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1.
Neuron ; 2024 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-38878768

RESUMO

NMDA receptors (NMDARs) are ionotropic receptors crucial for brain information processing. Yet, evidence also supports an ion-flux-independent signaling mode mediating synaptic long-term depression (LTD) and spine shrinkage. Here, we identify AETA (Aη), an amyloid-ß precursor protein (APP) cleavage product, as an NMDAR modulator with the unique dual regulatory capacity to impact both signaling modes. AETA inhibits ionotropic NMDAR activity by competing with the co-agonist and induces an intracellular conformational modification of GluN1 subunits. This favors non-ionotropic NMDAR signaling leading to enhanced LTD and favors spine shrinkage. Endogenously, AETA production is increased by in vivo chemogenetically induced neuronal activity. Genetic deletion of AETA production alters NMDAR transmission and prevents LTD, phenotypes rescued by acute exogenous AETA application. This genetic deletion also impairs contextual fear memory. Our findings demonstrate AETA-dependent NMDAR activation (ADNA), characterizing AETA as a unique type of endogenous NMDAR modulator that exerts bidirectional control over NMDAR signaling and associated information processing.

2.
Front Pharmacol ; 12: 769046, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34658899

RESUMO

Ionotropic glutamate receptors (iGluRs) are tetrameric ligand-gated ion channels that play a crucial role in excitatory synaptic transmission in the central nervous system. Each subunit contributes with three helical transmembrane segments (M1, M3, and M4) and a pore loop (M2) to form the channel pore. Recent studies suggest that the architecture of all eukaryotic iGluRs derives from a common prokaryotic ancestral receptor that lacks M4 and consists only of transmembrane segments M1 and M3. Although significant contribution has emerged in the last years, the role of this additionally evolved transmembrane segment in iGluR assembly and function remains unclear. Here, we have investigated how deletions and mutations of M4 in members of the NMDA receptor (NMDAR) subfamily, the conventional heteromeric GluN1/GluN2 and glycine-gated GluN1/GluN3 NMDARs, affect expression and function in Xenopus oocytes. We show that deletion of M4 in the GluN1, GluN2A, or GluN3A subunit, despite retained receptor assembly and cell surface expression, results in nonfunctional membrane receptors. Coexpression of the corresponding M4 as an isolated peptide in M4-deleted receptors rescued receptor function of GluN1/GluN2A NMDARs without altering the apparent affinity of glutamate or glycine. Electrophysiological analyses of agonist-induced receptor function and its modulation by the neurosteroid pregnenolone sulfate (PS) at mutations of the GluN1-M4/GluN2/3-transmembrane interfaces indicate a crucial role of position M813 in M4 of GluN1 for functional coupling to the core receptor and the negative modulatory effects of PS. Substitution of residues and insertion of interhelical disulfide bridges confirmed interhelical interactions of positions in M4 of GluN1 with residues of transmembrane segments of neighboring subunits. Our results show that although M4s in NMDARs are not important for receptor assembly and surface expression, the residues at the subunit interface are substantially involved in M4 recognition of the core receptor and regulation of PS efficacy. Because mutations in the M4 of GluN1 specifically resulted in loss of PS-induced inhibition of GluN1/GluN2A and GluN1/GluN3A NMDAR currents, our results point to distinct roles of M4s in NMDAR modulation and highlight the importance of the evolutionarily newly evolved M4 for selective in vivo modulation of glutamate- and glycine-activated NMDARs by steroids.

3.
Sci Rep ; 10(1): 16569, 2020 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-33024136

RESUMO

The glycine receptor (GlyR), a member of the pentameric ligand-gated ion channel family (pLGIC), displays remarkable variations in the affinity and efficacy of the full agonist glycine and the partial agonist taurine depending on the cell system used. Despite detailed insights in the GlyR three-dimensional structure and activation mechanism, little is known about conformational rearrangements induced by these agonists. Here, we characterized the conformational states of the α1 GlyR upon binding of glycine and taurine by microscale thermophoresis expressed in HEK293 cells and Xenopus oocytes after solubilization in amphipathic styrene-maleic acid copolymer nanodiscs. Our results show that glycine and taurine induce different conformational transitions of the GlyR upon ligand binding. In contrast, the variability of agonist affinity is not mediated by an altered conformational change. Thus, our data shed light on specific agonist induced conformational features and mechanisms of pLGIC upon ligand binding determining receptor activation in native environments.


