Solubilization and Partial Purification of α-Amino-3- Hydroxy-5-Methyl-4-Isoxazolepropionic Acid Binding Sites from Rat Brain

alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) binding sites were solubilized from rat brain membranes using 1% Triton X-100 in 0.5 M potassium phosphate buffer containing 20% glycerol. The solubilized binding sites were stable, permitting biochemical and pharmacological characterization as well as partial purification. Pharmacological and binding analyses indicated that the solubilized binding sites were similar to the membrane-bound sites. Both the solubilized and the membrane-bound preparations contained high- and low-affinity AMPA binding sites in the presence of potassium thiocyanate. A similar rank order for inhibition of [3H]AMPA binding by several excitatory amino acid analogs was obtained for the soluble and membrane-bound preparations. [3H]AMPA binding to both soluble and membrane-bound preparations was increased in the presence of potassium thiocyanate. The solubilized AMPA binding sites migrated as a single peak with gel filtration chromatography, with an Mr of 425,000. Beginning with the solubilized preparation, AMPA binding sites were purified 54-fold with ion-exchange chromatography and gel filtration. The characterization and purification of these soluble binding sites is potentially useful for the molecular characterization of this putative excitatory amino acid receptor subtype.     

Solubilization and Purification of an α-Amino-3-Hydroxy-5-Methylisoxazole-4-Propionic Acid Binding Protein from Bovine Brain

alpha-Amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) is a selective ligand for an excitatory amino acid receptor subtype in mammalian brain. We have solubilized an AMPA binding protein from bovine brain membranes with 1% Triton X-100 in 0.5 M phosphate buffer and 20% glycerol at 37 degrees C and purified the stable binding sites using a series of chromatographic steps. Scatchard analysis of the purified preparation showed a curvilinear plot with dissociation constants of 10.6 and 323 nM and Bmax values of 670 and 1,073 pmol/mg of protein for the high- and low-affinity sites, respectively. Inhibition constants for several excitatory amino acid analogues were similar to those obtained for other membrane and solubilized preparations. Gel filtration of the soluble AMPA binding protein showed a single peak of [3H]AMPA binding activity at Mr approximately 500,000. With sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified AMPA binding protein showed a single major band at Mr = 110,000. Previously, we have shown that a monoclonal antibody (KAR-B1) against a frog brain kainate binding protein selectively recognizes an unknown protein in mammalian brain migrating at Mr approximately 100,000. We now show that this antibody recognizes the major component of the purified AMPA binding protein, supporting a structural similarity between the frog brain kainate binding protein and the mammalian AMPA binding protein.     

Characterisation of an Allosteric Modulatory Protein Associated with α-[3H]Amino-3-Hydroxy-5-Methylisoxazolepropionate Binding Sites in Chick Telencephalon: Effects of High-Energy Radiation and Detergent Solubilisation

alpha-[3H]Amino-3-hydroxy-5-methylisoxazolepropionate ([3H]AMPA) binds to 1-day-old chick telencephalon membranes with KD and Bmax values of 138 nM and 2.56 pmol/mg of protein, respectively. High-energy radiation bombardment of intact frozen telencephalon resulted in a biphasic inactivation curve for [3H]AMPA binding. At a 5.8-Mrad radiation dose, the affinity of [3H]AMPA binding was increased (54 nM), but there was no apparent alteration in the Bmax value (2.76 pmol/mg of protein). We attribute this phenomenon to the inactivation of a high molecular weight modulatory protein that down-regulates the affinity of [3H]AMPA binding. The estimated molecular masses of the AMPA binding site and of the modulatory component were 59 and 108 kDa, respectively. Solubilisation with n-octyl-beta-glucopyranoside resulted in an increase in the Bmax (4.7 pmol/mg of protein) with no pronounced alteration in the affinity (109 nM) of [3H]AMPA binding. However, the solubilisation-induced increase in Bmax did not occur in telencephalon irradiated before solubilisation. In contrast, the increase in affinity induced by radiation treatment was still detected in solubilised extracts. These results suggest that the number and affinity of [3H]AMPA sites in chick telencephalon are closely regulated and that the modulatory systems involved are affected by both irradiation and solubilisation.     

