NMDA Receptor Gating Research Articles

Investigating the role of GluN1 Met813 on NMDA receptor gating

Investigating the role of GluN1 Met813 on NMDA receptor gating

An integrated approach to evaluate the functional effects of disease-associated NMDA receptor variants

The NMDA receptor (NMDAR) is a ubiquitously expressed glutamate-gated ion channel that plays key roles in brain development and function. Not surprisingly, a variety of disease-associated variants have been identified in genes encoding NMDAR subunits. A critical first step to assess whether these variants contribute to their associated disorder is to characterize their effect on receptor function. However, the complexity of NMDAR function makes this challenging, with many variants typically altering multiple functional properties. At synapses, NMDARs encode pre- and postsynaptic activity to carry a charge transfer that alters membrane excitability and a Ca2+ influx that has both short- and long-term signaling actions. Here, we characterized epilepsy-associated variants in GluN1 and GluN2A subunits with various phenotypic severity in HEK293 cells. To capture the complexity of NMDAR gating, we applied 10 glutamate pulses at 10 Hz to derive a charge integral. This assay is advantageous since it incorporates multiple gating parameters – activation, deactivation, and desensitization – into a single value. We then integrated this gating parameter with Mg2+ block and Ca2+ influx using fractional Ca2+ currents to generate indices of charge transfer and Ca2+ transfer over wide voltage ranges. This approach yields consolidated parameters that can be used as a reference to normalize channel block and allosteric modulation to better define potential patient treatment. This is especially true for variants in the transmembrane domain that affect not only receptor gating but also often Mg2+ block and Ca2+ permeation.

Direct M3-M4 Interactions Control NMDA Receptor Gating and Desensitization

N-Methyl-D-aspartate receptors (NMDARs) are glutamate-gated channels that mediate excitatory neurotransmission in the central nervous system. They are heterotetrameric channels composed of two GluN1 and two GluN2 subunits, each containing four (M1-M4) membrane-embedded helices. Glutamate and glycine binding results in closure of the ligand binding domain and results in opening of the Na+/Ca2+-permeable pore by direct tethering to the gate at the M3 helix. The role of the peripheral M4 helix in modulating channel gating is unclear. To determine if the M4 helix controls gating by direct allosteric interactions with the gate, we performed a cysteine mutagenesis scan and western blot analyses to identify putative regions of direct contact between GluN1 M4 and GluN2A M3. We identified strong crosslinking at GluN2A-F636 with GluN1-G815 and GluN1-L808. We performed alanine mutagenesis on these sites and recorded single-molecule steady-state currents using cell-attached patch clamp. We performed kinetic modelling and used estimated rate constants to execute a double mutant cycle analysis on these recordings to determine the functional impact of these interactions. Each mutant significantly impacted gating open probability (GluN1/GluN2A 0.53 ± 0.10; GluN1G815A/GluN2A 0.32 ± 0.11; GluN1/GluN2AF636A 0.81 ± 0.04; GluN1G815A/GluN2AF636A 0.88 ± 0.04). We found GluN1-G815 made contact with GluN2A-F636 and contributed 1.9 kJ to desensitization. This indicates a functional interaction between the residues that coincides with the physical interaction observed between GluN1-G815 on the M4 helix with GluN2A-F636 on the M3 helix. Our results suggest a model whereby the GluN1 M4 helix makes direct, functionally important contacts with the GluN2A channel gate. This may serve as a mechanism by which intracellular changes in the c-terminal domain can communicate with the channel gate. The GluN1 M4 also houses several disease-associated sites. Mutations that disrupt M3-M4 coupling may be a novel mechanism of disease pathology.

