Glu Binding Sites Research Articles

Subunit stoichiometry and arrangement in a heteromeric glutamate-gated chloride channel

The invertebrate glutamate-gated chloride-selective receptors (GluClRs) are ion channels serving as targets for ivermectin (IVM), a broad-spectrum anthelmintic drug used to treat human parasitic diseases like river blindness and lymphatic filariasis. The native GluClR is a heteropentamer consisting of α and β subunit types, with yet unknown subunit stoichiometry and arrangement. Based on the recent crystal structure of a homomeric GluClαR, we introduced mutations at the intersubunit interfaces where Glu (the neurotransmitter) binds. By electrophysiological characterization of these mutants, we found heteromeric assemblies with two equivalent Glu-binding sites at β/α intersubunit interfaces, where the GluClβ and GluClα subunits, respectively, contribute the "principal" and "complementary" components of the putative Glu-binding pockets. We identified a mutation in the IVM-binding site (far away from the Glu-binding sites), which significantly increased the sensitivity of the heteromeric mutant receptor to both Glu and IVM, and improved the receptor subunits' cooperativity. We further characterized this heteromeric GluClR mutant as a receptor having a third Glu-binding site at an α/α intersubunit interface. Altogether, our data unveil heteromeric GluClR assemblies having three α and two β subunits arranged in a counterclockwise β-α-β-α-α fashion, as viewed from the extracellular side, with either two or three Glu-binding site interfaces.

Probing Allosteric Relationships Between the Neurotransmitter-Binding Site and the Binding Pocket of the Anthelmintic Drug Ivermectin in Glutamate-Gated Chloride Channels

Ivermectin (IVM) is a broad-spectrum anthelmintic drug used to treat human parasitic diseases like onchocerciasis (river blindness) and elephantiasis (lymphatic filariasis). IVM acts on invertebrate Cys-loop receptors (GluClRs) that naturally bind the neurotransmitter glutamate (Glu) to open an intrinsic chloride-selective channel. Native heteropentameric GluClα/βRs are irreversibly stabilized in an open-channel conformation by IVM. Hence, IVM causes sustained inflow of chloride ions and hyperpolarization that suppress nervous impulses in nematodes. Consequently, vital physiological processes in the nematode are abolished. Upon expression in Xenopus oocytes, homomeric GluClαRs are activated (being opened) by IVM but not by Glu, whereas homomeric GluClβRs are activated by Glu but not by IVM (Cully et al., Nature, 1994). A heteromeric GluClα/βR carrying a mutation in the β-subunit that inhibits Glu binding is readily activated by IVM (Li et al., FEBS Lett, 2002). These differential responses suggest that the drug and the neurotransmitter binding sites are independent of each other. Yet, a recent X-ray crystal structure shows that IVM-bound homomeric GluClαR can accommodate Glu (Hibbs and Gouaux, Nature, 2011), in line with a study showing that robust activation of homomeric GluClαRs by IVM enables further (slight) activation by Glu (Etter et al., JBC, 1996). Here, we explored whether the structurally separated IVM and Glu binding sites are functionally coupled in heteromeric GluClα/βRs. Mutations introduced between the Glu and IVM binding pockets or inside the IVM binding pocket were examined for their influence on agonistic efficacies. We found mutations that improve IVM efficacy without changing Glu efficacy, and mutations that either reduce or increase the agonistic efficacies of IVM and Glu concomitantly. Our results strongly indicate that the IVM and Glu binding sites in heteromeric GluClRs are functionally coupled.