Assuntos
Canais Iônicos de Abertura Ativada por Ligante/fisiologia , Nanoestruturas , Polímeros , Receptores de Glicina , Animais , Glicina/metabolismo , Células HEK293 , Humanos , Oócitos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Receptores de Glicina/química , Receptores de Glicina/metabolismo , Sinapses/fisiologia , Transmissão Sináptica , Taurina/metabolismo , Xenopus
4.
ACS Sens ; 5(1): 234-241, 2020 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-31829017

RESUMO

Combining the stability of solid-state nanopores with the unique sensing properties of biological components in a miniaturized electrical hybrid nanopore device is a challenging approach to advance the sensitivity and selectivity of small-molecule detection in healthcare and environment analytics. Here, we demonstrate a simple method to design an electrical hybrid nanosensor comprising a bacterial binding protein tethered to a solid-state nanopore allowing high-affinity detection of phosphonates. The diverse family of bacterial substrate-binding proteins (SBPs) binds specifically and efficiently to various substances and has been implicated as an ideal biorecognition element for analyte detection in the design of hybrid bionanosensors. Here, we demonstrate that the coupling of the purified phosphonate binding protein PhnD via primary amines to the reactive NHS groups of P(DMAA-co-NMAS) polymers inside a single track-etched nanopore in poly(ethylene terephthalate) (PET) foils results in ligand-specific and concentration-dependent changes in the nanopore current. Application of the phosphonate 2-aminoethylphosphonate (2AEP) or ethylphosphonate (EP) induces a large conformational rearrangement in PnhD around the hinge in a venus flytrap mechanism resulting in a concentration depended on increase of the single pore current with binding affinities of 27 and 373 nM, respectively. Thus, the specificity and stability of this simple hybrid sensor concept combine the advantages of both, the diversity of ligand-specific substrate-binding proteins and solid-state nanopores encouraging further options to produce robust devices amenable to medical or environmental high-throughput-based applications in nanotechnology.


Assuntos
Técnicas Biossensoriais/métodos , Organofosfonatos/química , Proteínas de Transporte , Eletricidade , Humanos , Conformação Molecular , Nanoporos
5.
Anal Chim Acta ; 1086: 14-15, 2019 Dec 04.
Artigo em Inglês | MEDLINE | ID: mdl-31561789
6.
Cancers (Basel) ; 11(4)2019 Apr 09.
Artigo em Inglês | MEDLINE | ID: mdl-30970642

RESUMO

Glioblastoma is one of the most aggressive malignant brain tumors, with a survival time less than 15 months and characterized by a high radioresistance and the property of infiltrating the brain. Recent data indicate that the malignancy of glioblastomas depends on glutamatergic signaling via ionotropic glutamate receptors. In this study we revealed functional expression of Ca2+-permeable NMDARs in three glioblastoma cell lines. Therefore, we investigated the impact of this receptor on cell survival, migration and DNA double-strand break (DSB) repair in the presence of both, glutamate and NMDAR antagonists, and after clinically relevant doses of ionizing radiation. Our results indicate that treatment with NMDAR antagonists slowed the growth and migration of glutamate-releasing LN229 cells, suggesting that activation of NMDARs facilitate tumor expansion. Furthermore, we found that DSB-repair upon radiation was more effective in the presence of glutamate. In contrast, antagonizing the NMDAR or the Ca2+-dependent transcription factor CREB impaired DSB-repair similarly and resulted in a radiosensitizing effect in LN229 and U-87MG cells, indicating a common link between NMDAR signaling and CREB activity in glioblastoma. Since the FDA-approved NMDAR antagonists memantine and ifenprodil showed differential radiosensitizing effects, these compounds may constitute novel optimizations for therapeutic interventions in glioblastoma.

