Solution X-ray scattering evidence for agonist- and antagonist-induced modulation of cleft closure in a glutamate receptor ligand-binding domain

Agonist-induced conformational changes in the ligand-binding domains (LBD) of glutamate receptor ion channels provide the driving force for molecular rearrangements that mediate channel opening and subsequent desensitization. The resulting regulated transmembrane ion fluxes form the basis for most excitatory neuronal signaling in the brain. Crystallographic analysis of the GluR2 LBD core has revealed a ligand-binding cleft located between two lobes. Channel antagonists stabilize an open cleft, whereas agonists stabilize a closed cleft. The crystal structure of the apo form is similar to the antagonist-bound, open state. To understand the conformational behavior of the LBD in the absence of crystal lattice constraints, and thus better to appreciate the thermodynamic constraints on ligand binding, we have undertaken a solution x-ray scattering study using two different constructs encoding either the core or an extended LBD. In agreement with the GluR2 crystal structures, the LBD is more compact in the presence of agonist than it is in the presence of antagonist. However, the time-averaged conformation of the ligand-free core in solution is intermediate between the open, antagonist-bound state and the closed, agonist-bound state, suggesting a conformational equilibrium. Addition of peptide moieties that connect the core domain to the other functional domains in each channel subunit appears to constrain the conformational equilibrium in favor of the open state.     

Population Shift Mechanism for Partial Agonism of AMPA Receptor

α-amino-3-hydroxy-5-methyl-4-isoaxazolepropionic acid (AMPA) ionotropic glutamate receptors mediate fast excitatory neurotransmission in the central nervous system, and their dysfunction is associated with neurological diseases. Glutamate binding to ligand-binding domains (LBDs) of AMPA receptors induces channel opening in the transmembrane domains of the receptors. The T686A mutation reduces glutamate efficacy so that the glutamate behaves as a partial agonist. The crystal structures of wild-type and mutant LBDs are very similar and cannot account for the observed behavior. To elucidate the molecular mechanism inducing partial agonism of the T686A mutant, we computed the free-energy landscapes governing GluA2 LBD closure using replica-exchange umbrella sampling simulations. A semiclosed state, not observed in crystal structures, appears in the mutant during simulation. In this state, the LBD cleft opens slightly because of breaking of interlobe hydrogen bonds, reducing the efficiency of channel opening. The energy difference between the LBD closed and semiclosed states is small, and transitions between the two states would occur by thermal fluctuations. Evidently, glutamate binding to the T686A mutant induces a population shift from a closed to a semiclosed state, explaining the partial agonism in the AMPA receptor.     

Role of Cross-Cleft Contacts in NMDA Receptor Gating

In response to brief glutamate exposure, NMDA receptors produce excitatory currents that have sub-maximal amplitudes and characteristically slow kinetics. The activation sequence starts when glutamate binds to residues located on the upper lobe of extracellularly located ligand-binding domains (LBDs) and then contacts lower lobe residues to bridge the cleft between the two hinged lobes. This event stabilizes a narrow-cleft LBD conformation and may facilitate subsequent inter-lobe contacts that further stabilize the closed cleft. Agonist efficacy has been traced to the degree of agonist-induced cleft-closure and may also depend on the stability of the closed-cleft conformation. To investigate how cross-cleft contacts contribute to the amplitude and kinetics of NMDA receptor response, we examined the activation reaction of GluN1/GluN2A receptors that had single-residue substitutions at the interface between LBD lobes. We found that side-chain truncations at residues of putative contact between lobes increased glutamate efficacy through independent additive mechanisms in GluN1 and GluN2A subunits. In contrast, removing side-chain charge with isosteric substitutions at the same sites decreased glutamate efficacy. These results support the view that in GluN1/GluN2A receptors’ natural interactions between residues on opposing sides of the ligand-binding cleft encode the stability of the glutamate-bound closed-cleft conformations and limit the degree of cleft closure, thus contributing to the sub-maximal response and emblematically slow NMDA receptor deactivation after brief stimulation.     

