Isolation, localization, and cloning of a kainic acid binding protein from frog brain

Excitatory amino acids (EAA) are major neurotransmitters in the vertebrate central nervous system. EAA receptors have been divided into three major subtypes on the basis of electrophysiological and ligand binding studies: N-methyl-D-aspartate, kainate, and quisqualate receptors. To understand their molecular properties, we undertook a project aimed at isolation and cloning of these receptor subtypes. We purified a kainate binding protein (KBP) from frog brain, in which kainate binding sites are about fortyfold more abundant than in rat brain, using domoic acid affinity chromatography, and made monoclonal and polyclonal antibodies to the purified protein. These antibodies immunoprecipitate the frog KBP but not KBPs from other species. Immunocytochemical analyses show that KBP has a synaptic and extrasynaptic localization in frog optic tectum, with most labeling being extrasynaptic. The cDNA encoding frog brain KBP was isolated by screening a frog brain cDNA library with oligonucleotide probes that were based on the amino acid sequence of the purified protein. The deduced amino acid sequence of the KBP has a hydrophobic profile similar to those of other ligand-gated ion channel subunits, such as the nicotinic acetylcholine receptor, the GABAA receptor, and the glycine receptor. Frog brain KBP is very similar (36% amino acid identity to the carboxyl half) to rat brain kainate receptor, suggesting that these two proteins evolved from a common ancestor. The function of KBP in frog brain remains a major question. Preliminary results showed that Xenopus laevis oocytes injected with KBP RNA did not produce a detectable electrophysiological response when perfused with kainate. These results suggest that additional subunits may be required to form a functional receptor or that KBP is not functionally related to a neurotransmitter receptor.     

Spermine modulation of the glutamate(NMDA) receptor is differentially responsive to conantokins in normal and Alzheimer's disease human cerebral cortex

The pharmacology of the N -methyl-d-aspartate (NMDA) receptor site was examined in pathologically affected and relatively spared regions of cerebral cortex tissue obtained at autopsy from Alzheimer's disease cases and matched controls. The affinity and density of the [(3)H]MK-801 binding site were delineated along with the enhancement of [(3)H]MK-801 binding by glutamate and spermine. Maximal enhancement induced by either ligand was regionally variable; glutamate-mediated maximal enhancement was higher in controls than in Alzheimer's cases in pathologically spared regions, whereas spermine-mediated maximal enhancement was higher in controls in areas susceptible to pathological damage. These and other data suggest that the subunit composition of NMDA receptors may be locally variable. Studies with modified conantokin-G (con-G) peptides showed that Ala(7)-con-G had higher affinity than Lys(7)-con-G, and also defined two distinct binding sites in controls. Nevertheless, the affinity for Lys(7)-con-G was higher overall in Alzheimer's brain than in control brain, whereas the reverse was true for Ala(7)-con-G. Over-excitation mediated by specific NMDA receptors might contribute to localized brain damage in Alzheimer's disease. Modified conantokins are useful for identifying the NMDA receptors involved, and may have potential as protective agents.     

Differential Regulation of Molecular Subtypes of Muscarinic Receptors in Alzheimer's Disease

Abstract: Molecular subtypes of muscarinic receptors (m1–m5) are novel targets for cholinergic replacement therapies in Alzheimer's disease. However, the status of these receptors in human brain and Alzheimer's disease is incompletely understood. The m1–m5 receptors in brains from control subjects and Alzheimer's disease patients were examined using a panel of specific antisera and radioligand binding. Quantitative immunoprecipitation demonstrated a predominance of the m1, m2, and m4 receptor subtypes in cortical and subcortical regions in control subjects. In Alzheimer's disease, normal levels of m1 receptors measured by radioligand binding contrasted with decreased m1 receptor immunoreactivity, suggesting that the m1 receptor is altered in Alzheimer's disease. The m2 immunoreactivity was decreased, consistent with the loss of m2 binding sites and the location of this receptor subtype on presynaptic cholinergic terminals. The m4 receptor was up-regulated significantly and may offer a target for new memory-enhancing drugs. Differential alterations of molecular subtypes of muscarinic receptors may contribute to the cholinergic component of Alzheimer's disease dementia.     