7.
Commun Biol ; 2: 75, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30820470

RESUMO

Ionotropic glutamate receptors (iGluRs) mediate excitatory neuronal signaling in the mammalian CNS. These receptors are critically involved in diverse physiological processes; including learning and memory formation, as well as neuronal damage associated with neurological diseases. Based on partial sequence and structural similarities, these complex cation-permeable iGluRs are thought to descend from simple bacterial proteins emerging from a fusion of a substrate binding protein (SBP) and an inverted potassium (K+)-channel. Here, we fuse the pore module of the viral K+-channel KcvATCV-1 to the isolated glutamate-binding domain of the mammalian iGluR subunit GluA1 which is structural homolog to SBPs. The resulting chimera (GluATCV*) is functional and displays the ligand recognition characteristics of GluA1 and the K+-selectivity of KcvATCV-1. These results are consistent with a conserved activation mechanism between a glutamate-binding domain and the pore-module of a K+-channel and support the expected phylogenetic link between the two protein families.


Assuntos
Ácido Glutâmico/metabolismo , Canais de Potássio/metabolismo , Receptores Ionotrópicos de Glutamato/metabolismo , Proteínas Virais/metabolismo , Sequência de Aminoácidos , Animais , Feminino , Ácido Glutâmico/química , Ativação do Canal Iônico/genética , Modelos Moleculares , Mutação , Oócitos/citologia , Oócitos/metabolismo , Técnicas de Patch-Clamp , Canais de Potássio/química , Canais de Potássio/genética , Domínios Proteicos , Ratos , Receptores Ionotrópicos de Glutamato/química , Receptores Ionotrópicos de Glutamato/genética , Homologia de Sequência de Aminoácidos , Proteínas Virais/química , Proteínas Virais/genética , Xenopus laevis
8.
Cancers (Basel) ; 11(3)2019 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-30841565

RESUMO

The activation of Ca2+-permeable N-methyl-D-aspartic acid (NMDA) receptor channels (NMDARs) is crucial for the development and survival of neurons, but many cancers use NMDAR-mediated signaling as well, enhancing the growth and invasiveness of tumors. Thus, NMDAR-dependent pathways emerge as a promising target in cancer therapy. Here, we use the LN229 and U-87MG glioblastoma multiforme (GBM) cells and immunofluorescence staining of 53BP1 to analyze NMDAR-induced DNA double-strand breaks (DSBs), which represent an important step in the NMDAR signaling pathway in neurons by facilitating the expression of early response genes. Our results show that NMDAR activation leads to the induction of DSBs in a subpopulation of glioma cells. In a further analogy to neurons, our results demonstrate that the induction of DSBs in LN229 cells is dependent on the activity of topoisomerase IIß (Top2ß). Western blot analysis revealed that the inhibition of NMDARs, cAMP-responsive element binding transcription factor (CREB) and Top2ß decreased the expression of the proto-oncogene cFos. Knockdown of Top2ß with siRNAs resulted in a downregulation of cFos and increased the radiosensitivity of LN229 cells in clonogenic survival. We also observed impaired cFos expression upon NMDAR and Top2ß inhibition in a primary GBM cell line, suggesting that NMDAR signaling may be widely used by GBMs, demonstrating the potential of targeting NMDAR signaling proteins for GBM therapy.

9.
Eur J Pharmacol ; 844: 216-224, 2019 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-30553788

RESUMO

N-methyl D-aspartate (NMDA) receptors play a crucial role in normal brain function, pathogenesis of neurodegenerative and psychiatric disorders. Functional tetra-heteromeric NMDA receptor contains two obligatory GluN1 subunits and two identical or different non-GluN1 subunits that evolve from six different genes including four GluN2 (A-D) and two GluN3 (A-B) subunits. Since NMDA receptors confer varied physiological properties and spatiotemporal distributions in the brain, pharmacological agents that target NMDA receptors with specific GluN2 subunits have significant potential for therapeutic applications. In the present work, by using electrophysiology techniques, we have studied the role of ligand binding domain (LBD) interactions in determining the effect of well-characterized pharmacological agents including agonists, competitive antagonists, channel blockers and an allosteric modulator. Remarkably, point mutations at the distal end (site-II&III) of GluN1 LBD interface increased memantine potency up to sevenfold when co-expressed with wild type GluN2A receptors but exhibit no effect on Mg2+ activity. Conversely, mutations at the proximal end (site-I) of the LBD interface did not affect the memantine but altered Zn2+ and Mg2+ potency towards opposite directions. These results indicate that GluN1/2A LBD interface interactions play a key role in determining channel function. Further, subtle changes in LBD interaction can be readily translated to the downstream extracellular vestibule of channel pore to adopt a conformation that may affect memantine, Zn2+ and Mg2+ binding. Further studies on NMDA receptor LBD to transmembrane domain signal propagation mechanisms will help develop GluN2 subunit selective biomolecules that can be used for the treatment of neurological and psychiatric disorders.