Structural Mechanism of N-Methyl-D-Aspartate Receptor Type 1 Partial Agonism

N-methyl-D-aspartate (NMDA) receptors belong to a family of ionotropic glutamate receptors that contribute to the signal transmission in the central nervous system. NMDA receptors are heterotetramers that usually consist of two GluN1 and GluN2 monomers. The extracellular ligand-binding domain (LBD) of a monomer is comprised of discontinuous segments that form the functional domains D1 and D2. While the binding of a full agonist glycine to LBD of GluN1 is linked to cleft closure and subsequent ion-channel opening, partial agonists are known to activate the receptor only sub-maximally. Although the crystal structures of the LBD of related GluA2 receptor explain the mechanism for the partial agonism, structures of GluN1-LBD cannot distinguish the difference between full and partial agonists. It is, however, probable that the partial agonists of GluN1 alter the structure of the LBD in order to result in a different pharmacological response than seen with full agonists. In this study, we used molecular dynamics simulations to reveal an intermediate closure-stage for GluN1, which is unseen in crystal structures. According to our calculations, this intermediate closure is not a transient stage but an energetically stable conformation. Our results demonstrate that the partial agonist cannot exert firm GluN1-LBD closure, especially if there is even a small force that disrupts the LBD closure. Accordingly, this result suggests the importance of forces from the ion channel for the relationship between pharmacological response and the structure of the LBD of members of this receptor family.     

Structure,Dynamics, and Allosteric Potential of Ionotropic Glutamate Receptor N-Terminal Domains

Ionotropic glutamate receptors (iGluRs) are tetrameric cation channels that mediate synaptic transmission and plasticity. They have a unique modular architecture with four domains: the intracellular C-terminal domain (CTD) that is involved in synaptic targeting, the transmembrane domain (TMD) that forms the ion channel, the membrane-proximal ligand-binding domain (LBD) that binds agonists such as L-glutamate, and the distal N-terminal domain (NTD), whose function is the least clear. The extracellular portion, comprised of the LBD and NTD, is loosely arranged, mediating complex allosteric regulation and providing a rich target for drug development. Here, we briefly review recent work on iGluR NTD structure and dynamics, and further explore the allosteric potential for the NTD in AMPA-type iGluRs using coarse-grained simulations. We also investigate mechanisms underlying the established NTD allostery in NMDA-type iGluRs, as well as the fold-related metabotropic glutamate and GABA_B receptors. We show that the clamshell motions intrinsically favored by the NTD bilobate fold are coupled to dimeric and higher-order rearrangements that impact the iGluR LBD and ultimately the TMD. Finally, we explore the dynamics of intact iGluRs and describe how it might affect receptor operation in a synaptic environment.     

Structural Dynamics of Glutamate Signaling Systems by smFRET

Single-molecule Förster resonance energy transfer (smFRET) is a powerful technique for investigating the structural dynamics of biological macromolecules. smFRET reveals the conformational landscape and dynamic changes of proteins by building on the static structures found using cryo-electron microscopy, x-ray crystallography, and other methods. Combining smFRET with static structures allows for a direct correlation between dynamic conformation and function. Here, we discuss the different experimental setups, fluorescence detection schemes, and data analysis strategies that enable the study of structural dynamics of glutamate signaling across various timescales. We illustrate the versatility of smFRET by highlighting studies of a wide range of questions, including the mechanism of activation and transport, the role of intrinsically disordered segments, and allostery and cooperativity between subunits in biological systems responsible for glutamate signaling.     

A Conserved Mechanism for Gating in an Ionotropic Glutamate Receptor

Ionotropic glutamate receptor (iGluR) channels control synaptic activity. The crystallographic structure of GluA2, the prototypical iGluR, reveals a clamshell-like ligand-binding domain (LBD) that closes in the presence of glutamate to open a gate on the pore lining α-helix. How LBD closure leads to gate opening remains unclear. Here, we show that bending the pore helix at a highly conserved alanine residue (Ala-621) below the gate is responsible for channel opening. Substituting Ala-621 with the smaller more flexible glycine resulted in a basally active, nondesensitizing channel with ∼39-fold increase in glutamate potency without affecting surface expression or binding. On GluA2(A621G), the partial agonist kainate showed efficacy similar to a full agonist, and competitive antagonists CNQX and DNQX acted as a partial agonists. Met-629 in GluA2 sits above the gate and is critical in transmitting LBD closure to the gate. Substituting Met-629 with the flexible glycine resulted in reduced channel activity and glutamate potency. The pore regions in potassium channels are structurally similar to iGluRs. Whereas potassium channels typically use glycines as a hinge for gating, iGluRs use the less flexible alanine as a hinge at a similar position to maintain low basal activity allowing for ligand-mediated gating.     