Analysis of 3H-kainic acid binding to brain membranes in rats and frogs

It has been shown that H3-kainic acid (3H-KA) specifically binds with membrane preparations from various parts of rat brain or whole frog brain. The saturation isotherms of 3H-KA binding revealed the presence of two sites with a high and low affinity. An exception was for rat cerebellum where Scatchard analysis showed but one low affinity site. The density of 3H-KA binding sites in frog brain was 5 to 10 times higher than in rat brain. Among the drugs studied, KA itself, L-glutamate and folic acid were the most potent inhibitors of specific binding. Methyltetrahydrofolate, quinolinic acid, kynurenine, GABA, taurine, L-aspartate were ineffective in this respect. The kinetic analysis of the binding data in the presence or absence of L-glutamate and folic acid showed, however, that these drugs inhibited 3H-KA binding in a noncompetitive manner. In the light of these findings L-glutamate or folate cannot be considered as endogenous ligands for hypothetic "kainate receptors".     

Loss of [3H]kainate and of NMDA-displaceable [3H]glutamate binding sites in brain in thiamine deficiency: Results of a quantitative autoradiographic study

Previous studies suggest that alterations of brain glutamate synthesis and release occur in experimental thiamine deficiency. In order to assess the integrity of post-synaptic glutamatergic receptors in thiamine deficiency, binding sites for [³H]glutamate (displaced by NMDA), [³H]-kainate, and [³H]quisqualate (AMPA sites) were evaluated using Quantitative Receptor Autoradiography in rat brain following 14 days of treatment with the central thiamine antagonist pyrithiamine. Compared to pair-fed controls, brains of symptomatic thiamine-deficient animals contained significantly fewer NMDA-displaceable binding sites in cerebral cortex, medial septum and hippocampus. It has been suggested that NMDA-receptor mediated glutamate excitotoxicity plays a role in the pathogenesis of neuronal loss in thiamine deficiency. If such is the case, the selective loss of NMDA binding sites in cerebral cortex and hippocampus offers a possible explanation for the relative nonvulnerability of these brain regions to pyrithiamine-induced thiamine deficiency. [³H]quisqualate (AMPA) binding sites were unchanged in all brain regions of pyrithiamine-treated rats whereas [³H]kainate sites were significantly reduced in density in medial and lateral thalamus. The decline in these binding sites may be due to neuronal loss in pyrithiamine-induced thiamine deficiency. Alterations of glutamatergic synaptic function involving both NMDA and kainate receptor subclasses could contribute to the pathogenesis of neurological dysfunction in Wernicke's Encephalopathy in humans.     

Kainate Receptors in Xenopus Central Nervous System: Solubilisation with n-Octyl-β-d-Glucopyranoside

[3H]Kainate binding to membrane homogenates and detergent extracts prepared from Xenopus central nervous system was evaluated in 50 mM Tris-citrate buffer, pH 7.0. In membrane fragment preparations, [3H]kainate bound with a KD of 54.4 nM to a large number of sites (Bmax = 27.8 pmol/mg of protein). Up to 80% of the total number of membrane-bound binding sites were solubilised using the nonionic detergent n-octyl-beta-D-glucopyranoside. Values for the KD of [3H]kainate for solubilised binding sites were 46.0 nM and 53.6 nM derived from equilibrium and kinetic binding experiments, respectively. Competitive binding studies revealed that a variety of ligands had similar Ki values in both membranes and solubilised extracts, with domoate and kainate being the most potent inhibitors of [3H]kainate binding. The dissociation rate of [3H]kainate from solubilised binding sites was 0.022 min-1. The binding component migrated in sucrose density gradients in a single 8.6S peak. These results demonstrate that the kainate receptor in Xenopus central nervous system, although similar to the [3H]kainate binding site from goldfish brain, differs in a number of important respects. In particular, the slower dissociation rate and higher affinity of [3H]kainate suggest that Xenopus provides the most convenient model system yet investigated for biochemical analysis of kainate receptors.     

