p-chloromercuribenzenesulfonic acid inhibited Na(+)-dependent alpha-aminoisobutyric acid transport by 50%. The degree of inhibition was dramatically reduced in the presence of substrates specific for the A transport system. Using an inhibition index to address the efficacy of inhibition in presence and absence of substrates, it could be shown that an index of 1.0 in presence of p-chloromercuribenzenesulfonic acid was reduced to 0.84 with (methylamino)isobutyric acid alone and 0.05 in the presence of 100 mM Na+ and 5 mM (methylamino)isobutyric acid. Na+ alone offered no protection. The results show that sulfhydryl group(s) on the amino acid carrier may be directly involved in substrate binding and that substrate binding sites are functional in the disaggregated membrane state. Furthermore, Na+ directly affects (methylamino)isobutyrate binding, since the degree of protection by the amino acid analogue against p-chloromercuribenzenesulfonic acid inhibition was influenced by the presence of Na+.(ABSTRACT TRUNCATED AT 250 WORDS)     

Skin sulfhydryl oxidase. Purification and some properties

A sulfhydryl-oxidizing enzyme has been found in skin of young rats and a method for purifying the enzyme over 600-fold has been developed. Enzymatic activity was assayed either by its ability to oxidize dithiothreitol of by measuring its ability to renature reductively denatured ribonuclease A. Skin sulfhydryl oxidase catalyzed the oxidation of various thiols: dithiothreitol, dithioerythritol, D-penicillamine, and L-cysteine. Glutathione and 2-mercaptoethanol were very poor substrates for the enzyme. The enzyme also reactivated reductively denatured ribonuclease A, with neither the presence of a thiol nor prior reduction of the enzyme being necessary. The molecular weight of the enzyme was estimated to be 66 000 +/- 2000, and the isoelectric point was determined to be at pH 4.65. Alkylating reagents alone had some inhibiting effect on skin sulfhydryl oxidase; when the enzyme was preincubated with thiols which were substrates, inhibition by alkylating reagents was greatly increased. After preincubation with dithiothreitol, treatment of the enzyme with alkylating reagents or N-ethylmaleimide caused significant inhibition; preincubation with a poor substrate, reduced glutathione, did not enhance inhibition by alkylating reagents or N-ethylmaleimide.     

Inhibition of phosphate transport across the human erythrocyte membrane by chemical modification of sulfhydryl groups.

Effects of sulfhydryl-reactive reagents on phosphate transport across human erythrocyte membranes were examined using 31P NMR. Phosphate transport was significantly inhibited in erythrocytes treated with sulfhydryl modifiers such as N-ethylmaleimide, diamide, and Cu2+/o-phenanthroline. Quantitation of sulfhydryl groups in band 3 showed that the inhibition is closely associated with the decrease of sulfhydryl groups. Data from erythrocytes treated with diamide or Cu2+/o-phenanthroline demonstrated that intermolecular cross-linking of band 3 by oxidation of a sulfhydryl group, perhaps Cys-201 or Cys-317, decreases the phosphate influx by about 10%. The inhibition was reversed by reduction using dithiothreitol. These results suggest that sulfhydryl groups in the cytoplasmic domain of band 3 may play an important role in the regulation of anion exchange across the membrane.     

Novikoff hepatoma deoxyribonucleic acid polymerase. Sensitivity of the beta-polymerase to sulfhydryl blocking agents.

Unlike other beta-class eukaryotic DNA polymerases, the enzyme purified from the Novikoff hepatoma is inhibited by both sulfhydryl blocking agents N-ethylmaleimide (NEM) and p-hydroxymercuribenzoate (pHMB). The degree of sensitivity varies depending on the enzyme purity, pH of the reaction, and the presence of sulfhydryl reducing agents. Novikoff beta-polymerase activity is unaffected by the presence of 2-mercaptoethanol (2-Me) or dithiothreitol (DTT); however, the combination of 2-mercaptoethanol and NEM or pHMB acts to reverse the inhibition of the sulfhydryl blocking agent. The reversal of inhibition involves more than just a titration of NEM with 2-mercaptoethanol since a) the combination of these two reagents actually stimulates the DNA polymerase, and b) dithiothreitol did not reverse the inhibition. Binding of the polymerase to DNA did not affect the enzyme sensitivity to NEM.     

