Constitutive activity of ionotropic glutamate receptors via hydrophobic substitutions in the ligand-binding domain

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1. 

   Biophysics Colab

   Mar 14, 2024

   Evaluation statement (8 March 2024)

   Seljeset et al. investigate the mechanism by which NMDA receptors are activated by co-agonists glutamate and glycine. By mutating residue Asp732 in the glycine-binding site, they generate receptors activated by glutamate, and not glycine, but inhibited by glycine antagonists. Conventional and unnatural amino acid mutagenesis reveals that Asp732 interacts with nearby residues to influence channel gating as well as ligand binding. Furthermore, a homomeric receptor from Trichoplax adhaerens, which has a tyrosine in the homologous position, displays constitutive activity that becomes glycine-dependent when the tyrosine is mutated to aspartate. The study is valuable because it reveals the importance of position 732 for controlling ligand potency and channel activity in glutamate receptors, which should …

   Evaluation statement (8 March 2024)

   Seljeset et al. investigate the mechanism by which NMDA receptors are activated by co-agonists glutamate and glycine. By mutating residue Asp732 in the glycine-binding site, they generate receptors activated by glutamate, and not glycine, but inhibited by glycine antagonists. Conventional and unnatural amino acid mutagenesis reveals that Asp732 interacts with nearby residues to influence channel gating as well as ligand binding. Furthermore, a homomeric receptor from Trichoplax adhaerens, which has a tyrosine in the homologous position, displays constitutive activity that becomes glycine-dependent when the tyrosine is mutated to aspartate. The study is valuable because it reveals the importance of position 732 for controlling ligand potency and channel activity in glutamate receptors, which should lead to a better understanding of how these receptors are primed for channel opening.

   Biophysics Colab recommends this study to scientists interested in the structure and function of glutamate receptors

   Biophysics Colab has evaluated this study as one that meets the following criteria:

   • Rigorous methodology
   • Transparent reporting
   • Appropriate interpretation

   (This evaluation refers to version 2 of this preprint, which has been revised in response to peer review of version 1.)

   Read the original source
2. 

   Biophysics Colab

   Feb 22, 2024

   Authors’ response (11 February 2024)

   GENERAL ASSESSMENT

   Ionotropic glutamate receptors mediate the large majority of excitatory synaptic transmission in the brain. These receptors consist of four classes: AMPA, kainate, NMDA and delta receptors. NMDA receptors are obligate tetramers composed of two GluN1 and two GluN2 (or GluN3) subunits. Compared to other iGluRs, they have the particularity of requiring two different agonists for their channel to open: glycine binding on GluN1 and glutamate on GluN2.

   Seljeset et al. investigate the molecular determinants controlling ligand potency and NMDAR activity at the level of the ligand-binding domains (LBDs), where the agonists bind. They identify a specific position, D732, whose mutation to either leucine or phenylalanine leads to a constitutively active GluN1 subunit, and thus to NMDARs …

   Authors’ response (11 February 2024)

   GENERAL ASSESSMENT

   Ionotropic glutamate receptors mediate the large majority of excitatory synaptic transmission in the brain. These receptors consist of four classes: AMPA, kainate, NMDA and delta receptors. NMDA receptors are obligate tetramers composed of two GluN1 and two GluN2 (or GluN3) subunits. Compared to other iGluRs, they have the particularity of requiring two different agonists for their channel to open: glycine binding on GluN1 and glutamate on GluN2.

   Seljeset et al. investigate the molecular determinants controlling ligand potency and NMDAR activity at the level of the ligand-binding domains (LBDs), where the agonists bind. They identify a specific position, D732, whose mutation to either leucine or phenylalanine leads to a constitutively active GluN1 subunit, and thus to NMDARs activated solely by glutamate. This aspartate is well known in the field, since it is a highly conserved, signature residue in iGluRs that binds amino acid ligands, together with an arginine in the LBD upper lobe. Surprisingly, although glycine cannot further activate GluN1-D732L/GluN2Awt receptors, glycine site antagonists like 5,7-DCKA or CGP-78608 can still bind and inhibit NMDAR activity. This study is therefore very intriguing, as it raises new questions about something that was previously thought to be understood. By using a combination of unnatural amino acids and conventional mutagenesis, the authors propose that D732 contributes to glycine-mediated effects by changing local interactions with nearby residues. In addition, they show that this behavior is specific for the GluN1 subunit, since mutation of the equivalent aspartate in the GluN2 subunit does not yield constitutively activated GluN2 subunits. Finally, the authors identify a homomeric iGluR from the placozoan Trichoplax adhaerens, Trichoplax AKDF¹⁹³⁸³, in which this conserved aspartate is replaced by a tyrosine. When expressed in Xenopus oocytes, the channel shows constitutive activity. Mutation of the tyrosine into an aspartate, to convert Trichoplax AKDF¹⁹³⁸³ into a “classical” iGluR, decreases Trichoplax AKDF¹⁹³⁸³ constitutive current and allows this channel to be activated by glycine and D-serine. Interestingly, an adjacent residue that is a serine in most mammalian subunits is also a tyrosine in Trichoplax AKDF¹⁹³⁸³, and mutation of both tyrosines yields a glutamate-gated ion channel comparable to mammalian receptors. All of this suggests that the nature of the residue at position 732 influences not only ligand binding but also channel gating.

