Modulation of glycine potency in rat recombinant NMDA receptors containing chimeric NR2A/2D subunits expressed in Xenopus laevis oocytes (original) (raw)
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Principal role of NR3 subunits in NR1/NR3 excitatory glycine receptor function
Biochemical and Biophysical Research Communications, 2007
Calcium-permeable N-methyl-D-aspartate (NMDA) receptors are tetrameric cation channels composed of glycine-binding NR1 and glutamate-binding NR2 subunits, which require binding of both glutamate and glycine for efficient channel gating. In contrast, receptors assembled from NR1 and NR3 subunits function as calcium-impermeable excitatory glycine receptors that respond to agonist application only with low efficacy. Here, we show that antagonists of and substitutions within the glycine-binding site of NR1 potentiate NR1/ NR3 receptor function up to 25-fold, but inhibition or mutation of the NR3 glycine binding site reduces or abolishes receptor activation. Thus, glycine bound to the NR1 subunit causes auto-inhibition of NR1/NR3 receptors whereas glycine binding to the NR3 subunits is required for opening of the ion channel. Our results establish differential roles of the high-affinity NR3 and low-affinity NR1 glycinebinding sites in excitatory glycine receptor function.
The glycine coagonist site of the NMDA receptor
Advances in experimental medicine and biology, 1990
The discovery of the glycine binding site on the NMDA receptor (Johnson and Ascher, 1987) is recognized as a highly significa;1t event by those interested in receptor-channel mechanisms. It also offers a new target site in drug development for the treatment of neuropa-thologies associated with NMDA receptor activation. The list of neural dysfunctions mediated in part by synaptic activation of NMDA receptors very likely includes epileptiform seizures (Croucher et ai., 1982) and brain damage induced by ischemia or hypoxia in some models (Simon et ai., 1984). Growing evidence indicates however that NMDA receptor activation is important also for establishing certain neuronal connections during development (Tsumoto et ai., 1987; Kleinschmidt et ai., 1987; Lincoln et ai., 1988) and has been implicated in some types of learning or memory (Morris et ai., 1986). Thus both agonists and antagonists of the NMDA receptor may find clinical utility in the future. Currently available NMDA receptor blockers, acting either at the glutamate binding site or the open ion channel, either have difficulty penetrating the blood-brain barrier or have unacceptable side effects such as psychosis. The glycine site appears to present a pharmacology sufficiently different from that of the other binding sites on the NMDA receptor to offer a novel target for new drug discovery. The preparation we hilve used is the Xenopus oocyte injected with rat brain mRNA. It is a large (1 mm diameter), hardy cell that ciln be cultured easily for a week or more after injection, during which time transliltion of the foreign mRNA, ilssembly of multisubunit receptors and right-side-out insertion into the plasma membrane occurs. Receptor properties arc then studied under well-defined voltage cl<Jmp conditions. The reproducibility of this preparation offers a reliable bioassilY for the quantitiltive study of receptor-coupled ion channel properties. We have shown previously that NMDA and kainilte/quisqualate receptors with properties indistinguishable from those in neurons are expressed by oocytes (
British Journal of Pharmacology, 2000
The potency of two novel glycine site antagonists, GV150,526A and GV196,771A, was assessed by their ability to inhibit the binding of [3H]‐MDL105,519 to cell homogenates prepared from mammalian cells transfected with either NR1‐1a, NR1‐2a, NR1‐1a/NR2A, NR1‐1a/NR2B, NR1‐1a/NR2C or NR1‐1a/NR2D NMDA receptor clones. The inhibition constants (Kis) for GV150,526A displacement of [3H]‐MDL105,519 binding to either NR1‐1a or NR1‐2a expressed alone were not significantly different and were best fit by a one‐site binding model. GV150,526A inhibition to NR1‐1a/NR2 combinations was best fit by a two‐site model with the NR1‐1a/NR2C having an approximate 2–4 fold lower affinity compared to other NR1‐1a/NR2 receptors. The Kis for GV196,771A displacement of [3H]‐MDL105,519 binding to NR1‐1a, NR1‐2a and all NR1‐1a/NR2 combinations was best fit by a two‐site binding model. There was no significant difference between the Kis for the binding to NR1‐1a and NR1‐2a; NR1‐1a/NR2A receptors had an approximat...