Elucidation of a Novel Extracellular Calcium-binding Site on Metabotropic Glutamate Receptor 1α (mGluR1α) That Controls Receptor Activation*

Metabotropic glutamate receptor 1α (mGluR1α) exerts important effects on numerous neurological processes. Although mGluR1α is known to respond to extracellular Ca(2+) ([Ca(2+)](o)) and the crystal structures of the extracellular domains (ECDs) of several mGluRs have been determined, the calcium-binding site(s) and structural determinants of Ca(2+)-modulated signaling in the Glu receptor family remain elusive. Here, we identify a novel Ca(2+)-binding site in the mGluR1α ECD using a recently developed computational algorithm. This predicted site (comprising Asp-318, Glu-325, and Asp-322 and the carboxylate side chain of the receptor agonist, Glu) is situated in the hinge region in the ECD of mGluR1α adjacent to the reported Glu-binding site, with Asp-318 involved in both Glu and calcium binding. Mutagenesis studies indicated that binding of Glu and Ca(2+) to their distinct but partially overlapping binding sites synergistically modulated mGluR1α activation of intracellular Ca(2+) ([Ca(2+)](i)) signaling. Mutating the Glu-binding site completely abolished Glu signaling while leaving its Ca(2+)-sensing capability largely intact. Mutating the predicted Ca(2+)-binding residues abolished or significantly reduced the sensitivity of mGluR1α not only to [Ca(2+)](o) and [Gd(3+)](o) but also, in some cases, to Glu. The dual activation of mGluR1α by [Ca(2+)](o) and Glu has important implications for the activation of other mGluR subtypes and related receptors. It also opens up new avenues for developing allosteric modulators of mGluR function that target specific human diseases.

Glycine stimulation of glutamate binding to chick retinal pigment epithelium.

The effect of glycine (Gly) and taurine (Tau) on the biochemical and pharmacological properties of [3H]L-glutamate ([3H] Glu) binding to membranes from primary cultures of chick retinal pigment epithelium (RPE), as well as from intact tissue during development was studied. Gly and Tau increase Bmax of [3H]Glu binding to a high affinity site (KB = 300 nM) in membranes from 16 days in vitro (immature) cultures; additionally, Gly discloses a low affinity Glu-binding site (KB = 970 nM) at this stage. In membranes from 25 days in vitro (mature) cultures, the high affinity site is no longer present and Tau has no effect on Glu-binding; Gly still stimulates binding to the low affinity site by four fold, with an EC50 = 200 microM. Pharmacological profile using specific excitatory amino acid (EAA) receptor agonists and antagonists suggests that at 16 days in vitro Glu binds preferentially to metabotropic Glu receptors (mGluRs), and at 25 days in vitro to ionotropic receptors different from neuronal ones. The stimulatory effect of Gly and Tau was also observed in intact RPE, and decreased with increasing embryonic age. Glu binding was also stimulated in membranes from chick retina, but not in those from rat brain. Results support the possibility of EAA participation in several aspects of RPE physiology, including phagocytosis and cell division.

Preferential Induction by Stress of the N‐Methyl‐d‐Aspartate Recognition Domain in Discrete Structures of Rat Brain

Immobilization stress in water for 3 h was effective in inducing significant potentiation of [3H](+)-5-methyl-10,11-dihydro-5H- dibenzo [a,d] cyclohepten-5,10-imine ([3H]MK-801) binding 5 days after the stressful manipulation in rat hypothalamus and cerebellum when determined before equilibrium in the absence of any added agonists, in addition to resulting in marked reduction of rearing behaviors of animals. However, the stressful manipulation failed to modulate the [3H]MK-801 binding in other central regions examined, and binding of either [3H]DL-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid or [3H]kainic acid was not significantly affected in all brain structures studied 5 days after the stress application. In contrast, the stressful procedures potentiated binding of both L-[3H]glutamic ([3H]Glu) and [3H]DL-(E)-2-amino-4-propyl-5-phosphono-3-pentenoic ([3H]CGP-39653) acids in the hypothalamus and cerebellum 5 days later, without affecting binding of [3H]-glycine and 5,7-dichloro[3H]kynurenic acid. The systemic administration of corticosterone mimicked the stress manipulation at doses of 5-50 mg/kg in terms of inducing significant enhancement of binding of both [3H]Glu and [3H]CGP-39653 in the hypothalamus and cerebellum when determined 5 days after the single administration. The translation inhibitor cycloheximide was effective in preventing the stress-induced potentiation of [3H]Glu binding in the cerebellum, without altering that in the hypothalamus. Furthermore, the stressful handling significantly increased the densities of [3H]Glu binding sites in the hypothalamus and cerebellum, with the affinities being unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)

Structural model for the reaction mechanism of glutamine synthetase, based on five crystal structures of enzyme-substrate complexes.