   The study is technically sound, with appropriate controls, and uncovers intriguing properties of a position in GluN1 LBD at which specific side chain mutations can lock the subunit in an active state. Investigation of Trichoplast iGluR further reinforces these findings. This study should lead to a better understanding of how LBDs prime channel opening in iGluRs in the absence of agonists. In addition, co-agonist insensitive GluN1-D732L containing NMDARs could be used as tools to investigate the physiological consequences of NMDAR regulation by their co-agonist site. In contrast to previously engineered NMDARs activated solely by glutamate, which rely on the LBD being locked in its active state by cysteine bridges (Blanke and VanDongen, J Biol Chem 2008), GluN1-D37L/GluN2A NMDARs remain druggable (i.e. they can still be inhibited by glycine-site competitive antagonists). This is a great advantage when investigating the function of these receptors in a native context. The study identifies a few gaps that remain in our mechanistic understanding of D732’s role in channel gating. Particularly, it is unclear how subtle modification of residue side chains at position D732 lead to such drastic changes in function and why these effects are specific to GluN1 LBD. Also, why does mutation of D732 into isoleucine lead to a constitutively active GluN1 subunit, while mutation of a closely related leucine residue prevents activation of the receptor by glycine? The idea of a “hydrophobic plug” formed by D732L or D732F sidechains leading to constitutive activation would benefit from further validation since other hydrophobic substitutions (A, V, I, Y, and W) do not produce similar effects. Finally, it would be interesting to carry out further investigations of the role of the interaction between D732 and Q536 in open conformation stability. Thus, this paper puts forth interesting questions that could be addressed by future studies, for example molecular dynamics simulations and exploration of the LBD free energy landscapes (as in Yao et al., Structure 2013), to understand the impact of the GluN1-D732L mutation on GluN1 LBD conformational mobility.

   RECOMMENDATIONS

   Essential revisions:

   1. Page 2, “These data show that essentially all substitutions at the GluN1-732 position decrease glycine potency, but leucine and phenylalanine substitutions also remove the requirement for glycine co-agonism in GluN1/GluN2A NMDA receptors”: One other hypothesis for the lack of glycine dependence of GluN1-D732I and D732Y + GluN2A receptors could be that the mutated receptors have a glycine potency so high that GluN1 LBD is already saturated by contaminating, ambient glycine. At this point in the paper, the authors cannot distinguish between one hypothesis or the other, therefore we suggest that this sentence be rephrased. Later in the text, control experiments with GluN1-R523K mutations that kill glycine binding and competition with 5,7-DCKA show that glycine-independent activation of GluN1-D732L/GluN2A mutants is not due to constitutive occupancy of GluN1 LBD by contaminating glycine.

   ER1) We have now changed this to (page 4): “These data show that most substitutions at the GluN1-732 position decrease glycine potency, but leucine and phenylalanine substitutions alter GluN1 activity in such a way that leads to single-mutant NMDA receptors activated solely by glutamate.”

   1. Does glycine insensitivity in GluN1-D732L/GluN2A NMDARs reflect a constitutively active GluN1 subunit or is this subunit locked in another conformational state that cannot be further modified by glycine? This could be answered by estimating the maximum open probability of GluN1-D732L/GluN2A NMDARs compared to their wt counterparts. To estimate Po, the authors could measure the kinetics of NMDA receptor current inhibition by MK801 (the slower MK801 inhibition, the lower the Po; see Chen et al., J. Neurosci 1999; Blanke and VanDongen, JBC 2008) in the presence of saturating agonist concentrations (100 μM Glu, 100 μM Gly for wt and only 100 μM Glu for mutant).

   ER2) We have now assessed the rate of MK-801 block in glutamate-gated mutant and glycine + glutamate-gated WT receptors, and reshuffled text/figures, as this ties in well with ER4) below. MK-801 results now in Figure 3 on page 6, and main text on page 5: “In order to understand whether the glycine-insensitive GluN1-D732L subunit is in a constantly activated state or occupies a different conformation that may reflect an alternative to typical channel gating, we compared the kinetics of WT receptor and GluN1-D732L-containing receptor inhibition by the open-channel blocker MK-801, which can be used to evaluate maximum open probability of NMDARs ^26,30. We observed very similar kinetics of inhibition of WT and mutant receptors (Fig. 3A), indicating similar open probability in solely glutamate-gated GluN1-D732L-containing receptors and glutamate and glycine-gated WT receptors. This reflects unchanged maximum open probability in solely glutamate-gated NMDARs with disulfide-locked GluN1 LBDs assayed by single channel recordings ²⁷. This suggests that the GluN1-D732L subunit is in a constantly activated state.”

