Constructing inhibitory synapses

Control of nerve-cell excitability is crucial for normal brain function. Two main groups of inhibitory neurotransmitter receptors--GABA(A) and glycine receptors--fulfil a significant part of this role. To mediate fast synaptic inhibition effectively, these receptors need to be localized and affixed opposite nerve terminals that release the appropriate neurotransmitter at multiple sites on postsynaptic neurons. But for this to occur, neurons require intracellular anchoring molecules, as well as mechanisms that ensure the efficient turnover and transport of mature, functional inhibitory synaptic receptor proteins. This review describes the dynamic regulation of synaptic GABA(A) and glycine receptors and discusses recent advances in this rapidly evolving field.     

A role for glycine in the gating of plant NMDA-like receptors

The amino acid glycine has a well-established role in signalling in the mammalian central nervous system. For example, glycine acts synergistically with the major excitatory neurotransmitter, glutamate, to regulate the influx of ions such as calcium, through N-methyl-d-aspartate (NMDA) receptors. Plants possess NMDA-like receptors, generically referred to as glutamate receptors (GLRs), named on the basis of their presumed ligand, glutamate. Previously, glycine has not been implicated in plant GLR activity or any other aspect of plant signalling. Using transgenic Arabidopsis seedlings expressing aequorin to monitor ligand-mediated changes in the cytosolic concentration of Ca2+ ([Ca2+]cyt), the data presented herein show that glutamate and glycine act synergistically to control ligand-mediated gating of calcium in plants. Glutamate and glycine synergism also regulates hypocotyl elongation. Transient increases in [Ca2+]cyt mediated by glutamate and glycine, as well as hypocotyl elongation, were inhibited by 6,7-dinitroquinoxaline-2,3 dione (DNQX), a competitive inhibitor of animal GLRs. Using a multiscale docking algorithm in combination with a molecular model of the ligand-binding domain of plant GLRs, evidence is provided indicating that glycine, and not glutamate, is likely to be the natural ligand for most plant GLR subunits. These findings uncover a hitherto unconsidered role for glycine signalling in plants, and suggest that the synergistic action of glutamate and glycine at NMDA-like receptors predates the divergence of plants and animals.     

Synthesis and photochemistry of photolabile N-glycine derivatives and effects of one on the glycine receptor.

Three photolabile precursors of glycine containing a photosensitive 2-nitrobenzyl moiety attached to the amino group have been synthesized. When exposed to ultraviolet radiation between 308 and 350 nm, the compounds photolyze to release glycine, an important inhibitory neurotransmitter in the central nervous system. The identification of glycine as a photolysis product was determined by two different methods: separation of the photolyzed sample by thin-layer chromatography followed by a reaction with ninhydrin, and recognition of derivatized glycine using the Waters pico-tag method in conjunction with high-performance liquid chromatography. The photolysis of these compounds at 22 degrees C has been investigated, and the rate of decay of a transient intermediate in the reaction, which is assumed to reflect product release, has been measured. For N-(alpha-carboxy-2-nitrobenzyl)glycine this decay rate was found to be 940 s-1 at pH 6.8 and 600 s-1 at pH 7.5. Additionally, this compound was found to exhibit biological activity upon photolysis; cultured mouse spinal cord cells containing neuronal glycine receptors were used to detect the glycine liberation. The approach adopted here is useful in demonstrating the utility of photolabile precursors of neurotransmitters that have the protecting group linked to the neurotransmitter through the amino group. The rapid photolysis of such compounds to release free neurotransmitter is valuable in gaining access to chemical kinetic studies of neurotransmitter receptors. Previously, such studies have been limited because the available methods for neurotransmitter delivery did not give a sufficiently high time resolution.     

Tyrosine-1290 of tetanus neurotoxin plays a key role in its binding to gangliosides and functional binding to neurones

Tetanus toxin acts by blocking the release of glycine from inhibitory neurones within the spinal cord. An initial stage in the toxin's action is binding to acceptors on the nerve surface and polysialogangliosides are a component of these acceptor moieties. Using site-directed mutagenesis, we identify tyrosine-1290 of tetanus toxin as a key residue that is involved in ganglioside binding. This residue, which is located at the centre of a shallow pocket on the beta-trefoil domain of the tetanus H(c) fragment, is also shown to play a key role in the functional binding of tetanus toxin to spinal cord neurones leading to the inhibition of neurotransmitter release.     

