Stacking-interactions in the control-gear binding of kynurenic acid with NR2A- and GluR2-subunits of glutamate ionotropic receptors

Kynurenine products in tryptophan metabolism are of crucial importance in modulation of neurodegenerative processes in the CNS. Kynurenic acid (KYNA): the endogenous antagonist of ionotropic glutamate receptors, displays specific affinity towards glycine site ofNMDA-receptor NR1 subunit. Mechanisms for the selective interaction of KYNA and its derivatives with other glutamate receptor subtypes are studied insufficiently. Ab initio quantum chemical calculations for KYNA-imidazole dimer, as a model for ligand interaction with His88 fragment of NR2A-subunit, along with KYNA-phenol dimer, as a model for ligand interaction with Tyr61 fragment of GluR2-subunit, were carried out in order to investigate stacking-interaction role of KYNA binding by NR2A subunit of NMDA-receptor and GluR2 subunit of AMPA-receptor. Stacking-interaction energy of KYNA-H88 for the assumed ligand orientation in the binding site is 3.0-5.0 kcal/mol and 102. kcal/mol for the optimized dimer KYNA-imidazole geometry. Stacking-interaction energy of KYNA-Tyr61 for the assumed ligand orientation in the binding site is 6.7-8.5 kcal/mol. The obtained values are comparable with the energies of hydrogen bonds. Thus, stacking-interaction should be taken into account while studing ligand glutamate receptor binding mechanisms. Stacking-interaction is evidently important for the initial ligand orientation inside the receptor binding site after which the delicate tuning of hydrogen bonding pattern is realized. On the other hand, the specific affinity of KYNA derivatives to the receptor subunits could be explained by ligand-aromatic receptor aminoacid stacking-interaction geometry difference.     

Neurosteroid binding to the amino terminal and glutamate binding domains of ionotropic glutamate receptors

The endogenous neurosteroids, pregnenolone sulfate (PS) and 3α-hydroxy-5β-pregnan-20-one sulfate (PREGAS), have been shown to differentially regulate the ionotropic glutamate receptor (iGluR) family of ligand-gated ion channels. Upon binding to these receptors, PREGAS decreases current flow through the channels. Upon binding to non-NMDA or NMDA receptors containing an GluN2C or GluN2D subunit, PS also decreases current flow through the channels, however, upon binding to NMDA receptors containing an GluN2A or GluN2B subunit, flow through the channels increases. To begin to understand this differential regulation, we have cloned the S1S2 and amino terminal domains (ATD) of the NMDA GluN2B and GluN2D and AMPA GluA2 subunits. Here we present results that show that PS and PREGAS bind to different sites in the ATD of the GluA2 subunit, which when combined with previous results from our lab, now identifies two binding domains for each neurosteroid. We also show both neurosteroids bind only to the ATD of the GluN2D subunit, suggesting that this binding is distinct from that of the AMPA GluA2 subunit, with both leading to iGluR inhibition. Finally, we provide evidence that both PS and PREGAS bind to the S1S2 domain of the NMDA GluN2B subunit. Neurosteroid binding to the S1S2 domain of NMDA subunits responsible for potentiation of iGluRs and to the ATD of NMDA subunits responsible for inhibition of iGluRs, provides an interesting option for therapeutic design.     

Linking tricyclic antidepressants to ionotropic glutamate receptors

Although tricyclic antidepressants have been in existence since the 1940s when they were discovered upon screening iminodibenzyl derivatives for other potential therapeutic uses, their mechanism of action has remained unclear [A. Goodman Gilman, T.W. Rall, A.S. Nies, P. Taylor, Goodman and Gilman's The Pharmacological Basis of Therapeutics, eighth ed., Pergamon Press, New York, 1990]. In addition to their ability to hinder the reuptake of biogenic amines, there is mounting evidence that the tricyclic antidepressants inhibit glutamate transmission. Here, intrinsic tryptophan fluorescence spectroscopy is used to document the binding of desipramine, a member of the tricyclic antidepressant family, to a well-defined extracellular glutamate binding domain (S1S2) of the GluR2 subunit of the amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor. The binding is distinct from those of other known effectors of the receptor, including the endogenous sulfated neurosteroids pregnenolone sulfate and 3alpha-hydroxy-5beta-pregnan-20-one sulfate, and is consistent with a conformational change upon binding that is allosterically transmitted to the channel region of the receptor.     