   When viewed alongside high sensitivity of mutant subunits to DCKA - OS1) below - it’s difficult to conclude what sort of active state the mutant subunit adopts. We’ve assessed the best we can at the moment, and in this paper we’ll have to leave it at “here is the observation; here is some evidence ruling out various possibilities; and here is a receptor from another family that shows something remarkably consistent”. Future studies will have to establish exactly what state the mutant subunit adopts.

   1. Page 4: The term “hydrophobic plug” is not fully justified since other hydrophobic residues do not lock GluN1 LBD in its active state.

   ER3) We have replaced nearly all use of this term, in the title and in the main text, to e.g. “certain hydrophobic substitutions” or “L/F substitutions”.

   1. Figure 2, redox sensitivity of GluN1-D732L/GluN2Awt: It would be helpful to explain the point of this experiment – maybe to investigate if the D732L mutation has an impact on the receptor gate rather than on the LBD? In any case, the authors should investigate the effect of DTT on the activity of wt GluN1/GluN2A receptors to determine whether there is an absence of an effect of the D732L mutant on redox sensitivity.

   ER4) Indeed we were curious if D732L affected the gate via this allosteric route, rather than by just altering LBD conformation. And we have now shown the effect of DTT on WT receptors.

   In addition to re-writing to better explain the point, as suggested, we have also re-written to follow on from new data/text on the whether the D732L mutation affects LBD, gating, etc: “We next questioned if D732L/F substitutions affect channel gating, rather than simply altering the LBD conformation. The gating machinery is complex, but it includes the peptide segment linking the C-terminal end of the LBD to membrane-spanning helix 4 (LBD-M4 linker, (11)). The LBD and LBD-M4 linker are confined by a C744—C798 disulfide, just four helical turns after D732, whose disruption by reduction enhances channel gating (28)). We considered that if the D732L/F substitution is coupled to channel gating via this route, then removal of the C744—C798 disulfide via the C744A mutation might alter glutamate-gated currents in GluN1-D732L-containing receptors. Alternatively, the typical enhancement by the reducing agent dithiothreitol (DTT) might differ in GluN1-D732L compared to WT receptors.”

   And new Figure 3 now includes DTT effects on WT receptors.

   1. Page 6: The authors find that mutation of Q536 decreases glycine potency and conclude there is an interaction between D732 and Q536. However, the effects of D732 and Q536 mutations could be independent, therefore the authors should consider mutating both residues together to look at the additive/non-additive effects of the mutations. Or perhaps, note in the Discussion that some sort of mutant cycle analysis or molecular dynamics simulation would be needed to rigorously test these ideas.

   ER5) We have now made and tested a double mutant combining D732E and Q536N and performed mutant cycle analysis.

   (We also tried to do this for Q536 side chain (regular mutations) and A734 main chain (non-canonical substitutions), but double mutants involving non-canonical amino acids at A734 were not successful – Figure S1.)

   As is now shown in Figure 4D, the effects of the mutations are decidedly non-additive, yielding an Ω value of 0.05, corresponding to a reasonably high energetic coupling of ~7 kJ/mol. We have now added to the relevant section of the Results on page 8: “If an interaction between Q536 and D732 were energetically important for receptor activation, the effects of their mutations should be non-additive ³¹. We therefore tested glycine potency at double-mutant GluN1-Q536N/D732E-containing receptors and observed non-additive changes in EC₅₀, with a strong coupling value, Ω, of 0.05 (Fig. 4D). This deviation of Ω from unity, corresponding to an interaction energy of 7.4 kJ/mol is relatively high ³¹, confirming that Q536 and D732 are energetically coupled. We tried to analyse energetic coupling between Q536 and A734 via double mutants incorporating nonsense suppression at the A734 position, but unfortunately, attempts to incorporate Aah into such double mutants via nonsense suppression were unsuccessful (Fig. S1B).”

   1. Page 6, “A hydrophobic plug does not cause constitutive activity in all NMDA receptor subtypes”: This title is misleading as it raises the expectation that the effect of GluN1-D732L has been investigated in the context of GluN1/GluN2A, GluN1/GluN2B, etc NMDARs. Instead, the equivalent mutation is made in the GluN2 subunit. We suggest using the word “subunit” rather than “subtype”.

   ER6) We have changed this Results section title (page 8) to: “L/F substitutions do not cause constitutive activity in all NMDA receptor subunits”

   1. Page 7, effect of GluN1-D732L in the context of GluN1/GluN3 NMDARs: We would not expect current to be observed with GluN1-D732L/GluN3 NMDARs, since locking GluN1 LBD in its active state desensitizes the receptors. The effect of the D732L mutation seems therefore conserved between GluN1/GluN2 and GluN1/GluN3 NMDARs. In addition, when using CGP, please cite Grand et al., Nat. Commun. 2018 since they were the first to use CGP as a tool to record GluN1/GluN3 currents.

   ER7) We have now cited that paper specifically here (page 8) and inserted the following (page 8/9): “While this seems like inactivity of the mutant GluN1 subunit in GluN1(4a)/GluN3A, it could yet reflect the activity of constitutively active mutant GluN1 subunits in GluN1/GluN2A receptors, as GluN1 activity in GluN1/GluN3A receptors is known to cause more desensitization than activation (Grand et al 2018).”

