APP fragment controls both ionotropic and non-ionotropic signaling of NMDA receptors.

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-β precursor protein (APP) cleavage product, as an NMDAR modulator with the unique dual regulatory capacity to impact both signaling modes.

Analysis of M4 Transmembrane Segments in NMDA Receptor Function: A Negative Allosteric Modulatory Site at the GluN1 M4 is Determining the Efficiency of Neurosteroid Modulation.

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.

Thermophoretic analysis of ligand-specific conformational states of the inhibitory glycine receptor embedded in copolymer nanodiscs.

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.

Electrical Sensing of Phosphonates by Functional Coupling of Phosphonate Binding Protein PhnD to Solid-State Nanopores.

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.

NMDA Receptor-Mediated Signaling Pathways Enhance Radiation Resistance, Survival and Migration in Glioblastoma Cells-A Potential Target for Adjuvant Radiotherapy.

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 Ca-permeable NMDARs in three glioblastoma cell lines.

NMDA Receptor Signaling Mediates cFos Expression via Top2β-Induced DSBs in Glioblastoma Cells.

The activation of Ca-permeable -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.

Coupling of a viral K-channel with a glutamate-binding-domain highlights the modular design of ionotropic glutamate-receptors.

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.

Ligand binding domain interface: A tipping point for pharmacological agents binding with GluN1/2A subunit containing NMDA receptors.

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.

Distinct roles of secreted APP ectodomain variants APPsα and APPsβ in regulation of spine density, synaptic plasticity, and cognition.

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 APPsβ fragments, generated by non-amyloidogenic or amyloidogenic APP processing, respectively, remain however unclear. Here, we expressed APPsα or APPsβ in the adult brain of conditional double knockout mice (cDKO) lacking APP and the related APLP2.

Positive Modulatory Interactions of NMDA Receptor GluN1/2B Ligand Binding Domains Attenuate Antagonists Activity.

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.

encephalopathy: novel findings on phenotype, variant clustering, functional consequences and treatment aspects.

Background: We aimed for a comprehensive delineation of genetic, functional and phenotypic aspects of encephalopathy and explored potential prospects of personalised medicine.

Methods: Data of 48 individuals with de novo variants were collected from several diagnostic and research cohorts, as well as from 43 patients from the literature. Functional consequences and response to memantine treatment were investigated in vitro and eventually translated into patient care.

Delineating the GRIN1 phenotypic spectrum: A distinct genetic NMDA receptor encephalopathy.

Objective: To determine the phenotypic spectrum caused by mutations in GRIN1 encoding the NMDA receptor subunit GluN1 and to investigate their underlying functional pathophysiology.

Methods: We collected molecular and clinical data from several diagnostic and research cohorts. Functional consequences of GRIN1 mutations were investigated in Xenopus laevis oocytes.

The N-terminal domain of the GluN3A subunit determines the efficacy of glycine-activated NMDA receptors.

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.

Molecular basis for convergent evolution of glutamate recognition by pentameric ligand-gated ion channels.

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.

Comparative pharmacology of flatworm and roundworm glutamate-gated chloride channels: Implications for potential anthelmintics.

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.

Opposing effects of the anesthetic propofol at pentameric ligand-gated ion channels mediated by a common site.

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.

GRIN2B mutations in West syndrome and intellectual disability with focal epilepsy.

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.

Propofol modulation of α1 glycine receptors does not require a structural transition at adjacent subunits that is crucial to agonist-induced activation.

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.

Mutations in GRIN2A cause idiopathic focal epilepsy with rolandic spikes.

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.

Probing the pharmacological properties of distinct subunit interfaces within heteromeric glycine receptors reveals a functional ββ agonist-binding site.

Synaptic glycine receptors (GlyRs) are hetero-pentameric chloride channels composed of α and β 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 β 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 βα, αβ and ββ subunit interfaces present in synaptic heteromeric GlyRs by generating recombinant homomeric receptors.

Structure-activity relationships for allosteric NMDA receptor inhibitors based on 2-naphthoic acid.

Over-activation of N-methyl-d-aspartate (NMDA) receptors is critically involved in many neurological conditions, thus there has been considerable interest in developing NMDA receptor antagonists. We have recently identified a series of naphthoic and phenanthroic acid compounds that allosterically modulate NMDA receptors through a novel mechanism of action. In the present study, we have determined the structure-activity relationships of 18 naphthoic acid derivatives for the ability to inhibit the four GluN1/GluN2(A-D) NMDA receptor subtypes.

Prolonged zymosan-induced inflammatory pain hypersensitivity in mice lacking glycine receptor alpha2.

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.

Robust post-translocational N-glycosylation at the extreme C-terminus of membrane and secreted proteins in Xenopus laevis oocytes and HEK293 cells.

N-Glycosylation is normally a co-translational process that occurs as soon as a nascent and unfolded polypeptide chain has emerged ~12 residues into the lumen of the endoplasmic reticulum (ER). Here, we describe the efficient utilization of an N-glycosylation site engineered within the luminal extreme C-terminal residues of distinct integral membrane glycoproteins, a native ER resident protein and an engineered secreted protein. This N-glycan addition required that the acceptor asparagine within an Asn-Trp-Ser sequon be located at least four residues away from the C-terminus of the polypeptide and, in the case of membrane proteins, at least 13 residues away from the lumenal side of the transmembrane segment.

Mutations in GRIN2A and GRIN2B encoding regulatory subunits of NMDA receptors cause variable neurodevelopmental phenotypes.

N-methyl-D-aspartate (NMDA) receptors mediate excitatory neurotransmission in the mammalian brain. Two glycine-binding NR1 subunits and two glutamate-binding NR2 subunits each form highly Ca²(+)-permeable cation channels which are blocked by extracellular Mg²(+) in a voltage-dependent manner. Either GRIN2B or GRIN2A, encoding the NMDA receptor subunits NR2B and NR2A, was found to be disrupted by chromosome translocation breakpoints in individuals with mental retardation and/or epilepsy.