N-methyl-D-aspartate Receptor Gating Research Articles

Effects of ethanol on GluN1/GluN2A and GluN1/GluN2B NMDA receptor-ion channel gating kinetics.

The N-methyl-D-aspartate receptor (NMDAR) is a major molecular target of alcohol action in the central nervous system, yet many aspects of alcohol's modulation of the activity of this ion channel remain unclear. We and others have shown that ethanol inhibition of NMDAR involves alterations in gating, especially a reduction in mean open time. However, a full description of ethanol's effects on NMDAR kinetics, including fitting them to a kinetic model, has not been reported. To determine ethanol's effects on NMDAR kinetics, we used steady-state single-channel recording in outside-out patches from HEK-293 cells transfected with recombinant GluN1/GluN2A or GluN1/GluN2B NMDAR subunits. Very low glutamate concentrations were used to isolate individual activations of the receptor. In both subunit types, ethanol, at approximate whole-cell IC50 values (156 mM, GluN2A; 150 mM, GluN2B), reduced open probability (po ) by approximately 50% and decreased mean open time without changing the frequency of opening. Open and shut time distributions exhibited two and five components, respectively; ethanol selectively decreased the time constant and relative proportion of the longer open time component. In the GluN2A subunit, ethanol increased the time constants of all but the longest shut time components, whereas in the GluN2B subunit, shut times were unchanged by ethanol. Fitting of bursts of openings (representing individual activations of the receptor) to the gating portion of a kinetic model revealed that ethanol altered two rates: the rate associated with activation of the GluN2A or GluN2B subunit, and the rate associated with the closing of the longer of the two open states. These results demonstrate that ethanol selectively alters individual kinetic rates and thus appears to selectively affect distinct conformational transitions involved in NMDAR gating.

The structure-energy landscape of NMDA receptor gating.

N-methyl-D-aspartate (NMDA) receptors are the main calcium-permeable excitatory receptors in the mammalian central nervous system. The NMDA receptor gating is complex, exhibiting multiple closed, open, and desensitized states; however, the central questions regarding the conformations and energetics of the transmembrane domains as they relate to the gating states are still unanswered. Here, using single molecule Förster Resonance Energy Transfer (smFRET), we map the energy landscape of the first transmembrane segment of the Rattus norvegicus NMDA receptor under resting and various liganded conditions. These results show kinetically and structurally distinct changes associated with apo, agonist-bound, and inhibited receptors linked by a linear mechanism of gating at this site. Furthermore, the smFRET data suggest that allosteric inhibition by zinc occurs by an uncoupling of the agonist-induced changes at the extracellular domains from the gating motions leading to an apo-like state, while dizocilpine, a pore blocker, stabilizes multiple closely packed transmembrane states.

Two adjacent phenylalanines in the NMDA receptor GluN2A subunit M3 domain interactively regulate alcohol sensitivity and ion channel gating

The N-methyl-d-aspartate (NMDA) receptor is a key target of ethanol action in the central nervous system. Alcohol inhibition of NMDA receptor function involves small clusters of residues in the third and fourth membrane-associated (M) domains. Previous results from this laboratory have shown that two adjacent positions in the M3 domain, F636 and F637, can powerfully regulate alcohol sensitivity and ion channel gating. In this study, we report that these positions interact with one another in the regulation of both NMDA receptor gating and alcohol action. Using dual mutant cycle analysis, we detected interactions among various substitution mutants at these positions with respect to regulation of glutamate EC50, steady-state to peak current ratios (Iss:Ip), mean open time, and ethanol IC50. This interaction apparently involves a balancing of forces on the M3 helix, such that the disruption of function due to a substitution at one position can be reversed by a similar substitution at the other position. For example, tryptophan substitution at F636 or F637 increased or decreased channel mean open time, respectively, but tryptophan substitution at both positions did not alter open time. Interestingly, the effects of a number of mutations on receptor kinetics and ethanol sensitivity appeared to depend upon subtle structural differences, such as those between the isomeric amino acids leucine and isoleucine, as they could not be explained on the basis of sidechain molecular volume or hydrophilicity.

Single-Molecule Patch-Clamp FRET Anisotropy Microscopy Studies of NMDA Receptor Ion Channel Activation and Deactivation under Agonist Ligand Binding in Living Cells.

N-methyl-d-aspartate (NMDA) receptor ion channel is activated by the binding of two pairs of glycine and glutamate along with the application of action potential. Binding and unbinding of ligands changes its conformation that plays a critical role in the open-close activities of NMDA receptor. Conformation states and their dynamics due to ligand binding are extremely difficult to characterize either by conventional ensemble experiments or single-channel electrophysiology method. Here we report the development of a new correlated technical approach, single-molecule patch-clamp FRET anisotropy imaging and demonstrate by probing the dynamics of NMDA receptor ion channel and kinetics of glycine binding with its ligand binding domain. Experimentally determined kinetics of ligand binding with receptor is further verified by computational modeling. Single-channel patch-clamp and four-channel fluorescence measurement are recorded simultaneously to get correlation among electrical on and off states, optically determined conformational open and closed states by FRET, and binding-unbinding states of the glycine ligand by anisotropy measurement at the ligand binding domain of GluN1 subunit. This method has the ability to detect the intermediate states in addition to electrical on and off states. Based on our experimental results, we have proposed that NMDA receptor gating goes through at least one electrically intermediate off state, a desensitized state, when ligands remain bound at the ligand binding domain with the conformation similar to the fully open state.

