1,384 research outputs found
Phosphatidylinositol 3 kinase activation and AMPA receptor subunit trafficking underlie the potentiation of miniature EPSC amplitudes triggered by the activation of L-type calcium channels
Functional assessment of the NMDA receptor variant GluN2A (R586K)
Background: The N-methyl-D-aspartate receptor (NMDAR) is an ionotropic glutamate receptor that has important roles in synaptogenesis, synaptic transmission, and synaptic plasticity. Recently, a large number of rare genetic variants have been found in NMDAR subunits in people with neurodevelopmental disorders, and also in healthy individuals. One such is the GluN2AR586K variant (GRIN2AG1757A), found in a person with intellectual disability. Identifying the functional consequences, if any, of such variants allows their potential contribution to pathogenesis to be assessed. Here, we assessed the effect of the GluN2AR586K variant on NMDAR pore properties. Methods: We expressed recombinant NMDARs with and without the GluN2AR586K variant in Xenopus laevis oocytes and in primary cultured mouse neurons, and made electrophysiological recordings assessing Mg2+ block, single-channel conductance, mean open time and current density. Results: The GluN2AR586K variant was not found to influence any of the properties assessed. Conclusions: Our findings suggest it is unlikely that the GluN2AR586K variant contributes to the pathogenesis of neurodevelopmental disorder
Functional properties of in vitro excitatory cortical neurons derived from human pluripotent stem cells
The in vitro derivation of regionally defined human neuron types from patient‐derived stem cells is now established as a resource to investigate human development and disease. Characterization of such neurons initially focused on the expression of developmentally regulated transcription factors and neural markers, in conjunction with the development of protocols to direct and chart the fate of differentiated neurons. However, crucial to the understanding and exploitation of this technology is to determine the degree to which neurons recapitulate the key functional features exhibited by their native counterparts, essential for determining their usefulness in modelling human physiology and disease in vitro. Here, we review the emerging data concerning functional properties of human pluripotent stem cell‐derived excitatory cortical neurons, in the context of both maturation and regional specificity. [Image: see text
Mechanism of partial agonism at NMDA receptors for a conformationally restricted glutamate analog
Title Page Structural features of the glutamate binding site in recombinant NR1/NR2A N-methyl- D-aspartate receptors determined by site-directed mutagenesis and molecular modeling
Abbreviations: NMDA, N-methyl-D-aspartate; AMPA, α -amino-3-hydroxy-5-methyl-4-isoxazole propionic acid; TEVC, two electrode voltage clamp; D-AP5, D-2-amino-5-phosphonopentanoic acid; MTSEA, methanethiosulfonate ethylammonium. MOL 8185 3 Abstract We have used site-directed mutagenesis of amino acids located within the S1 and S2 ligand binding domains of the NR2A NMDA receptor subunit to explore the nature of ligand binding. Wild-type or mutated NR1/NR2A NMDA receptors were expressed in Xenopus laevis oocytes and studied using TEVC. We investigated the effects of mutations in the S1 and S2 regions on the potencies of the agonists L-glutamate, L-aspartate, RS-tetrazol-5-yl glycine and NMDA. Mutation of each of the corresponding residues found in the NR2A receptor subunit, suggested to be contact residues in the GluR2 AMPA receptor subunit, caused a rightward shift in the concentration-response curve for each agonist examined. None of the mutations examined altered the efficacy of glutamate as assessed by MTSEA potentiation of agonist-evoked currents. In addition none of the mutations altered the potency of glycine. Homology modeling and molecular dynamics were used to evaluate molecular details of ligand binding of both wild-type and mutant receptors, as well as to explore potential explanations for agonist selectivity between glutamate receptor subtypes. Our modeling studies support our interpretation of the mutagenesis data and indicate a similar binding strategy for L-glutamate and NMDA when they occupy the binding site in NMDA receptors as has been proposed for glutamate binding to the GluR2 AMPA receptor subunit. Furthermore, we offer an explanation as to why 'charge conserving' mutations of two residues in the binding pocket result in non-functional receptor-channels and suggest a contributing molecular determinant of why NMDA is not an agonist at AMPA receptors. MOL 8185 4 The NMDA receptor-channel is thought to be formed from the co-assembly of two NR1 subunits and two NR2 subunits in a dimer of dimers configuration (Schorge and Colquhoun, 2003). NMDA receptors are unique among