Modulation of α-Amino-3-Hydroxy-5-Methylisoxazole-4-Propionic Acid (AMPA) Binding Sites by Nitric Oxide

Abstract: Nitric oxide release is reported to be involved in physiological processes associated with altered sensitivity of the α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) class of glutamate receptor. A series of compounds liberating nitric oxide were therefore tested for their ability to modulate in vitro the characteristics of [³H]AMPA binding to sections of rat brain. Pretreatment of forebrain or cerebellar sections with sodium nitroprusside (1 m M ), S -nitroso- N -acetylpenicillamine (SNAP, 200 µ M ), glyceryl trinitrate (1 µ M ), or isosorbide dinitrate (0.5 m M ) all increased the binding of 3 n M [³H]AMPA by 15–30%. These actions were reproduced by 8-bromo-cyclic GMP (200 µ M ) in the cerebellum but not in the forebrain. In a similar manner, the effect of SNAP was attenuated by an inhibitor of cyclic GMP-dependent protein kinase in the cerebellum but not in the forebrain. The elevated [³H]AMPA binding observed after pretreatment with SNAP was caused by an increase in binding affinity, but the capacity of the sites was unchanged. Autoradiographic analysis showed that forebrain binding was enhanced in the cerebral cortex and hippocampus but not in the striatum. Nitric oxide therefore appears to be able to increase the affinity of AMPA binding sites via two distinct mechanisms in different brain areas. This action may contribute to synaptic plasticity associated with nitric oxide release.     

Nootropic Drugs Positively Modulate α-Amino-3-Hydroxy-5-Methyl-4-Isoxazolepropionic Acid-Sensitive Glutamate Receptors in Neuronal Cultures

Micromolar concentrations of piracetam, aniracetam, and oxiracetam enhanced alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-stimulated 45Ca2+ influx in primary cultures of cerebellar granule cells. Nootropic drugs increased the efficacy but not the potency of AMPA and their action persisted in the presence of the voltage-sensitive calcium channel blocker nifedipine. Potentiation by oxiracetam was specific for AMPA receptor-mediated signal transduction, as the drug changed neither the stimulation of 45Ca2+ influx by kainate or N-methyl-D-aspartate nor the activation of inositol phospholipid hydrolysis elicited by quisqualate or (+-)-1-aminocyclopentane-trans-1,3-dicarboxylic acid. Piracetam, aniracetam, and oxiracetam increased the maximal density of the specific binding sites for [3H]AMPA in synaptic membranes from rat cerebral cortex. Taken collectively, these results support the view that nootropic drugs act as positive modulators of AMPA-sensitive glutamate receptors in neurons.     

α-[3H]Amino-3-Hydroxy-5-Methyl-4-Isoxazolepropionic Acid Binding to Rat Striatal Membranes: Effects of Selective Brain Lesions

The binding of alpha-[3H]amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid ([3H]AMPA), a structural Glu analog, to rat striatal membranes was studied. In the absence of potassium thiocyanate and Cl-/Ca2+, saturation-curve analysis of [3H]AMPA binding suggested that a single class of noninteracting binding sites with a KD value of 340 +/- 27 nM was involved, although AMPA inhibition of [3H]AMPA binding set at a concentration of 100 nM suggested, in contrast, the presence of multiple populations of striatal binding sites. Several other excitatory amino acid receptor agonists and antagonists were tested, and the most potent and selective quisqualic acid (QA) receptor agonists (QA, L-Glu, and AMPA) were found to represent the most potent inhibitors of [3H]AMPA binding. N-Methyl-D-aspartate receptor agonists and antagonists were ineffective as displacers of the [3H]AMPA binding. Lesions of intrastriatal neurons (using kainic acid local injections) and of corticostriatal afferent fibers led 2-3 weeks later to large decreases (63 and 30%, respectively) in striatal [3H]AMPA binding, whereas selective lesion of the nigrostriatal dopaminergic pathway (using nigral injection of 6-hydroxy-dopamine) was without any influence. Taken together, these results suggest that [3H]AMPA binding is primarily associated with postsynaptic intrastriatal neurons. Some [3H]AMPA binding sites may also be located presynaptically on corticostriatal nerve endings. So, in addition to the possibility that [3H]AMPA binding sites may be involved in corticostriatal synaptic transmission, it is interesting that these putative QA-preferring excitatory amino acid receptor sites may also play some role in autoregulatory processes underlying this excitatory synaptic transmission.     