Role of Guanine Nucleotides as Endogenous Ligands of a Kainic Acid Binding Site Population in the Mammalian Cerebellum

Abstract: An endogenous inhibitor of the membrane binding of kainic acid was extracted from pig brain tissue and purified. The substance was identified as GMP by structural analysis: Most likely it corresponds to an inhibitor previously extracted from the rat brain. The nucleotide is active as an inhibitor for kainate binding on goldfish brain synaptosomes, probably owing to direct displacement on receptor sites; it is also active on a low-affinity kainate site population in membranes from rat cerebellum. The interaction of GMP with the latter sites leads to a concentration-dependent kainate binding increase or inhibition, thus demonstrating that these sites can bind the nucleotide and cooperatively increase their affinity. Other guanine nucleotides show interaction with these sites, by either an increase (GTP) or inhibition (cyclic GMP or GDP) of kainate binding. These findings support the view that a guanine nucleotide is the endogenous ligand of a receptor in the mammalian cerebellum similar to the kainate binding protein present with high density in the cerebellum of lower vertebrates, whose function is probably connected to the role of the glial cells in this zone.     

Characterization, distribution and plasticity of atrial natriuretic factor binding sites in brain

The [125I]iodotyrosyl derivative of atrial natriuretic factor [( 125I])ANF) apparently binds to a single class of high affinity sites in guinea pig brain membrane preparations. Ligand selectivity pattern reveals that the structural requirements of brain [125I]ANF binding sites are similar to those reported in most peripheral tissues. In vitro receptor autoradiographic studies demonstrate that the brain distribution of [125I]ANF binding sites is species dependent. In rat, high levels of binding are found in olfactory bulb, subfornical organ, area postrema, choroid plexus, and ependyma. In guinea pig, these regions are also enriched with [125I]ANF binding in addition to various thalamic nucleic, amygdala, hippocampus, and cerebellum. In monkey, high densities of sites are seen in the cerebellar cortex. This suggests that brain ANF receptor sites could mediate ANF effects related to the central integration of cardiovascular parameters, as well as other actions not associated with these systems. As in the periphery, it appears that brain [125I]ANF binding sites are associated with guanylate cyclase. Moreover, the density of [125I]ANF receptor binding sites is altered in certain brain regions in spontaneously hypertensive rats and in cardiomyopathic hamsters, demonstrating the plasticity of brain ANF receptors. Thus, ANF and ANF receptors are complementary facets of a new neurotransmitter-neuromodulator system present in mammalian brain.     

Isolation,immunochemical characterization and localization of the kainate sub-class of glutamate receptor from chick cerebellum.

The low-affinity kainate binding sites, present at high density in chick cerebellar membranes, were solubilized with Triton X-100 and purified 41-fold. The purified kainate binding sites, therein referred to as the kainate receptor, displayed the expected pharmacological specificity: domoate = kainate much greater than L-glutamate much greater than D-glutamate, quisqualate, N-methyl-D-aspartate. Analysed by SDS-PAGE under reducing conditions, a single polypeptide with a Mr = 49,000 was observed. Western blots of membranes prepared from different brain areas and animal species were analysed using a monoclonal antibody, named mAb IX-50, raised against the purified kainate receptor. The mAb IX-50 stained the 49,000 polypeptide in chick, goldfish and mammalian brain tissues indicating its conservation during evolution. The staining intensity correlated with the density of kainate binding sites. The mAb IX-50 stained also a 93,000 polypeptide but the latter did not copurify with the 49,000 polypeptide. The kainate binding activity was selectively immunoadsorbed on mAb IX-50 coupled to Sepharose which, upon elution, released a 49,000 polypeptide. The immunohistochemical localization of mAb IX-50 binding sites in the chick cerebellar molecular layer coincided with that of the kainate receptor. We conclude that the 49,000 polypeptide is part of the kainate receptor and carries the kainate recognition site.     