Dual action of amitriptyline on NMDA receptors: enhancement of Ca-dependent desensitization and trapping channel block

Although the tricyclic antidepressant amitriptyline (ATL) is widely used in the clinic, the mechanism underlying its high therapeutic efficacy against neuropathic pain remains unclear. NMDA receptors (NMDARs) represent a target for ATL and are involved in sensitization of neuropathic pain. Here we describe two actions of ATL on NMDARs: 1) enhancement of Ca2+-dependent desensitization and 2) trapping channel block. Inhibition of NMDARs by ATL was found to be dependent upon external Ca2+ concentration ([Ca2+]) in a voltage-independent manner, with an IC50 of 0.72 μM in 4 mM [Ca2+]. The ATL IC50 value increased exponentially with decreasing [Ca2+], with an e-fold change observed per 0.69 mM decrease in [Ca2+]. Loading neurons with BAPTA abolished Ca2+-dependent inhibition, suggesting that Ca2+ affects NMDARs from the cytosol. Since there is one known Ca2+-dependent process in gating of NMDARs, we conclude that ATL most likely promotes Ca2+-dependent desensitization. We also found ATL to be a trapping open-channel blocker of NMDARs with an IC50 of 220 µM at 0 mV. An e-fold change in ATL IC50 was observed to occur with a voltage shift of 50 mV in 0.25 mM [Ca2+]. Thus, we disclose here a robust dependence of ATL potency on extracellular [Ca2+], and demonstrate that ATL bound in the NMDAR pore can be trapped by closure of the channel.

Age-related shift in LTD is dependent on neuronal adenosine A2A receptors interplay with mGluR5 and NMDA receptors

Synaptic dysfunction plays a central role in Alzheimer’s disease (AD), since it drives the cognitive decline. An association between a polymorphism of the adenosine A2A receptor (A2AR) encoding gene—ADORA2A, and hippocampal volume in AD patients was recently described. In this study, we explore the synaptic function of A2AR in age-related conditions. We report, for the first time, a significant overexpression of A2AR in hippocampal neurons of aged humans, which is aggravated in AD patients. A similar profile of A2AR overexpression in rats was sufficient to drive age-like memory impairments in young animals and to uncover a hippocampal LTD-to-LTP shift. This was accompanied by increased NMDA receptor gating, dependent on mGluR5 and linked to enhanced Ca2+ influx. We confirmed the same plasticity shift in memory-impaired aged rats and APP/PS1 mice modeling AD, which was rescued upon A2AR blockade. This A2AR/mGluR5/NMDAR interaction might prove a suitable alternative for regulating aberrant mGluR5/NMDAR signaling in AD without disrupting their constitutive activity.

Quantifying Kinetic Transitions between NMDA Receptor Gating Modes

Quantifying Kinetic Transitions between NMDA Receptor Gating Modes

Septal Cholinergic Neuromodulation Tunes the Astrocyte-Dependent Gating of Hippocampal NMDA Receptors to Wakefulness

The activation of the N-methyl D-aspartate receptor (NMDAR) is controlled by a glutamate-binding site and a distinct, independently regulated, co-agonist-binding site. In most brain regions, the NMDAR co-agonist is the astrocyte-derived gliotransmitter D-serine. We found that D-serine levels oscillate in mouse hippocampus as a function of wakefulness, invitro and invivo. This causes a full saturation of the NMDAR co-agonist site in the dark (active) phasethat dissipates to sub-saturating levels during the light (sleep) phase, and influences learning performance throughout the day. We demonstrate that hippocampal astrocytes sense the wakefulness-dependent activity of septal cholinergic fibers through the α7-nicotinic acetylcholine receptor (α7nAChR), whose activation drives D-serine release. We conclude that astrocytes tune the gating of synaptic NMDARs to the vigilance state and demonstrate that this is directly relevant to schizophrenia, a disorder characterized by NMDAR and cholinergic hypofunctions. Indeed, bypassing cholinergic activity with a clinically tested α7nAChR agonist successfully enhances NMDAR activation. VIDEO ABSTRACT.