   1. Figure 5C: It is stated in the text that the aspartate position is “highly” conserved. However, no actual number or percentages are given for this statement. How does it compare to the residues in the highly conserved SYTANLAAF motif or other conserved positions? This sort of analysis does not need to be done for the entire receptor, but perhaps for glycine and glutamate binding residues and SYTANLAAF motif, to give a quantitative feel for statements about conservation. In addition, what other types of residues occupy this position in other species? And what was the number of species/subunits included in the analysis?

   ER8) To clarify the level of conservation, we have added Table 1 (page 10) listing the % conservation of amino acids at selected positions.

   In analyzing % conservation, we noticed that several iGluR sequences with gaps in the ligand-binding domain or channel-forming helices had escaped our filtering out incomplete sequences in our phylogenetic analysis. We therefore revisited our phylogenetic analysis, removed several incomplete sequences, and replaced Crassostrea gigas (a mollusc spiralian) iGluR sequences with Schmidtea mediterranea (a flatworm spiralian) sequences. This (1) means less sequences with gaps in the ligand-binding domain in our alignment/tree and (2) better covers the diversity of the lineage Spiralia now that we have sequences of Lingula anatina and Schmidtea mediterranea, which are more distantly related than Lingula anatina and Crassosttrea gigas (Laumer et al 2019, PMID:31690235; Marlétaz et al., 2019, PMID:30639106).

   The result is a phylogenetic and amino acid sequence analysis of 204 iGluR genes (previous version had 212 genes) with the same overall topology as the previous version, including lambda, NDMA, epsilon, and AKDF iGluR families (Fig. 5B, page 9).

   The number of subunits/genes used is stated in the Figure legend. The number of and reasoning behind the number of species used is described under Methods, Bioinformatic analyses: in exploring the conservation of the D732 residue, we have not tried to use as many iGluR sequences as possible; rather we have tried to assess this residue in a broad sample covering all (animal) iGluR families and from a careful selection of different animal lineages, while also avoiding fast-evolving species like Drosophila, which complicate tree topology. Hence our description of “two ctenophores, one poriferan, etc” under Methods, Bioinformatic analyses. In the main text (Results, page 9), we retain our original description: “We assembled diverse iGluR sequences, covering all animal lineages and animal iGluR families (Fig. 6A,B)…”

   1. Figure 5, panel F: From what we understand, the authors created dose-response curves for wt Trichoplast AKDF¹⁹³⁸⁶³ based on steady-state currents and for Y742D/Y743S mutants based on peak currents. If this is the case, one cannot compare the two dose-response curves since peak current potentiation and steady-state inhibition likely reflect different conformational transitions.

   ER9) We acknowledge this issue and that we can’t really say that ligand-activated D742 channels bind D-serine better than ligand-deactivated Y742 channels. But we think it’s fair to point out that mutant D742 channels react (by conducting current) to micromolar ligand concentrations whereas wildtype Y742 channels react (with decreased current) only to millimolar concentrations, and we have re-written to acknowledge the issue raised for this comparison (page 11): “Finally, we tried to assess whether position 742 determines ligand potency in addition to channel activity in AKDF¹⁹³⁸³ receptors. For these experiments we employed D-serine, as recovery from glycine-induced deactivation (Fig. 6C, far-left) and activation/desensitization (Fig. 6C, far-right) was very slow. Substantial deactivation of WT receptors was only induced by millimolar D-serine concentrations, whereas Y742D-containing mutants were activated by micromolar concentrations (Fig. 6D,E), with an EC₅₀ of 490 ± 120 µM at Y742D/Y743S (n = 4; Y742D EC₅₀ not assessed due to slow recovery from desensitization). Our measure of potency is confounded by the fact that deactivation (in WT channels) and activation (in mutant channels) are presumably coupled to D-serine binding via different conformational transitions. Nonetheless, we observe that a naturally occurring large hydrophobic side chain at the top of the β-strand preceding the αI helix leads to an AKDF homo-tetramer that shows constitutive activity and responds only to millimolar concentrations of D-serine. In contrast, “re-introducing” an aspartate to this position reinstates more typical ligand-dependent activation and sensitivity to micromolar concentrations of D-serine.”

   Optional suggestions:

   1. Figure 2, glycine/DCKA competition: It is difficult to understand how a GluN1 LBD-locked closed (active state) could still bind DCKA. If the open-to-close equilibrium of GluN1 LBD is displaced towards its closed state, then DCKA Ki should be shifted to the right compared to wt receptors. Additionally, DCKA inhibition kinetics should be slower if DCKA needs to “wait” for rare resting-like conformational changes to bind. Did the authors investigate DCKA potency and inhibition kinetics?