Dynamics and allostery of the ionotropic glutamate receptors and the ligand binding domain

The dynamics of the ligand‐binding domain (LBD) and the intact ionotropic glutamate receptor (iGluR) were studied using Gaussian Network Model (GNM) analysis. The dynamics of LBDs with various allosteric modulators is compared using a novel method of multiple alignment of GNM modes of motion. The analysis reveals that allosteric effectors change the dynamics of amino acids at the upper lobe interface of the LBD dimer as well as at the hinge region between the upper‐ and lower‐ lobes. For the intact glutamate receptor the analysis show that the clamshell‐like movement of the LBD upper and lower lobes is coupled to the bending of the trans‐membrane domain (TMD) helices which may open the channel pore. The results offer a new insight on the mechanism of action of allosteric modulators on the iGluR and support the notion of TMD helices bending as a possible mechanism for channel opening. In addition, the study validates the methodology of multiple GNM modes alignment as a useful tool to study allosteric effect and its relation to proteins dynamics. Proteins 2016; 84:267–277. © 2015 Wiley Periodicals, Inc.     

Bicarbonate-Independent Sodium Conductance of Na/HCO3 Cotransporter NBCn1 Decreases NMDA Receptor Function

The sodium bicarbonate cotransporter NBCn1 is an electroneutral transporter with a channel activity that conducts Na⁺ in a HCO₃^–-independent manner. This channel activity was suggested to functionally affect other membrane proteins which permeate Na⁺ influx. We previously reported that NBCn1 is associated with the NMDA receptors (NMDARs) at the molecular and physiological levels. In this study, we examined whether NBCn1 channel activity affects NMDAR currents and whether this effect involves the interaction between the two proteins. NBCn1 and the NMDAR subunits GluN1A/GluN2A were expressed in Xenopus oocytes, and glutamate currents produced by the receptors were measured using two-electrode voltage clamp. In the absence of CO₂/HCO₃^–, NBCn1 channel activity decreased glutamate currents mediated by GluN1A/GluN2A. NBCn1 also decreased the slope of the current–voltage relationships for the glutamate current. Similar effects on the glutamate current were observed with and without PSD95, which can cluster NBCn1 and NMDARs. The channel activity was also observed in the presence of CO₂/HCO₃^–. We conclude that NBCn1 channel activity decreases NMDAR function. Given that NBCn1 knockout mice develop a downregulation of NMDARs, our results are unexpected and suggest that NBCn1 has dual effects on NMDARs. It stabilizes NMDAR expression but decreases receptor function by its Na⁺ channel activity. The dual effects may play an important role in fine-tuning the regulation of NMDARs in the brain.     

Replica Exchange Molecular Dynamics Simulations Provide Insight into Substrate Recognition by Small Heat Shock Proteins

The small heat shock proteins (sHSPs) are a virtually ubiquitous and diverse group of molecular chaperones that can bind and protect unfolding proteins from irreversible aggregation. It has been suggested that intrinsic disorder of the N-terminal arm (NTA) of sHSPs is important for substrate recognition. To investigate conformations of the NTA that could recognize substrates we performed replica exchange molecular dynamics simulations. Behavior at normal and stress temperatures of the dimeric building blocks of dodecameric HSPs from wheat (Ta16.9) and pea (Ps18.1) were compared because they display high sequence similarity, but Ps18.1 is more efficient in binding specific substrates. In our simulations, the NTAs of the dimer are flexible and dynamic; however, rather than exhibiting highly extended conformations they retain considerable α-helical character and contacts with the conserved α-crystallin domain (ACD). Network analysis and clustering methods reveal that there are two major conformational forms designated either “open” or “closed” based on the relative position of the two NTAs and their hydrophobic solvent accessible surface area. The equilibrium constant for the closed to open transition is significantly different for Ta16.9 and Ps18.1, with the latter showing more open conformations at elevated temperature correlated with its more effective chaperone activity. In addition, the Ps18.1 NTAs have more hydrophobic solvent accessible surface than those of Ta16.9. NTA hydrophobic patches are comparable in size to the area buried in many protein-protein interactions, which would enable sHSPs to bind early unfolding intermediates. Reduced interactions of the Ps18.1 NTAs with each other and with the ACD contribute to the differences in dynamics and hydrophobic surface area of the two sHSPs. These data support a major role for the conformational equilibrium of the NTA in substrate binding and indicate features of the NTA that contribute to sHSP chaperone efficiency.     

