Differential effects of metal ligands on synaptic membrane glutamate binding and uptake systems

The high affinity, Na⁺-independentl-[³H]glutamate binding process in synaptic membranes and in the purified binding protein was shown to be inhibited to an almost equal extent by the metal ligands NaN₃, KCN, ando-phenanthroline, and by 2,4,5-trihydroxyphenylalanine (6-OH DOPA). The high affinity, Na⁺-dependent glutamate transport activity in these membranes was almost totally insensitive to NaN₃,o-phenanthroline, KCN, and 6-OH DOPA. These agents, especially 6-OH DOPA, may be useful tools in achieving a discrimination between putative physiologic receptors and uptake carrier sites forl-glutamate in synaptic membranes. The sensitivity of the glutamate binding sites to the effects of the metal ligands may be correlated to the presence of an iron-sulfur center in the purified glutamate binding protein. Some of the characteristics of this metallic center were explored by optical and paramagnetic resonance spectroscopic techniques and are described in this study.This research was supported by grants DAAG29-79-C-0156 from the Army Research Office and AA 04732 from NIAAA.     

Effects of amphipathic drugs onl-[3H]glutamate binding to synaptic membranes and the purified binding protein

Four amphipathic molecules with known local anesthetic activity, dibucaine, tetracaine, chlorpomazine, and quinacrine, inhibited the binding ofl-[³H]glutamic acid to rat brain synaptic plasma membranes and to the purified glutamate binding protein. Neither haloperidol nor diphenylhydantoin had significant inhibitory effects on the glutamate binding activity of the membranes or of the purified protein. The amphipathic drugs apparently inhibitedl-[³H]glutamate binding to synaptic membranes by a mixed type of inhibition. The inhibitory activity of quinacrine on glutamate binding to the synaptic membranes was greater in a low ionic strength, Ca²⁺-free buffer medium, than in a physiologic medium (Krebs-Henseleit buffer). Removal of Ca²⁺ from the Krebs solution enhanced quinacrine's inhibition of glutamate binding. Quinacrine up to 1 mM concentration did not inhibit the high affinity Na⁺-dependentl-glutamate transport in these membrane preparations. The importance of Ca²⁺ in the expression of quinacrine's effects on the glutamate binding activity of synaptic membranes and the observed tetracaine and chlorpromazine-induced increases in the transition temperature for the glutamate binding process of these membranes, were indicative of an interaction of the local anesthetics with the lipid environment of the glutamate binding sites.     

High-affinity binding ofl-glutamate to chick retinal membranes

Binding ofl-[³H]glutamate to membranes from whole chick retina and from subcellular fractions enriched with photoreceptor terminals (P₁), or terminals from the inner plexiform layer (P₂) was studied. Na⁺-dependent and Na⁺-independent binding to these membranes was demonstrated. Na⁺-independent binding was stereospecific. Kinetic analysis of the binding process indicated a single high-affinity system (K _B=0.55 M) with a capacity of approximately 20 pmoles/mg protein in all the membrane fractions. [³H]Glutamate binding to P₁ and P₂ fractions was effectively displaced by several structural analogues of glutamate. Glutamate diethyl-ester appreciably displaced binding, whereas kainic acid did not displace bound glutamate. Data indicate the binding of [³H]glutamate to physiologically relevant receptors in the chick retina.     

Age and Brain Structural Related Effects of Glutaric and 3-Hydroxyglutaric Acids on Glutamate Binding to Plasma Membranes During Rat Brain Development