Characterization of newly cloned variant of rat glycine receptor alpha1 subunit

Responses to glycine, a major inhibitory neurotransmitter within the nervous system, are mediated by glycine receptors (GlyRs). Here, we report the cloning and analysis of a novel splicing variant of the GlyRalpha1 subunit. This variant, named GlyRalpha1del, has a truncated cytoplasmic region between transmembrane domains (TM)3 and TM4, and compared to other variants, the truncation is contributed by a different acceptor site in exon 9. We transfected GlyRalpha1 or GlyRalpha1del into HEK293 cells, and then examined the glycine-activated currents using a whole-cell patch-clamp recording technique. Maximal currents and current-voltage relationships showed no clear difference between GlyRalpha1del and GlyRalpha1. Moreover, dose-response curves indicated that the EC50 values for glycine differed significantly between the two GlyRalpha1 derivatives, although their Hill coefficients were similar. When present with other isoforms, GlyRalpha1del might alter the response to glycine or to other agonists, as this variant expands the potential heterogeneity among glycine receptors.     

Phosphorylation of ligand-gated ion channels: a possible mode of synaptic plasticity.

Most neurotransmitter receptors examined to date have been shown either to be regulated by protein phosphorylation or to contain consensus sequences for phosphorylation by protein kinases. Neurotransmitter receptors that mediate rapid synaptic transmission in the nervous system are the ligand-gated ion channels and include the nicotinic acetylcholine receptors of muscle and nerve and the excitatory and inhibitory amino acid receptors: the glutamate, GABAA, and glycine receptors. These receptors are multimeric proteins composed of homologous subunits which each span the membrane several times and contain a large intracellular loop that is a mosaic of consensus sites for protein phosphorylation. Recent evidence has suggested that extracellular signals released from the presynaptic neuron, such as neurotransmitters and neuropeptides as well as an extracellular matrix protein, regulate the phosphorylation of ligand-gated ion channels. The functional effects of phosphorylation are varied and include the regulation of receptor desensitization rate, subunit assembly, and receptor aggregation at the synapse. These results suggest that phosphorylation of neurotransmitter receptors represents a major mechanism in the regulation of their function and may play an important role in synaptic plasticity.     

Distribution and colocalization of neurotransmitters and receptors in the pre-B?tzinger complex of rats.

The pre-B?tzinger complex (PBC), thought to be the center of respiratory rhythm generation, is a cell column ventrolateral to the nucleus ambiguus. The present study analyzed its cellular and neurochemical composition in adult rats. PBC neurons were mainly oval, fusiform, or multipolar in shape and small to medium in size. Neurokinin-1 receptor, a marker of the PBC, was present in the plasma membrane of mostly medium and small neurons and their associated processes and boutons. Among neurons immunoreactive for different neurotransmitter or receptor candidates, various numbers were colocalized with neurokinin-1 receptor. The highest ratio was with nitric oxide synthase (52.72%), and the lowest was with glycine receptors (31.93%). Glutamic acid decarboxylase- and glycine transporter 2-immunoreactive boutons, as well as GABA(A) receptor-immunoreactive plasma membrane processes and boutons, were also identified in the PBC. PBC neurons exhibited different levels of cytochrome oxidase activity, indicating their various energy demands. Our results suggest that synaptic interactions within the PBC of adult rats involve a variety of neurotransmitter and receptor types and that nitric oxide may play an important role in addition to glutamate, GABA, glycine, and neurokinin.     

Glycine in the Central Nervous System in Experimental Allergic Encephalomyelitis

: The content of glycine, a possibly inhibitory neurotransmitter was studied in central nervous system of guinea pigs with experimental allergic encephalomyelitis (EAE). The glycine level was increased in spinal cord, but not in the brain of animals with EAE. The greatest increase in glycine concentration was in lumbosacral cord, and at the time of appearance of clinical signs of disease. The results are discussed in terms of possible connection between the changes of glycine concentration and clinical signs of EAE.     

Anesthetic and ethanol effects on spontaneously opening glycine receptor channels

Strychnine-sensitive glycine receptors mediate inhibitory neurotransmission occurring in the brain stem and spinal cord. Alcohols, volatile anesthetics and inhaled drugs of abuse are positive allosteric modulators of glycine receptor function, normally enhancing function only in the presence of glycine. A complication in studying allosteric actions on ligand-gated ion channels is in the dissection of their effects on neurotransmitter binding from their effects on channel opening. Mutation of an aspartate residue at position 97 to arginine in the glycine receptor alpha1 subunit simulated the effects of glycine binding, producing receptors that exhibited tonic channel opening in the absence of neurotransmitter; i.e. these receptors demonstrated a dissociation of channel opening from neurotransmitter binding. In these receptors, ethanol, enflurane, chloroform, halothane, 1,1,1-trichloroethane and toluene elicited inward currents in the absence of glycine. We previously identified mutations on ligand-gated ion channels that eliminate ethanol, anesthetic and inhalant actions (such as S267I on alpha1 glycine receptors). The double mutant (D97R and S267I) receptors were both constitutively active and resistant to the enhancing effects of ethanol and enflurane. These data demonstrate that ethanol and volatile anesthetics can affect glycine receptor channel opening independently of their effects on enhancing neurotransmitter binding.     