Solubilization, Characterization, and Partial Purification of α-Amino-3-Hydroxy-5-Methyl-4-Isoxazolepropionic Acid-, Quisqualate-, Kainate-Sensitive l-Glutamate Binding Sites from Porcine Brain Synaptic Junctions

L-[3H]Glutamate binding sites were solubilized from porcine brain synaptic junctions by Triton X-114 in the presence of KCl. The solubilized binding sites bound L-[3H]glutamate reversibly with KD and Bmax values of 1.48 +/- 0.18 microM and 178.2 +/- 15.9 pmol/mg of protein, respectively. These binding sites appeared to be integral membrane glycoproteins, with sugar moieties recognized by wheat germ agglutinin. A 49.3-fold purification of these binding sites was achieved by Triton X-114 solubilization, anion-exchange chromatography, and affinity chromatography using wheat germ agglutinin-Sepharose. The apparent molecular mass of the partially purified binding sites was 620 +/- 50 kDa. L-[3H]Glutamate bound to the solubilized preparation could be effectively displaced by agonists of non-N-methyl-D-aspartate (NMDA) L-glutamate receptors but not by NMDA or alpha-amino-4-phosphonobutyrate. The rank order for the competitive ligands in displacing L-[3H]glutamate was: quisqualate greater than alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid greater than L-glutamate greater than kainate.     

High- and Low-Affinity α-[3H]Amino-3-Hydroxy-5-Methylisoxazole-4-Propionic Acid ([3H]AMPA) Binding Sites Represent Immature and Mature Forms of AMPA Receptors and Are Composed of Differentially Glycosylated Subunits

Abstract: Quantitative α-[³H]amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid ([³H]AMPA) binding autoradiography was performed on frozen-thawed sections from rat brain after preincubation at 0 or 35°C for 1 h. Preincubation at 35°C instead of 0°C resulted in a selective decrease of [³H]AMPA binding assayed at a low concentration of [³H]-AMPA (50 nM) and an enhancement of binding at a high concentration (500 nM). The decrease in [³H]AMPA binding after preincubation at 35°C was accompanied with the loss of the lighter organelles of P3 (microsomal) fractions. These organelles were found to contain a small subpopulation of AMPA/GluR receptors exhibiting a high affinity for [³H]AMPA(K_D～14 nM), whereas heavier organelles exhibited lower affinity for AMPA (K_D～190 nM). This small subpopulation of AMPA/GluR receptors contained almost exclusively a structurally distinct species of GluR2/3 subunits with an apparent molecular mass of 103.5 kDa (assessed with anti-GluR2/3, C-terminal antibodies). Experiments using two deglycosylating enzymes, N-glycopeptidase F and endoglycosidase H, clearly indicated that the 103.5-kDa species represented a partially unglycosylated form of GluR2/3 subunits containing the high-mannose type of oligosaccharide moiety, whereas receptors present in synaptosomal fractions were composed of subunits with complex oligosaccharides. A similar result was obtained by using an antibody recognizing the N-terminal domain of GluR2(4). The same enzymatic treatment indicated that GluR1 subunits also exhibited a partially glycosylated form. These data indicate that high-affinity [³H]AMPA binding sites represent nonsynaptic, intracellular membrane-bound AMPA receptors that differ from synaptic receptors by at least the glycosylation state of GluR2 (and GluR1) subunits. In addition, our results provide a relatively simple way of assessing changes in two spatially and structurally distinct [³H]AMPA binding/GluR sites.     

