Two extreme Loss-of-Function GRIN2B -mutations are detrimental to tri-heteromeric NMDAR-function, but rescued by pregnanolone-sulfate

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   Jun 28, 2023

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   Reply to the reviewers

   Reviewer #1 (Evidence, reproducibility and clarity (Required)):

   Summary: Kellner and Berlin present their research findings pertaining to the effect of GRIN2B variants that modify NMDA receptor function and pharmacology. While these mutations were published previously, the new manuscript provides a more thorough investigation into the effects that these variants pose when incorporated into heteromeric complexes with either wildtype GluN2B or GluN2A - NMDA receptors containing only a single mutated GluN2B subunits is more relevant to the disease cases because the associated patients are heterozygous for the variant. The authors achieved selective expression …

   Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.

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   Reply to the reviewers

   Reviewer #1 (Evidence, reproducibility and clarity (Required)):

   Summary: Kellner and Berlin present their research findings pertaining to the effect of GRIN2B variants that modify NMDA receptor function and pharmacology. While these mutations were published previously, the new manuscript provides a more thorough investigation into the effects that these variants pose when incorporated into heteromeric complexes with either wildtype GluN2B or GluN2A - NMDA receptors containing only a single mutated GluN2B subunits is more relevant to the disease cases because the associated patients are heterozygous for the variant. The authors achieved selective expression of receptor heteromeric complexes by utilising an established trafficking control system. The authors found that while a single variant subunit in the receptor complex is largely dominant in its effect on reducing glutamate potency of the NMDA receptor, it 's effect on receptor pharmacology varied. Unlike diheteromeric receptors containing mutated subunits, polyamine spermine potentiated GluN1/2B (but not GluN1/2A/2B) receptors that contained a single mutated GluN2B. In contrast, the neurosteroid, pregnenolone-sulfate (PS), was effective at potentiating the NMDA receptor currents (to varying degrees) regardless of the subunit composition. The potentiation of NMDA receptor currents by PS was also observed in neurons overexpressing the variants.

   The techniques used in this study were appropriate to address the objectives and the overall effects are large, and generally convincing. I like the way the results are presented, although have a few (easily addressable) comments.

   We thank the reviewer for the positive remarks on our manuscript.

   Major comments:

   #1 When incrementally adding drugs (e.g. traces in figures 5 and 6), it doesn't always appear like the response has plateaued before changing the solutions/drugs. Therefore, I am curious to what extent the effects observed are underestimated.

   The reviewer is correct to note that some responses do not necessarily reach a plateau, despite our efforts reach steady-state (as shown in most figures, e.g., Figs. 1-4, 6b, etc.), in particular when applying pregnenolone-sulfate (PS) (Fig. 5a, all traces in middle and bottom rows). However, in several instances, this was unobtainable due the very slow effect of the neurosteroid (its mode of action is from within the membrane) and the very large size of the cell (~1 mm). For these reasons, these experiments mandated excessively long exposures (~minutes) of oocytes to glutamate and PS (see scale bar- 20 secs) to try to reach steady-state, however this also caused deterioration to some cells (which did not return to baseline- and were therefore discarded). Thus, we eventually converged on settings whereby we did not expose oocytes to more than 4 minutes of the drug. Nevertheless, to try to estimate the extent of the underestimation (if any), we fitted the currents (standard mono-exponential fit, as previously reported1–3 (Suppl. 5a). We found that our application times of PS were, on average, three time the response’s time constants (tau) (Suppl. 5b), and we found a very weak relationship (R2 = 0.09) between the response to PS and time of its application (Suppl. 5c). These are now explicitly mentioned in the text (line #203), and in the legend of Suppl. 5. These thereby suggest that the reaction reached approximately 95% (1 - 1/e^3) of the steady-state value, and we are therefore confident that we have very small, if any, underestimation the extent of PS potentiation.

   #2 Also, in relation to figure 6, to what extent does agonist application cause desensitization here? Looking at traces in Figure 6b it appears that there is some desensitization and it isn’t clear to what extent this persists during the solution changes.

   Agonist desensitization of NMDARs-currents is a well-known phenomenon, but it is very well established that it is not always observed in cells, including neurons (e.g., 4–7). In general, we did not observe very frequent desensitization’s (we provide a larger variety of traces of desensitizing and non-desensitizing currents (Fig. 6b Suppl. 7e and Suppl. 8a). Nevertheless, we explicitly note that in neurons, currents that didn’t reach steady-state after application of 100 mM NMDA were excluded from analysis (Methods - Patch clamping of cultured neurons, line #474), and in most cases desensitization was minor (or absent) following application of 100 mM NMDA and 100 mM PS (Fig. 6b).

   #3 Could the authors conduct/show the controls where NMDA alone (for 50-60s), or NMDA followed by PE-S (without ifenprodil).

   These recordings are now shown in Fig. 6b and Suppl. 8a, (as opposed to Suppl. 7e).

   #4 Finally, figure 5 shows the effect of the neurosteroid (and ifenprodil) on NMDA-evoked currents in neurons overexpressing the GluN2B variants in neurons. However, there currents probably reflect a mixture of extrasynaptic and synaptic receptors. To what extent are synaptic NMDA receptors affected by the variants?

   To show the extent of the effect of the variants over synaptic receptors, we recorded miniature NMDA-dependent EPSCs; mEPSCNMDA), as described in our previous report8. We find that the varinats completely eliminate the appearance of mEPSCs (Suppl. 7a, b). Change in minis’ frequency is not the result of a presynaptic change or a change in synapse number9, as we have shown that AMPAR-mEPSC frequency was unaffected by the variants (i.e., synapse number and probability of presynaptic release are unchanged by the variants).

     To further address this, we also explored the relative synaptic vs. extrasynaptic distribution of the variants by using the established MK-801-protocol (to block all synaptic receptors during spontaneous activity, leaving extrasynaptic receptors unblocked)10,11. In neurons overexpressing the GluN2B-*wt* subunit, we obtained an extrasynaptic fraction of 38%, highly consistent with previous reports12,13. Overexpression of the variants, however, yielded a significantly and higher fraction (~50%) of the remaining current, supposedly suggesting more variant receptors at extrasynaptic loci (__Suppl. 8b, c__). However, due to the experimental settings we have chosen, the results from this experiment represent quite the inverse when involving extreme LoF variants. Firstly, 100 mM NMDA does not saturate variant receptors (whether pure, mixed di- or tri-heteromers, see __Table 1__). Secondly, normal neurotransmission does not open synaptic receptors containing mutant GluN2B-subunits, attested by the complete absence of mEPSCs (see __Suppl. 7a, b and __8,9). Thus, during the 10 minutes exposure to MK-801, only (mostly) purely *wt* receptors are blocked by spontaneous synaptic activity, and thus the second bout of 100 mM NMDA solely exposes the remaining *wt*-receptors. An increase in the number reflects more *wt*-receptors at the extrasynapse than the synapse. Thus, the observed increase in the fraction of extrasynaptic receptors in neurons overexpressing the variants, implies that the number of *wt*-receptors is necessarily decreased from the synapse and increases at the extrasynapse. We deem this to ensue due to the incorporation of the variants at the synapse. This increase cannot be explained by an overall increase in membrane expression of *wt*-receptors in neurons overexpressing the variants, as these cells show a strong reduction in Imax  (see __Fig. 6c and Suppl. 7e__). This is now detailed in the text (lines #270-290).
   

   Minor comments:

   #5 Looking at the fits in the graph of Figure 2b it appears that the slope on the concentration response curves is less steep for the mixed 2B-diheteromeric NMDA receptors. How much are the Hill coefficients changing and can this be interpreted to provide more mechanistic insight? Wouldn't it make sense to include the Hill coefficients in Table 1?