Direct interaction enables cross-talk between ionotropic and group I metabotropic glutamate receptors

Functional interplay between ionotropic and metabotropic receptors frequently involves complex intracellular signaling cascades. The group I metabotropic glutamate receptor mGlu5a co-clusters with the ionotropic N-methyl-d-aspartate (NMDA) receptor in hippocampal neurons. In this study, we report that a more direct cross-talk can exist between these types of receptors. Using bioluminescence resonance energy transfer in living HEK293 cells, we demonstrate that mGlu5a and NMDA receptor clustering reflects the existence of direct physical interactions. Consequently, the mGlu5a receptor decreased NMDA receptor current, and reciprocally, the NMDA receptor strongly reduced the ability of the mGlu5a receptor to release intracellular calcium. We show that deletion of the C terminus of the mGlu5a receptor abolished both its interaction with the NMDA receptor and reciprocal inhibition of the receptors. This direct functional interaction implies a higher degree of target-effector specificity, timing, and subcellular localization of signaling than could ever be predicted with complex signaling pathways.     

Differential regulation of ionotropic glutamate receptors

Ionotropic glutamate receptors (iGluRs), a family of ligand-gated ion channels, are responsible for the majority of fast excitatory neurotransmission in the central nervous system. Within this family, different members serve distinct roles at glutamatergic synapses. Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors mediate fast depolarization while N-methyl-D-aspartate (NMDA) receptors mediate the slower component of the excitatory postsynaptic potential. These disparate functions suggest alternate modes of regulation. In this work, we show that endogenous regulators of iGluRs have different abilities to bind to specific domains of NMDA NR1-1b and AMPA GluR2 subunits. We have previously shown that the sulfated neurosteroids pregnenolone sulfate and 3α-hydroxy-5β-pregnan-20-one sulfate bind to the extracellular glutamate-binding core (S1S2) of the GluR2 subunit. Here we show that neither neurosteroid binds to the S1S2 domain of the NMDA NR1-1b subunit. This NR1-1b NMDA domain does, however, bind to the endogenous polyamines spermine and spermidine as well as Zn(II). Binding of the polyamines and Zn(II) to the S1S2 domain of the GluR2 subunit was not observed. This binding of Zn(II) and polyamines to the S1S2 domain of the NR1-1b subunit defines a new binding site for each of these modulators.     

Dynamics and allostery of the ionotropic glutamate receptors and the ligand binding domain

The dynamics of the ligand‐binding domain (LBD) and the intact ionotropic glutamate receptor (iGluR) were studied using Gaussian Network Model (GNM) analysis. The dynamics of LBDs with various allosteric modulators is compared using a novel method of multiple alignment of GNM modes of motion. The analysis reveals that allosteric effectors change the dynamics of amino acids at the upper lobe interface of the LBD dimer as well as at the hinge region between the upper‐ and lower‐ lobes. For the intact glutamate receptor the analysis show that the clamshell‐like movement of the LBD upper and lower lobes is coupled to the bending of the trans‐membrane domain (TMD) helices which may open the channel pore. The results offer a new insight on the mechanism of action of allosteric modulators on the iGluR and support the notion of TMD helices bending as a possible mechanism for channel opening. In addition, the study validates the methodology of multiple GNM modes alignment as a useful tool to study allosteric effect and its relation to proteins dynamics. Proteins 2016; 84:267–277. © 2015 Wiley Periodicals, Inc.     

Facilitation of glutamate release by ionotropic glutamate receptors in osteoblasts

Constitutive expression of mRNA was seen for the vesicular glutamate transporter brain-specific Na(+)-dependent inorganic phosphate cotransporter (BNPI), but not differentiation-associated Na(+)-dependent inorganic phosphate cotransporter, in rat calvarial osteoblasts cultured for 7 and 21 days in vitro (DIV). Three different agonists for ionotropic glutamate receptors (iGluR) at 1mM, as well as 50mM KCl, significantly increased the release of endogenous L-glutamate from osteoblasts cultured for 7DIV when determined 5 min after the addition by using a high performance liquid chromatograph. The inhibitor of desensitization of DL-alpha-amino-3-hydroxy-5-methylisoxasole-4-propionate (AMPA) receptors cyclothiazide significantly potentiated and prolonged the release of endogenous L-glutamate evoked by AMPA in a dose-dependent manner. The release evoked by AMPA was significantly prevented by the addition of an AMPA receptor antagonist as well as by the removal of Ca(2+) ions. These results suggest that endogenous L-glutamate could be released from intracellular vesicular constituents associated with BNPI through activation of particular iGluR subtypes expressed in cultured rat calvarial osteoblasts.     