Glutamine synthetase brings nitrogen into metabolism by condensing ammonia and glutamate, with the aid of ATP, to yield glutamine, ADP, and inorganic phosphate. Here we present five crystal structures of GS complexed with each of two substrates, Glu and AMPPNP (an ATP analog), with a transition-state analogue, L-methionine-S-sulfoximine, and with each of two products, Gln and ADP. GS of the present study is from Salmonella typhimurium, has Mn2+ bound, and is fully unadenylylated. Protein-metal-substrate interactions and small but significant conformational changes induced by substrate binding are defined by Fourier maps. On the basis of these maps, we propose a tentative structure-based enzymatic mechanism of glutamine synthesis with these steps: (1) ATP binds first at the top of the funnel-shaped active site cavity, adjacent to the n2 Mn2+; Arg 359 moves toward the Glu binding site. (2) Glu binds adjacent to the n1 Mn2+ at the bottom of the active site near a flexible loop (residues 324-328). As proposed earlier by Meister and others, Glu attacks the gamma-phosphorus atom of ATP to produce gamma-glutamyl phosphate and ADP. (3) The presence of ADP (but not ATP) moves Arg 339 toward the Pi site, perhaps stabilizing the gamma-glutamyl phosphate, and moves Asp 50' of the adjacent subunit toward a putative ammonium ion site, enhancing binding of this third substrate. Deprotonation of the ammonium ion, perhaps by Asp 50', permits the resulting active species, ammonia, to attack the gamma-glutamyl phosphate, forming a tetrahedral intermediate.(ABSTRACT TRUNCATED AT 250 WORDS)

Synthesis of vitamin K‐dependent proteins

The unique and characteristic feature of vitamin K-dependent proteins is the presence of gamma-carboxyglutamyl (Gla) residues formed during the posttranslational processing of these proteins. The energy needed to drive this microsomal carboxylation event comes from the reoxidation of the reduced, hydronaphthoquinone form of vitamin K to its 2,3-epoxide. Recent studies have suggested that an intermediate epoxide alkoxide is the strong base needed to abstract a proton from the relatively unreactive methylene carbon of the glutamyl residue. The primary gene product of the vitamin K-dependent proteins contains a homologous propeptide extension between the amino terminus of the mature protein and signal peptide. This region, which is cleaved before secretion of these proteins, serves to dock the protein substrate to the enzyme catalyzing the carboxylation event, and to also alter the apparent Km of the Glu binding site of the enzyme. The order in which the multiple Glu sites on the substrate proteins are carboxylated is unknown, but elucidation of this property of the enzyme and further details of the bioorganic mechanism should be aided by recent reports of purification of this unique carboxylase.

Disclosure by triton X-100 of NMDA-sensitive [ 3H]glutamate binding sites in brain synaptic membranes

Pretreatment of brain synaptic membrane homogenates with Triton X-100 resulted in a drastic disclosure of [3H] glutamate (Glu) binding activity which was sensitive to one of the central Glu receptor agonists, N-methyl-D-aspartic acid (NMDA). The NMDA-sensitive binding was inversely dependent on the incubation temperature, and was a reversible and saturable process. Scatchard analysis revealed that Triton X-100 treatment yielded in a significant enhancement of the affinity with a concomitant increment of the density of binding sites. Electrophysiologically identified agonists and antagonists for the NMDA receptors all significantly inhibited the binding to Triton-treated membranes. These results suggest that Triton-treatment may disclose NMDA-sensitive [3H] Glu binding sites in brain synaptic membranes.