   We agree with the reviewer’s observation. Actually, the mixed di-heteromers have a similar Hill coefficient (nH) as the purely di-heteromeric GluN2Bwt receptors (see Table 1), and these show the typical near nH ~1 (e.g., 14–16). The only diverging groups are the purely di-heteromeric variant-containing channels (G689C/S only containing receptors; nH~2). Although these may suggest positive cooperation between the subunits, we are less inclined to infer insights from the latter owing to the fact that we limited our examination to 10 mM glutamate (we limit exposure of oocytes to 10 mM glutamate due to artifacts arising past this concentration, as discussed in Kellner et al.8: Fig. 2—figure supplement 1). (this description is now mentioned in page lines #149, 318, 319).

   #6 The authors illustrate the changes in potency by the shift in the concentration response curves, but is there any change in efficacy? A simple way to illustrate this would be also present a simple graph showing the maximum current amplitudes (i.e. to 10 mM glutamate) for each of the receptor complexes.

   We now provide these data in (Suppl. 2a, b). We would like to note however that the expression pattern of the tailed-receptors (i.e., subunits with carboxy-termini tagged with C1/C2 tails, see Fig. 1a) are less expressive in general when compared with the native subunits (Suppl. 2c). This description is detailed in lines# 162-166.

   #7 The authors characterize the 'apparent' affinity (or potency) of the receptor using concentration-response curves, but numerous points in the manuscript refer to changes in affinity. None of the experiments shown directly measure affinity (which would require ligand-binding assays) and so the use of the word affinity is inaccurate/misleading. I suggest the authors replace the instances of the word 'affinity' with 'potency'.

   We apologize for the confusion surrounding our use of the term affinity. In fact, we do initially define this term in introduction (page #4): “apparent glutamate affinity (EC50)” to differentiate from affinity (KD). Regardless, and to avoid confusion, we replaced all terms, as suggested by reviewer to potency.

   #8 In the third line of the abstract, the authors wrote, 'for which there are no treatments' in relation to GRINopathies. My understanding is that there are symptomatic treatments but that there are no disease-modifying treatments.

   Indeed, all current treatments are supportive, rather than provide a bona fide cure or disease-modifying. These are now better defined in the abstract.

   #9 The authors have interchangeably used the terms NMDAR or GluNRs throughout the manuscript. I suggest sticking to one of these terms. I would suggest NMDARs since this is less likely to be misread as a a specific NMDA receptor subunit.

   Agreed and corrected throughout manuscript.

   #10 Typos: 1) Results paragraph 2 sentence one: 'We thereby produced GluN2B-wt, GluN2B-G689C and GluN2B-G689S subunits tagged with C1 or C2, co-expressed these along with the GluN1a-wt subunits in...') Results paragraph 2: '...but these were mainly noticeable when oocytes are were exposed to high (saturating) glutamate concentrations...'

   1. Last sentence in the second to last paragraph of the results section entitled 'Mixed di-and tri-heteromeric channels...': 'This , PS may serve to rescue...'
   2. Last sentence in last paragraph of the results section entitled 'Mixed di-and tri-heteromeric channels...': 'Despite the latter, we found no evidence for any direct effect of three different physiologically relevant concentrations of the drug on di- or tri-heteromeric receptors'

   All typos corrected.

   #11 Figures 1e, 2b, 3b: it would be helpful to add a legend to the graph so that the curves can be interpreted without having to read through the figure legend.

   Corrected.

   #12 The bar graphs in Figure 6 show individual data points but those in figures 4b and 5b don't. Can the authors please add the data points to these graph.

   Individual data points have been added.

   #13 It would be helpful to reviewers that future manuscripts by the authors include page numbers and line numbers.

   Included.

   **Referees cross-commenting**

   #14 Reviewers 2 and 3 highlight an important issue concerning figure 6 and the extent to which the overexpressed variants subunits can compete and assemble with endogenous NMDA receptors (unlike the system where the surface expression of specific receptor complexes is controlled). Indeed in the recent paper by the same authors, the two variants differed in their surface expression (in HEK cells), with G689C expressing particularly poorly. With reference to the second minor comment of Reviewer 1, the maximum current amplitudes would of course need to be normalized to cell surface expression of the receptor to gain any insight into efficacy.

   We provide maximal current amplitudes (Imax) as a proxy for expression level as typically done (e.g.,8,17). These are now shown in __Suppl. 2a, b __(and see our response to comment #6, above). We would like to emphasize that we find it challenging to gain insights about efficacy of the variants in neuronal synapses, as we purposefully express non-C1/C2 tagged subunits in neurons (as we covet assembly of the variants with endogenous subunits). Moreover, the C1/C2-tagged subunits (whether wt or variants) are less expressive compared to their non-tagged NMDAR-counterparts. For instance, tagged GluN2B-wt subunits express at ~50% compared to non-tagged GluN2B wt subunits (Suppl. 2c). Thus, we find that efficacy of the C1/C2 tagged-subunits is less relatable to the non-tagged subunits (which are used in neurons and likely more relevant to the disease).

   Despite the latter, we deem that we have specifically addressed this issue by measuring miniature EPSCs (mEPSCs) (see our reply to comment #4, Suppl. 7a, b). Briefly, even though the non-tagged G689C expresses at ~40% compared to other subunits (in oocytes and mammalian cells8), in neurons it engenders a robust (and highly significant) negative effect over synaptic currents (mEPSCs), as strong as the G689S-variant which expresses much more robustly (non-tagged G689S expresses to same extent as wt subunits). This demonstrates that the reduced efficacy the tagged subunits is less relatable to the non-tagged subunits and, importantly, it does not hinder the variants’ ability to incorporate within the synapse and affect function (i.e., exert a dominant negative effect). Here, we extend these observations towards the major postnatal channel subtype, namely tri-heteromers (2A/2B*), and therefore demonstrate that the robust dominant negative effect of G689C and G689S variants is likely due to their ability to incorporate within the predominant receptor subtype at the synapse (Suppl. 8).

   Reviewer #1 (Significance (Required)):

   This study emphasizes the complex pattern of effects that variants can have on glutamate receptor function and pharmacology, especially considering the context of receptor subunit composition. The authors have followed up their previous findings on the same mutants (Kellner et al, 2021, Elife), but used a trafficking control system here to characterize properties of mutated receptor complexes that are most likely to exist in neurons. The authors show that the defective currents mediated by NMDA receptors containing a loss-of-function GluN2B variant can be enhanced by neurosteroids (and in the case of GluN1/2B receptors, polyamines also). Development and approval of neurosteroids for the clinic would be required for the findings to translate to a therapy for patients. Readers should also be aware that neurosteroids act on other receptors too (e.g. GABA receptors), which could complicate the outcome. The expertise of the reviewer is in glutamate receptors and synaptic transmission.

   We agree with the reviewer’s comment pertaining to challenges in translating PS to the clinic. Indeed, we explicitly mentioned its inhibitory effect on GABAA receptors (see line #366-367 and reference 18), as well as note its potential negative effect over GluN2C/D-containing receptors (line #365 and reference 19). We further describe alternative neurosteroids and means to bypass the limitations of PS, for instance by use of 24(S)-hydroxycholesterol6,18 or synthetic analogues (SGE-201, SGE-301)6. Lastly, we also propose a novel therapeutic approach, for which we did not find any mentions in the literature with regard to GRINopathies, consisting of the use of the FDA-approved Efavirenz (anti-retroviral compound20) to promote activity of cytochrome P450 46A1 (CYP46A1) to increase amounts of 24-S in the brain (discussion, lines #370-383).