ligand-gated ion channels in that the binding of two different ligands is required for the activation of the receptor-channel complex. Glycine, a co-agonist, binds to residues located in the NR1 subunits, whereas glutamate binds to residues located in NR2 subunits (reviewed by X-ray diffraction studies have described the glycine binding domain of the NR1 subunit In this study we have systematically mutated all six of the predicted contact residues in either the S1 or S2 domains together with the histidine residue thought to play the same sitecapping role as Tyr450 in GluR2 Materials and Methods Plasmid constructs, cRNA synthesis and receptor expression in oocytes. The wild-type pSP64T-derived expression plasmids for NR1 and NR2A NMDA receptor subunits were as described in Oocytes were obtained from Xenopus laevis, after administration of a lethal dose of anaesthetic (all procedures were carried out in accordance with current UK Home Office requirements). Prior to injection with cRNAs of interest, the follicular membranes of the oocytes were removed. After injection oocytes were placed in separate wells of 24-well plates hours to allow for receptor expression and then stored at 4˚C until they were used for electrophysiological measurements. Electrophysiological Recordings and Solutions. TEVC recordings were made, using a GeneClamp 500 amplifier (Axon Instruments, Union City, CA), from oocytes that were placed in a modified frog Ringer solution that contained (in mM): NaCl 115, KCl 2.5, HEPES 10, BaCl 2 1.8; pH 7.3 with NaOH (20 ºC) (Sigma-Aldrich, UK). Current and voltage electrodes were made from thin-walled borosilicate glass (GC150TF-7.5, Harvard Apparatus, Kent, UK) using a PP-830 electrode puller (Narashige Instruments, Japan) and when filled with 3 M KCl possessed resistances of between 0.5 and 1.5 MΩ. Oocytes were voltageclamped at potentials of -40 mV. For L-glutamate, L-aspartate, NMDA and RS-tetrazol-5-yl glycine concentration-response measurements, the recording solution was further supplemented with 20 µM glycine and for glycine dose-response measurements this solution was supplemented with either 30 µM glutamate for wild-type receptors or 10 mM glutamate for mutant receptors. Glutamate receptor agonists were purchased from Tocris Cookson (Avonmouth, UK). Application of solutions was controlled manually and data were filtered at 10 Hz and digitized at 100 Hz. Test solutions were applied for 20 s or until a plateau to the agonist-evoked response had been achieved. The maximum agonist concentration applied to any oocyte was 30 mM as concentrations higher than this produced considerable changes in the osmolarity of the recording solution. The current response to a maximal agonist concentration for all constructs used here was always less than 3 µA at a holding potential of -40 mV. The open probability of NMDA receptors containing mutations in either the S1 or MOL 8185 8 S2 binding site was assessed by co-expressing these subunits with NR1 subunits carrying the A652C mutation where n H is the Hill coefficient, I max is the maximum current, [A] is the concentration of agonist, and EC 50 is the concentration of agonist that produces a half-maximum response. Each data point was then normalized to the fitted maximum of the dose-response curve. The normalized values were then pooled and averaged for each construct and fitted again with the Hill equation, with the maximum and minimum for each curve being constrained to asymptote to 1 and 0 respectively. A similar protocol was used to determine the concentration of the NMDA antagonist, D-AP5 required to inhibit a glutamate-evoked response by 50 % (IC 50 ). In these experiments the glutamate concentration was fixed at the concentration required to evoke a half-maximal response in the construct being investigated. Modeling of the glutamate binding site in the NR2A NMDA receptor subunit. A homology model of the S1-S2 region of NR2A (GenBank Accession D13211) was constructed from the NR1-glycine crystal structure (1PB7; In order to generate a binding site without close contacts and with reasonable hydrogen bonds, the homology model was supplemented with MMFF94 force field charges and subjected to Sybyl (version 6.9) molecular dynamics (MD) for 5000 femtoseconds (fs) using an NTP ensemble (20 K and 1 atm). During the first stage of molecular dynamics only the backbone atoms of the protein were permitted to move. A second molecular dynamics run with identical parameters allowed only the side chain atoms to move. Finally, the agonist ligand and all amino acid residues within 6.0 Å of it were subjected to the same treatment. In this way, all poor contacts within the protein were removed, while the ligand adopted reasonable hydrogen-bonding interactions with the surrounding residues. This allowed the model to achieve a relatively strain-free