GluN1-specific redox effects on the kinetic mechanism of NMDA receptor activation

NMDA receptors are glutamate-activated ion channel complexes central to the functioning of the mammalian nervous system. Opening of the NMDA receptor ion channel pore is initiated by agonist-induced conformational changes in the extracellular ligand-binding domain (LBD) but the dynamic mechanism of this process remains unresolved. We studied how a disulfide bond in the obligatory GluN1 subunit—the sole site of redox modulation in NMDA receptors—controls this activation gating mechanism. This disulfide bond is located in the hinge region of the LBD, and presumably constrains agonist-induced cleft closure of the clamshell-like LBD. Elimination of this bond, by either DTT-mediated reduction or mutagenesis, enhances gating efficiency such that pore opening now occurs with higher frequency and longer duration. The most prominent effect was to shift opening modes to long duration openings reminiscent of a high P_o gating mode that the NMDA receptor exhibits under ambient oxidizing conditions. In terms of preopen gating steps, elimination of this bond has effects only on the fast gating step consistent with this step being GluN1-specific and reflecting GluN1 gating movements immediately before channel opening. Overall, our results suggest that the dynamics of the GluN1 LBD have strong effects on late pore opening steps including regulating the duration of pore opening. This redox-mediated gating modulation could be an underlying mechanism of NMDA receptor malfunction in redox-dependent disease states and presents a potential target of pharmacologic action.     

Glutamate Binding and Conformational Flexibility of Ligand-binding Domains Are Critical Early Determinants of Efficient Kainate Receptor Biogenesis

Intracellular glutamate binding within the endoplasmic reticulum (ER) is thought to be necessary for plasma membrane expression of ionotropic glutamate receptors. Here we determined the importance of glutamate binding to folding and assembly of soluble ligand-binding domains (LBDs), as well as full-length receptors, by comparing the secretion of a soluble GluR6-S1S2 protein versus the plasma membrane localization of GluR6 kainate receptors following mutagenesis of the LBD. The mutations were designed to either eliminate glutamate binding, thereby trapping the bilobate LBD in an “open” conformation, or “lock” the LBD in a closed conformation with an engineered interdomain disulfide bridge. Analysis of plasma membrane localization, medium secretion of soluble LBD proteins, and measures of folding efficiency suggested that loss of glutamate binding affinity significantly impacted subunit protein folding and assembly. In contrast, receptors with conformationally restricted LBDs also exhibited decreased PM expression and altered oligomeric receptor assembly but did not exhibit any deficits in subunit folding. Secretion of the closed LBD protein was enhanced compared with wild-type GluR6-S1S2. Our results suggest that glutamate acts as a chaperone molecule for appropriate folding of nascent receptors and that relaxation of LBDs from fully closed states during oligomerization represents a critical transition that necessarily engages other determinants within receptor dimers. Glutamate receptor LBDs therefore must access multiple conformations for efficient biogenesis.Cellular control over the biogenesis and trafficking of ionotropic glutamate receptors (iGluRs)² probably constitutes a major regulatory process that prevents aberrant excitatory neurotransmission within the central nervous system. Many receptor trafficking determinants are genetically encoded within the primary amino acid sequences of cytoplasmic carboxyl-terminal domains of iGluR subunits (1, 2). Association of these determinants with accessory or chaperone proteins enhances forward transit from the endoplasmic reticulum (ER), insertion into the plasma membrane (PM), synaptic targeting, or a variety of other trafficking processes (3–6). Ligand binding and gating domains upstream of carboxyl-terminal domains also are critical in biogenesis of iGluRs (7–11). Elucidating how these domains are engaged as quality control checkpoints is essential to developing a comprehensive understanding of the neuronal mechanisms for control of excitatory transmission in the central nervous system.Several recent studies have proposed that intracellular glutamate binding within the ER promotes proper folding and maturation of AMPA and kainate receptors and is required for forward trafficking of fully assembled receptors (10, 12–15). This hypothesis is based in large part on the observation that mutagenesis of key glutamate-binding residues in the LBD greatly reduces or eliminates PM localization (7, 10, 12, 13); the mechanistic basis of this putative quality control process is not understood. The critical receptor determinants involved in these cellular checkpoints could reside in the LBD, in other regions that undergo glutamate-dependent alterations in structure, or in intersubunit interactions associated with receptor desensitization (7–10, 16). It is possible that glutamate binding to iGluRs (and subsequent conformational rearrangements) act as tests of the functionality of fully assembled, export-ready receptors (8, 12, 16); alternatively, intact glutamate-binding sites may be required for earlier steps in receptor biogenesis, such as subunit folding and oligomeric assembly (7, 16).We tested here whether the glutamate-dependent conformational changes critical to GluR6a KAR cell surface expression are restricted solely to the bilobate LBD, formed by the S1 and S2 segments of the receptor protein, or instead involve additional domains in the KAR subunits. Toward that end, we compared the effect of an LBD mutation that eliminated binding affinity for glutamate, T690A, on the efficiency of secretion of a soluble GluR6-S1S2 protein versus PM localization of the full-length receptor; this served as one way to test the autonomy of the LBD in quality control processes, because soluble LBDs fold properly, form binding sites similar to those in the full-length receptor, and transit the secretory pathway to be released into culture media. Furthermore, we trapped LBDs of full-length receptors and soluble GluR6-S1S2 proteins in conformations that mimic closed, glutamate-bound states by introduction of a reversible, interdomain (D1-D2) disulfide bond, allowing us to test the role of LBD relaxation or flexibility in assembly and trafficking. We found that the ligand binding mutation resulted in reduced maturation and receptor surface expression in part through misfolding of the LBD. In addition, we found that locking the LBD into a closed state reduced receptor maturation and expression but that this engaged regions outside of the LBD. Although the locked, closed mutant also had assembly defects that occurred at the transition from dimers to tetramers, these did not result from apparent folding defects, suggesting that relaxation or opening of the LBD represents a critical step during oligomerization. In summary, we propose that LBDs must be able to access multiple conformations for efficient KAR biogenesis.     