Assuntos
Subunidades Proteicas/fisiologia , Receptores de N-Metil-D-Aspartato/fisiologia , Ligantes , Preparações Farmacêuticas/metabolismo , Domínios Proteicos
10.
EMBO J ; 37(11)2018 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-29661886

RESUMO

Increasing evidence suggests that synaptic functions of the amyloid precursor protein (APP), which is key to Alzheimer pathogenesis, may be carried out by its secreted ectodomain (APPs). The specific roles of APPsα and APPsß fragments, generated by non-amyloidogenic or amyloidogenic APP processing, respectively, remain however unclear. Here, we expressed APPsα or APPsß in the adult brain of conditional double knockout mice (cDKO) lacking APP and the related APLP2. APPsα efficiently rescued deficits in spine density, synaptic plasticity (LTP and PPF), and spatial reference memory of cDKO mice. In contrast, APPsß failed to show any detectable effects on synaptic plasticity and spine density. The C-terminal 16 amino acids of APPsα (lacking in APPsß) proved sufficient to facilitate LTP in a mechanism that depends on functional nicotinic α7-nAChRs. Further, APPsα showed high-affinity, allosteric potentiation of heterologously expressed α7-nAChRs in oocytes. Collectively, we identified α7-nAChRs as a crucial physiological receptor specific for APPsα and show distinct in vivo roles for APPsα versus APPsß. This implies that reduced levels of APPsα that might occur during Alzheimer pathogenesis cannot be compensated by APPsß.


Assuntos
Doença de Alzheimer/genética , Precursor de Proteína beta-Amiloide/genética , Cognição/fisiologia , Plasticidade Neuronal/genética , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Hipocampo/metabolismo , Hipocampo/patologia , Humanos , Camundongos , Camundongos Knockout , Neurônios/metabolismo , Neurônios/patologia , Coluna Vertebral/metabolismo , Coluna Vertebral/patologia , Transmissão Sináptica/genética , Receptor Nicotínico de Acetilcolina alfa7/genética
11.
Front Pharmacol ; 8: 229, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28536523

RESUMO

N-methyl D-aspartate receptors (NMDAR) play crucial role in normal brain function and pathogenesis of neurodegenerative and psychiatric disorders. Functional tetra-heteromeric NMDAR contains two obligatory GluN1 subunits and two identical or different non-GluN1 subunits that include six different gene products; four GluN2 (A-D) and two GluN3 (A-B) subunits. The heterogeneity of subunit combination facilities the distinct function of NMDARs. All GluN subunits contain an extracellular N-terminal Domain (NTD) and ligand binding domain (LBD), transmembrane domain (TMD) and an intracellular C-terminal domain (CTD). Interaction between the GluN1 and co-assembling GluN2/3 subunits through the LBD has been proven crucial for defining receptor deactivation mechanisms that are unique for each combination of NMDAR. Modulating the LBD interactions has great therapeutic potential. In the present work, by amino acid point mutations and electrophysiology techniques, we have studied the role of LBD interactions in determining the effect of well-characterized pharmacological agents including agonists, competitive antagonists, and allosteric modulators. The results reveal that agonists (glycine and glutamate) potency was altered based on mutant amino acid sidechain chemistry and/or mutation site. Most antagonists inhibited mutant receptors with higher potency; interestingly, clinically used NMDAR channel blocker memantine was about three-fold more potent on mutated receptors (N521A, N521D, and K531A) than wild type receptors. These results provide novel insights on the clinical pharmacology of memantine, which is used for the treatment of mild to moderate Alzheimer's disease. In addition, these findings demonstrate the central role of LBD interactions that can be exploited to develop novel NMDAR based therapeutics.