Functional Roles of a Conserved Tryptophan at Subunit-Subunit Interfaces in NMDA Receptor Membrane Regions

NMDA receptors (NMDARs) are ionotropic glutamate receptors (iGluRs) characterized by channel block by extracellular magnesium (Mg2+o) and slow deactivation kinetics. NMDARs are typically composed of two GluN1 and two GluN2 (GluN2A-GluN2D) subunits. The transmembrane domain (TMD) of each subunit contains three membrane-spanning regions (M1, M3, and M4) and a pore lining re-entrant loop (M2). The M2 region of each NMDAR subunit contains a tryptophan (W) that is highly conserved: nearly all known mammalian iGluR subunits, several potassium channels, and iGluR-like proteins in phylogenetically distant organisms contain W at the homologous site. This highly conserved W is located in NMDARs at the interface between the M2 region and the adjacent M3 region. Previous work demonstrated that mutation of this highly-conserved W in the GluN2B subunit (W607) drastically decreased block by Mg2+o, while mutation of the homologous residue in the GluN2A subunit (W606) had a relatively minor effect (Williams et al., 1998 Mol Pharmacol 53, 933). Interestingly, mutating the homologous residue in GluN1 subunits (W608) also has little effect on Mg2+o block (Siegler Retchless et al., 2012 Nat Neurosci 15, 406). To further explore the function of the conserved W at subunit-subunit interfaces we used whole-cell recordings from recombinant receptors expressed in tsA201 cells. We examined GluN2A(W606) and GluN2B(W607) mutants expressed with wild-type GluN1 subunits or GluN1 subunits with mutations of M3 residues at the M2-M3 interface. We found that receptors containing mutations at both GluN2A(W606) and a residue in the adjacent interface-lining GluN1 M3 region dramatically decreased block by Mg2+o. We also examined GluN1(W608) mutants and observed profoundly slowed deactivation kinetics. Overall, these results suggest that the conserved W is broadly involved in channel properties and gating of NMDARs.

Nonlinear AMPA Sensitivity to Glutamate and Recruitment of NMDA Activity in Dendritic Spines

Our lab has been developing and improving the optical and molecular tools and techniques to record EPSPs (excitatory postsynaptic potentials) in dendritic spines. With a dual laser 2-photon microscope we are able to record from voltage-sensitive dye (VSD) labeled spines from cortical pyramidal neurons while evoking unitary EPSPs using glutamate uncaging. By varying the uncaging laser intensity we observe a nonlinear response to glutamate when the resulting EPSPs amplitudes are observed at the soma. By optically recording EPSPs in spines we can determine that the observed nonlinearity is often present even when spine EPSPs are relatively small, i.e. < 10 mV. Pharmacology is used to block NMDA receptors and determine the recruitment of these receptors and their contribution to both voltage and calcium influx. Calcium imaging reveals that NMDA receptors are recruited in a very linear fashion according to the spine depolarization. This calcium influx however, does not appear to play a significant role in the spine depolarization. Finally, modeling is used to reconcile the observed phenomena with detailed models of AMPA and NMDA receptor gating, and glutamate binding.

Separate Intramolecular Targets for Protein Kinase A Control N-Methyl-d-aspartate Receptor Gating and Ca2+ Permeability

Protein kinase A (PKA) enhances synaptic plasticity in the central nervous system by increasing NMDA receptor current amplitude and Ca(2+) flux in an isoform-dependent yet poorly understood manner. PKA phosphorylates multiple residues on GluN1, GluN2A, and GluN2B subunits in vivo, but the functional significance of this multiplicity is unknown. We examined gating and permeation properties of recombinant NMDA receptor isoforms and of receptors with altered C-terminal domain (CTDs) prior to and after pharmacological inhibition of PKA. We found that PKA inhibition decreased GluN1/GluN2B but not GluN1/GluN2A gating; this effect was due to slower rates for receptor activation and resensitization and was mediated exclusively by the GluN2B CTD. In contrast, PKA inhibition reduced NMDA receptor-relative Ca(2+) permeability (PCa/PNa) regardless of the GluN2 isoform and required the GluN1 CTD; this effect was due primarily to decreased unitary Ca(2+) conductance, because neither Na(+) conductance nor Ca(2+)-dependent block was altered substantially. Finally, we show that both the gating and permeation effects can be reproduced by changing the phosphorylation state of a single residue: GluN2B Ser-1166 and GluN1 Ser-897, respectively. We conclude that PKA effects on NMDA receptor gating and Ca(2+) permeability rely on distinct phosphorylation sites located on the CTD of GluN2B and GluN1 subunits. This separate control of NMDA receptor properties by PKA may account for the specific effects of PKA on plasticity during synaptic development and may lead to drugs targeted to alter NMDA receptor gating or Ca(2+) permeability.