   OS1) We have now investigated DCKA potency. DCKA capably inhibits GluN1-D732L/GluN2A-WT activity, and perhaps surprisingly, potency of DCKA at the mutant is greater than at wildtype. We suspect this is due to (1) the introduction of a hydrophobic leucine residue right next to an aryl group of DCKA, increasing DCKA affinity directly, (2) the absence of glycine binding to this site, so no need for competition, and (3) potentially other mechanisms such as cooperativity between subunits. Again, establishing the precise nature of our mutant LBD conformation here is for future structural and molecular dynamics studies. But we have described the results, along with our following interpretation, (page 4): “Whether increased DCKA potency in GluN1-D732L subunits derives from the now non-competitive nature of the inhibition in mutant receptors or from the introduction of a favourable hydrophobic interaction with the dichlorobenzene moiety of the inhibitor is unclear. But the high DCKA potency would suggest that the constitutively active GluN1-D732L subunit is, unexpectedly, not due to a permanently clamshell-closed LBD in the mutant. This may reflect the fact that extent of LBD closure is poorly correlated with agonist efficacy in GluN1 subunits, in contrast to AMPA receptor GluA2 subunits ²¹.”

   1. The authors show in many panels that GluN1/GluN2A currents desensitize (e.g. Fig.1B, 3C, 4A). In Xenopus oocytes, NMDAR currents do not normally desensitize. We fear this desensitization might stem from contamination of the NMDA current by calcium-activated chloride channels, which can be activated by high quantities of barium when large NMDAR currents are measured. To avoid this problem, we advise that NMDA currents above 2 µA are avoided.

   OS2) We have moved forward presuming that potential changes in current amplitude due to a small chloride flux doesn’t affect our measures of potency or ligand-selectivity. But in our new experiments, we’ve especially tried to avoid large currents.

   1. Page 5, investigation of D732 state-dependent interactions: Mutation of residues near D732 to unnatural amino acids to replace the peptidic NH do not bring much information about the mechanisms of D732 action. The fact that the 734Aah and 735Vah cannot mimic the effect of the D732L mutation could be due to many factors, including the fact that changing the peptide bond probably changes the local structure of the LBD. Perhaps mention this in the discussion.

   OS3) We have now acknowledged this possibility in the Results, right after we describe the decrease in glycine potency caused by the 734Aah mutation (page 7): “Although this may be due to local conformational changes due to altered main chain structure,…”

   1. It is intriguing that the D732L mutation locks an active conformation of the GluN1 subunit but not the GluN2 subunit, suggesting two different mechanisms of LBD closure by glutamate and glycine. It would be interesting to look at the effect of the equivalent mutation on the GluN3 subunit to investigate if this locking effect is specific to glycine-binding LBDs or just to the GluN1 subunit.

   OS4) We have now made and tested mutant GluN3A subunits D485L and D485F. Simply decreases glycine activity altogether (reflecting the effects of the mutations in GluN2A). Described on page 9: “Similarly, at oocytes injected with GluN1(4a)-WT and GluN3A-D845L or -D845F mRNAs, we saw no response to glycine alone or glycine in the presence of CGP 78608 (Fig 5D). Together, these results indicate that the induction of a constitutively active state by the D732L/F substitution is an exclusive feature of the GluN1 subunit, and the only conserved feature of the mutation in different subunits is a decrease in agonist potency.”

   1. Page 9: Discussing the position of residue side chains from structures with 4 Å resolution does not seem relevant and would benefit from a caveat.

   OS5) We want to retain our comparison of experiments with available structural data, so we have kept this but re-written to more openly acknowledge the caveat (page 12): “Indeed, in a cryo-electron microscopy (cryo-EM) study of GluN1/GluN2B receptors, D732 has only swung toward the ligand and away from A734 in a second of two putative pre-gating step structural models, although this is speculative considering the poor resolution of D732 side chains in those cryo-EM maps (12).”

   1. Page 10: We don’t understand the point that the authors want to make with the activation of Aplysia californica. Please clarify.

   OS6) He we were trying to say that “not much is required to change NMDARs from requisite co-agonism to single-ligand agonism”, either (a) in the lab via the D732L mutation or (b) naturally, as invertebrate NMDA receptors apparently show single-ligand agonism (results on invertebrate NDMARs in the literature). Further, we want to say that “by extension, we wonder if (c) in certain physiological situations, vertebrate NMDARs might indeed need only a single ligand.” We acknowledge this was unclear and – although it’s still speculative – we have now changed to (page 13): “Our work shows that only small changes in the GluN1 LBD are required for solely glutamate-gated currents in vertebrate GluN1/GluN2 receptors, and previous work suggests that invertebrate Drosophila melanogaster and Aplysia californica GluN1/GluN2 receptors can be activated by single ligands ^50,51. This suggests that NMDA receptors’ requirement of co-agonism is easily alleviated by certain mutations or conditions. As iGluR-modulatory proteins vary across cell types or even across neuronal compartments ^52,53 and NMDA receptor sequence varies across animals, it is foreseeable that in certain physiological settings, certain NMDA receptors might be activated by glutamate alone. But in most settings, certainly in vertebrates, it seems that glutamate-induced activation of NMDA receptors relies on a system of ambient glycine or D-serine ^54,55.”