12.
Neuropharmacology ; 105: 133-141, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-26777280

RESUMO

N-methyl-d-aspartate (NMDA) receptors composed of glycine-binding GluN1 and GluN3 subunits function as excitatory glycine receptors that respond to agonist application only with a very low efficacy. Binding of glycine to the high-affinity GluN3 subunits triggers channel opening, whereas glycine binding to the low-affinity GluN1 subunits causes an auto-inhibition of the maximal glycine-inducible receptor current (Imax). Hence, competitive antagonists of the GluN1 subunit strongly potentiate glycine responses of wild type (wt) GluN1/GluN3 receptors. Here, we show that co-expression of N-terminal domain (NTD) deleted GluN1 (GluN1(ΔNTD)) and GluN3 (GluN3(ΔNTD)) subunits in Xenopus oocytes generates GluN1/GluN3 receptors with a large increase in the glycine-inducible Imax accompanied by a strongly impaired GluN1 antagonist-mediated potentiation. Affinity purification after metabolic or surface labeling revealed no differences in subunit stoichiometry and surface expression between wt GluN1/GluN3A and mutant GluN1(ΔNTD)/GluN3A(ΔNTD) receptors, indicating a specific effect of NTD deletions on the efficacy of receptor opening. Notably, GluN1/GluN3A(ΔNTD) receptors showed a similar increase in Imax and a greatly reduced GluN1 antagonist-mediated current potentiation as GluN1(ΔNTD)/GluN3A(ΔNTD) receptors, whereas the glycine-induced currents of GluN1(ΔNTD)/GluN3A receptors resembled those of wt GluN1/GluN3A receptors. Furthermore, oxidative crosslinking of the homophilic GluN3A NTD intersubunit interface in mutant GluN1/GluN3A(R319C) receptors caused both a decrease in the glycine-induced Imax concomitantly with a marked increase in GluN1 antagonist-mediated current potentiation, whilst mutations within the intrasubunit region linking the GluN3A NTD to the ligand binding domain had opposite effects. Together these results show that the GluN3A NTD constitutes a crucial regulatory determinant of GluN1/GluN3A receptor function.


Assuntos
Glicina/fisiologia , Receptores de Glicina/fisiologia , Receptores de N-Metil-D-Aspartato/fisiologia , Animais , Sítios de Ligação , Glicina/farmacologia , Estrutura Terciária de Proteína , Receptores de Glicina/agonistas , Receptores de Glicina/química , Xenopus laevis
13.
Sci Rep ; 5: 8558, 2015 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-25708000

RESUMO

Glutamate is an indispensable neurotransmitter, triggering postsynaptic signals upon recognition by postsynaptic receptors. We questioned the phylogenetic position and the molecular details of when and where glutamate recognition arose in the glutamate-gated chloride channels. Experiments revealed that glutamate recognition requires an arginine residue in the base of the binding site, which originated at least three distinct times according to phylogenetic analysis. Most remarkably, the arginine emerged on the principal face of the binding site in the Lophotrochozoan lineage, but 65 amino acids upstream, on the complementary face, in the Ecdysozoan lineage. This combined experimental and computational approach throws new light on the evolution of synaptic signalling.


Assuntos
Canais de Cloreto/metabolismo , Ácido Glutâmico/metabolismo , Sequência de Aminoácidos , Animais , Arginina/química , Arginina/metabolismo , Sítios de Ligação , Canais de Cloreto/classificação , Canais de Cloreto/genética , Evolução Molecular , Ácido Glutâmico/química , Humanos , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Filogenia , Estrutura Terciária de Proteína , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos
14.
Int J Parasitol Drugs Drug Resist ; 4(3): 244-55, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25516835

RESUMO

Pharmacological targeting of glutamate-gated chloride channels (GluCls) is a potent anthelmintic strategy, evidenced by macrocyclic lactones that eliminate numerous roundworm infections by activating roundworm GluCls. Given the recent identification of flatworm GluCls and the urgent need for drugs against schistosomiasis, flatworm GluCls should be evaluated as potential anthelmintic targets. This study sought to identify agonists or modulators of one such GluCl, SmGluCl-2 from the parasitic flatworm Schistosoma mansoni. The effects of nine glutamate-like compounds and three monoterpenoid ion channel modulators were measured by electrophysiology at SmGluCl-2 recombinantly expressed in Xenopus laevis oocytes. For comparison with an established anthelmintic target, experiments were also performed on the AVR-14B GluCl from the parasitic roundworm Haemonchus contortus. l-Glutamate was the most potent agonist at both GluCls, but l-2-aminoadipate, d-glutamate and d-2-aminoadipate activated SmGluCl-2 (EC50 1.0 ± 0.1 mM, 2.4 ± 0.4 mM, 3.6 ± 0.7 mM, respectively) more potently than AVR-14B. Quisqualate activated only SmGluCl-2 whereas l-aspartate activated only AVR-14B GluCls. Regarding the monoterpenoids, both GluCls were inhibited by propofol, thymol and menthol, SmGluCl-2 most potently by thymol (IC50 484 ± 85 µM) and least potently by menthol (IC50 > 3 mM). Computational docking suggested that agonist and inhibitor potency is attributable to particular interactions with extracellular or membrane-spanning amino acid residues. These results reveal that flatworm GluCls are pharmacologically susceptible to numerous agonists and modulators and indicate that changes to the glutamate γ-carboxyl or to the propofol 6-isopropyl group can alter the differential pharmacology at flatworm and roundworm GluCls. This should inform the development of more potent compounds and in turn lead to novel anthelmintics.