Kainate Receptor Post-translational Modifications Differentially Regulate Association with 4.1N to Control Activity-dependent Receptor Endocytosis

Kainate receptors exhibit a highly compartmentalized distribution within the brain; however, the molecular and cellular mechanisms that coordinate their expression at neuronal sites of action are poorly characterized. Here we report that the GluK1 and GluK2 kainate receptor subunits interact with the spectrin-actin binding scaffolding protein 4.1N through a membrane-proximal domain in the C-terminal tail. We found that this interaction is important for the forward trafficking of GluK2a receptors, their distribution in the neuronal plasma membrane, and regulation of receptor endocytosis. The association between GluK2a receptors and 4.1N was regulated by both palmitoylation and protein kinase C (PKC) phosphorylation of the receptor subunit. Palmitoylation of the GluK2a subunit promoted 4.1N association, and palmitoylation-deficient receptors exhibited reduced neuronal surface expression and compromised endocytosis. Conversely, PKC activation decreased 4.1N interaction with GluK2/3-containing kainate receptors in acute brain slices, an effect that was reversed after inhibition of PKC. Our data and previous studies therefore demonstrate that these two post-translational modifications have opposing effects on 4.1N association with GluK2 kainate and GluA1 AMPA receptors. The convergence of the signaling pathways regulating 4.1N protein association could thus result in the selective removal of AMPA receptors from the plasma membrane while simultaneously promoting the insertion and stabilization of kainate receptors, which may be important for tuning neuronal excitability and synaptic plasticity.     

The interaction of a kainate receptor from goldfish brain with a pertussis toxin-sensitive GTP-binding protein.

Kainate receptors are present in high concentrations in goldfish brain (Henley and Oswald, 1988a and b; Ziegra et al., 1990), possibly in neuronal and glial cells. In a number of systems, the kainate receptor has been assumed to be an integral ion channel (Watkins and Evans, 1981); but, for some kainate receptors, ion channel activity has not been demonstrated (Wada et al., 1989). This study presents evidence that a portion of the [3H]kainate-binding sites in goldfish brain is sensitive to guanine nucleotides, with a loss of high affinity binding in the presence of nonhydrolyzable GTP analogs. Pertussis toxin pretreatment of membranes causes a loss of high affinity [3H]kainate binding and of the guanine nucleotide-sensitive binding. Pertussis toxin catalyzes the specific [32P]ADP-ribosylation of a 40-kDa substrate in a kainate-sensitive manner. In addition, incorporation of [alpha-32P]GTP-gamma-azidoanilide by photoaffinity labeling was enhanced in the presence of kainate. These results indicate that a subpopulation of [3H]kainate-binding sites in goldfish brain may be coupled to G proteins.     

Endogenous inhibitor of [3H]kainate binding to synaptic membrane in rat brain

An understanding of the mechanism of kainic acid toxicity to neurons could provide important clues to pathogenesis of Huntington's chorea. The existence of high-affinity binding sites for kainate, a foreign compound, is suggestive of the existence of kainate-like substances in the brain. In addition to such neurotoxic kainate-like substances, and endogenous inhibitor of kainate binding may also exist in the brain to allow the synaptic function to operate normally. Based on this idea, the existence of molecules which inhibit [3H]kainate binding to synaptic membranes was examined in rat brain. An endogenous inhibitor of [3H]kainate binding to synaptic membranes was found in the supernatant obtained from synaptic membranes of rat brain. The inhibitor is a thermostable, basic protein with a relatively low molecular weight.     

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.     