conformation. Subsequently, separate protein-ligand models of L-glutamate and N-methyl-D-aspartate (NMDA) were created with ligand conformations that closely matched that of L-glutamate from the GluR2 site (protein data bank 1FTJ). These were loaded with MMFF94 charges and manually docked into the candidate agonist binding site using the match command and the GluR2 site as guidance. The individual ligand-protein systems were subjected to molecular dynamics simulation in Sybyl (version 6.9) using the TRIPOS force field (20 K, 1 atm, NTP ensemble, coupling constant 20), for MOL 8185 10 10,000 fs holding fixed all residues outside of a 6.0 angstrom radius around the ligand aggregate. To produce the final structures, the last 1000 fs of molecular dynamics was averaged and subsequently minimized with the Powell algorithm until an energy gradient of 0.01 kcal/mol was reached. The resulting structures were used to evaluate changes in binding site interactions caused by mutation of binding site residues. For the mutation studies of R499K and D712E, modified structures were created using Sybyl's "modify/mutate" command on the optimized protein-ligand complexes described above. The mutated and liganded protein complexes were then subjected to the same molecular dynamics and energy minimization procedures employed for the native protein. All models can be downloaded from www.pharm.emory/straynelis/downloads. Further analysis of both NMDA and L-glutamate in the GluR2 structure and the NR2A model was performed using the GROMACS molecular dynamics package Results Steady MOL 8185 11 with values obtained from various studies of native and recombinant NMDA receptors (for a review see Dingledine et al., 1999). In addition, the mean maximal currents evoked by each of the agonists were similar, these being 1.77 ± 0.34 µA (tetrazol-5yl-glycine), 1.42 ± 0.23 µA (glutamate), 1.64 ± 0.20 µA (aspartate) and 1.66 ± 0.17 µA (NMDA). Evaluation of responses to maximally effective concentrations suggests that agonist efficacy relative to glutamate for all compounds ranged between 89 -97 % (data not shown). The ligand binding site in the NR2A receptor subunit. We created a homology model of the NR2A S1-S2 ligand binding domain using the NR1-glycine crystal structure as a template Glutamate and its analogues were docked into the NR2A model using the original description of glutamate binding to AMPA receptor S1-S2 (Armstrong and Gouaux, 2000) as a guide and molecular dynamics as a provisional refinement protocol (see Materials and Methods). We subsequently docked NMDA into our model of NR2A and found several notable differences in binding. The Tyr711 orientation is assisted by H-bonding to Glu394. Given that in GluR2 Met708 NMDA receptor subunits is conserved in the GluR2 AMPA receptor subunit. It has been reported previously NMDA receptor subunit to glycine (S664G) reduces glutamate potency by 180-fold. Thus, we first made the equivalent serine to glycine mutation in the NR2A NMDA receptor subunit. Previous studies have characterized the properties of glutamate-activated NR1/NR2A(T671A) receptors and the homologous mutation in NR2D-containing NMDA receptors at both the whole-cell and single-channel level We interpret the effect on potency of the glycine, but not alanine, substitution at NR2A Ser670 as a likely indication of alterations in the geometry of the backbone chain. Glycine, which increases chain flexibility, may allow the chain to adopt conformations that hinder the ability of a hydrogen bond to form between the Ser670 chain nitrogen and the α- carboxyl. This interaction may help stabilize the other interactions of Ser670 and Thr671 with the γ-carboxyl (see MOL 8185 17 between the potency of NMDA and glutamate for NR2A(T671A) containing receptors. Given a possible reduction in degrees of freedom and different orientations in the cleft, we predict that shorter D-aspartate based ligands may require less stabilization of their γ-carboxyl. Mutation of residues in the S1 binding domain. Three uncharged residues in the S1 binding domain (H466, S492 and T494) were separately replaced with alanine by mutagenesis. Each of these residues is conserved in the four NR2 NMDA receptor subunits, while in the corresponding GluR2 AMPA subunit a tyrosine residue replaces histidine and a proline is found at the homologous position occupied by serine. Thr494 is conserved in each of these subunits as well as in the NR1 subunit (see The biggest shift in potency for tetrazol-5yl-glycine was seen with the H466A mutation, T494A gave the biggest shifts in glutamate and aspartate potencies, while the biggest shift in NMDA potency was observed upon mutation of the Ser492 residue. In general these three mutations had less effect on tetrazol-5yl-glycine potency with only between 21-and 42-fold shifts in potency being observed when compared to wild-type receptors. We