15.
J Neurosci ; 34(6): 2155-9, 2014 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-24501356

RESUMO

Propofol is an intravenous general anesthetic that alters neuronal excitability by modulating agonist responses of pentameric ligand-gated ion channels (pLGICs). Evidence suggests that propofol enhancement of anion-selective pLGICs is mediated by a binding site between adjacent subunits, whereas propofol inhibition of cation-selective pLGICs occurs via a binding site contained within helices M1-M4 of individual subunits. We considered this idea by testing propofol modulation of homomeric human glycine receptors (GlyRs) and nematode glutamate-gated chloride channels (GluCls) recombinantly expressed in Xenopus laevis oocytes with electrophysiology. The Haemonchus contortus AVR-14B GluCl was inhibited by propofol with an IC50 value of 252 ± 48 µM, providing the first example of propofol inhibition of an anion-selective pLGIC. Remarkably, inhibition was converted to enhancement by a single I18'S substitution in the channel-forming M2 helix (EC50 = 979 ± 88 µM). When a previously identified site between adjacent subunits was disrupted by the M3 G329I substitution, both propofol inhibition and enhancement of GluCls were severely impaired (IC50 and EC50 values could not be calculated). Similarly, when the equivalent positions were examined in GlyRs, the M2 S18'I substitution significantly altered the maximum level of enhancement by propofol, and the M3 A288I substitution abolished propofol enhancement. These data are not consistent with separate binding sites for the opposing effects of propofol. Instead, these data suggest that propofol enhancement and inhibition are mediated by binding to a single site in anion-selective pLGICs, and the modulatory effect on channel gating depends on the M2 18' residue.


Assuntos
Anestésicos Intravenosos/metabolismo , Canais Iônicos de Abertura Ativada por Ligante/química , Canais Iônicos de Abertura Ativada por Ligante/metabolismo , Propofol/metabolismo , Anestésicos Intravenosos/farmacologia , Animais , Sítios de Ligação/fisiologia , Proteínas de Caenorhabditis elegans/agonistas , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/metabolismo , Cristalização , Feminino , Humanos , Canais Iônicos de Abertura Ativada por Ligante/agonistas , Propofol/farmacologia , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Xenopus laevis
16.
Ann Neurol ; 75(1): 147-54, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24272827

RESUMO

OBJECTIVE: To identify novel epilepsy genes using a panel approach and describe the functional consequences of mutations. METHODS: Using a panel approach, we screened 357 patients comprising a vast spectrum of epileptic disorders for defects in genes known to contribute to epilepsy and/or intellectual disability (ID). After detection of mutations in a novel epilepsy gene, we investigated functional effects in Xenopus laevis oocytes and screened a follow-up cohort. RESULTS: We revealed de novo mutations in GRIN2B encoding the NR2B subunit of the N-methyl-D-aspartate (NMDA) receptor in 2 individuals with West syndrome and severe developmental delay as well as 1 individual with ID and focal epilepsy. The patient with ID and focal epilepsy had a missense mutation in the extracellular glutamate-binding domain (p.Arg540His), whereas both West syndrome patients carried missense mutations within the NR2B ion channel-forming re-entrant loop (p.Asn615Ile, p.Val618Gly). Subsequent screening of 47 patients with unexplained infantile spasms did not reveal additional de novo mutations, but detected a carrier of a novel inherited GRIN2B splice site variant in close proximity (c.2011-5_2011-4delTC). Mutations p.Asn615Ile and p.Val618Gly cause a significantly reduced Mg(2+) block and higher Ca(2+) permeability, leading to a dramatically increased Ca(2+) influx, whereas p.Arg540His caused less severe disturbance of channel function, corresponding to the milder patient phenotype. INTERPRETATION: We identified GRIN2B gain-of-function mutations as a cause of West syndrome with severe developmental delay as well as of ID with childhood onset focal epilepsy. Severely disturbed channel function corresponded to severe clinical phenotypes, underlining the important role of facilitated NMDA receptor signaling in epileptogenesis.