Characterization of [3H]glutamate binding sites on frozen sections from rat adrenal

Some biochemical characteristics of [(3)H]glutamate (Glu) binding sites on frozen sections from the rat adrenal glands were studied. Adrenal frozen sections exhibited stereo-selective, saturable and temperature-dependent binding of [(3)H]Glu. An agonist for one of the subclasses of central Glu receptors, quisqualic acid (QA), elicited a significant inhibition of the binding, whereas neither N-methyl-d-aspartic acid nor kainic acid, agonists for other subclasses of the receptors, had such a significant effect on the binding at the concentration range similar to QA. In vitro addition of sodium acetate (100 mM) resulted in a significant inhibition of [(3)H]Glu binding to frozen sections of the rat adrenal glands. It thus appears that there exist QA-sensitive binding sites of [(3)H]Glu in the rat adrenal glands which exhibit pharmacological characteristics distinctly different from those in the brain.

Functional and biochemical characteristics of a putative quisqualate-type receptor in rat striatum: effect of brain lesions.

Excitatory amino acids such as L-glutamate (Glu) and quisqualate (QUIS) markedly potentiated K+-evoked release of exogenous [3H]dopamine (DA) from rat striatal slices. Intrastriatal kainic acid injections resulted in a total disappearance of the stimulatory effects of Glu on evoked-release of [3H]DA as well as in a parallel reduction in the maximal number (Bmax) of a D-aspartate-insensitive L-[3H]Glu binding site in striatal particulate fractions. Following cortical ablation, the potentiating effect of Glu on [3H]DA release in decorticated striatal slices lasted longer, compared to normal slices, and occurred during the 2nd min following K+-depolarization. However, the extent (%) of Glu stimulation on [3H]DA release remained the same in decorticated and normal striatal slices. Cortical ablation produced also a significant decrease in the Bmax and in the KD' of the D-aspartate-insensitive binding site towards L-[3H]Glu. These results support the proposal that the D-aspartate-insensitive Glu binding site is somehow related to an amino acid receptor-mediated modulation of dopaminergic transmission in the rat corpus striatum.

Development of a micromethod to study the Na +-independent l-[ 3H]glutamic acid binding to rat striatal membranes. II. Effects of selective striatal lesions and deafferentations

An apparent single class of Na +-independent l-[ 3H]glutamate ( l-[ 3H]Glu) binding sites was biochemically and pharmacologically identified on rat striatal tissue. The K d value was 1.75 μM and the B max3.89 nmol/g protein. In order to further elucidate the putative physiological role of these binding sites and to valid our binding assay, experiments were conducted to determine the anatomical location of the l-[ 3H]Glu binding sites in the striatum. Local injection of the neurotoxin, kainic acid into the striatum caused degeneration of target cells in the structure followed by an important decrease (-37%) in the number of these binding sites, with no significant change in the affinity constant. Lesions of the cortical frontal and parietal areas projecting to the striatum via putative glutamatergic fibers led, on the other hand, after 3 weeks to one month, to a significant increase (+23%) in the number of l-[ 3H]Glu binding sites. The K d value does not significantly change after decortication. Finally, suppression either of the nigrostriatal dopaminergic input or of the partially cholinergic thalamostriatal tract do not affect [ 3H]Glu binding to striatal tissue. These results suggest that about 40% of our [ 3H]Glu binding sites are located on striatal target cell sensitive to the neurotoxic effect of kainic acid. Therefore, they seem to be partly postsynaptic. The existence of a relation between these binding sites and the corticostriatal glutamatergic input was shown by the development of a supersensitivity response after suppression of this cortical input. These sites might therefore constitute one of the receptor subclass for Glu linked to the excitatory action of the corticostriatal afferent. Since degeneration of either the nigrostriatal dopaminergic or the thalamostriatal fibres did not affect the binding of l-[ 3H]Glu characterized in our study, the binding sites will probably not occur at the presynaptic level on these nerve terminals in the striatum.

Specific binding of L-[3H]-glutamic acid to rat substantia nigra synaptic membranes.