predict that tetrazol-5yl-glycine will show additional hydrogen bonds between the tetrazol ring and residues within the binding pocket (modeling data not shown), suggesting this agonist utilizes additional features of the binding pocket to stabilize its interactions. We interpret these additional interactions as stabilizing forces that counteract the loss of Ser492 and Thr494 interactions. NR2A(S492A S670G) has additive effects on glutamate potency. We also investigated the effects of introducing two mutations in different lobes into the binding site of MOL 8185 18 the NR2A NMDA receptor subunit. The two double mutations chosen were NR2A(S492A S670G) and NR2A(T494A T671A). As is shown in essentially abolished glutamate sensitivity, as only very small currents could be evoked by glutamate even when the concentration of agonist was raised to 30 mM. The ability of the double mutant to reduce potency further than each single mutant in isolation suggests that each of these mutations, individually, may not dramatically alter contacts of the other serine and threonine residues within the binding site. Mutations in S1 and S2 domains do not affect agonist efficacy. Changes in the potency (EC 50 ) of an agonist at receptors carrying mutations are difficult to ascribe to changes in agonist binding if the mutation also affects the agonist's efficacy i.e. its ability to gate the channel (see . For this reason we present several lines of evidence as to why we consider the main effects of the mutations we have described above are to alter the ability of glutamate (and other agonists) to bind to the NMDA receptor. First, to examine the ability of mutations in the S1 and S2 binding domains to affect the ability of glutamate to gate the channel once bound to the receptor we co-expressed wildtype and mutant NR2A subunits with NR1 subunits carrying the A652C mutation MOL 8185 19 residues located in the S1 Second and in contrast to their effects on the potency for glutamate analogues, none of the mutations studied here affected the potency of glycine Finally, for each of the mutations made in either the S1 or S2 domain we did not observe > 5-fold changes in the maximal currents that were evoked in mutated NR2 subunits when compared to wild-type responses. The fact that large differences were not observed and also given that there were no significant decreases in the Hill Slopes (see Mutation of NR2A S1 residues to the homologous residues in NR1. Two of the proposed residues in the S1 domain, thought to participate in the binding of glutamate, vary between the NR2 and NR1 subunits; namely a serine at position 492 in NR2A (a proline residue in NR1 and GluR2) and a histidine at position 466 in NR2A (a phenylalanine residue in NR1). Our modeling studies suggest that the amino group of NMDA forms a hydrogen bond with the backbone oxygen of Ser492 His466 occupies a position analogous to Tyr450 in GluR2, and likely provides the binding pocket with a similar electron polarizable ring. MOL 8185 22 effects may arise as a result of ring twisting. The lower potency shift in the case of the H466F mutation may owe its origin to such a phenomenon. NR2A(R499K) and NR2A(D712E) produce non-functional receptor-channels. Two of the six hypothetical contact residues in the binding site are charged. We introduced charge-conserving mutations at position 499 (arginine to lysine; R499K) and position 712 (aspartate to glutamate; D712E). When co-injected with cRNA for the NR1 NMDA receptor subunit neither of these constructs gave currents when glutamate was applied up to 10 mM. We also investigated whether any of the other three ligands used in this study might activate either NR2A(R499K)-or NR2A(D712E)-containing NMDA receptor-channels. In all cases each of these ligands failed to evoke a response in oocytes injected with these two constructs. It has previously been reported that both the equivalent arginine to lysine mutation in the NR2B NMDA receptor subunit (R493K; Laube et al., 1997) and the equivalent aspartate to glutamate mutation in NR2A/NR2B NMDA receptor subunits (D731E and D732E; In a separate vein, the NR2A model provides a satisfying interpretation of the consequences of replacing Asp712 with Glu. In the wild-type receptor Asp712 makes three coupled hydrogen bonds, two of which are to the neutral OH groups in the side chains of Ser492 and Tyr742 (not shown in subunit to an Asn residue also abolishes receptor function. Thus it appears that conservative substitution (with Glu) or substitution with either small non-polar residues or polar residues each result in non-functional receptors. Hence these two non-functional mutations serve to illustrate important geometric constraints within the binding pocket. Discussion Effects of S1 and S2 mutations on agonist potency. Several structure-function studies of ionotropic glutamate receptors