   1. In iGluRs, constitutive currents are often induced by mutations in the gate region, near the SYTANLAAF motif (e.g. lurcher mutations). Does the sequence around the gate of Trichoplast AKDF¹⁹³⁸⁶³ predict channel constitutive activity?

   OS7) Our results with WT, single mutant Y742D, and double mutant Y742D/Y743S Trichoplax AKDF¹⁹³⁸³ receptors already show convincing evidence that the constitutive activity is via the Y742 and Y743 position: the tyrosine residues are unique to this leaky channel, and their mutation to more typical residues removes the leak current (Fig. 7B, page 11, revised manuscript).

   But a look at upper M3 is warranted. As shown in Fig. 6C, AKDF¹⁹³⁸³ (YTANMAAFL) is quite similar to typical iGluRs (e.g. GluA2 YTANLAAFL). But one might ask about the single M/L difference in that motif, and we have therefore made and tested the M657L AKDF¹⁹³⁸³ mutant, comparing it with WT. Results show that this small M3 difference has little effect on channel activity. We have added this data in new Figure 7D and described it (page 11): “As channel activity of iGluRs also relies on the upper segment of the third membrane-spanning helix (M3, (34)), we also examined this segment in AKDF¹⁹³⁸³. AKDF¹⁹³⁸³ differs only subtly from most iGluRs with a methionine residue (M657) instead of leucine here (Fig. 6C), but we tested potential effects of this difference by mutating M657 to leucine. M657L activity was much like WT (Fig. 7D), however, confirming that divergence at Y742/Y743 and not the upper M3 segment determines the unique activity of AKDF¹⁹³⁸³.”

   1. D-serine is another co-agonist that binds the GluN1 subunit. Compared to glycine, D-serine can make additional interactions with the lower lobe of GluN1 LBD. It would be interesting to look at D-serine dose-response curves in GluN1-D732L/GluN2A receptors: are these receptors also D-serine insensitive or can they be further activated by D-serine?

   OS8) We have now measured the effects of D-serine on GluN1-D732L/GluN2A-WT receptors. As we now show in Figure 1B (green symbols), D-serine at increasing concentrations (100 nM through 100 μM) activates no additional current on top of the glutamate-gated current in mutant receptors. We have added to the end of the first Results paragraph (page 3): “Similarly, large currents were activated in mutant GluN1-D732L/GluN2A-WT receptors when 100 nM through 100 μM D-Serine was applied the presence of 100 µM glutamate (green in Fig. 1B).”

   (This is a response to peer review conducted by Biophysics Colab on version 1 of this preprint.)

   Read the original source
3. 

   Version published to 10.1101/2023.08.03.551817v2 on bioRxiv

   Feb 12, 2024
4. 

   Biophysics Colab

   Dec 8, 2023

   Consolidated peer review report (28 September 2023)

   GENERAL ASSESSMENT

   Ionotropic glutamate receptors mediate the large majority of excitatory synaptic transmission in the brain. These receptors consist of four classes: AMPA, kainate, NMDA and delta receptors. NMDA receptors are obligate tetramers composed of two GluN1 and two GluN2 (or GluN3) subunits. Compared to other iGluRs, they have the particularity of requiring two different agonists for their channel to open: glycine binding on GluN1 and glutamate on GluN2.

   Seljeset et al. investigate the molecular determinants controlling ligand potency and NMDAR activity at the level of the ligand-binding domains (LBDs), where the agonists bind. They identify a specific position, D732, whose mutation to either leucine or phenylalanine leads to a constitutively active GluN1 subunit, and thus …

   Consolidated peer review report (28 September 2023)

   GENERAL ASSESSMENT

   Ionotropic glutamate receptors mediate the large majority of excitatory synaptic transmission in the brain. These receptors consist of four classes: AMPA, kainate, NMDA and delta receptors. NMDA receptors are obligate tetramers composed of two GluN1 and two GluN2 (or GluN3) subunits. Compared to other iGluRs, they have the particularity of requiring two different agonists for their channel to open: glycine binding on GluN1 and glutamate on GluN2.