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.

Identification of a single amino acid in GluN1 that is critical for glycine-primed internalization of NMDA receptors

BackgroundNMDA receptors are ligand-gated ion channels with essential roles in glutamatergic synaptic transmission and plasticity in the CNS. As co-receptors for glutamate and glycine, gating of the NMDA receptor/channel pore requires agonist binding to the glycine sites, as well as to the glutamate sites, on the ligand-binding domains of the receptor. In addition to channel gating, glycine has been found to prime NMDA receptors for internalization upon subsequent stimulation of glutamate and glycine sites.ResultsHere we address the key issue of identifying molecular determinants in the glycine-binding subunit, GluN1, that are essential for priming of NMDA receptors. We found that glycine treatment of wild-type NMDA receptors led to recruitment of the adaptor protein 2 (AP-2), and subsequent internalization after activating the receptors by NMDA plus glycine. However, with a glycine-binding mutant of GluN1 – N710R/Y711R/E712A/A714L – we found that treating with glycine did not promote recruitment of AP-2 nor were glycine-treated receptors internalized when subsequently activated with NMDA plus glycine. Likewise, GluN1 carrying a single point mutation – A714L – did not prime upon glycine treatment. Importantly, both of the mutant receptors were functional, as stimulating with NMDA plus glycine evoked inward currents.ConclusionsThus, we have identified a single amino acid in GluN1 that is critical for priming of NMDA receptors by glycine. Moreover, we have demonstrated the principle that while NMDA receptor gating and priming share a common requirement for glycine binding, the molecular constraints in GluN1 for gating are distinct from those for priming.

Modulating the Intrinsic Disorder in the Cytoplasmic Domain Alters the Biological Activity of the N-Methyl-d-aspartate-sensitive Glutamate Receptor

The NMDA-sensitive glutamate receptor is a ligand-gated ion channel that mediates excitatory synaptic transmission in the nervous system. Extracellular zinc allosterically regulates the NMDA receptor by binding to the extracellular N-terminal domain, which inhibits channel gating. Phosphorylation of the intrinsically disordered intracellular C-terminal domain alleviates inhibition by extracellular zinc. The mechanism for this functional effect is largely unknown. Proline is a hallmark of intrinsic disorder, so we used proline mutagenesis to modulate disorder in the cytoplasmic domain. Proline depletion selectively uncoupled zinc inhibition with little effect on receptor biogenesis, surface trafficking, or ligand-activated gating. Proline depletion also reduced the affinity for a PDZ domain involved in synaptic trafficking and affected small molecule binding. To understand the origin of these phenomena, we used single molecule fluorescence and ensemble biophysical methods to characterize the structural effects of proline mutagenesis. Proline depletion did not eliminate intrinsic disorder, but the underlying conformational dynamics were changed. Thus, we altered the form of intrinsic disorder, which appears sufficient to affect the biological activity. These findings suggest that conformational dynamics within the intrinsically disordered cytoplasmic domain are important for the allosteric regulation of NMDA receptor gating.