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   Reviewer #2 (Evidence, reproducibility and clarity (Required)):

   #1 The objective of this paper is to assess whether a single mutated subunit of GRIN can affect the function of various forms of NMDA receptors. In particular, this study investigates the functional consequences of a GRIN variant when assembled within tri-heteromers, containing 2 GluN1, 1 GluN2A and 1 GluN2B subunits, the major postnatal receptor type. For this purpose, the authors artificially forced the subunits to associate in predefined complexes, using chimeras of GRIN subunits fused to GABAb receptor retention control sites at the endoplasmic reticulum. This trick allows to control the stoichiometry of the channels at the membrane and thus to focus on the function of a single type of NMDA receptor.The take home message of the paper is that a single GluN2B‐variant, whether assembled with a GluN2B‐wt subunit to form mixed di‐heteromer or with a GluN2A‐wt‐subunit (tri‐heteromer), strongly impairs the receptor functioning, as reported by a decrease of the apparent glutamate affinity of the receptor.

   Altogether, this is a straightforward study of great interest for the GRIN community.

   We greatly appreciate the reviewer’s comment about the relevance of our work towards the GRIN-community.

   #2 However, the way the background and purpose of the study (title and abstract) are presented is a bit confusing for non-specialists and could be easily improved. Technical information, which is crucial to validate the conclusions drawn from data analysis, should be added to the article. Some additional experiments are suggested to consolidate the work. Finally, additional discussion points are strongly encouraged.

   We apologize for not making the paper more accessible to a broader readership. We did so for the sake of brevity. Nevertheless, we have re-written major parts of the manuscript to address this issue and retitled the report: “Rescuing Tri-Heteromeric NMDA Receptor Function: The potential of Pregnenolone-Sulfate in Loss-of-Function GRIN2B Mutations”.

   Specific comments

   Abstract / Title:

   #3 This work shows that a single GRIN variant impairs the function of various forms of NMDA receptors. Several sentences in the title and the abstract are confusing for a non-specialized audience. "Two extreme Loss‐of‐Function GRIN2B‐mutations are detrimental to triheteromeric NMDAR‐function, but rescued by pregnanolone‐sulfate." "Here, we have systematically examined how two de novo GRIN2B variants (G689C and G689S) affect the function of di‐ and tri‐heteromers." The number of variants tested is not of capital importance in the title, especially because one could believe that both are tested at the same time; similarly, when variants are named in the abstract, the fact that only 1 variant is studied at a time should be clarified (G689C OR G689S). Indeed, the problem is obvious to those familiar with GRIN disorders, but if this paper is to be published in a journal reaching a large audience rather than a specialized audience, the title of the paper should be modified. As noted in our reply to comment #1 of this reviewer, we apologize for not making the paper more accessible and have therefore changed the title and re-written major parts of the manuscript to address this issue. We would like to note that we appreciate the reviewer’s comment and intent to increase the readership of our manuscript.

   #4 "We find that the inclusion of a single GluN2B‐variant within mixed di‐ or tri‐heteromeric channels is sufficient to prompt a strong reduction in the receptors' glutamate affinity, but these reductions are not as drastic as in purely di‐heterometric receptors containing two copies of the variants. This observation is supported by the ability of a GluN2B‐selective potentiator (spermine) to potentiate mixed diheteromeric channels." Please, clarify the link between the two sentences. How do spermin potentiation of mixed diheteromeric channels supports the observation that the inclusion of a single GluN2B‐variant has less effect than the inclusion of two variants?

   Our intention was to highlight that mixed di-heteromeric channels (2B/2B*) are less “damaged” (this is the link) than purely di-heteromeric channels (2B*/2B*).Explicitly mixed di-heteromers show less reduction in glutamate potency AND are also spermine-responsive, whereas purely mutant di-heteromers (2B*/2B*) show reduced potency, BUT do not respond to spermine at all. We have rephrased the sentences in our current manuscript to be clearer:

   For instance: The positive responses of mixed di-heteromers, compared to the null effect over pure di-heteromers, is likely the result of the restored pH-sensitivity of mixed di-heteromers (Suppl. 3). This was surprising as the minimal and essential rules of engagement for potentiation by spermine are not well established, particularly in the case of tri-heteromers21,22 (see discussion, lines #341-353).

   Methods

   #5 All this study is based on the use of a unique ER‐retention technique to limit expression of a desired receptor‐population at the membrane of cells. According to the ER system retention of GABAb receptor, used in this study, while C1/C1-fused subunits are retained in the ER, C2/C2 reach the cell surface and the association of C1/C2 in the ER enables cell-surface targeting of the heterodimer. However, GB2 does not contain any retention signal and can reach the cell surface in the absence of GB1, as a functionally inactive homo-dimer (doi: 10.1042/BJ20041435). If there is an experimental trick that prevents the addressing of C2/C2 to the cell membrane, it should be specified and explained. This is critically important for understanding which receptor populations the data are derived from: receptors containing C1/C2 fused subunits only as stated by the authors, or C1/C2 and C2/C2 fused subunits?

   We base our experiments on two seminal reports—23,24—that have developed this unique method (which we also refer to in the text, lines# 112-116). Briefly, the method employs the binding motifs of GABAB1 (GB1) and GB2 subunits and ER-retention motifs (these are now better detailed in Methods section, line # 448). Previous reports explicitly demonstrate that C1/C1- OR C2/C2-containing receptors do not reach the plasma (or very minimally) and we have reproduced these data with our variants (C1/C1: Suppl. 1a-d; C2/C2: Fig. 1a-c).

   Figures #6 NMDA-receptor current amplitude should be normalized by the membrane expression of the receptors. A preliminary experiment should measure the effective cell surface expression of each of the subunits in the different transfection conditions.

   To address the effective cell surface expression, we employed Imax as a proxy for functional expression (e.g.,8,17). These are now shown in __Suppl. 2a, b __(and see our response to reviewer 1, comments #6 and 14). Expectedly, we find significantly reduced efficacy by the varinats compared to wt-receptors, and the purely mutant di-heteromeric receptors exhibit the weakest efficacy. We have also addressed this issue by measuring miniature EPSCs (mEPSCs) (see our reply to reviewer 1, comment #4,). We find the variants to abolish mEPSCNMDA frequency (Suppl. 7a, b). This shows that the variants’ reduced efficacy translates to elimination of synaptic activity (dominant negative effect) (also seen in Suppl. 8).

   #7 Fig.1a

   The scheme should include C2-C2 complexes and mention whether these complexes are expressed at the cell surface (see previous and following comments).

   As noted in our reply to comment #5 of this reviewer (above), C2/C2-containing receptors do not reach the plasma membrane (Fig. 1a-c). To avoid confusion, we have now added this scheme to the cartoon presented in Fig. 1a and have provided a more detailed description of the method and clones produced in the Methods section (line # 448).

   #8 Fig.1b and c

   Current from cells transfected with GluN2B‐wt‐C1 and GluN2B‐wt‐C2 should be compared to current expressed in cells expressing untagged receptor subunits: GluN2B‐wt Current from cells transfected with GluN2B‐wt‐C1 alone should be shown as well (although expected to be retained in the ER) (as performed for GluN2A‐wt‐C1 GluN2B‐wt‐C1 in suppl Fig. 1a)

   Current comparisons of oocytes expressing tagged GluN2B‐wt‐C1 and GluN2B‐wt‐C2 and non-tagged GluN2B‐wt are now demonstrated in Suppl. 2c. The results indicate that the “tags” (C1 and C2) affect the expression of the subunits. We have also added a sample trace of current from a cell expressing the GluN2B‐wt‐C1 alone (Fig. 1b).