Assuntos
Epilepsias Parciais/genética , Deficiência Intelectual/genética , Mutação/genética , Receptores de N-Metil-D-Aspartato/genética , Espasmos Infantis/genética , Animais , Criança , Pré-Escolar , Cristalografia por Raios X , Epilepsias Parciais/complicações , Epilepsias Parciais/diagnóstico , Feminino , Humanos , Recém-Nascido , Deficiência Intelectual/complicações , Deficiência Intelectual/diagnóstico , Ratos , Receptores de N-Metil-D-Aspartato/química , Espasmos Infantis/complicações , Espasmos Infantis/diagnóstico , Xenopus laevis
17.
ACS Chem Neurosci ; 4(11): 1469-78, 2013 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-23992940

RESUMO

Pentameric glycine receptors (GlyRs) couple agonist binding to activation of an intrinsic ion channel. Substitution of the R271 residue impairs agonist-induced activation and is associated with the human disease hyperekplexia. On the basis of a homology model of the α1 GlyR, we substituted residues in the vicinity of R271 with cysteines, generating R271C, Q226C, and D284C single-mutant GlyRs and R271C/Q226C and R271C/D284C double-mutant GlyRs. We then examined the impact of interactions between these positions on receptor activation by glycine and modulation by the anesthetic propofol, as measured by electrophysiological experiments. Upon expression in Xenopus laevis oocytes, D284C-containing receptors were nonfunctional, despite biochemical evidence of successful cell surface expression. At R271C/Q226C GlyRs, glycine-activated whole-cell currents were increased 3-fold in the presence of the thiol reductant dithiothreitol, whereas the ability of propofol to enhance glycine-activated currents was not affected by dithiothreitol. Biochemical experiments showed that mutant R271C/Q226C subunits form covalently linked pentamers, showing that intersubunit disulfide cross-links are formed. These data indicate that intersubunit disulfide links in the transmembrane domain prevent a structural transition that is crucial to agonist-induced activation of GlyRs but not to modulation by the anesthetic propofol and implicate D284 in the functional integrity of GlyRs.


Assuntos
Propofol/química , Propofol/farmacologia , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Receptores de Glicina/química , Receptores de Glicina/metabolismo , Rigidez Muscular Espasmódica/genética , Rigidez Muscular Espasmódica/metabolismo , Anestésicos Intravenosos , Animais , Dissulfetos/química , Ditiotreitol/química , Humanos , Canais Iônicos de Abertura Ativada por Ligante/agonistas , Canais Iônicos de Abertura Ativada por Ligante/química , Canais Iônicos de Abertura Ativada por Ligante/metabolismo , Mutação/efeitos dos fármacos , Neurotransmissores/química , Neurotransmissores/metabolismo , Oócitos/química , Oócitos/efeitos dos fármacos , Propofol/agonistas , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/genética , Estrutura Terciária de Proteína/efeitos dos fármacos , Subunidades Proteicas/genética , Rigidez Muscular Espasmódica/fisiopatologia , Regulação para Cima/efeitos dos fármacos , Xenopus laevis
18.
Nat Genet ; 45(9): 1067-72, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23933819

RESUMO

Idiopathic focal epilepsy (IFE) with rolandic spikes is the most common childhood epilepsy, comprising a phenotypic spectrum from rolandic epilepsy (also benign epilepsy with centrotemporal spikes, BECTS) to atypical benign partial epilepsy (ABPE), Landau-Kleffner syndrome (LKS) and epileptic encephalopathy with continuous spike and waves during slow-wave sleep (CSWS). The genetic basis is largely unknown. We detected new heterozygous mutations in GRIN2A in 27 of 359 affected individuals from 2 independent cohorts with IFE (7.5%; P = 4.83 × 10(-18), Fisher's exact test). Mutations occurred significantly more frequently in the more severe phenotypes, with mutation detection rates ranging from 12/245 (4.9%) in individuals with BECTS to 9/51 (17.6%) in individuals with CSWS (P = 0.009, Cochran-Armitage test for trend). In addition, exon-disrupting microdeletions were found in 3 of 286 individuals (1.0%; P = 0.004, Fisher's exact test). These results establish alterations of the gene encoding the NMDA receptor NR2A subunit as a major genetic risk factor for IFE.