α-[3H]Amino-3-Hydroxy-5-Methylisoxazole-4-Propionic Acid Binding to Human Cerebral Cortical Membranes: Minimal Changes in Postmortem Brains of Chronic Schizophrenics

The binding of alpha-[3H]amino-3-hydroxy-5-methylisoxazole-4-propionic acid ([3H]AMPA), a selective ligand for the ion channel-linked quisqualate receptor, was evaluated in Triton X-100-treated membranes of human cerebral cortex. The presence of chaotropic ions produced divergent effects on specific [3H]AMPA binding: A twofold increase in the binding was observed with thiocyanide at 100 mM, although iodide (100 mM) and perchlorate (100 mM) reduced the binding. Chemical modifications of the sulfhydryl group with p-chloromercuriphenylsulfonic acid (PCMBS) produced threefold increases in specific [3H]-AMPA binding in the absence of KSCN as well as in the presence of KSCN. Treatment with dithiothreitol restored the enhanced specific [3H]AMPA binding by PCMBS to the basal level. Although specific [3H]AMPA binding in the absence of KSCN showed a single site (KD = 220 nM, Bmax = 235 fmol/mg of protein), curvilinear Scatchard plots of specific [3H]AMPA binding in the presence of 100 mM KSCN can be resolved into two binding sites with the following parameters: KD1 = 5.82 nM, Bmax1 = 247 fmol/mg of protein; KD2 = 214 nM, Bmax2 = 424 fmol/mg of protein. Quisqualate and AMPA were the most potent inhibitors of the [3H]AMPA binding in the presence of KSCN. Potent inhibitors of the binding included beta-N-oxalylamino-L-alanine (L-BOAA), cysteine-S-sulfate, L-glutamate, 6-cyano-7-nitroquinoxaline-2,3-dione, and 6,7-dinitroquinoxaline-2,3-dione. Kainate, L-homocysteine sulfinic acid, and L-homocysteic acid were active with an IC50 value of a micromolar concentration, whereas L-cysteic acid and L-cysteine sulfinic acid were weakly active.(ABSTRACT TRUNCATED AT 250 WORDS)     

Solubilisation and Characterisation of a Putative Quisqualate-Type Glutamate Receptor from Chick Brain

The brains of 1-day-old chicks were shown to be a rich source of binding sites with the pharmacological characteristics expected of a quisqualate-type glutamate receptor. alpha-[3H]Amino-3-hydroxy-5-methylisoxazolepropionate ([3H]AMPA) bound with KD and Bmax values, measured at 0 degree C in the presence of the chaotrope potassium thiocyanate, of 55 nM and 2.6 pmol/mg protein. The regional localisations of [3H]AMPA and [3H]kainate binding sites were manifestly different. The membrane-bound [3H]AMPA binding sites were efficiently solubilised by N-octyl-beta-D-glucopyranoside (1%) in the presence of 0.2 M thiocyanate. In the detergent extract the affinity was 69 nM and there was an apparent increase in the number of sites (Bmax, 4.6 pmol/mg protein). The rank order of potency for competitive ligands in displacing [3H]AMPA binding was quisqualate approximately AMPA greater than 6-cyano-7-nitroquinoxaline-2,3-dione greater than L-glutamate greater than kainate and was identical for the membrane-bound and solubilised sites. Dissociation was biphasic with rate constants of 0.117 min-1 and 0.015 min-1. The association rate constants for [3H]AMPA at the solubilised sites were 1.45 x 10(6) M-1 min-1 and 6.55 x 10(6) M-1 min-1. The kinetically derived KD values were 80.7 nM and 2.3 nM. The detection of higher affinity binding sites by kinetic analysis but not by equilibrium binding may be explained by the greater sensitivity of dissociation data to small populations of high-affinity sites.(ABSTRACT TRUNCATED AT 250 WORDS)     