(1) In the present study we determined the effects of glutaric (GA, 0.01–1 mM) and 3-hydroxyglutaric (3-OHGA, 1.0–100 μM) acids, the major metabolites accumulating in glutaric acidemia type I (GA I), on Na⁺-independent and Na⁺-dependent [³H]glutamate binding to synaptic plasma membranes from cerebral cortex and striatum of rats aged 7, 15 and 60 days. (2) GA selectively inhibited Na⁺-independent [³H]glutamate binding (binding to receptors) in cerebral cortex and striatum of rats aged 7 and 15 days, but not aged 60 days. In contrast, GA did not alter Na⁺-dependent glutamate binding (binding to transporters) to synaptic membranes from brain structures of rats at all studied ages. Furthermore, experiments using the glutamatergic antagonist CNQX indicated that GA probably binds to non-NMDA receptors. In addition, GA markedly inhibited [³H]kainate binding to synaptic plasma membranes in cerebral cortex of 15-day-old rats, indicating that this effect was probably directed towards kainate receptors. On the other hand, experiments performed with 3-OHGA revealed that this organic acid did not change Na⁺-independent [³H]glutamate binding to synaptic membranes from cerebral cortex and striatum of rats from all ages, but inhibited Na⁺-dependent [³H]glutamate binding to membranes in striatum of 7-day-old rats, but not in striatum of 15- and 60-day-old rats and in cerebral cortex of rats from all studied ages. We also provided some evidence that 3-OHGA competes with the glutamate transporter inhibitor L-trans-pyrrolidine-2,4-dicarboxylate, suggesting a possible interaction of 3-OHGA with glutamate transporters on synaptic membranes. (3) These results indicate that glutamate binding to receptors and transporters can be inhibited by GA and 3-OHGA in cerebral cortex and striatum in a developmentally regulated manner. It is postulated that a disturbance of glutamatergic neurotransmission caused by the major metabolites accumulating in GA I at early development may possibly explain, at least in part, the window of vulnerability of striatum and cerebral cortex to injury in patients affected by this disorder.     

Analysis of glutamate receptors in primary cultured neurons from fetal rat forebrain

In order to further analyze the development of glutamatergic pathways in neuronal cells, the expression of excitatory amino acid receptors was studied in a model of neurons in primary culture by measuring the specific binding of L-[³H]glutamate under various incubation conditions in 8-day-old intact living neurons isolated from the embryonic rat forebrain, as well as in membrane preparations from these cultures and from newborn rat forebrain. In addition, the receptor responsiveness to glutamate was assessed by studying the uptake of tetraphenylphosphonium (TPP⁺) which reflects membrane polarization. In the presence of a potent inhibitor of glutamate uptake, the radioligand bound to a total number of sites of 36.7 pmol/mg protein in intact cells incubated in a Tris buffer containing Na⁺, Ca²⁺, and Cl^–, with a Kd around 2 M. In the absence of the above ions, [³H]glutamate specific binding diminished to 14.2 pmol/mg protein with a Kd-value of 550 nM. Under both of the above conditions, similar Kd were obtained in membranes isolated from cultures and from the newborn brain. However, Bmax-values were significantly lower in culture membranes than in intact cells or newborn membranes. Displacement studies showed that NMDA was the most potent compound to inhibit [³H]glutamate binding in membranes obtained from cultured neurons as well as from the newborn brain, whereas quisqualate, AMPA, kainate andtrans-ACPD were equally effective. According to these data and to the ionic dependence of glutamate binding, it was concluded that cultured neurons from the rat embryo forebrain express various glutamate receptor subtypes, mainly L-AP4 and NMDA receptors, with characteristics close to those in the newborn brain, and which display functional properties since a transient cell exposure to glutamate led to a 70% inhibition of [³H]TPP⁺ uptake.     

Purification, Biochemical Characterization, Binding Activity, and Selectivity of a Glutamate Binding Protein from Bovine Brain

A glutamate binding protein was purified from bovine brain to apparent homogeneity. The procedure used for the purification of this protein involved extraction of a crude synaptic membrane fraction with Na-cholate, followed by solubilization of the binding protein from the membranes by Triton X-100, and, finally, affinity batch separation of the protein on L-glutamate-loaded glass fiber. The molecular characteristics of the purified protein were similar to those previously described for the glutamate binding protein from rat brain synaptic membranes and included the following: small Mr (14,000), acidic (pI = 4.7) protein with a single NH2-terminal amino acid (tyrosine), and significant absorption at wave-lengths greater than 300 nm. Complete amino acid analysis of the protein was not achieved, either because of destruction of some amino acids or of incomplete hydrolysis of the protein. The protein bound L-glutamate with high affinity (KD = 0.87 microM), exhibited one class of L-glutamate binding sites, and bound glutamate with a stoichiometry of 0.7 mol ligand/mol protein. The displacement of protein-bound L-glutamic acid by other neuroactive amino acids had characteristics similar to those observed for the displacement of L-glutamate from rat brain synaptic membrane or purified protein binding sites. Finally, the metal ligand formers KCN and NaN3 inhibited the activity of this protein just as they have been shown to do in rat brain synaptic membranes or the purified protein.     