   #9 How could you explain the null current from cells transfected with GluN2B‐wt‐C2 alone (Fig.1b middle, and 1c)? since GB2 does not contain any retention signal and can reach the cell surface in the absence of GB1, GluN2B‐wt‐C2 is supposed to reach the cell surface. This is a very important point to clarify (I am probably missing a technical detail) because if the sub-unit tagged with C2 does reach the cell surface, then all the results and conclusions drawn from the C1-C2 conditions are wrong and could be attributed to a mix of complexes containing either C1-C2 or C2-C2.

   We now realize that the reviewer was missing a crucial technical detail, namely how the clones are designed. Briefly, all clones have ER retention motifs and cannot reach plasma membrane unless they necessarily assemble as C1/C223,24. Also, please see our replies to comments #5, 7 to this reviewer (and Methods section, line # 448).

   My following comments are based on the assumption that only receptors containing C1-C2 tagged subunits reach the membrane (as assumed by the authors and suggested in Figure 1b middle), but explanations should absolutely be provided to convince the reader. Fig. 4a and 5a (see our above replies to comments #5, 7 and 9; and references 23,24).

   #10 Please, keep the current scale constant between all current illustrations within the same figure (4a and 5a). Indeed, not only the Spermin- or SP- induced potentiation is an important data (which is presently quantified on the histograms of fig. 4b and 5b) but also knowing whether the amount of current recorded in cells expressing one mutant subunit in presence of SP (for example GluN2A‐wt‐C1 GluN2B‐G689S‐C2) is comparable to the current recorded in wt receptor-expressing cells (GluN2A‐wt‐C1 GluN2B‐wt‐C2) in absence of SP would be an excellent added value for the paper. A special figure could quantify this rescue effect of SP, measuring and comparing the mean currents recorded in these conditions (one current illustration is not sufficient given variations between similar samples). By the way 5mM glutamate might be an excessive concentration. At 1mM, the expected synaptic concentration of glutamate following action potential, according to figures 3 and Suppl1 the response of the mutated receptor is much lower than that of the WT which is already almost maximal. In these conditions, SP-induced potentiation by a factor of two of GluN2A‐wt‐C1 GluN2B‐G689S‐C2 current could be equivalent to control currents recorded in GluN2A‐wt‐C1 GluN2B‐wt‐C2 cells.

   We have rescaled all current amplitudes in Figs. 4 and 5 to be identical in size for easier comparison.

   We have added all current amplitudes to try to examine the rescue effect of the two drugs in cell overexpressing a specific channel subtype, as requested (Suppl. 4). We find that; indeed, the potentiated currents of the mutant receptors reach (or even surpass) the basal Imax (i.e., current before potentiation) of the non-mutated receptors (Suppl. 4, dashed statistics bar).

   In neurons, we address this in two ways. First, we show that the total NMDA-current is reduced by expression of the variants, and this current is “normalized” by PS (Fig. 6a-c). Similar reductions in Imax (by the variants) are shown in __Suppl. 7e __(to provide more examples). Secondly, neurotransmission (i.e., 1 mM glutamate25,26) is not sufficient for activating mutant receptors, certainly not pre-di-heteromers (see Table1, EC50 and Suppl. 7a, b- no mEPSCs)27–29. Therefore, 5 mM was required. Together, these strongly suggest that PS may normalize the currents of different receptors that respond to PS (under physiological settings and not 1- or 5mM NMDA). As suggested by the reviewer, there are many subtypes, and some may be activated by ambient glutamate (as suggested by application of PS onto neurons without opening the receptors by NMDA; see Suppl. 7c, d).

   #11 Fig. 6

   Figure 6 is not convincing because cultured hippocampal neurons do express endogenous NMDA receptors. To what extent the recording currents are affected by endogenous, non-mutated GluN2B subunits? Western Blots showing an extinction of endogenous subunits expression when transfected tagged subunits are competitively expressed would be required.

   We have previously shown that the two variants incorporate very efficiently within synapses, causing a very robust elimination of synaptic currents (by measuring miniature NMDA-dependent EPSCs; minis) [see Fig. 8 in Kellner et al. eLIFE, 202127, and see review by Sabo et al.9 ). Change in minis’ frequency can be interpreted as either a presynaptic change or a change in synapse number, however we observed that AMPAR-mEPSC frequency was unaffected by these variants. These imply that synapse number and probability of release are unchanged by the variants. As the experiments are performed in wild-type neurons, (which express wild-type GluN2A and -2B), the dramatic effects we observed on minis suggests a dominant-negative effect of these disease-associated GluN2B variants. These are consistent with our observations that mutant subunits can co-assemble with wild-type GluN2B and/or GluN2A subunits. We have now reproduced this experiment (in fact, we employ this strategy prior each experiment to ensure expression of the variants) (Suppl. 7a, b). This thereby shows that there are no available wt-receptors at the synapse.

   As there are various pools of NMDARs at synaptic and extrasynaptic sites, we did not think that a western blot would sufficiently differentiate between the latter, and thereby would not provide insight about extinction of wt-receptors (which could be simply pushed to other sites compared to synapse). Moreover, the intracellular pool of receptors is much larger than the amount of NMDARs that can be detected at the membrane (e.g., 30,31), and therefore electrophysiology seemed to be a better means to monitor membrane receptors only:

   Thus, to examine the distribution of the variants between synaptic- and extrasynaptic loci, we employed a standard procedure consisting of the use of the activity-dependent blocker MK-801 (Methods). Briefly, neurons were persistently bathed in TTX during which they were probed for Imax using 100 mM NMDA (to refrain from activating other GluRs), followed by application of MK-801 for 10 minutes to exclusively blocks synaptic receptors (that open following action-potential independent miniature neurotransmission). This thereby spares all extrasynaptic receptors from being blocked by MK-801, which are subsequently revealed by a second application of 100 mM NMDA (Suppl. 8a, inset)12. In neurons overexpressing the GluN2B-wt subunit, we obtained an extrasynaptic fraction of 38%, highly consistent with previous reports12,13. Overexpression of the variants, however, yielded a significantly and higher fraction (~50%) of remaining current (Suppl. 8b, c), but instead of reflecting a larger pool of extrasynaptic receptors, the experiment represents quite the inverse when involving LoF variants. Firstly, 100 mM NMDA does not saturate variant receptors (whether pure, mixed di- or tri-heteromers, see Table 1). Secondly, normal neurotransmission does not open synaptic receptors containing mutant GluN2B-subunits, attested by the complete absence of mEPSCs (see Suppl. 7). Thus, during the 10 minutes exposure to MK-801, only wt receptors are blocked by spontaneous synaptic activity, and thus the second bout of 100 mM NMDA solely exposes the remaining wt-receptors at the extrasynapse. Thus, the observed increase in the fraction of extrasynaptic receptors, in neurons overexpressing the variants, implies that the number of wt-receptors is necessarily decreased from the synapse and increases at the extrasynapse, most likely due to the incorporation of the variants at the synapse. This increase cannot be explained by an overall increase in membrane expression of wt-receptors in neurons overexpressing the variants, as these cells show, yet again, a strong reduction in Imax as seen above (see Fig. 6c and Suppl. 7e) (lLines #270-291). These thereby suggest that purely wt-receptors are not necessarily eliminated from the membrane (extinct), rather pushed outside of the synapse.

   #12 Fig.6b “PE-S” on the graph should be replaced by “PS”

   Typo corrected.

   Discussion #13 The authors are surprised by the fact (Fig.2) that 1 variant reduces the apparent glutamate affinity of the receptor, but not as much as 2 variants, despite the fact that "NMDARs opening requires all four subunits to be liganded (i.e., occupied by a ligand) which implies that the least affine subunit should have dominated the final affinity of the receptor". I agree that the difference is noticeable, however the glutamate affinity for receptors containing 1 variant is much closer to that of receptors containing 2 variants than that of wild-type receptors. Hence, the results obtained do not seem so surprising and could result, as rightly explained by the authors from a possible cooperativity between the subunits.