Autoradiographic Characterization and Localization of Quisqualate Binding Sites in Rat Brain Using the Antagonist [3H]6-Cyano-7-Nitroquinoxaline-2,3-Dione: Comparison with (R,S)-[3H]α-Amino-3-Hydroxy-5-Methyl-4-Isoxazolepropionic Acid Binding Sites

Using quantitative autoradiography, we have investigated the binding sites for the potent competitive non-N-methyl-D-aspartate (non-NMDA) glutamate receptor antagonist [3H]6-cyano-7-nitro-quinoxaline-2,3-dione ([3H]-CNQX) in rat brain sections. [3H]CNQX binding was regionally distributed, with the highest levels of binding present in hippocampus in the stratum radiatum of CA1, stratum lucidum of CA3, and molecular layer of dentate gyrus. Scatchard analysis of [3H]CNQX binding in the cerebellar molecular layer revealed an apparent single binding site with a KD = 67 +/- 9.0 nM and Bmax = 3.56 +/- 0.34 pmol/mg protein. In displacement studies, quisqualate, L-glutamate, and kainate also appeared to bind to a single class of sites. However, (R,S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) displacement of [3H]CNQX binding revealed two binding sites in the cerebellar molecular layer. Binding of [3H]AMPA to quisqualate receptors in the presence of potassium thiocyanate produced curvilinear Scatchard plots. The curves could be resolved into two binding sites with KD1 = 9.0 +/- 3.5 nM, Bmax = 0.15 +/- 0.05 pmol/mg protein, KD2 = 278 +/- 50 nM, and Bmax = 1.54 +/- 0.20 pmol/mg protein. The heterogeneous anatomical distribution of [3H]CNQX binding sites correlated to the binding of L-[3H]glutamate to quisqualate receptors and to sites labeled with [3H]AMPA. These results suggest that the non-NMDA glutamate receptor antagonist [3H]CNQX binds with equal affinity to two states of quisqualate receptors which have different affinities for the agonist [3H]AMPA.     

Similarity Between Rat Brain Nicotinic α-Bungarotoxin Receptors and Stably Expressed α-Bungarotoxin Binding Sites

Abstract: The present results demonstrate stable expression of α-bungarotoxin (α-BGT) binding sites by cells of the GH₄C₁ rat pituitary clonal line. Wild-type GH₄C₁ cells do not express α-BGT binding sites, nor do they contain detectable mRNA for nicotinic receptor α2, α3, α4, α5, α7, β2, or β3 subunits. However, GH₄C₁ cells stably transfected with rat nicotinic receptor α7 cDNA (α7/GH₄C₁ cells) express the transgene abundantly as mRNA, and northern analysis showed that the message is of the predicted size. The α7/GH₄C₁ cells also express saturable, high-affinity binding sites for ¹²⁵I-labeled α-BGT, with a K_D of 0.4 nM and B_max of 3.2 fmol/10⁶ intact cells. ¹²⁵I-α-BGT binding affinities and pharmacological profiles are not significantly different for sites in membranes prepared either from rat brain or α7/GH₄C₁ cells. Furthermore, K_D and K_i values for ¹²⁵I-α-BGT binding sites on intact α7/GH₄C₁ cells are essentially similar to those for hippocampal neurons in culture. Sucrose density gradient analysis showed that the size of the α-BGT binding sites expressed in α7/GH₄C₁ cells was similar to that of the native brain α-BGT receptor. Chronic exposure of α7/GH₄C₁ cells in culture to nicotine or an elevated extracellular potassium concentration induces changes in the number of α-BGT binding sites comparable to those observed in cultured neurons. Collectively, the present results show that the properties of α-BGT binding sites in transfected α7/GH₄C₁ cells resemble those for brain nicotinic α-BGT receptors. If the heterologously expressed α-BGT binding sites in the present study are composed solely of α7 subunits, the results could suggest that the rat brain α-BGT receptor has a similar homooligomeric structure. Alternatively, if α-BGT binding sites exist as heterooligomers of α7 plus some other previously identified or novel subunit(s), the data would indicate that the α7 subunits play a major role in determining properties of the α-BGT receptor.     