   Seljeset et al. investigate the molecular determinants controlling ligand potency and NMDAR activity at the level of the ligand-binding domains (LBDs), where the agonists bind. They identify a specific position, D732, whose mutation to either leucine or phenylalanine leads to a constitutively active GluN1 subunit, and thus to NMDARs activated solely by glutamate. This aspartate is well known in the field, since it is a highly conserved, signature residue in iGluRs that binds amino acid ligands, together with an arginine in the LBD upper lobe. Surprisingly, although glycine cannot further activate GluN1-D732L/GluN2Awt receptors, glycine site antagonists like 5,7-DCKA or CGP-78608 can still bind and inhibit NMDAR activity. This study is therefore very intriguing, as it raises new questions about something that was previously thought to be understood. By using a combination of unnatural amino acids and conventional mutagenesis, the authors propose that D732 contributes to glycine-mediated effects by changing local interactions with nearby residues. In addition, they show that this behavior is specific for the GluN1 subunit, since mutation of the equivalent aspartate in the GluN2 subunit does not yield constitutively activated GluN2 subunits. Finally, the authors identify a homomeric iGluR from the placozoan Trichoplax adhaerens, Trichoplax AKDF19383, in which this conserved aspartate is replaced by a tyrosine. When expressed in Xenopus oocytes, the channel shows constitutive activity. Mutation of the tyrosine into an aspartate, to convert Trichoplax AKDF19383 into a "classical" iGluR, decreases Trichoplax AKDF19383 constitutive current and allows this channel to be activated by glycine and D-serine. Interestingly, an adjacent residue that is a serine in most mammalian subunits is also a tyrosine in Trichoplax AKDF19383, and mutation of both tyrosines yields a glutamate-gated ion channel comparable to mammalian receptors. All of this suggests that the nature of the residue at position 732 influences not only ligand binding but also channel gating.

   The study is technically sound, with appropriate controls, and uncovers intriguing properties of a position in GluN1 LBD at which specific side chain mutations can lock the subunit in an active state. Investigation of Trichoplast iGluR further reinforces these findings. This study should lead to a better understanding of how LBDs prime channel opening in iGluRs in the absence of agonists. In addition, co-agonist insensitive GluN1-D732L containing NMDARs could be used as tools to investigate the physiological consequences of NMDAR regulation by their co-agonist site. In contrast to previously engineered NMDARs activated solely by glutamate, which rely on the LBD being locked in its active state by cysteine bridges (Blanke and VanDongen, J Biol Chem 2008), GluN1-D37L/GluN2A NMDARs remain druggable (i.e. they can still be inhibited by glycine-site competitive antagonists). This is a great advantage when investigating the function of these receptors in a native context. The study identifies a few gaps that remain in our mechanistic understanding of D732's role in channel gating.Particularly, it is unclear how subtle modification of residue side chains at position D732 lead to such drastic changes in function and why these effects are specific to GluN1 LBD. Also, why does mutation of D732 into isoleucine lead to a constitutively active GluN1 subunit, while mutation of a closely related leucine residue prevents activation of the receptor by glycine? The idea of a "hydrophobic plug" formed by D732L or D732F sidechains leading to constitutive activation would benefit from further validation since other hydrophobic substitutions (A, V, I, Y, and W) do not produce similar effects. Finally, it would be interesting to carry out further investigations of the role of the interaction between D732 and Q536 in open conformation stability. Thus, this paper puts forth interesting questions that could be addressed by future studies, for example molecular dynamics simulations and exploration of the LBD free energy landscapes (as in Yao et al., Structure 2013), to understand the impact of the GluN1-D732L mutation on GluN1 LBD conformational mobility.

   RECOMMENDATIONS

   Essential revisions:

   1. Page 2, "These data show that essentially all substitutions at the GluN1-732 position decrease glycine potency, but leucine and phenylalanine substitutions also remove the requirement for glycine co-agonism in GluN1/GluN2A NMDA receptors": One other hypothesis for the lack of glycine dependence of GluN1-D732I and D732Y + GluN2A receptors could be that the mutated receptors have a glycine potency so high that GluN1 LBD is already saturated by contaminating, ambient glycine. At this point in the paper, the authors cannot distinguish between one hypothesis or the other, therefore we suggest that this sentence be rephrased. Later in the text, control experiments with GluN1-R523K mutations that kill glycine binding and competition with 5,7-DCKA show that glycine-independent activation of GluN1-D732L/GluN2A mutants is not due to constitutive occupancy of GluN1 LBD by contaminating glycine.
   2. Does glycine insensitivity in GluN1-D732L/GluN2A NMDARs reflect a constitutively active GluN1 subunit or is this subunit locked in another conformational state that cannot be further modified by glycine? This could be answered by estimating the maximum open probability of GluN1-D732L/GluN2A NMDARs compared to their wt counterparts. To estimate Po, the authors could measure the kinetics of NMDA receptor current inhibition by MK801 (the slower MK801 inhibition, the lower the Po; see Chen et al., J. Neurosci 1999; Blanke and VanDongen, JBC 2008) in the presence of saturating agonist concentrations (100 μM Glu, 100 μM Gly for wt and only 100 μM Glu for mutant).
   3. Page 4: The term "hydrophobic plug" is not fully justified since other hydrophobic residues do not lock GluN1 LBD in its active state.
   4. Figure 2, redox sensitivity of GluN1-D732L/GluN2Awt: It would be helpful to explain the point of this experiment – maybe to investigate if the D732L mutation has an impact on the receptor gate rather than on the LBD? In any case, the authors should investigate the effect of DTT on the activity of wt GluN1/GluN2A receptors to determine whether there is an absence of an effect of the D732L mutant on redox sensitivity.
   5. Page 6: The authors find that mutation of Q536 decreases glycine potency and conclude there is an interaction between D732 and Q536. However, the effects of D732 and Q536 mutations could be independent, therefore the authors should consider mutating both residues together to look at the additive/non-additive effects of the mutations. Or perhaps, note in the Discussion that some sort of mutant cycle analysis or molecular dynamics simulation would be needed to rigorously test these ideas.
   6. Page 6, "A hydrophobic plug does not cause constitutive activity in all NMDA receptor subtypes": This title is misleading as it raises the expectation that the effect of GluN1-D732L has been investigated in the context of GluN1/GluN2A, GluN1/GluN2B, etc NMDARs. Instead, the equivalent mutation is made in the GluN2 subunit. We suggest using the word "subunit" rather than "subtype".
   7. Page 7, effect of GluN1-D732L in the context of GluN1/GluN3 NMDARs: We would not expect current to be observed with GluN1-D732L/GluN3 NMDARs, since locking GluN1 LBD in its active state desensitizes the receptors. The effect of the D732L mutation seems therefore conserved between GluN1/GluN2 and GluN1/GluN3 NMDARs. In addition, when using CGP, please cite Grand et al., Nat. Commun. 2018 since they were the first to use CGP as a tool to record GluN1/GluN3 currents.
   8. Figure 5C: It is stated in the text that the aspartate position is "highly" conserved. However, no actual number or percentages are given for this statement. How does it compare to the residues in the highly conserved SYTANLAAF motif or other conserved positions? This sort of analysis does not need to be done for the entire receptor, but perhaps for glycine and glutamate binding residues and SYTANLAAF motif, to give a quantitative feel for statements about conservation. In addition, what other types of residues occupy this position in other species? And what was the number of species/subunits included in the analysis?
   9. Figure 5, panel F: From what we understand, the authors created dose-response curves for wt Trichoplast AKDF193863 based on steady-state currents and for Y742D/Y743S mutants based on peak currents. If this is the case, one cannot compare the two dose-response curves since peak current potentiation and steady-state inhibition likely reflect different conformational transitions.