and related kainate binding proteins have identified residues located in the S1 and S2 binding domains that, when mutated, lead to a reduction in the ability of the ligand to remain bound to its binding site (for example see MOL 8185 24 While shifts in the EC 50 of a ligand for the activation of a receptor or a change in 'apparent affinity' of a ligand measured in a binding assay cannot indicate, definitively, that the mutation has had a direct effect on the microscopic rate constants determining binding (see Further evidence that the S1 and S2 mutations studied have their major effect on agonist Taken together these results support our hypothesis that the shifts in EC 50 we report largely reflect changes in binding rates. Each of the point mutations investigated in this study caused a decrease in potency for all agonists examined. However the degrees of shift observed were, in general, greater for Even with knowledge that a residue is located in the binding pocket and is likely to hydrogen bond directly with glutamate, structure-function studies may miss, or place lesser emphasis on, a residue when only a small decrease in agonist potency is observed. This is exemplified when we consider the Ser670 residue in the S2 domain. In our present study we have shown that mutation of Ser670 to glycine, in the NR2A NMDA receptor subunit causes a far greater shift in potency than that seen when the residue is mutated to an alanine residue MOL 8185 26 Modeling of the binding site in the NR2A NMDA receptor subunit. A primary goal of this study is to provide functional data supporting the emerging idea that the glutamate binding site shares similar molecular features across the glutamate receptor family. The increasing availability of crystallographic data and homology modeling has increased the need for functional studies to substantiate and interpret data. Our experiments represent a systematic exploration of all proposed contact residues in a single NMDA subunit (NR2A). Furthermore, we studied four ligands at all mutant receptors, which provides additional insight into the nature of the binding pocket. Several clear themes emerge from these data. First, we find that the major molecular determinants of glutamate binding to GluR2 (see This is most notable when we mutated the two charged residues in the binding site. Shortening the side chain length of Arg499 by changing this to lysine increases the distance between its side chain and the α-carboxyl of glutamate to such an extent that it cannot hydrogen bond. Similarly increasing the amount to which the γ-carboxyl of the residue at position 712 protrudes into the binding site by swapping the aspartate residue for glutamate indicates that length of the side chain at this position is critical for a functional receptor. Third, we describe several hypothetical features of the NMDA receptor binding pocket that define its selectivity for aspartate derivatives compared to AMPA receptors. Finally we propose that the differential contribution of residues to stabilization of different ligands MOL 8185 27 explains the differing effects of mutants on the rank order of potency -for example the relatively small shifts in potency of tetrazol-5yl-glycine may result from its ability to hydrogen bond with (additional) groups in the binding site which counteract the loss of the sites utilized by the other ligands. Summary. Although these conclusions appear straightforward and predictive, we are aware of the limitations of homology modeling, particularly for the glutamate binding pocket which shows a number of interesting features we have neglected. Specifically, we have not included potential water bridged interactions in the pocket, and at times needed to choose between one of several possible orientations for side chains. Despite these caveats, the data presented here answer several longstanding questions about glutamate receptor pharmacology, and make a number of predictions that will help focus additional questions and experiments on the glutamate receptor-binding domain
Identification of Antigens Specific to Non-Tuberculous Mycobacteria: The Mce Family of Proteins as a Target of T Cell Immune Responses
The lack of an effective TB vaccine hinders current efforts in combating the TB pandemic. One theory as to why BCG is less protective in tropical countries is that exposure to non-tuberculous mycobacteria (NTM) reduces BCG efficacy. There are currently several new TB vaccines in clinical trials, and NTM exposure may also be relevant in this context. NTM exposure cannot be accurately evaluated in the absence of specific antigens; those which are known to be present in NTM and absent from M. tuberculosis and BCG. We therefore used a bioinformatic pipeline to define proteins which are present in common NTM and absent from the M. tuberculosis complex, using protein BLAST, TBLASTN and a short sequence protein BLAST to ensure the specificity of this process. We then assessed immune responses to these proteins, in healthy South Africans and in patients from the United Kingdom and United States with documented exposure to NTM. Low level responses were detected to a cluster of proteins from the mammalian cell entry family, and to a cluster of hypothetical proteins, using ex vivo ELISpot and a 6 day proliferation assay. These early findings may provide a basis for characterising exposure to NTM at a population level, which has applications in the field of TB vaccine design as well as in the development of diagnostic tests