Differential Effects of Tetanus Toxin on Inhibitory and Excitatory Neurotransmitter Release from Mammalian Spinal Cord Cells in Culture

The effect of tetanus toxin on depolarization-evoked and spontaneous synaptic release of inhibitory and excitatory neurotransmitters was examined in murine spinal cord cell cultures. Toxin action on the release of radiolabeled glycine and glutamate was followed over time intervals corresponding to the early phase of convulsant activity through the later phase of electrical quiescence. Tetanus toxin inhibited potassium-evoked release of [3H]glycine and [3H]glutamate in a time- and dose-dependent manner. Ninety minutes after the application of toxin (6 x 10(-10) M), the stimulated release of [3H]glycine was blocked completely, whereas stimulated release of [3H]glutamate was not blocked completely until 150-210 min after toxin application. Fragment C, the binding portion of the tetanus toxin molecule, had no effect on stimulated release of either transmitter. The spontaneous synaptic release of [3H]glycine was blocked totally within 90 min of toxin exposure. In contrast, the spontaneous release of [3H]glutamate, in toxin-exposed cultures, was elevated to nearly twice that of control cultures at this time. Thus, toxin-induced convulsant activity is characterized by a reduction in the spontaneous synaptic release of inhibitory neurotransmitter with a concomitant increase in the release of excitatory neurotransmitter, as well as the more rapid onset of blockade of depolarization-evoked release of inhibitory versus excitatory neurotransmitter.     

Amino acid and putative neurotransmitter transport in human Y79 retinoblastoma cells. Effect of insulin and insulin-like growth factor

The binding of insulin and insulin-like growth factor I (IGF-I) and their effect on amino acid and neurotransmitter transport was studied in cultured human Y79 retinoblastoma cells. Y79 cells possess specific receptors for both insulin and IGF-I. Insulin binding to Y79 cells is characterized by a curvilinear Scatchard plot suggesting a two-site or two-affinity binding system. In contrast, IGF-I binding has a linear plot indicative of a one-site, one-affinity binding system. The uptake of glycine, a putative neurotransmitter in the retina occurs by a specific transport system in Y79 cells, independent of the uptake of other neutral amino acids. The uptake of glycine was increased 25-50% by either insulin or IGF-I. The response to insulin or IGF-I on glycine uptake is gradual and concentration dependent. The accumulation of other amino acids and putative retinal neurotransmitters by Y79 cells was not significantly affected by insulin of IGF-I. In addition, the activity of Na+/K+-ATPase was not influenced. The analysis of high affinity glycine uptake indicates that insulin and IGF-I are stimulating glycine transport by increasing the V'max without significantly affecting the K'm. Further analysis suggests that insulin and IGF-I are causing a recruitment of additional glycine transporters at the cell surface or activating otherwise nonfunctional transporters by an unexplained mechanism. Because of the implication that glycine responds as a neuroactive amino acid in Y79 cells these studies suggest that insulin and IGF-I may influence neuroactivity in the human retina by regulating the transport of glycine.     

Molecular basis for substrate discrimination by glycine transporters

Glycine is an inhibitory neurotransmitter in the spinal cord and brain stem, where it acts on strychnine-sensitive glycine receptors, and is also an excitatory neurotransmitter throughout the brain and spinal cord, where it acts on the N-methyl-d-aspartate family of receptors. There are two Na(+)/Cl(-)-dependent glycine transporters, GLYT1 and GLYT2, which control extracellular glycine concentrations and these transporters show differences in substrate selectivity and blocker sensitivity. A bacterial Na(+)-dependent leucine transporter (LeuT(Aa)) has recently been crystallized and its structure determined. When the amino acid residues within the leucine binding site of LeuT(Aa) are aligned with residues of the two glycine transporters there are a number of identical residues and also some key differences. In this report, we demonstrate that the LeuT(Aa) structure represents a good working model of the Na(+)/Cl(-)-dependent neurotransmitters and that differences in substrate selectivity can be attributed to a single difference of a glycine residue in transmembrane domain 6 of GLYT1 for a serine residue at the corresponding position of GLYT2.     