Functional chloride channels by mammalian cell expression of rat glycine receptor subunit

Cultured human cells were transfected with cloned rat glycine receptor (GlyR) 48 kd subunit cDNA. In these cells glycine elicited large chloride currents (up to 1.5 nA), which were blocked by nanomolar concentrations of strychnine. However, no corresponding high-affinity binding of [3H]strychnine was detected in membrane preparations of the transfected cells. Analysis by monoclonal antibodies specific for the 48 kd subunit revealed high expression levels of this membrane protein. After solubilization, the 48 kd subunit behaved as a macromolecular complex when analyzed by sucrose density centrifugation. Approximately 50% of the solubilized complex bound specifically to a 2-aminostrychnine affinity column, indicating the existence of low-affinity antagonist binding sites on most of the expressed GlyR protein. Thus, the 48 kd strychnine binding subunit efficiently assembles into high molecular weight complexes, resembling the native spinal cord GlyR. However, formation of functional receptor channels of high affinity for strychnine occurs with low efficiency.     

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.     

Arginine and cysteine residues in the ligand binding site of alpha 2-adrenergic receptors

alpha 2-Adrenergic receptors were identified in calf brain, human platelet and human uterus membranes by [3H]-rauwolscine binding. The reagents phenylglyoxal (selective for guanidino groups), p- hydroxy mercuribenzoate and N-ethylmaleimide (selective for sulfhydryl groups) caused a time- and dose- dependent decrease in the number of receptor sites. alpha 2-Adrenergic agonists and antagonists mediated efficient protection of the receptors against these reagents. These data suggest that essential arginine and cysteine residues are present at or near the alpha 2-adrenergic binding site.     

A single amino acid exchange alters the pharmacology of neonatal rat glycine receptor subunit

Agonist activation of the inhibitory glycine receptor (GlyR) in the adult vertebrate CNS is efficiently antagonized by the alkaloid strychnine. Here, we describe a novel rat GlyR alpha subunit cDNA (alpha 2*) that generates chloride channels of low strychnine sensitivity upon expression in Xenopus oocytes. Comparison with the highly homologous human alpha 2 polypeptide and site-directed mutagenesis identified a single amino acid exchange at position 167 that causes the altered pharmacology of alpha 2* receptors. Amplification by the polymerase chain reaction revealed a strong decrease in alpha 2* mRNA abundancy during postnatal spinal cord development. These data indicate that alpha 2* represents a ligand binding subunit of the previously identified neonatal GlyR isoform of low strychnine affinity.     

Effects of sulfhydryl reagents and other inhibitors on Ca2+ transport and inositol trisphosphate-induced Ca2+ release from human platelet membranes

The effects of modifiers of Ca2+ uptake and release in sarcoplasmic reticulum were studied in human platelet membranes. AgNO3,p-chloromercuribenzoate (pClHgBzO), N-ethylmaleimide (MalNEt), quercetin, vanadate, A23187, and caffeine all had the same effects on Ca2+ uptake in platelet membranes as had been observed for sarcoplasmic reticulum. These results strengthen our earlier conclusion that the Ca2+-pump proteins from internal human platelet membranes and muscle sarcoplasmic reticulum are very similar in functional properties. The sulfhydryl reagents Ag+ and pClHgBzO elicited rapid release of Ca2+ from platelet membranes in the presence of ATP, whereas MalNEt induced slow release. Quercetin also caused slow release of Ca2+ from platelet membranes in the presence of ATP. The effects of all three sulfhydryl reagents could be reversed by dithiothreitol, and Ag+-induced release was also reversed by ruthenium red. These effects are similar to those observed in sarcoplasmic reticulum, but in contrast caffeine did not induce Ca2+ release. In the absence of ATP, passively loaded platelet membranes did not release Ca2+ when exposed to sulfhydryl reagents. However, AgCl and pClHgBzO inhibited inositol trisphosphate (InsP3)-induced Ca2+ release from platelet membranes and this effect was reversed by dithiothreitol. Ruthenium red also inhibited InsP3-induced release, but ATP was found not to be required for InsP3-mediated release. LiCl enhanced Ca2+ release from platelet membranes. These results demonstrate that the InsP3-gated Ca2+ release channel is a separate entity from the Ca2+-pump and that essential protein sulfhydryls are involved in the release process.     