Molecular aspects of glutamate receptors and sodium-calcium exchange carriers in mammalian brain: Implications for neuronal development and degeneration

N-Methyl-d-aspartate (NMDA) andl-glutamate activate membrane receptor that produce substantial permeation of Na⁺, K⁺ and Ca²⁺ through the neuronal membrane. These ionic fluxes are intimately linked to processes that regulate neuronal survival, growth and differentiation. Intracellular free Ca²⁺ concentrations are thought to be particularly important determinants of the vulnerability of neurons to excessive excitatory stimulation produced through activation of NMDA receptors. In order to understand the molecular events involved in both NMDA receptor activation and regulation of intracellular Ca²⁺ levels, we have purified and reconstituted the protein complexes that form the NMDA/glutamate receptors in rat brain synaptic membranes and those that constitute the Na⁺-Ca²⁺ antiporters in bovine brain synaptic membranes. The molecular properties of these protein complexes are described, and information from the most recent studies of exploration of the molecular structures of these receptors and transport carriers is summarized.Special issue dedicated to Dr. Frederick E. Samson     

HIGH AFFINITY l-[3H]GLUTAMATE BINDING TO POSTSYNAPTIC RECEPTOR SITES ON RAT CEREBELLAR MEMBRANES

l [³H]glutamate binding was investigated in membrane preparations derived from rat cerebellum, an area of the brain where it is likely that a high density of postsynaptic glutamate receptors occurs. Glutamate was hound specifically and, in freshly prepared membranes, was optimal under physiological conditions of pH and temperature and was associated with the synaptic membrane fraction of the cell. Specific binding occurred through a single, high-affinity process with a K_D, of 744 nM and a capacity of 73 pmol/mg protein. Unlike the findings reported for GABA, the specific binding of glutamate to fresh membranes did not involve an uptake site. Comparison of the potencies of a wide range of compounds with known pharmacological activities, demonstrated that their ability to displace specific glutamate binding was consistent with specific interactions with glutamate receptors.     

Interference of S-Alkyl Derivatives of Glutathione with Brain Ionotropic Glutamate Receptors

The effects of glutathione, glutathione sulfonate and S-alkyl derivatives of glutathione on the binding of glutamate and selective ligands of ionotropic N-methyl-D-aspartate (NMDA) and non-NMDA receptors were studied with mouse synaptic membranes. The effects of glutathione and its analogues on ⁴⁵Ca²⁺ influx were also estimated in cultured rat cerebellar granule cells. Reduced and oxidized glutathione, glutathione sulfonate, S-methyl-, -ethyl-, -propyl-, -butyl- and -pentylglutathione inhibited the Na⁺-independent binding of L-[³H]glutamate. They strongly inhibited also the binding of (S)-2-amino-3-hydroxy-5-[³H]methyl-4-isoxazolepropionate [³H]AMPA (IC₅₀ values: 0.8–15.9 M). S-Alkylation of glutathione rendered the derivatives unable to inhibit [³H]kainate binding. The NMDA-sensitive binding of L-[³H]glutamate and the binding of 3-[(R)-2-carboxypiperazin-4-yl][1,2-³H]propyl-1-phosphonate ([³H]CPP, a competitive antagonist at NMDA sites) were inhibited by the peptides at micromolar concentrations. The strychnine-insensitive binding of the NMDA coagonist [³H]glycine was attenuated only by oxidized glutathione and glutathione sulfonate. All peptides slightly enhanced the use-dependent binding of [³H]dizocilpine (MK-801) to the NMDA-gated ionophores. This effect was additive with the effect of glycine but not with that of saturating concentrations of glutamate or glutamate plus glycine. The glutamate- and NMDA-evoked influx of ⁴⁵Ca²⁺ into cerebellar granule cells was inhibited by the S-alkyl derivatives of glutathione. We conclude that besides glutathione the endogenous S-methylglutathione and glutathione sulfonate and the synthetic S-alkyl derivatives of glutathione act as ligands of the AMPA and NMDA receptors. In the NMDA receptor-ionophore these glutathione analogues bind preferably to the glutamate recognition site via their -glutamyl moieties.     