Transmembrane domain III plays an important role in ion binding and permeation in the glycine transporter GLYT2

The neuronal glycine transporter GLYT2 takes up glycine from the extracellular space by an electrogenic process where this neurotransmitter is co-transported with sodium and chloride ions. We report in this paper that tyrosine at position 289 of GLYT2a is crucial for ion coupling, glycine affinity and sodium selectivity, stressing the essential role played by this residue of transmembrane domain III in the mechanism of transport. Substitution to tryptophan (Y289W), phenylalanine (Y289F), or serine (Y289S), renders transporters unable to catalyze glycine uptake. Measurements of glycine evoked steady-state currents in transfected HEK-293 cells reveal EC(50) values for glycine 17-fold (Y289F) and 45-fold (Y289S) higher than that of the wild type transporter. Sodium dependence is severely altered in tyrosine 289 mutants, both at the level of apparent affinity and cooperativity, with the more dramatic change corresponding to the less conservative substitution (Y289S). Accordingly, sodium selectivity is gradually lost in Y289F and Y289S mutants, and chloride dependence of glycine evoked currents is markedly decreased in Y289F and Y289S mutants. In the absence of three-dimensional information from these transporters, these results provide experimental evidence supporting the hypothesis of transmembrane domain III being part of a common permeation pathway for substrate and co-transported ions.     

Levels,uptake, and release of glycine and glutamate in the rat pontine reticular formation

In this work we have determined the levels of glycine, glutamate, and other amino acids in the rat pontine reticular formation (PRF), in addition to some properties of the uptake and release of labeled glycine and glutamate in slices of this region. Glutamate was the most concentrated amino acid in the PRF, although its content was about half that of the striatum. Surprisingly, glycine levels in the PRF were 3.2-fold higher than in the striatum, whereas GABA content was similar in both regions. The uptake of both glycine and glutamate by PRF slices was strictly Na⁺-dependent. Their release was stimulated by K⁺-depolarization, but only the release of glycine was Ca²⁺-dependent. These findings suggest that glycine is a strong candidate for a neurotransmitter role in the PRF and that glutamate might also play such a role in this region.Special issue dedicated to Dr. Morris H. Aprison     

gamma-Aminobutyric acid-containing terminals can be apposed to glycine receptors at central synapses

The distributions of terminals containing gamma-aminobutyric acid (GABA) and of endings apposed to glycine receptors were investigated cytochemically in the ventral horn of the rat spinal cord. For this purpose, a polyclonal antibody raised to recognize glutamic acid decarboxylase (GAD), a synthetic enzyme for GABA, and three monoclonal antibodies (mAb's) directed against the glycine receptor were used. Double immunofluorescence showed that, surprisingly, GAD-positive terminals are closely associated in this system with glycine receptors at all the investigated cells, most of which were spinal motoneurons. Furthermore, double labeling was performed with immunoenzymatic recognition of GAD and indirect marking of mAb's with colloidal gold. With this combined approach, it was found, at the electron microscopic level, that all GAD-positive terminals are in direct apposition with glycine receptors while, on the other hand, not all glycine receptors are in front of GABA-containing boutons. This result is not due to a cross-reactivity of mAb's with GABA receptors as shown by using as a control synapses known to use GABA as a neurotransmitter in the cerebellar cortex. Indeed, no glycine receptor immunoreactivity was detected on Purkinje cells facing basket axon terminals. However, Purkinje neurons can express glycine receptor immunoreactivity at other synaptic contacts. Assuming that the presence of postsynaptic receptors for glycine indicates that this amino acid is used for neurotransmission at a given synapse, our results strongly support the notion that GABA and glycine, two classical inhibitory transmitters, coexist at some central connections. However, such is not always the case; in the cerebellum, Golgi terminals impinging on the dendrites of granule cells are either GAD-positive or face glycine receptors, in a well-segregated manner.     

Coumarin-caged glycine that can be photolyzed within 3 microseconds by visible light

The synthesis and characterization of a new photolabile precursor of glycine (coumarin-caged glycine) are reported. The new compound is suitable for rapid chemical kinetic investigations of the membrane-bound neurotransmitter receptor activated by glycine. Unlike previously used caging groups for glycine, this precursor can be photolyzed rapidly and efficiently in the visible wavelength region. This allows the use of a relatively inexpensive light source. The alpha-carboxyl group of glycine was covalently coupled to the 7-(diethylamino)coumarin (DECM) caging group. The caged compound has a major absorption band with a maximum at 390 nm (epsilon390 = 13,900 M-1 cm-1). Photolysis was performed at wavelengths of >or=400 nm (epsilon400 = 12,400 M-1 cm-1). Under physiological conditions, DECM-caged glycine is water soluble and stable. In the visible wavelength region, it photolyzes rapidly to release glycine with a half-life of approximately 2.5 micrometers and a quantum yield of 0.12 +/- 0.01. The experimental results demonstrated that neither DECM-caged glycine nor its byproduct inhibits or activates human alpha1 glycine receptors expressed on the surface of HEK 293 cells.     