The specific binding of L-[3H]-glutamic acid (GLU) was investigated in synaptic membranes from rat substantia nigra. L-[3H]-GLU binding to the membrane preparations occurred in a reversible and saturable way. The specific binding was stimulated by the presence of CaCl2 and was reduced by freezing and thawing the membranes. Scatchard analysis of the saturation isotherms yielded a non-linear plot suggesting that the binding reaction does not occur through a simple bimolecular association. Assuming non-interacting binding sites, a high (KD1, 139 nM; Bmax1, 3.5 pmoles/mg protein) and a low (KD2, 667 nM; Bmax2, 15.1 pmoles/mg protein) affinity L-[3H]-GLU binding site were obtained. The kinetics of dissociation of bound L-[3H]-GLU was biphasic; the respective dissociation rate constant (k-1) being 0.20 min-1 and 0.013 min-1. A series of amino acid receptor agonists and antagonists were tested as inhibitors of L-[3H]-GLU specific binding. Quisqualic acid, L-GLU and D-alpha-aminoadipate (D-alpha-AA) were the most potent inhibitors. DL-2-amino-4-phosphonobutyrate (APB), N-Methyl-D-aspartate (NMDA) and D-GLU were moderate inhibitors, whereas diaminopimelic acid (DAPA) and glutamate diethyl ester (GDEE) exhibited the lowest relative potency. Kainic acid (KA), gamma-aminobutyric acid (GABA) and bicuculline were not able to modify at any concentration used the specific binding of L-[3H]-GLU. These data demonstrate the presence of specific GLU binding sites in synaptic structures at substantia nigra level and support the idea that excitatory amino acids may play a role in synaptic transmission in this brain region.

Possible interaction of [3H]glutamate binding sites with anion channels in rat neural tissues

The effect of various ions on [(3)H]l-glutamic acid (Glu) binding was examined using crude synaptic membrane preparations from the rat brain. In vitro addition of sodium acetate (1-100 mM) exhibited a significant enhancement of the binding in a concentration dependent manner. Ammonium chloride (20 mM) prevented the potentiation by sodium acetate at 2 degrees C, whereas sodium acetate exerted an inhibitory action on the ammonium chloride-induced augmentation of the binding at 30 degrees C. Ammonium chloride (1-100 mM) itself elicited a temperature dependent stimulation of the binding, which was invariably attenuated by an antagonist for the anion channels such as picrotoxinin (10(?3) M) as well as by inhibitors of anion transport including ethacrynic acid (10(?3) M) and 4,4?-diisothiocyanatostilbene-2,2?-disulfonic acid (10(?4)?10(?3) M), respectively. The later two inhibitors also caused a significant additional raise of the sodium acetate-induced enhancement of the binding. A significant augmentation of the binding resulted from the addition (20 mM) of various anions known to penetrate the anion channels such as bromide, iodide, nitrate, bicarbonate and thiocyanate in a permeability related manner, while that of non-permeable anions including fluoride, sulfate, acetate, formate, phosphate, oxalate, lactate, succinate and tartarate had no such a profound effect on the binding. Addition of d-aspartic acid resulted in the complete abolition of the Na(+)-dependent binding while sparing the Cl(?)-dependent binding. Scatchard analysis revealed that Cl(?) ions induced a two-fold increase in the number of the binding sites without affecting their affinity, whereas Na(+) ions reduced the affinity with a concomitant increase of the number of the binding sites. Addition of quisqualic acid (10(?5)?10(?3) M) inhibited the Cl(?)-dependent binding of [(3)H]Glu to a significantly greater extent than the inhibition on Na(+)-dependent binding. N- Methyl- d -aspartic acid and kainic acid exerted no preventive action on the basal, Cl(?)-dependent and Na(+)-dependent binding. respectively. The highest basal binding activity was found in the retina among various central structures examined. A significant basal binding activity of [(3)H]Glu was also detected in the pituitary and adrenal but not in the kidney. Chloride ions exhibited a significant facilitation of [(3)H]Glu binding to central regions without altering that to peripheral tissues such as pituitary and adrenal. In contrast, Na(+) ions induced significant attenuation of the binding to the pituitary, adrenal and retina despite the occurrence of augmentation of the binding to other central structures. These results suggest the Glu binding sites may be linked to the anion channels in the rat central nervous system and that this linkage may be absent from the pituitary, adrenal and retina.