   We agree with the reviewer that glutamate potency of receptors containing 1 variant subunit is much closer to that of receptors containing 2 variant subunits. However, we maintain our surprise because we expected it to equal (not just close) to the potency of the least affine subunit (the limiting factor). This is based on the notion that all four subunits need to be liganded for channel opening4,32–34. We gently raise the possibility of potential cooperativity (Table 1, see Hill-coefficient and 33,35,36), as well as mention that this may also stem from the variants’ lower proton sensitivity (Suppl. 3), which has also been shown to promote motions of the ATD (amino terminal domain) and increase open probability (positive cooperativity)36. Nevertheless, we are very careful with interpreting the Hill coefficient , as we limited exposure of oocytes to 10 mM glutamate due to artifacts arising past this concentration (see Kellner et al.8: Fig. 2—figure supplement 1). This description is now mentioned in page lines #149, 318, 319. Thus, even the slightest underestimation of the maximal reposnse would surely affect the slope.

   #14 On the other hand, the data in Figure 6 are much more difficult to interpret and reconcile with the nature of the expressed receptor subunits (which this time is not controlled) nor their association within the same receptor. However, this aspect, which is essential to the understanding of the consequences of 1 variant on neuronal signalling, is not discussed: Whatever the stoichiometry of the complexes in the heterozygous disease, the mutated and wild type GluN2B subunits coexist in the same cell: Either within the same di-heteromeric complexes GluN2B-wt + GluN2B-mutant, or in separate complexes but nevertheless expressed in the same cell, in di heteromeric (GluN2B-wt + GluN2B-wt and GluN2B-mutant + GluN2B-mutant); or tri-heteromeric (GluN2A-wt + GluN2B-wt and GluN2A-wt + GluN2B-mutant) complexes. Assuming that half of the complexes remain wild-type, e.g. (GluN2A-wt + GluN2B-wt and GluN2A-wt + GluN2B-mutant) we would expect (Fig. 6) a small decrease in NMDA current (carried only by the half that expresses the mutated subunit, and whose function is not zero but only decreased by about 20% in response to 5 mM Glutamate, Fig. 3b). The same reasoning applies to the di-heteromeric conditions (GluN2B-wt + GluN2B-wt; GluN2B-mutant + GluN2B-mutant), here again the decrease observed Fig. 6b is difficult to reconcile with the responses measured Fig. 2b.

   In other words, how to explain a 50% decrease of the currents, instead of the 10% expected by the previous reasoning. In this experiment we do not know which subunits are expressed, their proportions, nor how they are associated in functional complexes, which makes the interpretation of the data impossible. The only explanation, far-fetched, for 50 % decrease would be that the complexes were to contain all (or the vast majority) 1 wild-type subunits associated with 1 variant, then a homogeneous 50% reduction in current could be expected. But this extreme condition could only be possible in the case of di-heteromers, which is unlikely the case in Fig.6 as GluN2A currents are measured in presence of Ifenprodil. To conclude

   1. the comparison of the currents in transfected and non-transfected neurons does not make sense in figure 6b which is not convincing because we do not know the nature of the currents actually measured. A comparison in controlled condition would make more sense (as I suggested in the criticism of figures 4, 5).

   1. The reality of the combinations of expression and association between subunits within different complexes expressed in the same cell must be considered and taken into account in the interpretation of the data. Undoubtedly, the means of restoring the NMDA current will be different depending on the presence of mutated subunits in all functional channels or not.

   Indeed, neurons express a variety of different combinations of channel stoichiometry, including following transfection with the variants. We do find find that the effect on whole-cell current is indeed ~50% (Fig. 6b, c), thereby safe to assume that 50% remain “wt”, but we do not know how they distribute between synaptic and extrasynaptic loci. Our results however argue against 50% remaining receptors at the synapse. First, mEPSCNMDA disappear (Suppl. 7a, b and see reply to comment #11 of this reviewer), but wt-receptors are still at the membrane, and they seem to be moving out of synapse (Suppl. 8). Thus, we can only state with higher certainty that the variant subunits are very efficient in incorporating within mixed or pure receptors, especially at the synapse.

     We also consider that the reduction in the whole-cell current observed in __Fig. 6b, c__ is not due to the remaining 50% GluN2B-*wt*-containing receptors, rather likely due to other variants, notably GluN2A, which are more prominent at postnatal stages37, such as in our case. In support, we see a large remaining current after saturating ifenprodil application (__Suppl. 7 e, f__)38. Thus, the variants incorporate within all 2A/2B membrane receptors, at the synapse and outside it (i.e., extrasynaptic) (see __Suppl. 8, c__).
   

   **Referees cross-commenting**

   The referees' comments are highly relevant. In particular, referee 3's comment 1 seems very interesting because it may help to better understand the discrepancy in the results observed in neurons, i.e. a 50% decrease in the currents induced by the expression of the mutant and wild type subunits in the same cells, whereas theoretically one would expect a 10% decrease of this current (cf. referee 2's 2nd comment in the discussion section). This comment 1 of referee 3 indeed stresses the fact that the control (non-transfected neurons) to which the heterozygous condition is compared is not the correct control, which should rather be neurons transfected with wild type receptor subunits. More generally, this comment underlines the importance of monitoring the effective membrane expression of the different subunits in each of the experimental conditions in order to be able to compare conditions and draw conclusions.

   We initially did not perform this control as the literature paints a clear picture whereby expression of the GluN2B-subunit (without adding excess of the GluN1 subunit) does not instigate a robust increase in surface expression of NMDARs (and thus current remains the ~same) 4,39–43, and see our reply to comment #14 (above), and reviewer 3 comment 1 (below). Nevertheless, we have now performed this test by overexpressing GluN2B-wt. In support of previous reports, we do not find any statistical difference in current size between non-transfected neurons and neurons solely overexpressing the GluN2B-wt subunit (Fig. 6a, b). Furthermore, application of PS onto naïve or GluN2Bwt expressing neurons yields identical currents (Imax) and potentiation (Fig. 6c, d). These argue that we did not obtain “overexpression”.

   We suggest that the 50% reduction in current size between neurons expressing the mutant and wt expressing neurons stems from the integration of mutant subunits and their dominant negative effect. Evidence for this incorporation is provided by the very strong reduction in synaptic currents (suppl 7a, b), and the supposedly higher abundance of wt-containing receptors in extrasynaptic regions (see reviewer 1 comment 4 and suppl 8). This is

   Reviewer #2 (Significance required):

   The novelty of the study, is to evaluate the consequences of a single mutated subunit within NMDA receptors affected by GRIN variant, to mimic the heterozygous condition of GRIN encephalopathies, this is of potential value for the field and the interest could also be extended to other genetic diseases (at least the experimental way to study the functioning of only one desired stoichiometric configuration). The strength of this paper is precisely to isolate technically and to study the functioning of a desired stoichiometric configuration only. The main limitation of the paper is the interpretation that the authors make of their data in a physio-pathological context. This work could be of interest for general audience, providing the title and summary are slightly modified. My area of expertise could not be closer to the topic of the article: Glutamate receptors; GRIN; molecular tinkering, cell culture, electrophysiology, receptor stoichiometry...

   We thank the reviewer for noting the value in our work and its potential contribution and interest to the field and other diseases. Per reviewer’s suggestion, we have modified the title and text to suit a larger audience.