Modal gating of GluN1/GluN2D NMDA receptors

GluN2D-containing NMDA receptors are characterized by an unusually low open probability (0.023), even in the presence of saturating glutamate and glycine. Here, we show that recombinant GluN1/GluN2D NMDA receptors can enter brief periods with exceptionally high open probability (0.65) in excised outside-out and cell-attached single channel recordings. GluN1/GluN2D channels during the enhanced gating mode have similar open durations as occurs outside of the high open probability burst of activity. However, the periods in the high gating mode only exhibit 4 brief closed duration exponential components similar to the briefest observed for openings outside the burst. GluN1/GluN2D receptors also open to a more prominent subconductance level compared to activity outside the high open probability burst. Evaluation of a five-state NMDA receptor gating model suggests that both the opening and closing rate constants differ for the periods of higher open probability compared to the high open probability arm of a gating model previously published for GluN1/GluN2D fit to a representative full length single channel recording. These data demonstrate that GluN2D-containing NMDA receptors can enter a conformation or mode that allows the pore to gate with high probability.

Rebuttal from James Howe

MacLean (2013) suggests an alternative approach to explain the effects of transmembrane AMPA receptor regulatory proteins (TARPs), namely that TARPs may promote closed-cleft ligand binding domain (LBD) conformations that are distinct from those frequented without TARPs. Evidence for multiple closed LBD conformations exists (Birdsey-Benson et al. 2010; Landes et al. 2011), and if they are correlated with structural differences elsewhere in the receptor this might lead to changes in multiple rate constants. Mechanistically, such a scenario could be represented with tiered reaction mechanisms similar to those employed to describe features of NMDA receptor gating (Zhang et al. 2008b; Vance et al. 2012). Both MacLean and I agree that kinetic models provide a useful focus for thinking about mechanisms. Although structural data are required to lend physical reality to the discrete states in such models, relating functional kinetic data to explicit mechanisms gives insight into the temporal sequence of conformational changes the receptor undergoes and the energy landscape through which it transits. Such an approach also avoids the danger of drawing mechanistic conclusions from measurements of operationally defined changes in population responses. For example, everyone agrees that TARPs slow the rate at which ensemble currents decay in response to a rapid application of near-saturating glutamate, which by definition represents a slowing of desensitization. However, the available evidence argues strongly against a significant effect of TARPs on δ, the rate at which receptors enter desensitized states (Fig. 1 in Howe (2013). If this rate primarily reflects the rate at which the dimer interface separates (Sun et al. 2002), then by extension TARPs do not primarily act by stabilizing this interface. The T686 mutants MacLean refers to, which disrupt a molecular latch that engages when the LBD closes, are an interesting case in point in this regard. The mutations both slow the rate at which ensemble currents decay during a sustained application of glutamate and increase the amplitude of the steady-state current as a fraction of the initial peak current (Robert et al. 2005). However, single-channel data show that both effects result directly from destabilization of the closed-cleft LBD (increases in CO; Fig. 1 in Howe (2013)), which prolongs latencies-to-first-opening and desynchronizes activation (Zhang et al. 2008a). Desynchronizing activation both slows the decay of ensemble currents and substantially reduces peak Popen (Colquhoun & Hawkes, 1995), and the larger relative sustained currents in the T686 mutants are not due to an increase in open probability late in the application but rather a reduction in the amplitude of peak currents.