N-Linked Glycosylation of the α-Amino-3-Hydroxy-5-Methylisoxazole-4-Propionate(AMPA)-Selective Glutamate Receptor Channel α2 Subunit Is Essential for the Acquisition of Ligand-Binding Activity

Abstract: The N-linked glycosylation of the α2 subunit of the mouse α-amino-3-hydroxy-5-methylisoxazole-4-propionate(AMPA)-selective glutamate receptor (GluR) channel was characterized. The receptor subunit protein has five putative N -glycosylation sites. The recombinant receptor proteins were identified by [³⁵S]methionine/[³⁵S]cysteine metabolic labeling, western blot analysis, immunocytochemical detection, and [³H]AMPA binding experiments when expressed in insect Spodoptera frugiperda cells using a baculovirus system. The effect of tunicamycin on the metabolic labeling and immunoblots suggested that the two products, a major protein species of ∼102 kDa and a minor species of ∼98 kDa, correspond to glycosylated and unglycosylated forms, respectively, which was also supported by the enzymic deglycosylation experiments. Immunofluorescence staining of tunicamycin-treated cells expressing only the unglycosylated form differed little from that of tunicamycin-nontreated cells expressing both glycosylated and unglycosylated forms. The lack of AMPA-binding activity of the unglycosylated form expressed in the presence of tunicamycin suggested that N -glycosylation is required, directly or indirectly, for functional expression in insect cells for ligand binding. These results demonstrate that occupancy of at least one N -glycosylation site is required for the formation and maintenance of the GluRα2 subunit protein in an active conformation for ligand binding. Possible roles of N -glycosylation of GluRα2 subunit protein are discussed.     

Interactions Between N-Acetylaspartylglutamate and AMPA, Kainate, and NMDA Binding Sites

Abstract: The structure of N -acetylaspartylglutamate (NAAG) suggests this neuronal dipeptide as a candidate for interaction with discrete subclasses of ionotropic and metabotropic acidic amino acid receptors. A substantial difficulty in the assay of these interactions is posed by membrane-bound peptidase activity that converts the dipeptide to glutamate and N -acetylaspartate, molecules that will interfere with receptor assays. We have developed two sets of unique receptor assay conditions and applied one standard assay to measure the interactions, under equilibrium binding conditions, of [³H]kainate, [³H]amino-3-hydroxy-5-methylisoxazole-4-propionic acid ([³H]AMPA), and [³H]CGS-19755 with the three classes (kainate, quisqualate, and N -methyl- d -aspartate) of ionotropic glutamate receptors, while inhibiting peptidase activity against NAAG. Under these conditions, NAAG exhibits apparent inhibition constants (IC₅₀) of 500, 790, and 8.8 µ M in the kainate, AMPA, and CGS-19755 receptor binding assays, respectively. Glutamate was substantially more effective and less specific in these competition assays, with inhibition constants of 0.36, 1.1, and 0.37 µ M . These data support the hypothesis that, relative to glutamate, NAAG functions as a specific, low potency agonist at N -methyl- d -aspartate subclass of ionotropic acidic amino acid receptors, but the peptide is not likely to activate directly the kainate or quisqualate subclasses of excitatory ionotropic receptors under physiologic conditions.     

Release of Endogenous and Newly Synthesized Glutamate and of Other Amino Acids Induced by Non-N-Methyl-D-Aspartate Receptor Activation in Cerebellar Granule Cell Cultures