Assuntos
Epilepsias Parciais/genética , Mutação , Receptores de N-Metil-D-Aspartato/genética , Substituição de Aminoácidos , Epilepsias Parciais/diagnóstico , Feminino , Humanos , Masculino , Modelos Moleculares , Mutação de Sentido Incorreto , Linhagem , Conformação Proteica , Receptores de N-Metil-D-Aspartato/química , Receptores de N-Metil-D-Aspartato/metabolismo
19.
J Neurochem ; 122(1): 38-47, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22486198

RESUMO

Synaptic glycine receptors (GlyRs) are hetero-pentameric chloride channels composed of α and ß subunits, which are activated by agonist binding at subunit interfaces. To examine the pharmacological properties of each potential agonist-binding site, we substituted residues of the GlyR α(1) subunit by the corresponding residues of the ß subunit, as deduced from sequence alignment and homology modeling based on the recently published crystal structure of the glutamate-gated chloride channel GluCl. These exchange substitutions allowed us to reproduce the ßα, αß and ßß subunit interfaces present in synaptic heteromeric GlyRs by generating recombinant homomeric receptors. When the engineered α(1) GlyR mutants were expressed in Xenopus oocytes, all subunit interface combinations were found to form functional agonist-binding sites as revealed by voltage clamp recording. The ßß-binding site displayed the most distinct pharmacological profile towards a range of agonists and modulators tested, indicating that it might be selectively targeted to modulate the activity of synaptic GlyRs. The mutational approach described here should be generally applicable to heteromeric ligand-gated ion channels composed of homologous subunits and facilitate screening efforts aimed at targeting inter-subunit specific binding sites.


Assuntos
Subunidades Proteicas/metabolismo , Receptores de Glicina/agonistas , Receptores de Glicina/genética , Sequência de Aminoácidos , Animais , Sítios de Ligação/efeitos dos fármacos , Simulação por Computador , Cobre , Etanol/farmacologia , Glicinérgicos/farmacologia , Concentração Inibidora 50 , Ivermectina/farmacologia , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/genética , Microinjeções , Modelos Moleculares , Mutagênese , Mutação/genética , Nortropanos/farmacologia , Oócitos , Técnicas de Patch-Clamp , Subunidades Proteicas/química , Subunidades Proteicas/genética , Receptores de Glicina/química , Receptores de Glicina/metabolismo , Xenopus laevis , Zinco/farmacologia
20.
Behav Brain Res ; 226(1): 106-11, 2012 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-21924294

RESUMO

Glycinergic synapses play a major role in shaping the activity of spinal cord neurons under normal conditions and during persistent pain. However, the role of different glycine receptor (GlyR) subtypes in pain processing has only begun to be unraveled. Here, we analysed whether the GlyR alpha2 subunit might be involved in the processing of acute or persistent pain. Real-time RT-PCR and in situ hybridization analyses revealed that GlyR alpha2 mRNA is enriched in the dorsal horn of the mouse spinal cord. Mice lacking GlyR alpha2 (Glra2(-/-) mice) demonstrated a normal nociceptive behavior in models of acute pain and after peripheral nerve injury. However, mechanical hyperalgesia induced by peripheral injection of zymosan was significantly prolonged in Glra2(-/-) mice as compared to wild-type littermates. We conclude that spinal GlyRs containing the alpha2 subunit exert a previously unrecognized role in the resolution of inflammatory pain.


Assuntos
Hiperalgesia/genética , Receptores de Glicina/genética , Medula Espinal/metabolismo , Zimosan/farmacologia , Animais , Hiperalgesia/induzido quimicamente , Hiperalgesia/metabolismo , Inflamação/genética , Inflamação/metabolismo , Camundongos , Camundongos Knockout , Neurônios/metabolismo , Receptores de Glicina/metabolismo , Sinapses/metabolismo
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