   Reviewer #3 (Evidence, reproducibility and clarity (Required):

   This paper is a follow up of an earlier paper published by the group (Kellner et al., eLife 2021), which aimed at characterizing the functional properties of two de novo GluN2B mutations in patients suffering from severe pediatric diseases, GluN2B-G689C and -G689S. NMDA receptors (NMDARs) are tetramers composed of two GluN1 and two GluN2 subunits. A single receptor can incorporate either two identical GluN2 subunits (di-heteromers) or two different GluN2 subunits (tri-heteromers), leading to a large diversity of NMDAR subtypes. The main NMDAR subtypes in the adult forebrain are GluN1/GluN2A and GluN1/GluN2B di-heteromers, as well as GluN1/GluN2A/GluN2B tri-heteromers. While the exact proportions of these three subtypes are still contentious, there are evidence that in the adult N1/2A/2B tri-heteromers form the major population of synaptic NMDARs in the adult forebrain. In addition, patients bearing pathogenic mutations are often heterozygous for the mutation, giving rise to mixed NMDARs incorporating one mutated and one intact GluN2 subunit. In their previous paper, Kellner et al. had shown that purely di-heteromeric GluN1/GluN2B-G689C and -G689S mutants display a drastic (> 1,000-fold) decrease of glutamate sensitivity and a decrease of surface expression. In the current paper, the authors characterize the effects of the -G689C and -G689S mutations on N1/2A/2B tri-heteromeric receptors, as well as on mixed di-heteromeric GluN1/GluN2B receptors containing one copy of the wild-type GluN2B subunit and one copy of the mutated GluN2B subunit. They show that one copy of the mutant subunit, either within mixed diheteromeric or tri-heteromeric receptors, is sufficient to decrease drastically glutamate sensitivity, although the shift in glutamate EC50 is not as strong as in pure di-heteromeric receptors (≈ 500-fold). They furthermore explore strategies to counteract the hypofunction induced by these mutations by testing the effect of positive allosteric modulators (PAMs). They show that spermine, a GluN2B-specific PAM, can potentiate the activity of mixed diheteromeric N1/2B but not N1/2A/2B tri-heteromers. However pregnenolone sulfate (a 2A/2B-specific PAM) can potentiate both the activity of mixed diheteromeric and tri-heteromeric NMDAR populations, either in oocytes or cultured neurons.I have very few major comments to make. The experiments are straightforward and the adequate controls have been made. Here are my two only major comments:

   We thank the reviewer for the very detailed overview of our work and for appreciating our controls and methods.

   #1 About the experiment on cultured neurons. The authors compare the currents of cultured neurons transfected with GluN2B-G689C and -G689S to non transfected neurons. The adequate control is rather neurons transfected with the wild-type GluN2B subunit to even out any phenomenon linked to transfection of the neuron. Given the overexpression that can occur after transfection, the effect of the mutations on the size of NMDAR currents might be even stronger than what the authors show. However in that case PS might not completely rescue mutant NMDAR currents to wild-type levels.

   We initially did not perform this control as the literature paints a clear picture whereby expression of the GluN2B-subunit (without adding excess of the GluN1 subunit) does not instigate a robust increase in surface expression of NMDARs (and thus current remains the ~same) 4,39–43, and see our reply to comment #14 (above), and reviewer 3 comment 1 (below). Nevertheless, we have now performed this test by overexpressing GluN2B-wt. In support of previous reports, we do not find any statistical difference in current size between non-transfected neurons and neurons solely overexpressing the GluN2B-wt subunit (Fig. 6a, b). Furthermore, application of PS onto naïve or GluN2Bwt expressing neurons yields identical currents (Imax) and potentiation (Fig. 6c, d). These argue that we did not obtain “overexpression”. Thus, our results and interpretations hold true, and are therefore not underestimation of the effects of PS in neurons.

   #2 How come high concentrations of glutamate (>100µM) produce additional current on wt GluN1/GluN2B (with retention signals) compared to 100 µM glutamate, which is supposed to be saturating? It does not seem to stem from an osmotic effect since 10 mM glutamate does not produce any current on uninjected oocytes. Knowing that this "artefactual" effect might also occur in the mutant receptors, how do you take this effect into account when calculating the glutamate EC50s of the mutants? Given the drastic shift in EC50 produced by the mutant, taking into account this artefact is not going to change the conclusion, but the actual EC50s will be affected.

   GluN1/GluN2B-wt receptors (with or without retention signals) are indeed saturated at 100 mM glutamate. However, excessively large concentrations of glutamate (>100 mM) may yield artefacts even in non-injected oocytes (in 10 mM, this occurs in ~20% of the cells, see Kellner et al 20218—Fig. 2 and Suppl. 1c, d) as well as in GluN2B-wt injected oocytes (supplementary Table 1 in 44). This is not due to osmolarity, as rightly mentioned by the reviewer (and below), rather possibly by endogenous glutamate receptors and transporters that do not readily contribute to current amplitude (these are extremely small currents), but can cause deterioration of the cell (and enhance ‘leak’) when activated for prolonged times by very large concentrations (e.g.,45). In fact, we explicitly report these to highlight potential artefacts, as these are often overlooked in the field. Regardless, most reports do no go past 100 mM glutamate, not even when describing GRIN2 mutations since most mutations do not cause such drastic shifts in potency as we observed (to the best of our knowledge only one report describes such an extreme LoF mutation for a GluN2A variant46). Of note, these effects are not seen when glycine is applied at high concentrations (supporting lack of effect by osmolarity)47. Thus, we refrained from testing concentrations past 10 mM, aware that it may yield a slight underestimation of glutamate potency (and perhaps the reason for the larger Hill coefficient, nH; see our reply to reviewer #1, comment #5). Importantly, despite the potential underestimation of the EC50, it does not change our conclusions as all groups are measured side-by-side (thus, the same underestimation equally applies to all other groups as well). We now mention this more in detail in the methods under the section – “Two Electrode Voltage Clamp recordings in Xenopus Laevis oocytes”.

   Minor comments:

   #3 In the first paragraph of the "Results" section, when describing the design of the constructs used to force a heteromeric stoichiometry in recombinant systems, the authors do as if they had designed the constructs themselves "Briefly, we tagged...are retained in the ER (Fig. 1a)". Please rewrite this paragraph to show that you used constructs that had been previously designed by another group (Hansen et al., 2014).

   We apologize. We did not mean to express that we have developed the method and indeed refer readers to the seminal works of those who did (Stroebel et al., 2014 and Hansen et al. 2014, lines #109-116). We did not go into details for the sake of brevity. We have rewritten this part to give proper acknowledgement to the method’s developers (also see Methods, line# 448).

   #4 I do not see any evidence of "positive cooperativity" between subunits in ref. 32. Ref. 32, to the best of my knowledge, states that in N1/2A/2B tri-heteromers, the 2A subunit sets the biophysical properties of the tri-heteromer. But there is no account of mixed di-heteromers. In addition, the cooperativity between the glutamate and glycine binding sites is negative.

   The reviewer is correct, and we apologize for the mis-citation. Indeed, the cooperativity between glutamate- and glycine-binding is typically reported as negative48,49, and our intention was to highlight the strong cooperativity (whether positive or negative) observed between NMDAR-subunits and meant to cite the works of: 33,35,50 (lines . We have now rephrased the sentence: The divergence from this scenario suggests that the slight amelioration in potency could stem from positive cooperativity between the subunits50 (but see Hill coefficients in Table 1). Indeed, mixed receptors show restored proton sensitivity (Suppl. 3), which has been suggested to be coupled to other receptor features, notably increase in open probability.