Amino acid release studies were performed by an HPLC procedure using differentiated rat cerebellar granule cell cultures. Kainic acid (KA; 50 microM) caused an increase (about threefold) in the release of endogenous glutamate and a lesser, but statistically significant, increase in the release of glutamine, glycine, threonine, taurine, and alanine. Quisqualic acid (QA) and, to a lesser degree, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) (both 50 microM) enhanced the release of the following amino acids in the order glutamate greater than aspartate greater than or equal to taurine, whereas the release of other amino acids was either unaffected or affected in a statistically nonsignificant way. The release of glutamate induced by KA was partially (43%) Ca2+ dependent. The other release-inducing effects of KA and QA were not Ca2+ dependent. In all cases, the evoked release could be prevented by the non-N-methyl-D-aspartate (non-NMDA) receptor antagonist 6-cyano-2,3-hydroxy-7-nitroquinoxaline, and thus appeared to be receptor mediated. NMDA (5 and 50 microM) had no release-inducing activity. The KA-, QA-, and AMPA-evoked release of newly synthesized [3H]glutamate and [3H]aspartate (formed in the cells exposed to [3H]glutamine) was very similar to the evoked release of endogenous glutamate and aspartate. On the other hand, the release of preloaded D-[3H]aspartate (purified by HPLC in the various fractions analyzed, before radioactivity determination) induced by 50 microM KA was twice as high as that of endogenous glutamate. In the case of high [K+] depolarization, in contrast, the release of preloaded D-[3H]aspartate was approximately 30% lower than that of endogenous glutamate.(ABSTRACT TRUNCATED AT 250 WORDS)     

The α5(H105R) mutation impairs α5 selective binding properties by altered positioning of the α5 subunit in GABAA receptors containing two distinct types of α subunits

GABA_A receptors are pentameric ligand-gated ion channels that are major mediators of fast inhibitory neurotransmission. Clinically relevant GABA_A receptor subtypes are assembled from α5(1-3, 5), β1-3 and the γ2 subunit. They exhibit a stoichiometry of two α, two β and one γ subunit, with two GABA binding sites located at the α/β and one benzodiazepine binding site located at the α/γ subunit interface. Introduction of the H105R point mutation into the α5 subunit, to render α5 subunit-containing receptors insensitive to the clinically important benzodiazepine site agonist diazepam, unexpectedly resulted in a reduced level of α5 subunit protein in α5(H105R) mice. In this study, we show that the α5(H105R) mutation did not affect cell surface expression and targeting of the receptors or their assembly into macromolecular receptor complexes but resulted in a severe reduction of α5-selective ligand binding. Immunoprecipitation studies suggest that the diminished α5-selective binding is presumably due to a repositioning of the α5(H105R) subunit in GABA_A receptor complexes containing two different α subunits. These findings imply an important role of histidine 105 in determining the position of the α5 subunit within the receptor complex by determining the affinity for assembly with the γ2 subunit.     

The loop between β-strands β2 and β3 and its interaction with the N-terminal α-helix is essential for biogenesis of α7 nicotinic receptors

Recently, we have shown that the α-helix present at the N-termini of α7 nicotinic acetylcholine receptors plays a crucial role in their biogenesis. Structural data suggest that this helix interacts with the loop linking β-strands β2 and β3 (loop 3). We studied the role of this loop as well as its interaction with the helix in membrane receptor expression. Residues from Asp62 to Val75 in loop 3 were mutated. Mutations of conserved amino acids, such as Asp62, Leu65 and Trp67 abolished membrane receptor expression in Xenopus oocytes. Others mutations, at residues Asn68, Ala69, Ser70, Tyr72, Gly74, and Val 75 were less harmful although still produced significant expression decreases. Steady state levels of wild-type and mutant α7 receptors (L65A, W67A, and Y72A) were similar but the formation of pentameric receptors was impaired in the latter (W67A). Mutation of critical residues in subunits of heteromeric nicotinic acetylcholine receptors (α3β4) also abolished their membrane expression. Complementarity between the helix and loop 3 was evidenced by studying the expression of chimeric α7 receptors in which these domains were substituted by homologous sequences from other subunits. We conclude that loop 3 and its docking to the α-helix is an important requirement for receptor assembly.     