Biophysical Coupling Mechanisms in NMDA Receptor Gating

NMDA receptors (NMDARs) are heterotetrameric glutamate-gated ion channels, typically composed of 2 GluN1 and 2 GluN2 subunits. The central function of NMDARs is gating - the process of converting agonist binding energy into pore opening by repositioning M3, the major pore-lining transmembrane helix. For glutamate receptors, it is largely assumed, though lacking direct evidence, that gating occurs through tension: ligand-binding domain (LBD) closure around an agonist generates tension in the linkers connecting the LBD to M3 (the M3-S2 linker), shifting M3 away from the central pore axis. To directly test this model, we altered the tension of the GluN1 and GluN2A M3-S2 linkers through residue insertions (to decrease tension) or deletions (to increase tension). Based on single-channel analyses, we find that these manipulations specifically affect pore opening (as opposed to ligand binding) and that they alter gating more dramatically in GluN2A than in GluN1. All-atom molecular dynamics simulations on a modeled GluN1/GluN2A receptor showed that this subunit-specific difference may arise, in part, from the disparate extensions and orientations of the GluN1 vs GluN2A M3-S2 linkers. Our functional data also suggests that the GluN1 M3-S2 linker gates primarily through tension. using rate equilibrium free energy relationship (REFER) analysis and length-tension analysis, we find that for the GluN1 M3-S2 linker, tension arises primarily during the C1-O1 transition (assuming a linear kinetic scheme) with a spring constant of ∼7.2 pN/nm, agreeing well with other biological springs. In contrast, our functional data suggests that mechanisms other than tension mainly mediate the role of the GluN2A M3-S2 linker in gating. Alternative mechanisms may include twisting and changes in electrostatic interactions.

Conformational Mobility of S1-M1 and S2-M4 Linkers Influence NMDA Receptor Gating

NMDA receptors are tetrameric glutamate-gated ion channels pivotal to excitatory neurotransmission in the mammalian central nervous system. Each subunit of the NMDA receptor harbors an extracellular ligand-binding domain (LBD) connected by three linkers to the transmembrane segments that form the ion channel. Activation gating of NMDAR involves the propagation of ligand-induced conformational changes in the LBD into ion channel pore opening via linker peptide segments. The M3-S2 linker, which is attached to the pore-lining M3 transmembrane segment, has been shown to be a critical gating element. Constraining the conformational freedom of M3-S2 relative to the adjacent S2-M4 linker of the same subunit drastically reduced the efficacy of gating with a 30-75-fold reduction in channel open probability. Here we report the gating effects of limiting the relative conformational mobility of the S1-M1 and S2-M4 linkers with crosslinking disulfide bonds. Both intra- and inter- subunit crosslinking strategies in GluN1/GluN2A-containing receptors were used to dissect the linkers’ roles in gating transduction. We found that although the S1-M1 and S2-M4 linkers are connected respectively to the M1 and M4 transmembrane segment located peripheral to the pore, constraining their relative movements significantly hampered gating efficacy, as assayed using single channel recording. These results suggest that all the linkers in a single receptor complex function as an intricate unit in the gating transduction process and have important implications for potential drug targeting as unique noncompetitive antagonists are known to bind to the S1-M1 and S2-M4 linkers of certain glutamate receptor subtypes, although their mechanisms of action are yet undefined.

Zinc Effects on NMDA Receptor Gating Kinetics

Zinc accumulates in the synaptic vesicles of certain glutamatergic forebrain neurons and modulates neuronal excitability and synaptic plasticity by multiple poorly understood mechanisms. Zinc directly inhibits NMDA-sensitive glutamate-gated channels by two separate mechanisms: high-affinity binding to N-terminal domains of GluN2A subunits reduces channel open probability, and low-affinity voltage-dependent binding to pore-lining residues blocks the channel. Insight into the high-affinity allosteric effect has been hampered by the receptor's complex gating; multiple, sometimes coupled, modulatory mechanisms; and practical difficulties in avoiding transient block by residual Mg 2+. To sidestep these challenges, we examined how nanomolar zinc concentrations changed the gating kinetics of individual block-resistant receptors. We found that block-insensitive channels had lower intrinsic open probabilities but retained high sensitivity to zinc inhibition. Binding of zinc to these receptors resulted in longer closures and shorter openings within bursts of activity but had no effect on interburst intervals. Based on kinetic modeling of these data, we conclude that zinc-bound receptors have higher energy barriers to opening and less stable open states. We tested this model for its ability to predict zinc-dependent changes in macroscopic responses and to infer the impact of nanomolar zinc concentrations on synaptic currents mediated by 2A-type NMDA receptors.

