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.
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. Sequencing of GRIN2B in 468 individuals with mental retardation revealed four de novo mutations: a frameshift, a missense and two splice-site mutations. In another cohort of 127 individuals with idiopathic epilepsy and/or mental retardation, we discovered a GRIN2A nonsense mutation in a three-generation family. In a girl with early-onset epileptic encephalopathy, we identified the de novo GRIN2A mutation c.1845C>A predicting the amino acid substitution p.N615K. Analysis of NR1-NR2AN615K (NR2A subunit with the p.N615K alteration) receptor currents revealed a loss of the Mg2+ block and a decrease in Ca2+ permeability. Our findings suggest that disturbances in the neuronal electrophysiological balance during development result in variable neurological phenotypes depending on which NR2 subunit of NMDA receptors is affected.
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. 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 Ca
-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.
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 Kcv
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 Kcv
. 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.
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.
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. 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 IIβ (Top2β). Western blot analysis revealed that the inhibition of NMDARs, cAMP-responsive element binding transcription factor (CREB) and Top2β decreased the expression of the proto-oncogene cFos. Knockdown of Top2β 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 Top2β 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.
Transmitter-gated ion channels mediate rapid synaptic transmission in the CNS and constitute important targets for many neuroactive drugs. Inhibitory glycine receptors (GlyRs) are members of the ...nicotinic acetylcholine receptor superfamily and inhibit neuronal firing by opening Cl− channels following agonist binding. In this article, we discuss recent developments in GlyR pharmacology, delineate the receptor domains that are involved in binding of agonists and allosteric modulators, and present a molecular model of the extracellular architecture of the receptor. The recent discovery of compounds that act preferentially on specific GlyR isoforms and the differential expression of these isoforms in distinct regions of the developing and adult CNS show considerable promise towards the development of drugs that act in defined glycine-mediated pathways. In particular, compounds that can potentiate GlyR function should provide leads for novel muscle relaxants in addition to sedative and analgesic agents.
The recent discovery of compounds that act preferentially on specific glycine receptor (GlyR) isoforms and the differential expression of these isoforms in distinct regions of the developing and adult CNS show considerable promise towards the development of novel muscle relaxant and analgesic drugs.
Collybistin (Cb) is a brain‐specific guanine nucleotide exchange factor that has been implicated in plasma membrane targeting of the postsynaptic scaffolding protein gephyrin found at glycinergic and ...GABAergic synapses. Here we show that Cb‐deficient mice display a region‐specific loss of postsynaptic gephyrin and GABAA receptor clusters in the hippocampus and the basolateral amygdala. Cb deficiency is accompanied by significant changes in hippocampal synaptic plasticity, due to reduced dendritic GABAergic inhibition. Long‐term potentiation is enhanced, and long‐term depression reduced, in Cb‐deficient hippocampal slices. Consistent with the anatomical and electrophysiological findings, the animals show increased levels of anxiety and impaired spatial learning. Together, our data indicate that Cb is essential for gephyrin‐dependent clustering of a specific set of GABAA receptors, but not required for glycine receptor postsynaptic localization.
The glycine transporter subtype 2 (GlyT2) is localized in the axon terminals of glycinergic neurons. Mice deficient in GlyT2 are normal at birth but during the second postnatal week develop a lethal ...neuromotor deficiency that resembles severe forms of human hyperekplexia (hereditary startle disease) and is characterized by spasticity, tremor, and an inability to right. Histological and immunological analyses failed to reveal anatomical or biochemical abnormalities, but the amplitudes of glycinergic miniature inhibitory currents (mIPSCs) were strikingly reduced in hypoglossal motoneurons and dissociated spinal neurons from GlyT2-deficient mice. Thus, postnatal GlyT2 function is crucial for efficient transmitter loading of synaptic vesicles in glycinergic nerve terminals, and the GlyT2 gene constitutes a candidate disease gene in human hyperekplexia patients.
J. Neurochem. (2012) 122, 38–47.
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 α1 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. When the engineered α1 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 ββ‐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.
The pharmacological profiles of the distinct subunit interfaces found in heteromeric synaptic GlyRs (scheme, left) have not been characterized in detail. By exchange substitutions of the agonist binding loops, the βα, αβ and ββ interfaces present in native synaptic GlyRs were individually generated in recombinant homomeric receptors (scheme, right). This approach should be generally applicable to the analysis of ligand binding sites located at subunit interfaces of heteromeric receptors composed of homologous subunits.
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