Kinetic Contributions to Gating by Interactions Unique to N-methyl-d-aspartate (NMDA) Receptors

Among glutamate-gated channels, NMDA receptors produce currents that subside with unusually slow kinetics, and this feature is essential to the physiology of central excitatory synapses. Relative to the homologous AMPA and kainate receptors, NMDA receptors have additional intersubunit contacts in the ligand binding domain that occur at both conserved and non-conserved sites. We examined GluN1/GluN2A single-channel currents with kinetic analyses and modeling to probe these class-specific intersubunit interactions for their role in glutamate binding and receptor gating. We found that substitutions that eliminate such interactions at non-conserved sites reduced stationary gating, accelerated deactivation, and imparted sensitivity to aniracetam, an AMPA receptor-selective positive modulator. Abolishing unique contacts at conserved sites also reduced stationary gating and accelerated deactivation. These results show that contacts specific to NMDA receptors, which brace the heterodimer interface within the ligand binding domain, stabilize actively gating receptor conformations and result in longer bursts and slower deactivations. They support the view that the strength of the heterodimer interface modulates gating in both NMDA and non-NMDA receptors and that unique interactions at this interface are responsible in part for basic differences between the kinetics of NMDA and non-NMDA currents at glutamatergic synapses.

Is cholesterol good or bad for your brain?--NMDARs have a say.

Is cholesterol good or bad for your brain?--NMDARs have a say.

Genome-wide association study of NMDA receptor coagonists in human cerebrospinal fluid and plasma.

The N-methyl-D-aspartate receptor (NMDAR) coagonists glycine, D-serine and L-proline play crucial roles in NMDAR-dependent neurotransmission and are associated with a range of neuropsychiatric disorders. We conducted the first genome-wide association study of concentrations of these coagonists and their enantiomers in plasma and cerebrospinal fluid (CSF) of human subjects from the general population (N=414). Genetic variants at chromosome 22q11.2, located in and near PRODH (proline dehydrogenase), were associated with L-proline in plasma (β=0.29; P=6.38 × 10(-10)). The missense variant rs17279437 in the proline transporter SLC6A20 was associated with L-proline in CSF (β=0.28; P=9.68 × 10(-9)). Suggestive evidence of association was found for the D-serine plasma-CSF ratio at the D-amino-acid oxidase (DAO) gene (β=-0.28; P=9.08 × 10(-8)), whereas a variant in SRR (that encodes serine racemase and is associated with schizophrenia) constituted the most strongly associated locus for the L-serine to D-serine ratio in CSF. All these genes are highly expressed in rodent meninges and choroid plexus, anatomical regions relevant to CSF physiology. The enzymes and transporters they encode may be targeted to further construe the nature of NMDAR coagonist involvement in NMDAR gating. Furthermore, the highlighted genetic variants may be followed up in clinical populations, for example, schizophrenia and 22q11 deletion syndrome. Overall, this targeted metabolomics approach furthers the understanding of NMDAR coagonist concentration variability and sets the stage for non-targeted CSF metabolomics projects.

Inhibition of GluN2A-Containing N-Methyl-d-Aspartate Receptors by 2-Naphthoic Acid

N-Methyl-D-aspartate (NMDA) receptors mediate excitatory synaptic transmission in the central nervous system and play important roles in synaptic development and plasticity, but also mediate glutamate neurotoxicity. Recently, 2-naphthoic acid (NPA) and its derivatives have been identified as allosteric, noncompetitive NMDA receptor inhibitors. The selectivity of NPA derivatives among NMDA receptor subtypes was mapped structurally to the ligand-binding domain, and was proposed to be mediated by residues on the S1 segment. To delineate the kinetic mechanism by which NPA inhibits NMDA receptor activity, we examined its effects on the NMDA receptor gating reaction. Using whole-cell patch clamping on human embryonic kidney 293 cells expressing recombinant NMDA family of glutamate receptor subunits, GluN1/GluN2A, we found that NPA has a 50% inhibitory effect at 1.9 mM. Further, from one-channel current recordings, we found that 4 mM NPA caused a 62% decrease in open probability by decreasing mean open time 2.5-fold and by increasing mean closed time 2-fold. Kinetic modeling suggested that NPA binding stabilized NMDA receptor closed states and increased the energy barriers toward open states, causing NMDA receptors to dwell longer in pre-open states along the activation pathway. The reaction mechanisms we derived provide quantitative insight into the inhibitory mechanism of NPA and help anticipate its effects on GluN1/GluN2A receptors during both physiologic and pathologic activation modalities.