Subunit Structure of α-Bungarotoxin Binding Component in Mouse Brain

Abstract: The α-bungarotoxin binding component in mouse brain was purified by affinity chromatography with toxin-Sepharose, gel-chromatography on Sepharose 6B, and ion-exchange chromatography with DE52 resin. The iodinated product of the last step produced one major and one minor band on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight of the minor peak was twice as large as that of the major one. The iodinated product could bind α-bungarotoxin, and this binding was inhibited by a nicotinic antagonist, d -tubocurarine, which demonstrated that the iodinated product was a true α-bungarotoxin binding component. The molecular structure of the product was analysed by cross-linking followed by SDS-PAGE. The results fitted the model for an α-bungarotoxin binding component in the mouse brain composed of six identical or very similar subunits of 51,000-52,000. One subunit carrying the binding site for toxin bound one molecule of toxin. This subunit structure of an α-bungarotoxin binding component in the brain is discussed in comparison with that of a nicotinic acetylcholine receptor in the electric organ.     

Nicotinic Agonists Regulate α-Bungarotoxin Binding Sites of TE671 Human Medulloblastoma Cells

The TE671 human medulloblastoma cell line expresses a variety of characteristics of human neurons. Among these characteristics is the expression of membrane-bound high-affinity binding sites for alpha-bungarotoxin, which is a potent antagonist of functional nicotinic acetylcholine receptors on these cells. These toxin binding sites represent a class of nicotinic receptor isotypes present in mammalian brain. Treatment of TE671 cells during proliferative growth phase with nicotine or carbamylcholine, but not with muscarine or d-tubocurarine, induced up to a five-fold increase in the density of radiolabeled toxin binding sites in crude membrane fractions. This effect was blocked by co-incubation with the nicotinic antagonists d-tubocurarine and decamethonium, but not by mecamylamine or by muscarinic antagonists. Following a 10-13 h lag phase upon removal of agonist, recovery of the up-regulated sites to control values occurred within an additional 10-20 h. These studies indicate that the expression of functional nicotinic acetylcholine receptors on TE671 cells is subject to regulation by nicotinic agonists. Studies of the murine CNS have consistently indicated nicotine-induced up-regulation of nicotinic acetylcholine receptors, thereby supporting the identification of the toxin binding site on these cells as the functional nicotinic receptor. Although a mechanism for this effect is not apparent, nicotine-induced receptor blockade does not appear to be involved.     

Role of Ca2+ in α-Amino-3-Hydroxy-5-Methyl-4-Isoxazolepropionic Acid-Mediated Polyphosphoinositide Turnover in Primary Neuronal Cultures

Abstract: Excitatory amino acid (EAA) receptors and EAA-mediated stimulation of polyphosphoinositide (poly-PI) turnover were studied in cultured neurons at different days in vitro (DIV). Six main observations have emerged from these studies: (a) Neurons increased their sensitivity to EAAs as a function of time in culture, indicated by increasing EAA-mediated poly-PI turnover, (b) Extracellular Ca²⁺ concentration played an important role in α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-stimulated poly-PI turnover in cells at 4 DIV, whereas poly-PI turnover mediated by l -glutamate and trans -1-amino-cyclopentane-1,3-dicarboxylic acid was not Ca²⁺-dependent. (c) A marked stimulation of poly-PI turnover by AMPA was seen in the cultured neurons at 4 DIV, but not at 17 DIV, suggesting that a distinct EAA receptor sensitive to AMPA is transiently expressed, (d) The Ca²⁺ ionophore A23187 increased poly-PI turnover in cultured neurons, suggesting that Ca²⁺ entry is involved in stimulating poly-PI turnover, (e) Stimulation of poly-PI turnover by carbachol was greater in neurons at 17 DIV as compared with −4 DIV, and appeared to be Ca²⁺-dependent across DIV. (f) 6-Cyano-7-nitroquinoxaline-2,3-dione, an antagonist for non- N -methyl- d -aspartate ionotropic EAA receptors, inhibited 100% and 35% of AMPA-and quisqualate-induced poly-PI turnover, respectively, suggesting an involvement of ionotropic AMPA/quisqualate receptors in stimulating poly-PI turnover.