Postnatal Development of Synaptic Glycine Receptors in Normal and Hyperglycinemic Rats

The postnatal development of glycine synaptic receptors has been studied. Strychnine binding to the synaptic membrane fraction is very low at birth, increases thereafter, and reaches adult values at the 15th day in the brain, and at the 30th day in the spinal cord. Throughout postnatal development, there are more glycine receptors in the spinal cord than in the brain. The development of receptors in the spinal cord displays a pattern similar to that reported previously for the glycine reuptake system in spinal cord slices and in the activity of spinal cord glycine synthase. In rats with experimental hyperglycinemia strychnine binding to spinal cord glycine receptors increases much more rapidly, reaching a level 1.5 times the control value by day 10. When the hyperglycinemia was induced after the 10th postnatal day, however, no effect on the glycine receptors was observed. This increased number of receptors could be explained by an effect of glycine on the synaptic stabilisation process. No changes in the KD for strychnine were observed either during postnatal development or in hyperglycinemic rats. The KD remained approximately 10 nM in the spinal cord and 50 nM in the brain. Results are discussed with respect to the ontogeny of glycinergic synapses and the pathogenesis of nonketotic hyperglycinemia.     

Conserved high affinity ligand binding and membrane association in the native and refolded extracellular domain of the human glycine receptor alpha1-subunit

The strychnine-sensitive glycine receptor (GlyR) is a ligand-gated chloride channel composed of ligand binding alpha- and gephyrin anchoring beta-subunits. To identify the secondary and quaternary structures of extramembraneous receptor domains, the N-terminal extracellular domain (alpha1-(1-219)) and the large intracellular TM3-4 loop (alpha1-(309-392)) of the human GlyR alpha1-subunit were individually expressed in HEK293 cells and in Escherichia coli. The extracellular domain obtained from E. coli expression was purified in its denatured form and refolding conditions were established. Circular dichroism and Fourier-transform-infrared spectroscopy suggested approximately 25% alpha-helix and approximately 48% beta-sheet for the extracellular domain, while no alpha-helices were detectable for the TM3-4 loop. Size exclusion chromatography and sucrose density centrifugation indicated that isolated glycine receptor domains assembled into multimers of distinct molecular weight. For the extracellular domain from E. coli, we found an apparent molecular weight compatible with a 15mer by gel filtration. The N-terminal domain from HEK293 cells, analyzed by sucrose gradient centrifugation, showed a bimodal distribution, suggesting oligomerization of approximately 5 and 15 subunits. Likewise, for the intracellular domain from E. coli, a single molecular mass peak of approximately 49 kDa indicated oligomerization in a defined native structure. As shown by [(3)H]strychnine binding, expression in HEK293 cells and refolding of the isolated extracellular domain reconstituted high affinity antagonist binding. Cell fractionation, alkaline extraction experiments, and immunocytochemistry showed a tight plasma membrane association of the isolated GlyR N-terminal protein. These findings indicate that distinct functional characteristics of the full-length GlyR are retained in the isolated N-terminal domain.     

The unique extracellular disulfide loop of the glycine receptor is a principal ligand binding element.

A loop structure, formed by the putative disulfide bridging of Cys198 and Cys209, is a principal element of the ligand binding site in the glycine receptor (GlyR). Disruption of the loop's tertiary structure by Ser mutations of these Cys residues either prevented receptor assembly on the cell surface, or created receptors unable to be activated by agonists or to bind the competitive antagonist, strychnine. Mutation of residues Lys200, Tyr202 and Thr204 within this loop reduced agonist binding and channel activation sensitivities by up to 55-, 520- and 190-fold, respectively, without altering maximal current sizes, and mutations of Lys200 and Tyr202 abolished strychnine binding to the receptor. Removal of the hydroxyl moiety from Tyr202 by mutation to Phe profoundly reduced agonist sensitivity, whilst removal of the benzene ring abolished strychnine binding, thus demonstrating that Tyr202 is crucial for both agonist and antagonist binding to the GlyR. Tyr202 also influences receptor assembly on the cell surface, with only large chain substitutions (Phe, Leu and Arg, but not Thr, Ser and Ala) forming functional receptors. Our data demonstrate the presence of a second ligand binding site in the GlyR, consistent with the three-loop model of ligand binding to the ligand-gated ion channel superfamily.