Receptor Interactions of β-N-Oxalyl-L-α,β-Diaminopropionic Acid,the Lathyrus sativus Putative Excitotoxin,with Synaptic Membranes

Direct evidence for the excitotoxicity of -N-oxalyl-L-,-diaminopropionic acid (ODAP), the Lathyrus sativus neurotoxin has been studied by examining the binding of chemically synthesized [2,3 ³H]ODAP ([³H]ODAP) to synaptic membranes. [³H]ODAP binding to membranes was mostly nonspecific, with only a very low specific binding (15–20% of the total binding) and was also not saturable. The low specific binding of [³H]ODAP remained unaltered under a variety of assay conditions. A low B_max of 3.2 ± 0.4 pmol/mg and K_d 0.2 ± 0.08 M could be discerned for the high affinity interactions under conditions wherein more than 80–90% of the binding was nonspecific. While ODAP could inhibit the binding of [³H]glutamate to chick synaptic membranes with a K_i of 10 ± 0.9 M, even L-DAP, a non neurotoxic amino acid was also equally effective in inhibiting the binding of [³H]glutamate. The very low specific binding of [³H]ODAP to synaptic membranes thus does not warrant considering its interactions at glutamate receptors as a significant event. The results thus suggest that the reported in vitro excitotoxic potential of ODAP may not reflect its true mechanism of neurotoxicity.     

[3H]Aniracetam Binds to Specific Recognition Sites in Brain Membranes

Abstract: [³H]Aniracetam bound to specific and saturable recognition sites in membranes prepared from discrete regions of rat brain. In crude membrane preparation from rat cerebral cortex, specific binding was Na⁺ independent, was still largely detectable at low temperature (4°C), and underwent rapid dissociation. Scatchard analysis of [³H]aniracetam binding revealed a single population of sites with an apparent K_D value of ～70 nM and a maximal density of 3.5 pmol/mg of protein. Specifically bound [³H]aniracetam was not displaced by various metabolites of aniracetam, nor by other pyrrolidinone-containing nootropic drugs such as piracetam or oxiracetam. Subcellular distribution studies showed that a high percentage of specific [³H]aniracetam binding was present in purified synaptosomes or mitochondria, whereas specific binding was low in the myelin fraction. The possibility that at least some [³H]aniracetam binding sites are associated with glutamate receptors is supported by the evidence that specific binding was abolished when membranes were preincubated at 37°C under fast shaking (a procedure that substantially reduced the amount of glutamate trapped in the membranes) and could be restored after addition of either glutamate or α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) but not kainate. The action of AMPA was antagonized by DNQX, which also reduced specific [³H]aniracetam binding in unwashed membranes. High levels of [³H]aniracetam binding were detected in hippocampal, cortical, or cerebellar membranes, which contain a high density of excitatory amino acid receptors. Although synaptosomal aniracetam binding sites may well be associated with AMPA-sensitive glutamate receptors, specifically bound [³H]aniracetam could not be displaced by cyclothiazide or GYKI 52466, which act as a positive and negative modulator of AMPA receptors, respectively.     

Glutamate receptor binding in insects and mammals

Summary High affinity stereospecific binding sites for L-glutamate have been reported in several regions of mammalian brain. The binding sites in the hippocampus and cerebellum have been studied more extensively than binding in other brain regions. The hippocampal and cerebellar binding sites show similar properties with respect to their pharmacology and their independence of Na⁺. There is evidence, particularly good in the case of hippocampus, of mechanisms that may regulate the availability of the binding sites in both brain areas. Some progress has been made with the isolation of the hippocampal binding site but the protein has not been extensively characterised.In the case of insect muscle, high-affinity stereospecific binding of L-glutamate to whole membrane preparations, to detergent-solubilised membranes and to isolated proteolipids has been reported. Much greater variability in the binding characteristics is seen than is the case with the mammalian brain preparations. Preliminary experiments suggest that at least four distinct binding sites may be present on insect muscle.The complete characterisation of glutamate binding sites is at present precluded by a lack of potent agonists and antagonists. However, recent advances in the pharmacological classification of receptor sites for the excitatory amino acids in mammalian brain could provide sufficient information to permit the identification of the binding sites as synaptic receptors. Invertebrate toxins whose site of action is the insect neuromuscular junction may well prove to be useful tools with which to isolate and characterise the synaptic receptor proteins.     