Functional architecture of olfactory ionotropic glutamate receptors

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Characterization of ionotropic glutamate receptors in insect neuro-muscular junction

Pharmacological properties of ionotropic glutamate receptors from Calliphora vicina larvae neuro-muscular junction (C. vicina iGlurs) were studied by two-electrode voltage-clamp technique. Characteristics of the ion channel pore were analyzed using a 26-member series of channel blockers, which includes mono- and dicationic derivatives of adamantane and phenylcyclohexyl. Structure-activity relationships were found to be markedly similar to the Ca2+-permeable AMPA receptors (AMPAR) but not NMDA receptors (NMDAR) channel subtype seen in vertebrates. Like AMPARs the channels of C. vicina iGlurs are sensitive mainly to dicationic compounds with 6-7 spacers between hydrophobic headgroup and terminal aminogroup. Study of the voltage dependence of block demonstrated that, like AMPARs, the C. vicina iGlur channels, are permeable to organic cations with dimensions exceeding 10 A. Concentration dependence of block suggests the presence of two distinct channel populations with approximately 20-fold different sensitivity to cationic blockers. The recognition domain properties are more complex. Besides glutamate, the channels can be activated by kainate, quisqualate and domoate. Competitive antagonists of AMPAR and NMDAR are virtually inactive against the C. vicina iGlurs as well as allosteric modulators GYKI 52466 and PEPA. Surprisingly, the responses were potentiated 3 times by 100 mkM of cyclothiazide. We conclude that the channel-forming domain of C. vicina iGlurs is AMPAR-like, whereas the recognition domain is specific.     

The role of ionotropic receptors of glutaminic acid in cardiovascular system

Summary.  The aim of our study was to estimate the involvement of the peripheral N-methyl-D-aspartate receptors in regulation of cardiovascular function. For this purpose we examined the effects of intravenous injection of the agonists – NMDA (0.025; 0.05 and 1.0 mg/kg iv) and 1R-3R-ACPD (0.025; 0.05 and 1.0 mg/kg iv) – and antagonist of NMDA receptors DL-AP7 (0.02; 0.07 and 0.2 mg/kg iv). To determine if the effects of NMDA come from central or peripheral action we observed the effect during blockade of autonomic ganglion by using the nicotinic receptor antagonist – chlorisondamine (1.25 mg/kg iv). Administration of NMDA in three doses evoked slight hypotension after injection of the medium dose, 0.05 mg/kg. In the condition of pretreatment with 1.25 mg/kg chlorisondamine the hypotensive effect of NMDA was markedly reduced, what might suggest that NMDA-induced hypotension raised from the action within the brain. The competetive NMDA receptor antagonist DL-AP7 slightly increased the blood pressure. None of the injected drug had an influence on the heart rate in our in vivo study. It is concluded that the peripherally localized NMDA receptors may take a part in regulation of cardiovascular system, since their stimulation or blockade evoked the changes of systemic pressure. Received August 6, 2002 Accepted October 10, 2002 Published online January 20, 2003 Aknowledgments This study was supported by a grant No 3-10871 from the State Committee for Scientific Research, Warszawa, Poland. The authors thank Ms. A. Barwińska and Ł. Stalenczyk for technical help. Authors' address: Prof. Konstanty Wiśniewski, Department of Pharmacology, Medical University of Białystok, Mickiewicza 2c, PL-15-222 Białystok, Poland     

The origin and molecular evolution of ionotropic glutamate receptors

A review of the main approaches to the revealing molecular evolution of glutamate receptors is presented. Large amount of evidences concerning the homology of glutamate-binding proteins forming the membrane channels has been accumulated. However, the knowledge of amino acid sequences of these proteins is the necessary but not sufficient condition for clarification of their origin and the changes in the course of molecular evolution. The natural selection estimated and secured the functional validity ofligand-gated channels. Therefore the functional and molecular approaches should supplement each other. It has been shown by and example of glutamate receptor channels of vertebrate and invertebrate animals that the combined analysis of the structure and function allows to reveal the main routes of molecular evolution of this kind of synaptic receptors.     

