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. 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

Potentiation of Glycine-Gated NR1/NR3A NMDA Receptors Relieves Ca2+-Dependent Outward Rectification

Glycine has diverse functions within the mammalian central nervous system. It inhibits postsynaptic neurons via strychnine-sensitive glycine receptors (GlyRs) and enhances neuronal excitation through co-activation of N-methyl-D-aspartate (NMDA) receptors. Classical Ca2+-permeable NMDA receptors are composed of glycine-binding NR1 and glutamate-binding NR2 subunits, and hence require both glutamate and glycine for efficient activation. In contrast, recombinant receptors composed of NR1 and the glycine binding NR3A and/or NR3B subunits lack glutamate binding sites and can be activated by glycine alone. Therefore these receptors are also named “excitatory glycine receptors”. Co-application of antagonists of the NR1 glycine-binding site or of the divalent cation Zn2+ markedly enhances the glycine responses of these receptors. To gain further insight into the properties of these glycine-gated NMDA receptors, we investigated their current-voltage (I–V) dependence. Whole-cell current-voltage relations of glycine currents recorded from NR1/NR3B and NR1/NR3A/NR3B expressing oocytes were found to be linear under our recording conditions. In contrast, NR1/NR3A receptors displayed a strong outwardly rectifying I–V relation. Interestingly, the voltage-dependent inward current block was abolished in the presence of NR1 antagonists, Zn2+ or a combination of both. Further analysis revealed that Ca2+ (1.8 mM) present in our recording solutions was responsible for the voltage-dependent inhibition of ion flux through NR1/NR3A receptors. Since physiological concentrations of the divalent cation Mg2+ did not affect the I–V dependence, our data suggest that relief of the voltage-dependent Ca2+ block of NR1/NR3A receptors by Zn2+ may be important for the regulation of excitatory glycinergic transmission, according to the Mg2+-block of conventional NR1/NR2 NMDA receptors

Molecular Determinants of Agonist Discrimination by NMDA Receptor Subunits: Analysis of the Glutamate Binding Site on the NR2B Subunit

AbstractNMDA receptors require both L-glutamate and the coagonist glycine for efficient channel activation. The glycine binding site of these heteromeric receptor proteins is formed by regions of the NMDAR1 (NR1) subunit that display sequence similarity to bacterial amino acid binding proteins. Here, we demonstrate that the glutamate binding site is located on the homologous regions of the NR2B subunit. Mutation of residues within the N-terminal domain and the loop region between membrane segments M3 and M4 significantly reduced the efficacy of glutamate, but not glycine, in channel gating. Some of the mutations also decreased inhibition by the glutamate antagonists, D-AP5 and R-CPP. Homology-based molecular modeling of the glutamate and glycine binding domains indicates that the NR2 and NR1 subunits use similar residues to ligate the agonists' α-aminocarboxylic acid groups, whereas differences in side chain interactions and size of aromatic residues determine ligand selectivity

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. 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

Hyperekplexia Phenotype of Glycine Receptor α1 Subunit Mutant Mice Identifies Zn2+ as an Essential Endogenous Modulator of Glycinergic Neurotransmission

SummaryZn2+ is thought to modulate neurotransmission by affecting currents mediated by ligand-gated ion channels and transmitter reuptake by Na+-dependent transporter systems. Here, we examined the in vivo relevance of Zn2+ neuromodulation by producing knockin mice carrying the mutation D80A in the glycine receptor (GlyR) α1 subunit gene (Glra1). This substitution selectively eliminates the potentiating effect of Zn2+ on GlyR currents. Mice homozygous for Glra1(D80A) develop a severe neuromotor phenotype postnatally that resembles forms of human hyperekplexia (startle disease) caused by mutations in GlyR genes. In spinal neurons and brainstem slices from Glra1(D80A) mice, GlyR expression, synaptic localization, and basal glycinergic transmission were normal; however, potentiation of spontaneous glycinergic currents by Zn2+ was significantly impaired. Thus, the hyperekplexia phenotype of Glra1(D80A) mice is due to the loss of Zn2+ potentiation of α1 subunit containing GlyRs, indicating that synaptic Zn2+ is essential for proper in vivo functioning of glycinergic neurotransmission