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.     

Why glycine transporters have different stoichiometries

In the brain, neurons and glial cells compete for the uptake of the fast neurotransmitters, glutamate, GABA and glycine, through specific transporters. The relative contributions of glia and neurons to the neurotransmitter uptake depend on the kinetic properties, thermodynamic coupling and density of transporters but also on the intracellular metabolization or sequestration of the neurotransmitter. In the case of glycine, which is both an inhibitory transmitter and a neuromodulator of the excitatory glutamatergic transmission as a co-agonist of N-methyl D-aspartate receptors, the glial (GlyT1b) and neuronal (GlyT2a) transporters differ at least in three aspects: (i) stoichiometries, (ii) reverse uptake capabilities and (iii) pre-steady-state kinetics. A 3 Na(+)/1 Cl(-)/gly stoichiometry was established for GlyT2a on the basis of a 2 charges/glycine flux ratio and changes in the reversal potential of the transporter current as a function of the extracellular glycine, Na(+) and Cl(-) concentrations. Therefore, the driving force available for glycine uphill transport in neurons is about two orders of magnitude larger than for glial cells. In addition, GlyT2a shows a severe limitation for reverse uptake, which suggests an essential role of GlyT2a in maintaining a high intracellular glycine pool, thus facilitating the refilling of synaptic vesicles by the low affinity, low specificity vesicular transporter VGAT/VIAAT. In contrast, the 2 Na(+)/1 Cl(-)/gly stoichiometry and bi-directional transport properties of GlyT1b are appropriate for the control of the extracellular glycine concentration in a submicromolar range that can modulate N-methyl D-aspartate receptors effectively. Finally, analysis of the pre-steady-state kinetics of GlyT1b and GlyT2a revealed that at the resting potential neuronal transporters are preferentially oriented outward, ready to bind glycine, which suggests a kinetic advantage in the uptake contest.     

Glycine neurotransmitter transporters: an update

Glycine accomplishes several functions as a transmitter in the central nervous system (CNS). As an inhibitory neurotransmitter, it participates in the processing of motor and sensory information that permits movement, vision, and audition. This action of glycine is mediated by the strychnine-sensitive glycine receptor, whose activation produces inhibitory post-synaptic potentials. In some areas of the CNS, glycine seems to be co-released with GABA, the main inhibitory amino acid neurotransmitter. In addition, glycine modulates excitatory neurotransmission by potentiating the action of glutamate at N-methyl-D-aspartate (NMDA) receptors. It is believed that the termination of the different synaptic actions of glycine is produced by rapid re-uptake through two sodium-and-chloride-coupled transporters, GLYT1 and GLYT2, located in the plasma membrane of glial cells or pre-synaptic terminals, respectively. Glycine transporters may become major targets for therapeutic of pathological alterations in synaptic function. This article reviews recent progress on the study of the molecular heterogeneity, localization, function, structure, regulation and pharmacology of the glycine transporter proteins.     

Glycine neurotransmitter transporters: an update

Glycine accomplishes several functions as a transmitter in the central nervous system(CNS). As an inhibitory neurotransmitter, it participates in the processing of motor and sensory information that permits movement, vision, and audition. This action of glycine is mediated by the strychnine-sensitive glycine receptor, whose activation produces inhibitory post-synaptic potentials. In some areas of the CNS, glycine seems to be co-released with GABA, the main inhibitory amino acid neurotransmitter. In addition, glycine modulates excitatory neurotransmission by potentiating the action of glutamate at N-methyl-D-aspartate (NMDA) receptors. It is believed that the termination of the different synaptic actions of glycine is produced by rapid reuptake through two sodium-and-chloride-coupled transporters, GLYT1 and GLYT2, located in the plasma membrane of glial cells or pre-synaptic terminals, respectively. Glycine transporters may become major targets for therapeutic of pathological alterations in synaptic function. This article reviews recent progress on the study of the molecular heterogeneity, localization, function, structure, regulation and pharmacology of the glycine transporter