Neurosteroid modulation of ionotropic glutamate receptors and excitatory synaptic transmission

Ionotropic glutamate receptors function can be affected by neurosteroids, both positively and negatively. N-methyl-D-aspartate (NMDA) receptor responses to exogenously applied glutamate are potentiated or inhibited (depending on the receptor subunit composition) by pregnenolone sulphate (PS) and inhibited by pregnanolone sulphate (3alpha5betaS). While PS effect is most pronounced when its application precedes that of glutamate, 3alpha5betaS only binds to receptors already activated. Synaptically activated NMDA receptors are inhibited by 3alpha5betaS, though to a lesser extent than those tonically activated by exogenous glutamate. PS, on the other hand, shows virtually no effect on any of the models of synaptically activated NMDA receptors. The site of neurosteroid action at the receptor molecule has not yet been identified, however, the experiments indicate that there are at least two distinct extracellularly located binding sites for PS mediating its potentiating and inhibitory effects respectively. Experiments with chimeric receptors revealed the importance of the extracellular loop connecting the third and the fourth transmembrane domain of the receptor NR2 subunit for the neurosteroid action. alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)/kainate receptors are inhibited by both PS and 3alpha5betaS. These neurosteroids also affect AMPA receptors-mediated synaptic transmission, however, in a rather indirect way, through presynaptically located targets of action.     

Pharmacology of ionotropic glutamate receptors: A structural perspective

The impact of structural biology on the design of ligands (agonists, antagonists and modulators) for ionotropic glutamate receptors is reviewed.     

Activation of ionotropic receptors and thermodynamics of binding

The structure, thermodynamics and activation mechanism of Cys-loop ionotropic receptors such as glycine, nicotinic acetylcholine, 5-HT3-type serotonin and A-type gamma-aminobutyric acid receptors are discussed. Based on the interrelationship of receptor binding and ionophore function, a ternary displacement mechanism of binding including the activation of ionophores is outlined. This displacement model can explain the enigmatic thermodynamic discrimination of agonists versus antagonists of Cys-loop ionotropic receptors. Binding of both agonists and antagonists is exothermic while activation is endothermic driven by large increases in entropy. Closure of the binding cavities around agonists in concert with subunit rotations and/or removal of water-filled crevices between transmembrane (TM) regions can account for entropy increases. Recombinant glycine and gamma-aminobutyric acidA receptors and their point mutations support the predominant role of entropy in receptor activation.     

Fleeting activation of ionotropic glutamate receptors sensitizes cortical neurons to complement attack

Insidious attack of cortical neurons by complement has been implicated in Alzheimer's and other neurodegenerative diseases. Excitotoxicity, triggered by excessive activation of glutamate receptors, has been implicated in neuronal death following diverse insults, including ischemia and seizures. Clinical studies suggested that a minimal excitotoxic insult might sensitize neurons to complement attack. We found that fleeting activation of ionotropic glutamate receptors sensitizes neurons but not astrocytes to complement attack. The complement molecule effecting cytotoxicity was the membrane attack complex. The site within the complement cascade at which sensitization was effected was the membrane attack pathway. Sensitization mediated by glutamate receptor activation required Ca(2+)(o) and generation of reactive oxygen species. These in vitro findings predict that a fleeting excitotoxic insult could act synergistically with complement to destroy cortical neurons and accelerate neurological deterioration.     

Involvement of ionotropic glutamate receptors in the genesis of arecoline-induced tremor

The muscarinic agonist arecoline (6 mg/kg, subcutaneously in mice) induced a long-lasting tremor. The inhibitory potency of non-competitive antagonists of ionotropic glutamate receptors has been studied. These antagonists are the derivatives of adamantane and phenylcyclohexyl. A part of them: monocationic compounds, selectively block the NMDA-receptor channels, their dicationic analogues affecting both channels of the NMDA- and the AMPA-glutamate receptors. Monocationic blockers effectively reduced the arecoline-evoked tremor and their potency correlated with ability to block the NMDA-receptor channels. Dicationic blockers revealed protective effect only in low range doses (0.0001-0.01 microM/kg). Further increase of the dose reduced or completely abolished this effect. This suggests that the NMDA-receptors are involved in the genesis of arecoline-evoked tremor. The only moderate blockade of the AMPA-receptors potentiates the drug blocking action but the prevalent blockade of these receptors impedes the effect on arecoline-evoked tremor.