   Optional suggestions:

   1. Figure 2, glycine/DCKA competition: It is difficult to understand how a GluN1 LBD-locked closed (active state) could still bind DCKA. If the open-to-close equilibrium of GluN1 LBD is displaced towards its closed state, then DCKA Ki should be shifted to the right compared to wt receptors. Additionally, DCKA inhibition kinetics should be slower if DCKA needs to "wait" for rare resting-like conformational changes to bind. Did the authors investigate DCKA potency and inhibition kinetics?
   2. The authors show in many panels that GluN1/GluN2A currents desensitize (e.g. Fig.1B, 3C, 4A). In Xenopus oocytes, NMDAR currents do not normally desensitize. We fear this desensitization might stem from contamination of the NMDA current by calcium-activated chloride channels, which can be activated by high quantities of barium when large NMDAR currents are measured. To avoid this problem, we advise that NMDA currents above 2 µA are avoided.
   3. Page 5, investigation of D732 state-dependent interactions: Mutation of residues near D732 to unnatural amino acids to replace the peptidic NH do not bring much information about the mechanisms of D732 action. The fact that the 734Aah and 735Vah cannot mimic the effect of the D732L mutation could be due to many factors, including the fact that changing the peptide bond probably changes the local structure of the LBD. Perhaps mention this in the discussion.
   4. It is intriguing that the D732L mutation locks an active conformation of the GluN1 subunit but not the GluN2 subunit, suggesting two different mechanisms of LBD closure by glutamate and glycine. It would be interesting to look at the effect of the equivalent mutation on the GluN3 subunit to investigate if this locking effect is specific to glycine-binding LBDs or just to the GluN1 subunit.
   5. Page 9: Discussing the position of residue side chains from structures with 4 Å resolution does not seem relevant and would benefit from a caveat.
   6. Page 10: We don't understand the point that the authors want to make with the activation of Aplysia californica. Please clarify.
   7. In iGluRs, constitutive currents are often induced by mutations in the gate region, near the SYTANLAAF motif (e.g. lurcher mutations). Does the sequence around the gate of Trichoplast AKDF193863 predict channel constitutive activity?
   8. D-serine is another co-agonist that binds the GluN1 subunit. Compared to glycine, D-serine can make additional interactions with the lower lobe of GluN1 LBD. It would be interesting to look at D-serine dose-response curves in GluN1-D732L/GluN2A receptors: are these receptors also D-serine insensitive or can they be further activated by D-serine?

   REVIEWING TEAM

   Reviewed by:

   Sudha Chakrapani, Professor, Case Western Reserve University, USA: Structure, function and modulation of neurotransmitter receptors

   Laetitia Mony, Permanent Researcher, Institute of Biology of École Normale Supérieure, France: NMDA receptor structure-function relationships and pharmacology

   Lonnie Wollmuth, Professor, Stony Brook University, USA: NMDA receptor structure-function relationships and pharmacology

   Curated by:

   Sudha Chakrapani, Professor, Case Western Reserve University, USA

   Read the original source
5. 

   Version published to 10.1101/2023.08.03.551817v1 on bioRxiv

   Aug 6, 2023