Na+-independent binding of GABA to the triton X-100 treated synaptic membranes from cerebellum of rat brain

The treatment of the membranes from cerebellum of rat brain with 0.5% Triton X-100 increases both the affinity and the density of the Na⁺-independent binding sites for ³H-GABA (γ-aminobutyric acid) from the values obtained from membranes of rat brain after an extensive freezing and thawing treatment (Young $\text{et al.}$, 1976). Upon repeated washings of the Triton-treated membranes, the binding of ³H-GABA is further increased and follows biphasic kinetics which indicates two binding components having dissociation constants of 5.9 and 27 nM and densities of 1.35 and 3.9 pmole/mg protein, respectively. GABA agonist, imidazoleacetic acid, and the GABA antagonists, bicuculline and d-tubocurarine, inhibit 50% of ³H-GABA binding at 1, 47 and 85 μM concentrations (IC₅₀ values), respectively. The IC₅₀ values for these compounds are unchanged by Na⁺. Thus, the Na⁺-independent binding of ³H-GABA to the Triton-treated membranes may represent binding to the synaptic GABA receptors.     

Monitoring of the mitochondrial and plasma membrane potentials in human fibroblasts by tetraphenylphosphonium ion distribution

The lipophilic cation tetraphenylphosphonium (TPP⁺) is accumulated by human skin fibroblasts across both the plasma and mitochondrial membranes. We show here that TPP⁺ uptake is indeed greatly decreased under conditions leading to de-energization of mitochondria. The TPP⁺ accumulation in the presence of the proton ionophore FCCP has been used for determination of the plasma membrane potential across the plasma membrane, after correction for potential-independent binding of TPP⁺ to cellular components. Following this procedure, a value of 75 mV has been obtained. Through the amount of TPP⁺ released by FCCP treatment, an estimate of thein situ mitochondrial membrane potential has been made. Furthermore, we report that the mitochondrial component of TPP⁺ accumulation decreases with aging of fibroblast cultures.Abbreviations _m membrane potential across thein situ mitochondria - _p membrane potential across the plasma membrane - TPP⁺ tetraphenylphosphonium - HEPES N-2-hydroxyethylpiperazineN-2-ethanesulfonic acid - FCCP carbonyl cyanidep-trifluoromethoxyphenylhydrazone     

gamma-Aminobutyric acid receptors in cerebellar membranes of rat brain after a treatment with Triton X-100.

Abstract— The treatment of cerebellar membranes of rat brain with a low concentration of Triton X-100 followed by sufficient washing results in an increase of the Na⁺-independent binding of [³H]GABA and a total loss of the Na ⁺-dependent binding of [³H]GABA. The Na⁺-independent binding of [³H]GABA was more abundant in membranes of cerebellum than in membranes of other rat brain regions and mainly localized in the synaptic membrane fraction of a cerebellar homogenate. In the Triton-treated membranes, the Na⁺-independent binding of [³H]GABA was a saturable process, which could be resolved into two components, a high and a low affinity component with dissociation constants of 4.5 and 30 nm , respectively. The neurophysiological agonists, muscimol, GABA, and imidazole acetic acid, and the antagonist, bicuculline, inhibited the high affinity Na⁺-independent binding of [³H]GABA by 50% at 0.003, 0.012, 0.3 and 10 μm respectively. These data suggest that the Na⁺-independent binding of [³H]GABA in the Triton-treated cerebellar membranes represents the synaptic receptors of GABA. It is emphasized that extensive washing of the membranes after a Triton treatment is necessary in order to detect the high affinity Na⁺-independent binding of [³H]GABA.     