Glycine-dependent activation of NMDA receptors
The Journal of general physiology, 2015
N-methyl-d-aspartate (NMDA) receptors are the only neurotransmitter receptors whose activation requires two distinct agonists. Heterotetramers of two GluN1 and two GluN2 subunits, NMDA receptors are broadly distributed in the central nervous system, where they mediate excitatory currents in response to synaptic glutamate release. Pore opening depends on the concurrent presence of glycine, which modulates the amplitude and time course of the glutamate-elicited response. Gating schemes for fully glutamate- and glycine-bound NMDA receptors have been described in sufficient detail to bridge the gap between microscopic and macroscopic receptor behaviors; for several receptor isoforms, these schemes include glutamate-binding steps. We examined currents recorded from cell-attached patches containing one GluN1/GluN2A receptor in the presence of several glycine-site agonists and used kinetic modeling of these data to develop reaction schemes that include explicit glycine-binding steps. Based...
1998
The NMDA type of ligand-gated glutamate receptor requires the presence of both glutamate and glycine for gating. These receptors are hetero-oligomers of NR1 and NR2 subunits. Previously it was thought that the binding sites for glycine and glutamate were formed by residues on the NR1 subunit. Indeed, it has been shown that the effects of glycine are controlled by residues on the NR1 subunit, and a "Venus flytrap" model for the glycine binding site has been suggested by analogy with bacterial periplasmic amino acid binding proteins. By analysis of 10 mutant NMDA receptors, we now show that residues on the NR2A subunit control glutamate potency in recombinant NR1/NR2A receptors, without affecting glycine potency. Furthermore, we provide evidence that, at least for some mutated residues, the reduced potency of glutamate cannot be explained by alteration of gating but has to be caused primarily by impairing the binding of the agonist to the resting state of the receptor. One NR2A mutant, NR2A(T671A), had an EC 50 for glutamate 1000-fold greater than wild type and a 255-fold reduced affinity for APV, yet it had single-channel openings very similar to those of wild type. Therefore we propose that the glutamate binding site is located on NR2 subunits and (taking our data together with previous work) is not on the NR1 subunit. Our data further imply that each NMDA receptor subunit possesses a binding site for an agonist (glutamate or glycine).
The Journal of neuroscience : the official journal of the Society for Neuroscience, 2000
We have used site-directed mutagenesis in conjunction with homologous recombination to generate two mouse lines carrying point mutations in the glycine binding site of the NMDAR1 subunit (Grin1). Glycine concentration-response curves from acutely dissociated hippocampal neurons revealed a 5- and 86-fold reduction in receptor glycine affinity in mice carrying Grin1(D481N) and Grin1(K483Q) mutations, respectively, whereas receptor glutamate affinity remained unaffected. Homozygous mutant Grin1(D481N) animals are viable and fertile and appear to develop normally. However, homozygous mutant Grin1(K483Q) animals are significantly lighter at birth, do not feed, and die within a few days. No gross abnormalities in CNS anatomy were detected in either Grin1(D481N) or Grin1(K483Q) mice. Interestingly, in situ hybridization and Western blot analysis revealed changes in the expression levels of NMDA receptor subunits in Grin1(D481N) mice relative to wild type that may represent a compensatory r...