   #5 Interpretation of spermine action within the Results section: it is striking indeed to observe that the mutations in the context of a mixed di-heteromer still allow spermine potentiation, while they abolish this potentiation in pure di-heteromers. As rightly said in the discussion, the regain of spermine potentiation in the mixed compared to the pure diheteromers is likely due to a more favorable transduction of spermine signaling to the pore, likely via a higher pH sensitivity of mixed di-heteromers compared to di-heteromers. I would thus avoid the terms of "one single intact interface" for the mixed di-heteromer, since both spermine binding sites are likely intact in this NMDAR configuration. How is pH sensitivity affected in the mixed di-heteromers?

   We have performed a detailed pH dose-responses for the various channel types (Suppl 3). We find that GluN2B mixed di-heteromers exhibit similar IC­50 as pure GluN2B-wt di-heteromers, thus explaining their ability to undergo potentiation by spermine via alleviation of proton inhibition. We therefore further suggest that mixed di-heteromers’ have higher pH-sensitivity compared to pure mutant di-heteromers and this mat also contributes to their higher spermine sensitivity. Lastly, we observed that all GluN2A-wt-containing tri-heteromeric receptors were non-responsive to spermine (Fig. 4a). In fact, under our experimental conditions tri-heteromers underwent slight inhibition by spermine, regardless the identity of the GluN2B subunit (whether wt or variant) (Fig. 4b). Thus, as the tri-heteromers used here exhibit identical pH-sensitivity as 2B-di-heteromers, the only diverging aspect is the missing interface between the GluN1a and GluN2B subunits, demonstrating that potentiation by spermine requires at least one GluN2B-subunit with an intact proton sensitivity, and mandates two intact interfaces between GluN1-wt and GluN2B-wt subunits (Table 1)21.

   #6 In the methods section, the oocyte recording solution (likely Ringer and not Barth) does not contain any potassium. This is probably a typo. Could you correct the composition of your Ringer?

   Corrected. We record NMDARs currents by use of a Barth solution containing (in mM): 100 NaCl, 0.3 BaCl2, 5 HEPES, pH 7.3 (adjusted with KOH, at ~2.5 mM) (as in 4,51).

   #7 There are several typos, especially in the Discussion.

   We have corrected the typos throughout the publication.

   **Referees cross-commenting**

   I overall agree with the comments of reviewers 1 and 2. In particular, I agree that it is pointless to compare the absolute currents of non transfected neurons vs mutant-transfected neurons without an idea of receptor cell-surface expression.

   We have performed this experiment (Fig. 6) and please see our reply to this reviewer’s comment #1.

   I would like however to give some precisions about some comments of Reviewer 2. About the ER retention technique to express tri-heteromers: I didn't know that the C2 signal could be addressed to the membrane on its own. The lack of leak current stemming from C1-C1 or C2-C2 combinations has been demonstrated in the paper establishing the technique (Hansen et al, 2014), as well as in another paper that developed an analog technique based on GABAB retention signals (Stroebel et al., J Neurosci 2014). So it is fair to consider that the authors were not surprised by the lack of current when co-expressing two GluN2B subunits carrying the C2 signal.

   We thank you for this addition and support for our observations.

   About the comparison about absolute currents wt vs mutants, +/- spermine (Fig. 4a and 5a). I agree with reviewer 2 that being able to compare absolute currents of wt without spermine to mutant + spermine would be very interesting to see if spermine can actually rescue mutant hypofunction. However, to the defense of the authors, comparing absolute current values of recordings from Xenopus oocytes is meaningless. Indeed the variability of currents for the same construct and same day of experiment is too high (there can be up to a ten-fold difference between the lowest and the highest current of oocytes expressing the same construct the same experimental day). A way to investigate this aspect would be to estimate the open probability of the different constructs with or without spermine via the inhibition kinetics of an open channel blocker (e.g. MK801) and measure surface expression by Western blot, but I am not sure these experiments are worth it for the spermine experiment.

   We agree with this reviewer about current size. It is quite variable among cells and would therefore introduce an additional variable and variability: the expression of these modified (C1/C2-tagged) subunits is dually affected by the mutation itself (Kellner et al. 2021) and by the introduction of the tagging (which really hampers there trafficking to membrane, Suppl. 2c); with unknown contribution of each variable. We thereby do not think these provide an added value to our conclusions, yet to grant reviewers’ no 2 request we have added __Suppl. 4 __which shows the rescue effect of the different drugs.

   Reviewer #3 (Significance (Required)):

   This paper is not of high significance since most of the characterization of the 2B-G689C and -G689S de novo mutants found in patients has already been published (Kellner et al., eLife 2021). However, this paper is worth publishing since it brings new data on the effect of the mutations on tri-heteromeric and mixed di-heteromeric NMDAR populations, which are likely the most abundant NMDAR populations in the patient's brain at adult stage. Tri-heteromeric and mixed NMDAR populations have often been overlooked when studying pathogenic NMDAR mutations due to the difficulty to express them specifically in recombinant systems. This paper (in addition to other papers in the field, see for instance Elmasri et al., Brain Sci. 2022; Li et al., Hum. Mutat. 2019) shows that the effect of the mutations on the receptor biophysical and pharmacological properties (but also on trafficking) differ whether the receptor contains one or two copies of the mutant subunit. This paper is of interest to scientists interested in NMDA receptor structure-function and pharmacology, as well as clinicians interested in GRINopathies (pathologies linked to NMDAR mutations).

   I, the reviewer, am an expert in NMDAR structure-function and pharmacology. I believe I have sufficient expertise to evaluate the entirety of the paper.

   We thank the reviewer for appreciating and acknowledging the merits of our work for publication.

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   46. Swanger, S. A. et al. Mechanistic Insight into NMDA Receptor Dysregulation by Rare Variants in the GluN2A and GluN2B Agonist Binding Domains. Am. J. Hum. Genet. 99, 1261–1280 (2016).
   47. Madry, C., Betz, H., Geiger, J. R. P. & Laube, B. Supralinear potentiation of NR1/NR3A excitatory glycine receptors by Zn2+ and NR1 antagonist. Proc. Natl. Acad. Sci. 105, 12563–12568 (2008).
   48. Regalado, M. P., Villarroel, A. & Lerma, J. Intersubunit Cooperativity in the NMDA Receptor. Neuron 32, 1085–1096 (2001).
   49. Durham, R. J. et al. Conformational spread and dynamics in allostery of NMDA receptors. Proc. Natl. Acad. Sci. 117, 3839–3847 (2020).
   50. Vyklicky, V., Stanley, C., Habrian, C. & Isacoff, E. Y. Conformational rearrangement of the NMDA receptor amino-terminal domain during activation and allosteric modulation. Nat. Commun. 12, 2694 (2021).
   51. Kellner, S. et al. Two de novo GluN2B mutations affect multiple NMDAR-functions and instigate severe pediatric encephalopathy. eLife 10, e67555 (2021).