An NMDA Receptor Gating Mechanism Developed from MD Simulations Reveals Molecular Details Underlying Subunit-Specific Contributions

N-methyl-D-aspartate (NMDA) receptors are obligate heterotetrameric ligand-gated ion channels that play critical roles in learning and memory. Here, using targeted molecular dynamics simulations, we developed an atomistic model for the gating of the GluN1/GluN2A NMDA receptor. Upon agonist binding, lobe closure of the ligand-binding domain produced outward pulling of the M3-D2 linkers, leading to outward movements of the C-termini of the pore-lining M3 helices and opening of the channel. The GluN2A subunits, similar to the distal (B/D) subunits in the homotetrameric GluA2 α-amino-3-hydroxy-5-methyl-4-isoxazoleproprionate receptor, had greater M3 outward movements and thus contributed more to channel gating than the GluN1 subunits. Our gating model is validated by functional studies, including cysteine modification data indicating wider accessibility to the GluN1 M3 helices than to the GluN2A M3 helices from the lumen of the open channel, and reveals why the Lurcher mutation in GluN1 has a stronger ability in maintaining channel opening than the counterpart in GluN2A. The resulting structural model for the open state provides an explanation for the Ca2+ permeability of NMDA receptors, and the structural differences between the closed and open states form the basis for drug design.

Contribution of the M1 Transmembrane Helix and Pre-M1 Region to Positive Allosteric Modulation and Gating of N-Methyl-d-Aspartate Receptors

N-methyl-D-aspartate (NMDA) receptors are glutamate-gated ion channels whose function is critical for normal excitatory synaptic transmission in the brain and whose dysfunction has been implicated in several neurologic conditions. NMDA receptor function is subject to extensive allosteric regulation both by endogenous compounds and by exogenous small molecules. Elucidating the structural determinants and mechanism of action by which allosteric regulators control gating will enhance our understanding of NMDA receptor activation and facilitate the development of novel therapeutics. Here, we investigated the structural determinants for (3-chlorophenyl)(6,7-dimethoxy-1-((4-methoxyphenoxy)methyl)-3,4-dihydroisoquinolin-2(1H)-yl)methanone (CIQ), a GluN2C/2D-selective positive allosteric modulator. We show that CIQ does not bind to the amino-terminal domain of the NMDA receptor and does not share structural determinants with modulators acting at the agonist-binding domain dimer interface or ion channel pore. Rather, we identified critical determinants of CIQ modulation in the region near the first transmembrane helix of GluN2D, including in a putative pre-M1 cuff helix that may influence channel gating. We also show that mutations within the GluN2D pre-M1 region alter open probability of the NMDA receptor. These results suggest a novel site of action for potentiation of NMDA receptors by small molecules and implicate the pre-M1 region in NMDA receptor gating.

Molecular determinants of proton-sensitive N-methyl-D-aspartate receptor gating.

Extracellular protons inhibit N-methyl-D-aspartate (NMDA) receptors with an IC50 value in the physiological pH range. To identify the molecular determinants of proton sensitivity, we used scanning mutagenesis of the NR1 subunit to search for residues that control proton inhibition of NMDA receptors. Homology modeling of the extracellular domains suggested that residues at which mutations perturbed pH sensitivity were localized in discrete regions. The majority of mutations that strongly affected proton sensitivity were clustered in the extracellular end of the second transmembrane domain (M3) and adjacent linker leading to the S2 portion of the glycine-binding domain of NR1. Mutations in NR2A confirmed that the analogous region controls the pH sensitivity of this subunit and also identified the linker region between the third transmembrane domain (M4) and the S2 portion of the NR2 glutamate binding domain as an additional determinant of proton sensitivity. One mutant receptor, NR1(A649C)/NR2A(A651T), showed a 145-fold reduction in the IC50 for protons (IC50, 17.3 microM corresponding to pH 4.9). The M3-S2 linker region has been suggested to control NMDA receptor gating, leading to the hypothesis that the proton sensor and receptor gate may be structurally and functionally integrated.

Modal gating of NMDA receptors and the shape of their synaptic response.

N-methyl-D-aspartate receptor (NMDAR) channels mediate the slow component of excitatory potentials at glutamatergic synapses. They have complex kinetic behavior, and much remains to be understood about NMDAR gating mechanisms and the molecular events that shape the synaptic current. Here we show that an individual NMDAR produces at least three stable patterns of activity. For all modes, channels gate by the same mechanism and can occupy either of two open states. The relative stability of the open states differs across modes because of a common perturbation to the NMDAR structure that may be subject to cellular control. Simulations indicate that native NMDAR-mediated synaptic responses arise mainly from the most common mode, and that the slow rise and decay of the current can be attributed to multiple transitions between fully liganded open and closed states rather than to agonist dissociation.