Involvement of Amino-Acid Side Chains of Membrane Proteins in the Binding of Glutathione to Pig Cerebral Cortical Membranes

Glutathione (GSH), a general antioxidant and detoxifying compound, is the most abundant thiol-containing peptide in the central nervous system. It has been earlier shown to regulate the functions of glutamate receptors and to possess specific binding sites in both neurons and glial cells. The possible involvement of disulfide bonds, cysteinyl, arginyl, lysyl, glutamyl, and aspartyl residues in the binding of tritiated GSH to specific sites in pig cerebral cortical synaptic membranes was now studied after covalent modification of membrane proteins. Treatment of synaptic membranes with the thiol-modifying reagents 5,5-dithio-bis(2-nitrobenzoate) (DTNB) and 4,4-dithiodipyridine (DDP) dramatically enhanced the binding of [³H]GSH in a dose-dependent manner. Dithiothreitol (DTT) alone reduced the binding, but pretreatment of the membranes with DTT potentiated the enhancing effect of DTNB. On the other hand, when the modification with DTNB was followed by treatment with DTT, the enhancement by DTNB was completely reversed. N-ethylmaleimide, a thiol alkylating agent, and phenylisothiocyanate, a thiol- and amino-group modifying compound, reduced the binding, and their effects were additive. The guanidino-modifying agent phenylglyoxal reduced the binding but the carboxyl-modifying reagent 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide had no significant effect. The results indicate that cysteinyl side chains and disulfide bonds are essential in the binding of GSH to membrane proteins and that arginyl and lysyl side chains may also be directly involved in this process.     

Isolation of rat brain subcellular fraction enriched in putative neurotransmitter receptors and synaptic junctions

A simple reliable method was developed for the rapid isolation of a synaptic plasma membrane-enriched fraction from rat brain. The procedure involves the direct lysis of a crude mitochondrial fraction followed by a combined flotation-sedimentation density gradient centrifugation in a fixed-angle centrifuge rotor. All fractions have been characterized with respect to relative enrichment of (Na⁺–K⁺) ATPase activity as well as putative cholinergic neurotransmitter receptors determined by [¹²⁵I]-bungarotoxin and [³H]quinuclidinyl benzilate binding. The 2-to 4-fold relative enrichment of putative receptor binding sites correlated well with the 4-fold enrichment of morphologically identifiable synaptic junctions in the synaptic plasma membrane enriched fraction.     

Lack of specificity in cation effects on solubilized benzodiazepine receptor

Receptors for benzodiazepines (BZ) and -carboline-carboxylic acid ethyl ester (-CCE) has been solubilized with decanoly-N-methylglucamide (DMG), a new kind of nonionic detergent. The apparent dissociation constants of diazepam and -CCE for solubilized receptor were similar to those for synaptic membranes. Sucrose density gradient centrifugation of the solubilized receptor protein revealed that the binding profile of [³H]-CCE essentially parallels that of [³H]diazepam and that both sedimentation coefficients were 10.5S. Co²⁺ and Ni²⁺, which increase [³H]diazepam binding and decrease [³H]-CCE binding to synaptic membranes, remarkably increased the binding of both to the solubilized receptor. Mg²⁺ and Ca²⁺, which had no effect on membrane receptor binding, also enhanced [³H]diazepam and [³H]-CCE binding to the solubilized receptor. The increase in binding in the presence of these divalent cations was due to a change in the apparent number of binding sites, with no change in binding affinities. The relative lack of specificity in divalent cation effects on solubilized BZ receptor may be caused by separation or destruction of the cation recognition site or channel of the BZ receptor complex by solubilization of the synaptic membrane with DMG.     

Evidence for the presence of specific binding sites for corticoids in mouse liver plasma membranes

Summary The specific binding of [³H]cortisol to plasma membranes purified from mouse liver, studied by the ultrafiltration method, shows the existence of specific binding sites for cortisol. The kinetic parameters of this binding areK _D=4.4nm andB _max=685 fmol/mg protein in presence of 1 m of corticosterone. With respect to the binding of 4nm [³H]cortisol to the membrane, the affinities of the steroids decreased in the following order: deoxycorticosterone>corticosterone>progesterone>cortisol >prednisolone>testosterone>20-hydroxyprogesterone >cortisone. Estradiol, dexamethasone, ouabain and triamcinolone acetonide do not have affinity for this binding site. Neither Ca²⁺ nor Mg²⁺ affected the binding of [³H]cortisol to the plasma membranes. Likewise, the presence of agonists and antagonists of alpha and beta-adrenergic receptors did not modify the binding of [³H]cortisol. The results suggest that the plasma membrane binding site characterized is more specific for corticoids and is different from nuclear glucocorticoid and progesterone receptors.