Engineered disulfide reveals structural dynamics of locked SARS-CoV-2 spike

The spike (S) protein of SARS-CoV-2 has been observed in three distinct pre-fusion conformations: locked, closed and open. Of these, the function of the locked conformation remains poorly understood. Here we engineered a SARS-CoV-2 S protein construct “S-R/x3” to arrest SARS-CoV-2 spikes in the locked conformation by a disulfide bond. Using this construct we determined high-resolution structures confirming that the x3 disulfide bond has the ability to stabilize the otherwise transient locked conformations. Structural analyses reveal that wild-type SARS-CoV-2 spike can adopt two distinct locked-1 and locked-2 conformations. For the D614G spike, based on which all variants of concern were evolved, only the locked-2 conformation was observed. Analysis of the structures suggests that rigidified domain D in the locked conformations interacts with the hinge to domain C and thereby restrains RBD movement. Structural change in domain D correlates with spike conformational change. We propose that the locked-1 and locked-2 conformations of S are present in the acidic high-lipid cellular compartments during virus assembly and egress. In this model, release of the virion into the neutral pH extracellular space would favour transition to the closed or open conformations. The dynamics of this transition can be altered by mutations that modulate domain D structure, as is the case for the D614G mutation, leading to changes in viral fitness. The S-R/x3 construct provides a tool for the further structural and functional characterization of the locked conformations of S, as well as how sequence changes might alter S assembly and regulation of receptor binding domain dynamics.     

Sustained calcium signalling and caspase-3 activation involve NMDA receptors in thymocytes in contact with dendritic cells

L-glutamate, the major excitatory neurotransmitter, also has a role in non-neuronal tissues and modulates immune responses. Whether NMDA receptor (NMDAR) signalling is involved in T-cell development is unknown. In this study, we show that mouse thymocytes expressed an array of glutamate receptors, including NMDARs subunits. Sustained calcium (Ca²⁺) signals and caspase-3 activation in thymocytes were induced by interaction with antigen-pulsed dendritic cells (DCs) and were inhibited by NMDAR antagonists MK801 and memantine. NMDARs were transiently activated, triggered the sustained Ca²⁺ signal and were corecruited with the PDZ-domain adaptor postsynaptic density (PSD)-95 to thymocyte-DC contact zones. Although T-cell receptor (TCR) activation was sufficient for relocalization of NMDAR and PSD-95 at the contact zone, NMDAR could be activated only in a synaptic context. In these T-DC contacts, thymocyte activation occurred in the absence of exogenous glutamate, indicating that DCs could be a physiological source of glutamate. DCs expressed glutamate, glutamate-specific vesicular glutamate transporters and were capable of fast glutamate release through a Ca²⁺-dependent mechanism. We suggest that glutamate released by DCs could elicit focal responses through NMDAR-signalling in T cells undergoing apoptosis. Thus, synapses between T and DCs could provide a functional platform for coupling TCR activation and NMDAR signalling, which might reflect on T-cell development and modulation of the immune response.     

