Bodo Laube

Evidence for a Tetrameric Structure of Recombinant NMDA Receptors

The amino acids l-glutamate and glycine are essential agonists of the excitatory NMDA receptor, a subtype of the ionotropic glutamate receptor family. The native NMDA receptor is composed of two types of homologous membrane-spanning subunits, NR1 and NR2. Here, the numbers of glycine-binding NR1 and glutamate-binding NR2 subunits in the NMDA receptor hetero-oligomer were determined by coexpressing the wild-type (wt) NR1 with the low-affinity mutant NR1Q387K, and the wt NR2B with the low-affinity mutant NR2BE387A, subunits in Xenopusoocytes. In both cases, analysis of the resulting dose–response curves revealed three independent components of glycine and glutamate sensitivity. These correspond to the respective wild-type and mutant affinities and an additional intermediate hybrid affinity, indicating the existence of three discrete receptor populations. Binomial analysis of these data indicates the presence of two glycine and two glutamate binding subunits in the functional receptor. In addition, we analyzed the inhibitory effects of the negative dominant NR1R505K and NR2BR493K mutants on maximal inducible whole-cell currents of wt NR1/NR2B receptors. The inhibition profiles obtained on expression of increasing amounts of these mutant proteins again were fitted best by assuming an incorporation of two NR1 and two NR2 subunits into the receptor hetero-oligomer. Our data are consistent with NMDA receptors being tetrameric proteins that are composed of four homologous subunits.

Mutational analysis of the glycine-binding site of the NMDA receptor: structural similarity with bacterial amino acid-binding proteins.

Activation of the NMDA subtype of ionotropic glutamate receptors requires binding of both L-glutamate and the coagonist glycine. Site-directed mutagenesis of the NMDAR1 (NR1) subunit revealed that aromatic residues at positions 390, 392, and 466 are crucial determinants of glycine binding. Glutamate efficacy was little affected by mutations at these positions; however, inhibition of channel gating by the glycine antagonist 7-chlorokynurenic acid was drastically reduced. In addition, glutamine (Q387), valine (V666), and serine (S669) substitutions were found to reduce glycine efficacy. Since the mutated residues correspond to positions forming the binding site of homologous bacterial amino acid-binding proteins, a common amino acid-binding fold appears to be conserved from prokaryotic periplasmic proteins to glutamate receptors in the mammalian brain.

The β Subunit Determines the Ligand Binding Properties of Synaptic Glycine Receptors

Inhibitory glycine receptors (GlyRs) regulate motor coordination and sensory signal processing in spinal cord and other brain regions. GlyRs are pentameric proteins composed of membrane-spanning alpha and beta subunits. Here, site-directed mutagenesis combined with homology modeling based on the crystal structure of the acetylcholine binding protein identified key ligand binding residues of recombinant homooligomeric alpha1 and heterooligomeric alpha1beta GlyRs. This disclosed two highly conserved, oppositely charged residues located on adjacent subunit interfaces as being crucial for agonist binding. In addition, the beta subunit was found to determine the ligand binding properties of heterooligomeric GlyRs. Expression of an alpha1beta tandem construct and affinity purification of metabolically labeled GlyRs confirmed a subunit stoichiometry of 2alpha3beta. Because the beta subunit anchors GlyRs at synaptic sites, our results have important implications for the biosynthesis, clustering, and pharmacology of synaptic GlyRs.

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. 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.

Deletion of the Mouse Glycine Transporter 2 Results in a Hyperekplexia Phenotype and Postnatal Lethality

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.

Glycine receptors: recent insights into their structural organization and functional diversity

Strychnine‐sensitive glycine receptors (GlyRs) are known to mediate synaptic inhibition in spinal cord, brainstem and other regions of the CNS. During the past 5 years, considerable progress has been made in delineating structural determinants of ligand binding and channel activation in recombinant GlyRs. Furthermore, immunohistochemical and gene inactivation studies have disclosed distinct distributions and functions of differentially expressed GlyR subtypes in retina, hippocampus and the dorsal horn of the spinal cord. Accordingly, GlyRs regulate not only the excitability of motor and sensory neurones, but are also essential for the processing of photoreceptor signals, neuronal development and inflammatory pain sensitization. Hence, these receptors constitute promising targets for the development of clinically useful compounds.

Modulation of glycine receptor function: a novel approach for therapeutic intervention at inhibitory synapses?

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.

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 (7.5%; P = 4.83 × 10−18, Fishers 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, Fishers exact test). These results establish alterations of the gene encoding the NMDA receptor NR2A subunit as a major genetic risk factor for IFE.

Modulation by zinc ions of native rat and recombinant human inhibitory glycine receptors.

1. The effect of the divalent cation Zn2+ on inhibitory glycine receptor (GlyR) currents was investigated in rat embryonic spinal cord neurons and Xenopus oocytes expressing recombinant GlyRs. 2. In cultured spinal neurons, Zn2+ potentiated glycine‐induced whole‐cell currents about 3‐fold when applied extracellularly at concentrations of 0.5‐10 microM. In contrast, higher concentrations (> 100 microM) of Zn2+ decreased the glycine response. 3. A similar biphasic modulation of glycine‐induced currents by Zn2+ was also found with recombinant homo‐ and hetero‐oligomeric GlyRs generated in Xenopus oocytes. Dose‐response analysis showed that both the potentiating and inhibitory effects of Zn2+ result from changes in apparent agonist affinity. 4. Analysis of chimeric constructs of the GlyR alpha 1‐ and beta‐subunits revealed that the positive and negative modulatory effects of Zn2+ are mediated by different regions of the alpha 1‐subunit. 5. Our data indicate the existence of distinct high‐ and low‐affinity Zn2+ binding sites on the ligand‐binding alpha‐subunits of the GlyR. These sites may be implicated in the regulation of synaptic efficacy within glycinergic pathways.

Assembly of the inhibitory glycine receptor: identification of amino acid sequence motifs governing subunit stoichiometry.

The inhibitory glycine receptor (GlyR) is a pentameric protein composed of two types (alpha and beta) of membrane-spanning subunits. Coexpression in Xenopus oocytes of a low affinity mutant of the alpha 2 subunit with the alpha 1 and beta subunits indicated that GlyRs assembled from alpha 1 and alpha 2 polypeptides contain variable subunit ratios, whereas alpha/beta hetero-oligomers have an invariant (3:2) stoichiometry. Analysis of different alpha/beta chimeric constructs revealed that this difference in assembly behavior is mediated by the N-terminal extracellular regions of the receptor subunits. Substitution of residues diverging between the alpha and beta subunits identified combinations of sequence motifs determining subunit stoichiometry.