Neuron, 1997
stimulation resulting in high Ca 2ϩ influx causes neuronal cell death in ischemia and other neurodegenerative dis-Heinrich Betz, and Jochen Kuhse orders (Choi, 1988). Third, simultaneous binding of glu-Department of Neurochemistry tamate and the coagonist glycine is required for efficient Max-Planck-Institute for Brain Research activation of NMDA receptors (Johnson and Ascher, Deutschordenstrae 46 1987; Kleckner and Dingledine, 1988). The mechanism 60528 Frankfurt of coagonist action is not entirely clear but may involve Federal Republic of Germany allosteric modulation of agonist binding affinities (Mayer *Symphony Pharmaceuticals Inc. et al., 1989). Pharmacological and electrophysiological Frazer, Pennsylvania 19355 studies indicate that glutamate and glycine occupy distinct sites of the receptor protein (Reynolds et al., 1987; Henderson et al., 1990). Summary Molecular studies have shown that NMDA receptors are oligomeric membrane proteins composed of homol-NMDA receptors require both L-glutamate and the coogous NMDAR1 (NR1) and NR2 subunits (reviewed by agonist glycine for efficient channel activation. The Hollmann and Heinemann, 1994). The NR1 and NR2 subglycine binding site of these heteromeric receptor prounits are synthesized as precursor proteins with a cleavteins is formed by regions of the NMDAR1 (NR1) subable leader sequence followed by a long N-terminal unit that display sequence similarity to bacterial amino extracellular domain and, in the second half of the polyacid binding proteins. Here, we demonstrate that the peptides, four hydrophobic membrane segments (M1glutamate binding site is located on the homologous M4). The M1, M3, and M4 segments are transmembrane regions of the NR2B subunit. Mutation of residues spanning, whereas segment M2 is thought to form a within the N-terminal domain and the loop region bereentrant loop (Hollmann and Heinemann, 1994; Bennett tween membrane segments M3 and M4 significantly and Dingledine, 1995; Wo and Oswald, 1994; Hirai et al., reduced the efficacy of glutamate, but not glycine, in 1996) that lines the ion channel (Kuner et al., 1996). channel gating. Some of the mutations also decreased The NR1 subunit is expressed in several distinct splice inhibition by the glutamate antagonists, D-AP5 and variants throughout the central nervous system (Durand R-CPP. Homology-based molecular modeling of the et al., 1992; Nakanishi et al., 1992; Sugihara et al., 1992; glutamate and glycine binding domains indicates that Hollmann et al., 1993). The NR2 subunit exists in four the NR2 and NR1 subunits use similar residues to liisoforms encoded by different genes (NR2A-D), which gate the agonists' ␣-aminocarboxylic acid groups, create further functional and regional heterogeneity of whereas differences in side chain interactions and size NMDA receptors (Kutsuwada et al., 1992; Meguro et al., of aromatic residues determine ligand selectivity. 1992; Monyer et al., 1992). Heterologous expression in Xenopus laevis oocytes has suggested that the NR1
2007
The N-methyl-D-aspartate (NMDA) subtype of ionotropic glutamate receptors (iGluRs) is a tetrameric complex composed of homologous NR1 and NR2 subunits, which require the binding of glycine and glutamate, respectively, for efficient channel gating. The extracellular N-terminal domains (NTDs) of iGluR subunits show sequence homology to the bacterial periplasmic leucine/isoleucine/valine binding protein (LIVBP) and have been implicated in iGluR assembly, trafficking and function. Here, we investigated how deletion of the NR1-and NR2-NTDs affects the expression and function of NMDA receptors. Both proteolytic cleavage of the NR1-NTD from assembled NR1/NR2 receptors and co-expression of the NTD-deleted NR1 subunit with wildtype or NTD-deleted NR2 subunits resulted in agonist-gated channels that closely resembled wild-type receptors. This indicates that the NTDs of both NMDA receptor subunits are not essential for receptor assembly and function. However, deletion of either the NR1 or the NR2 NTD eliminated high-affinity, allosteric inhibition of agonist-induced currents by Zn 2+ and ifenprodil, consistent with interdomain interactions between these domains being important for allosteric receptor modulation. Furthermore, by replacing the NR2A-NTD with the NR2B NTD, and vice versa, the different glycine affinities of NR1/NR2A and NR1/NR2B receptors were found to be determined by their respective NR2-NTDs. Together, these data show that the NTDs of both the NR1 and NR2 subunits determine allosteric inhibition and glycine potency but are not required for NMDA receptor assembly.