Subunit Specific Impact of Lurcher Motif Residues on NMDA Receptor Gating

In all glutamate-activated channels, the outward portion of the M3 transmembrane helix consists of a conserved nine amino acid residue stretch (lurcher motif): SYTANALAAF. Substitution of position 8 alanine with threonine (A8T, the lurcher position) in a wide range of glutamate receptors result in constitutively active channels. Scanning mutagenesis and cysteine modification experiments, implicated the position 7 alanine (A7) of GluN1 and GluN2 subunits as critical elements in gating, perhaps the equivalent of the ‘bundle crossing’ in potassium-selective channels. To investigate how A7 and A8 residues affect NMDA receptor gating, we used single channel recordings and kinetic models of receptor activity from HEK 293 cells. We found that substitutions at these positions in GluN1 or GluN2A resulted indeed in channels with modified gating. Compared to wild-type receptors (PO = 0.51 ± 0.05, MOT = 8.6 ± 1 ms, MCT = 7.5 ± 0.7 ms), A7C and A8T resulted in distinct gating patterns depending on the subunit in which they were introduced. GluN1(A7C) had shorter openings (Po = 0.02 ± 0.01, MOT= 3.2 ± 0.5 ms, MCT = 143 ± 17 ms) but GluN2A A7C) had significantly prolonged openings (PO = 0.17 ± 0.02, MOT = 13 ± 1 ms, MCT = 65 ± 7 ms). Further, A8T prolonged openings in GluN1 (PO = 0.41 ± 0.07, MOT = 21 ± 4 ms, MCT = 30 ± 6.4 ms) but had no effect on gating when introduced in GluN2A (PO = 0.57 ± 0.08, MOT = 11.2 ± 1 ms, MCT = 9.5 ± 2.6 ms). The implication is that the A7 and A8 positions play distinct gating roles in GluN1 vs. GluN2A subunits, supporting the view that in NMDA receptors, subunits are staggered along M3 into the channel's external vestibule.

Contributions of Carboxy-Terminal Domains to NMDA Receptor Gating

The intracellularly located carboxy terminal domain (CTD) of NMDA receptors represents almost half of these receptors’ mass; it interacts with a wide variety of regulatory proteins; and it serves to control receptor targeting and function. Calcium-activated proteins such as calmodulin, actinin and calcineurin, bind to or modify residues on CTDs and results in run-down, inactivation or desensitization of macroscopic responses. The mechanisms by which these kinetic changes occur are unknown. As a prelude into investigations of calcium-dependent modulation mechanism, we first asked how do CTDs of GluN1 and GluN2A subunits contribute to the receptor's gating reaction. We used cell-attached single-channel recordings, kinetic analyses and modeling to characterize the stationary gating kinetics of NMDA receptors lacking CTDs: GluN1(K838Stop) and/or GluN2(K844Stop). We found that compared to wild-type receptors (Po, .53 ± 0.03; MOT, 6.0 ± 0.6 ms; MCT, 5.4 ± 0.7; n = 17) receptors lacking CTD of GluN1 subunits had similar open probabilities but with slightly longer openings and closures (Po, .5±0.05 , MOT, 8.5 ± 1.2 ms, MCT, 11.0 ±2.4 ms; n = 9). In contrast, receptors lacking the CTD of GluN2A subunits were ∼2.5-fold less active (Po, .2±0.06; n = 9), with slightly longer openings (MOT, 10.7±1.3 ms,) and ∼17-fold longer closures. Receptors completely devoid of CTD, were much less active then either of the single-subunit CTD deletions, mainly due to substantially longer closures (Po, 0.1 ± 0.03, MOT, 6.5±1.4 ms, MCT, 175.2±53.4; n=8). These results demonstrate that the CTDs of both the GluN1 and GluN2A subunits contribute to normal gating kinetics, with the CTD of GluN2A receptors playing a major role in maintaining high open probabilities.