Interplay between structural rigidity and electrostatic interactions in the ligand binding domain of GluR2

Using molecular dynamics (MD) simulations, computational protein modifications, and a novel theoretical methodology that determines structural rigidity/flexibility (the FIRST algorithm), we investigate how molecular structure and dynamics of the glutamate receptor ligand binding domain (GluR2 S1S2) facilitate its conformational transition. S1S2 is a two-lobe protein, which undergoes a cleft closure conformational transition upon binding an agonist in the cleft between the two lobes; hence it is expected that the mechanism of this conformational transition can be characterized as a hinge-type. However, in the rigidity analysis one lobe of the protein is identified as a single rigid cluster while the other one is structurally flexible, inconsistent with a presumed mechanical hinge mechanism. Instead, we characterize the cleft-closing transition as a load and lock mechanism. We find that when two cross-cleft hydrogen bonds are disrupted the protein undergoes a rapid cleft opening transition. At the same time, the dynamical behavior of the cleft in the presence of the glutamate ligand is only weakly affected by the S652 peptide bond in its flipped conformation observed in the crystal structure. The residue E705 plays significant role in stabilization of the closed conformation via electrostatic interactions. The presence of the E705-K730 salt bridge seems to correlate strongly withthe cleft opening transition in the MD simulations.     

Role of Conformational Dynamics in α-Amino-3-hydroxy-5-methylisoxazole-4-propionic Acid (AMPA) Receptor Partial Agonism

We have investigated the range of cleft closure conformational states that the agonist-binding domains of the α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors occupy when bound to a series of willardiine derivatives using single-molecule FRET. These studies show that the agonist-binding domain exhibits varying degrees of dynamics when bound to the different willardiines with differing efficacies. The chlorowillardiine- and nitrowillardiine-bound form of the agonist-binding domain probes a narrower range of cleft closure states relative to the iodowillardiine bound form of the protein, with the antagonist (αS)-α-amino-3-[(4-carboxyphenyl)methyl]-3,4-dihydro-2,4-dioxo-1(2H)-pyrimidinepropanoic acid (UBP-282)-bound form exhibiting the widest range of cleft closure states. Additionally, the average cleft closure follows the order UBP-282 > iodowillardiine > chlorowillardiine > nitrowillardiine-bound forms of agonist-binding domain. These single-molecule FRET data, along with our previously reported data for the glutamate-bound forms of wild type and T686S mutant proteins, show that the mean currents under nondesensitizing conditions can be directly correlated to the fraction of the agonist-binding domains in the “closed” cleft conformation. These results indicate that channel opening in the AMPA receptors is controlled by both the ability of the agonist to induce cleft closure and the dynamics of the agonist-binding domain when bound to the agonist.     

Constitutive activation of the N-methyl-D-aspartate receptor via cleft-spanning disulfide bonds

Although the N-methyl-D-aspartate (NMDA) receptor plays a critical role in the central nervous system, many questions remain regarding the relationship between its structure and functional properties. In particular, the involvement of ligand-binding domain closure in determining agonist efficacy, which has been reported in other glutamate receptor subtypes, remains unresolved. To address this question, we designed dual cysteine point mutations spanning the NR1 and NR2 ligand-binding clefts, aiming to stabilize these domains in closed cleft conformations. Two mutants, E522C/I691C in NR1 (EI) and K487C/N687C in NR2 (KN) were found to exhibit significant glycine- and glutamate-independent activation, respectively, and co-expression of the two subunits produced a constitutively active channel. However, both individual mutants could be activated above constitutive levels in a concentration-dependent manner, indicating that cleft closure does not completely prevent agonist association. Interestingly, whereas the NR2 KN disulfide was found to potentiate channel gating and M3 accessibility, NR1 EI exhibited the opposite phenotype, suggesting that the EI disulfide may trap the NR1 ligand-binding domain in a lower efficacy conformation. Furthermore, both mutants affected agonist sensitivity at the opposing subunit, suggesting that closed cleft stabilization may contribute to coupling between the subunits. These results support a correlation between cleft stability and receptor activation, providing compelling evidence for the Venus flytrap mechanism of glutamate receptor domain closure.