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Investigating the utility of human embryonic stem cell-derived neurons to model ageing and neurodegenerative disease using whole-genome gene expression and splicing analysis
A major goal in regenerative medicine is the predictable manipulation of human embryonic stem cells (hESCs) to defined cell fates that faithfully represent their somatic counterparts. Directed differentiation of hESCs into neuronal populations has galvanized much interest into their potential application in modelling neurodegenerative disease. However, neurodegenerative diseases are age-related, and therefore establishing the maturational comparability of hESC-derived neural derivatives is critical to generating accurate in vitro model systems. We address this issue by comparing genome-wide, exon-specific expression analyses of pluripotent hESCs, multipotent neural precursor cells and a terminally differentiated enriched neuronal population to expression data from post-mortem foetal and adult human brain samples. We show that hESC-derived neuronal cultures (using a midbrain differentiation protocol as a prototypic example of lineage restriction), while successful in generating physiologically functional neurons, are closer to foetal than adult human brain in terms of molecular maturation. These findings suggest that developmental stage has a more dominant influence on the cellular transcriptome than regional identity. In addition, we demonstrate that developmentally regulated gene splicing is common, and potentially a more sensitive measure of maturational state than gene expression profiling alone. In summary, this study highlights the value of genomic indices in refining and validating optimal cell populations appropriate for modelling ageing and neurodegeneration
Cellular and synaptic phenotypes lead to disrupted information processing in Fmr1-KO mouse layer 4 barrel cortex
Sensory hypersensitivity is a common and debilitating feature of neurodevelopmental disorders such as Fragile X Syndrome (FXS). How developmental changes in neuronal function culminate in network dysfunction that underlies sensory hypersensitivities is unknown. By systematically studying cellular and synaptic properties of layer 4 neurons combined with cellular and network simulations, we explored how the array of phenotypes in Fmr1-knockout (KO) mice produce circuit pathology during development. We show that many of the cellular and synaptic pathologies in Fmr1-KO mice are antagonistic, mitigating circuit dysfunction, and hence may be compensatory to the primary pathology. Overall, the layer 4 network in the Fmr1-KO exhibits significant alterations in spike output in response to thalamocortical input and distorted sensory encoding. This developmental loss of layer 4 sensory encoding precision would contribute to subsequent developmental alterations in layer 4-to-layer 2/3 connectivity and plasticity observed in Fmr1-KO mice, and circuit dysfunction underlying sensory hypersensitivity.</p
The Subtype of GluN2 C-terminal Domain Determines the Response to Excitotoxic Insults
It is currently unclear whether the GluN2 subtype influences NMDA receptor (NMDAR) excitotoxicity. We report that the toxicity of NMDAR-mediated Ca(2+) influx is differentially controlled by the cytoplasmic C-terminal domains of GluN2B (CTD(2B)) and GluN2A (CTD(2A)). Studying the effects of acute expression of GluN2A/2B-based chimeric subunits with reciprocal exchanges of their CTDs revealed that CTD(2B) enhances NMDAR toxicity, compared to CTD(2A). Furthermore, the vulnerability of forebrain neurons in vitro and in vivo to NMDAR-dependent Ca(2+) influx is lowered by replacing the CTD of GluN2B with that of GluN2A by targeted exon exchange in a mouse knockin model. Mechanistically, CTD(2B) exhibits stronger physical/functional coupling to the PSD-95-nNOS pathway, which suppresses protective CREB activation. Dependence of NMDAR excitotoxicity on the GluN2 CTD subtype can be overcome by inducing high levels of NMDAR activity. Thus, the identity (2A versus 2B) of the GluN2 CTD controls the toxicity dose-response to episodes of NMDAR activity
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