Biochemical characterization of an autoradiographic method for studying excitatory amino acid receptors using L-[3H]glutamate

A method was developed for radiolabeling excitatory amino acid receptors of rat brain with L-[3H]glutamate. Effective labeling of glutamate receptors in slide-mounted 10-microns sections was obtained using a low incubation volume (0.15 ml) and rapid washing: a procedure where high ligand concentrations were achieved with minimal waste. Saturation experiments using [3H]glutamate revealed a single binding site of micromolar affinity. The Bmax was trebled in the presence of Ca2+ (2.5 mM) and Cl- (20 mM) with no change in the Kd. Binding was rapid, saturable, stereospecific, and sensitive to glutamate receptor agonists. The proportions of [3H]glutamate binding sensitive to N-methyl-D-aspartate (NMDA), kainate, and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) were 34, 54, and 51%, respectively. NMDA inhibited binding at a distinct subset of L-[3H]glutamate sites, whereas AMPA and kainate competed for some common sites. Labeling of sections with L-[3H]glutamate in the presence of the selective agonists allowed autoradiographic visualization of glutamate receptor subtypes in brain tissue.     

Chemical and Surgical Lesions of Rat Olfactory Bulb: Changes in Thyrotropin-Releasing Hormone and Other Systems

Stereotaxic injection of kainic acid (15 micrograms) into rat olfactory bulbs was accompanied by a 53% (n = 4; p less than 0.02) depletion of endogenous thyrotropin-releasing hormone (TRH) as compared to sham-operated controls 2 weeks postlesion. TRH levels remained unaltered in three other caudal regions. Bulbar kainate lesions produced a 58% (n = 5; p less than 0.001) decrease in TRH receptor binding capacity without affecting the receptor affinity. Kainate lesions also reduced bulbar muscarinic and benzodiazepine receptors by 60% and 48%, respectively. Again, no changes in TRH receptors were apparent in six other brain areas after bulbar kainate treatment. Injection of the dopaminergic neurotoxin, 6-hydroxydopamine (8 micrograms), into rat bulbs decreased TRH receptors by 35% (n = 4; p less than 0.05) 1 week postlesion. One month after surgical bulbectomy, TRH and TRH receptor levels in a number of brain areas were unaltered compared to those of control animals. These studies suggest that TRH in the olfactory bulb originates intrinsically and may be produced predominantly for local use. Secondly, TRH receptors in the bulb appear to be postsynaptically localized on intrinsic neurons, although a small proportion are also associated with presynaptic elements of dopaminergic noradrenergic neurons. Bulbar TRH receptors exhibited nanomolar affinity and a pharmacological selectivity akin to that of the pituitary gland and other brain regions.     

The distribution of [3H]kainate binding sites in primate hippocampus is similar to the distribution of both Ca2+-sensitive and Ca2+-insensitive [3H]kainate binding sites in rat hippocampus

The distribution of [³H]kainate binding sites was determined by quantitative autoradiography in three vertebrate species: rat, monkey, and human. These animals displayed a similar pattern of binding site density in the hippocampus. Highest levels were found within the stratum lucidum and moderate levels in the inner portion of the dentate gyrus molecular layer. Although the distribution is similar, there is a lower density of binding sites in the stratum lucidum of primates than in rodents. Experiments using rat brain synaptic plasma membrane fractions indicated that inclusion of Ca²⁺ ions results in a selective reduction in binding at the high affinity sites. The Ca²⁺-inhibited and Ca²⁺-insensitive binding sites in the rat hippocampus exhibited a similar distribution. Together, these results suggest that in a variety of mammalian species kainate receptors exhibit similar regional distributions, and that the high anf loe affinity kainate binding sites also exhibit similar regional distributions.Special Issue dedicated to Prof. Eduardo De Robertis.     