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.Results:We identified heterozygous de novo GRIN1 mutations in 14 individuals and reviewed the phenotypes of all 9 previously reported patients. These 23 individuals presented with a distinct phenotype of profound developmental delay, severe intellectual disability with absent speech, muscular hypotonia, hyperkinetic movement disorder, oculogyric crises, cortical blindness, generalized cerebral atrophy, and epilepsy. Mutations cluster within transmembrane segments and result in loss of channel function of varying severity with a dominant-negative effect. In addition, we describe 2 homozygous GRIN1 mutations (1 missense, 1 truncation), each segregating with severe neurodevelopmental phenotypes in consanguineous families.Conclusions:De novo GRIN1 mutations are associated with severe intellectual disability with cortical visual impairment as well as oculomotor and movement disorders being discriminating phenotypic features. Loss of NMDA receptor function appears to be the underlying disease mechanism. The identification of both heterozygous and homozygous mutations blurs the borders of dominant and recessive inheritance of GRIN1-associated disorders.Johannes R. Lemke (32EP30_136042/1) and Peter De Jonghe (G.A.136.11.N and FWO/ESF-ECRP) received financial support within the EuroEPINOMICS-RES network (www.euroepinomics.org) within the Eurocores framework of the European Science Foundation (ESF). Saskia Biskup and Henrike Heyne received financial support from the German Federal Ministry for Education and Research (BMBF IonNeurONet: 01 GM1105A and FKZ: 01EO1501). Katia Hardies is a PhD fellow of the Institute for Science and Technology (IWT) Flanders. Ingo Helbig was supported by intramural funds of the University of Kiel, by a grant from the German Research Foundation (HE5415/3-1) within the EuroEPINOMICS framework of the European Science Foundation, and additional grants of the German Research Foundation (DFG, HE5415/5-1, HE 5415/6-1), German Ministry for Education and Research (01DH12033, MAR 10/012), and grant by the German chapter of the International League against Epilepsy (DGfE). The project also received infrastructural support through the Institute of Clinical Molecular Biology in Kiel, supported in part by DFG Cluster of Excellence "Inflammation at Interfaces" and "Future Ocean." The project was also supported by the popgen 2.0 network (P2N) through a grant from the German Ministry for Education and Research (01EY1103) and by the International Coordination Action (ICA) grant G0E8614N. Christel Depienne, Caroline Nava, and Delphine Heron received financial support for exome analyses by the Centre National de Genotypage (CNG, Evry, France)

Potentiation of inhibitory glycinergic neurotransmission by Zn2+: a synergistic interplay between presynaptic P2X2 and postsynaptic glycine receptors.

The divalent cation zinc is known to modulate chloride currents carried by native and recombinant mammalian glycine receptors (GlyRs). To unravel the effect of Zn2+ on glycinergic neurotransmission, inhibitory postsynaptic currents (IPSC) of rat spinal neurons grown in culture were analysed in the absence and presence of Zn2+. Low concentrations of Zn2+ (0.5 and 5 micro m) augmented the mean amplitude of miniature IPSCs by approximately 40% over values obtained in the absence of zinc, whereas higher concentrations of Zn2+ (50 micro m) significantly decreased mean amplitude values. Remarkably, low concentrations of Zn2+ also significantly increased the mean frequency of miniature IPSCs. This effect was blocked by the P2X receptor antagonists PPADS and suramin, indicating the presence of Zn2+-sensitive presynaptic P2X receptors on glycinergic terminals. Immunostaining with antibodies against different P2X receptor subtypes revealed that P2X2 receptors partially colocalize with the GlyR. Potentiating concentrations of Zn2+ also affected the kinetics of miniature and evoked IPSCs by significantly prolonging their decay time constants. Electrophysiological analysis of heterologously expressed glycine transporters (GlyT) revealed for GlyT2 zero, and for GlyT1 a modest (< 20%), reduction of glycine uptake in the presence of 5 micro m Zn2+, indicating that prolongation of glycinergic IPSCs by Zn2+ is not due to inhibition of glycine removal from the synaptic cleft. Together, these results suggest that Zn2+ is a potent modulator of glycinergic synaptic transmission which increases in a synergistic manner the agonist affinity of both presynaptic P2X2 receptors and postsynaptic GlyRs

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. Here, we fuse the pore module of the viral K+-channel KcvATCV-1 to the isolated glutamatebinding 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