Influence of age on NMDA receptor complex in rat brain studied by in vitro autoradiography

N-methyl-D-aspartate (NMDA) receptors are known to play an important role in learning and memory and to be involved in neuron cell death accompanying cerebral ischemia, seizures, and Alzheimer's disease. The NMDA receptor complex has been considered to consist of an L-glutamate recognition site, a strychnine-insensitive glycine modulatory site, and a voltage-dependent cation channel. In the present study, effects of age on an L-glutamate recognition site and a glycine site were examined in rat brain by quantitative in vitro autoradiography with [3H]-CPP and [3H]-glycine. Both [3H]-glycine and [3H]-CPP binding sites were most abundant in the hippocampus and cerebral cortex, and they showed a similar distribution pattern throughout the brain. [3H]-glycine binding sites were severely decreased in the telencephalic regions, including the hippocampus and cerebral cortex, in aged brain. Conversely, [3H]-CPP binding sites were well preserved in these brain areas. In the mid-brain regions and cerebellum, neither [3H]-glycine nor [3H]-CPP binding sites changed in the aged brain. Our results indicate that within the NMDA receptor complex, glycine receptors are primarily affected in the aging process.     

The physiological role of kainate receptors in the amygdala

The kainate subtype of glutamate receptors has received considerable attention in recent years, and a wealth of knowledge has been obtained regarding the function of these receptors. Kainate receptors have been shown to mediate synaptic transmission in some brain regions, modulate presynaptic release of glutamate and gamma-aminobutyric acid (GABA), and mediate synaptic plasticity or the development of seizure activity. This article focuses on the function of kainate receptors in the amygdala, a brain region that plays a central role in emotional behavior and certain psychiatric illnesses. Evidence is reviewed indicating that postsynaptic kainate receptors containing the glutamate receptor 5 kainate receptor (GLUk5) subunit are present on interneurons and pyramidal cells in the basolateral amygdala and mediate a component of the synaptic responses of these neurons to glutamatergic input. In addition, GLUk5-containing kainate receptors are present on presynaptic terminals of GABAergic neurons, where they modulate the release of GABA in an agonist concentration-dependent, bidirectional manner. GLUk5-containing kainate receptors also mediate a longlasting synaptic facilitation induced by low-frequency stimulation in the external capsule to the basolateral nucleus pathway, and they appear to be partly responsible for the susceptibility of the amygdala to epileptogenesis. Taken together, these findings have suggested a prominent role of GLUk5-containing kainate receptors in the regulation of neuronal excitability in the amygdala.     

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.     

Pharmacology of putative glutamate receptors from insect skeletal muscles, insect central nervous system and rat brain

Binding of [3H]glutamate to housefly brain and honeybee brain and thoracic muscle membranes as well as to the American cockroach nerve cord was measured in Na+-free Tris-citrate buffer, 2.5 mM CaCl2, pH 7.4. The dissociation constants (KDS) ranged from 0.16 to 1.36 microM, and thoracic muscles had 2-4-fold higher density of receptors than brain tissue. The potent inhibitors of housefly brain binding were in decreasing order of effectiveness: L-glutamate greater than L-aspartate = L-cysteate = ibotenate greater than quisqualate greater than L-homocysteate greater than L-APB greater than L-APV greater than NMDA greater than D-APB greater than D-glutamate, with no inhibition by 100 microM of GDEE, dihydrokainate, D-APV, D-homocysteate or D-aspartate. The drug specificity of [3H]glutamate binding sites in housefly brain was generally similar to that of binding sites in housefly muscle, except that the former had a slightly higher affinity for L-APB, L-homocysteate and NMDA. [3H]Glutamate binding to insect tissues differed in its drug sensitivity from binding to rat brain. Binding to insect membranes was much less sensitive to L-APB, D-APB, APV, homocysteate, L-cysteate, quisqualate and ibotenate. However, the insect binding site was much more stereoselective for the L than D isomers of glutamate and aspartate, while the rat brain site was more stereoselective for APB. It is suggested that the observed [3H]glutamate binding to insect tissue is not to NMDA or kainate receptors.(ABSTRACT TRUNCATED AT 250 WORDS)