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   Referee #3

   Evidence, reproducibility and clarity

   This paper is a follow up of an earlier paper published by the group (Kellner et al., eLife 2021), which aimed at characterizing the functional properties of two de novo GluN2B mutations in patients suffering from severe pediatric diseases, GluN2B-G689C and -G689S. NMDA receptors (NMDARs) are tetramers composed of two GluN1 and two GluN2 subunits. A single receptor can incorporate either two identical GluN2 subunits (di-heteromers) or two different GluN2 subunits (tri-heteromers), leading to a large diversity of NMDAR subtypes. The main NMDAR subtypes in the adult forebrain are GluN1/GluN2A and GluN1/GluN2B di-heteromers, as well as …

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   Referee #3

   Evidence, reproducibility and clarity

   This paper is a follow up of an earlier paper published by the group (Kellner et al., eLife 2021), which aimed at characterizing the functional properties of two de novo GluN2B mutations in patients suffering from severe pediatric diseases, GluN2B-G689C and -G689S. NMDA receptors (NMDARs) are tetramers composed of two GluN1 and two GluN2 subunits. A single receptor can incorporate either two identical GluN2 subunits (di-heteromers) or two different GluN2 subunits (tri-heteromers), leading to a large diversity of NMDAR subtypes. The main NMDAR subtypes in the adult forebrain are GluN1/GluN2A and GluN1/GluN2B di-heteromers, as well as GluN1/GluN2A/GluN2B tri-heteromers. While the exact proportions of these three subtypes are still contentious, there are evidence that in the adult N1/2A/2B tri-heteromers form the major population of synaptic NMDARs in the adult forebrain. In addition, patients bearing pathogenic mutations are often heterozygous for the mutation, giving rise to mixed NMDARs incorporating one mutated and one intact GluN2 subunit. In their previous paper, Kellner et al. had shown that purely di-heteromeric GluN1/GluN2B-G689C and -G689S mutants display a drastic (> 1,000-fold) decrease of glutamate sensitivity and a decrease of surface expression. In the current paper, the authors characterize the effects of the -G689C and -G689S mutations on N1/2A/2B tri-heteromeric receptors, as well as on mixed di-heteromeric GluN1/GluN2B receptors containing one copy of the wild-type GluN2B subunit and one copy of the mutated GluN2B subunit. They show that one copy of the mutant subunit, either within mixed diheteromeric or tri-heteromeric receptors, is sufficient to decrease drastically glutamate sensitivity, although the shift in glutamate EC50 is not as strong as in pure di-heteromeric receptors (≈ 500-fold). They furthermore explore strategies to counteract the hypofunction induced by these mutations by testing the effect of positive allosteric modulators (PAMs). They show that spermine, a GluN2B-specific PAM, can potentiate the activity of mixed diheteromeric N1/2B but not N1/2A/2B tri-heteromers. However pregnenolone sulfate (a 2A/2B-specific PAM) can potentiate both the activity of mixed diheteromeric and tri-heteromeric NMDAR populations, either in oocytes or cultured neurons.

   I have very few major comments to make. The experiments are straightforward and the adequate controls have been made. Here are my two only major comments:

   1. About the experiment on cultured neurons. The authors compare the currents of cultured neurons transfected with GluN2B-G689C and -G689S to non transfected neurons. The adequate control is rather neurons transfected with the wild-type GluN2B subunit to even out any phenomenon linked to transfection of the neuron. Given the overexpression that can occur after transfection, the effect of the mutations on the size of NMDAR currents might be even stronger than what the authors show. However in that case PS might not completely rescue mutant NMDAR currents to wild-type levels.
   2. How come high concentrations of glutamate (>100µM) produce additional current on wt GluN1/GluN2B (with retention signals) compared to 100 µM glutamate, which is supposed to be saturating? It does not seem to stem from an osmotic effect since 10 mM glutamate does not produce any current on uninjected oocytes. Knowing that this "artefactual" effect might also occur in the mutant receptors, how do you take this effect into account when calculating the glutamate EC50s of the mutants? Given the drastic shift in EC50 produced by the mutant, taking into account this artefact is not going to change the conclusion, but the actual EC50s will be affected.

   Minor comments:

   1. In the first paragraph of the "Results" section, when describing the design of the constructs used to force a heteromeric stoichiometry in recombinant systems, the authors do as if they had designed the constructs themselves "Briefly, we tagged...are retained in the ER (Fig. 1a)". Please rewrite this paragraph to show that you used constructs that had been previously designed by another group (Hansen et al., 2014).
   2. I do not see any evidence of "positive cooperativity" between subunits in ref. 32. Ref. 32, to the best of my knowledge, states that in N1/2A/2B tri-heteromers, the 2A subunit sets the biophysical properties of the tri-heteromer. But there is no account of mixed di-heteromers. In addition, the cooperativity between the glutamate and glycine binding sites is negative.
   3. Interpretation of spermine action within the Results section: it is striking indeed to observe that the mutations in the context of a mixed di-heteromer still allow spermine potentiation, while they abolish this potentiation in pure di-heteromers. As rightly said in the discussion, the regain of spermine potentiation in the mixed compared to the pure diheteromers is likely due to a more favorable transduction of spermine signaling to the pore, likely via a higher pH sensitivity of mixed di-heteromers compared to di-heteromers. I would thus avoid the terms of "one single intact interface" for the mixed di-heteromer, since both spermine binding sites are likely intact in this NMDAR configuration. How is pH sensitivity affected in the mixed di-heteromers?
   4. In the methods section, the oocyte recording solution (likely Ringer and not Barth) does not contain any potassium. This is probably a typo. Could you correct the composition of your Ringer?
   5. There are several typos, especially in the Discussion.

   Referees cross-commenting

   I overall agree with the comments of reviewers 1 and 2. In particular, I agree that it is pointless to compare the absolute currents of non transfected neurons vs mutant-transfected neurons without an idea of receptor cell-surface expression.

   I would like however to give some precisions about some comments of Reviewer 2. About the ER retention technique to express tri-heteromers: I didn't know that the C2 signal could be addressed to the membrane on its own. The lack of leak current stemming from C1-C1 or C2-C2 combinations has been demonstrated in the paper establishing the technique (Hansen et al, 2014), as well as in another paper that developed an analog technique based on GABAB retention signals (Stroebel et al., J Neurosci 2014). So it is fair to consider that the authors were not surprised by the lack of current when co-expressing two GluN2B subunits carrying the C2 signal. About the comparison about absolute currents wt vs mutants, +/- spermine (Fig. 4a and 5a). I agree with reviewer 2 that being able to compare absolute currents of wt without spermine to mutant + spermine would be very interesting to see if spermine can actually rescue mutant hypofunction. However, to the defense of the authors, comparing absolute current values of recordings from Xenopus oocytes is meaningless. Indeed the variability of currents for the same construct and same day of experiment is too high (there can be up to a ten-fold difference between the lowest and the highest current of oocytes expressing the same construct the same experimental day). A way to investigate this aspect would be to estimate the open probability of the different constructs with or without spermine via the inhibition kinetics of an open channel blocker (e.g. MK801) and measure surface expression by Western blot, but I am not sure these experiments are worth it for the spermine experiment.

   Significance

   This paper is not of high significance since most of the characterization of the 2B-G689C and -G689S de novo mutants found in patients has already been published (Kellner et al., eLife 2021). However, this paper is worth publishing since it brings new data on the effect of the mutations on tri-heteromeric and mixed di-heteromeric NMDAR populations, which are likely the most abundant NMDAR populations in the patient's brain at adult stage. Tri-heteromeric and mixed NMDAR populations have often been overlooked when studying pathogenic NMDAR mutations due to the difficulty to express them specifically in recombinant systems. This paper (in addition to other papers in the field, see for instance Elmasri et al., Brain Sci. 2022; Li et al., Hum. Mutat. 2019) shows that the effect of the mutations on the receptor biophysical and pharmacological properties (but also on trafficking) differ whether the receptor contains one or two copies of the mutant subunit. This paper is of interest to scientists interested in NMDA receptor structure-function and pharmacology, as well as clinicians interested in GRINopathies (pathologies linked to NMDAR mutations). I, the reviewer, am an expert in NMDAR structure-function and pharmacology. I believe I have sufficient expertise to evaluate the entirety of the paper.

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   Referee #2

   Evidence, reproducibility and clarity

   The objective of this paper is to assess whether a single mutated subunit of GRIN can affect the function of various forms of NMDA receptors. In particular, this study investigates the functional consequences of a GRIN variant when assembled within tri-heteromers, containing 2 GluN1, 1 GluN2A and 1 GluN2B subunits, the major postnatal receptor type. For this purpose, the authors artificially forced the subunits to associate in predefined complexes, using chimeras of GRIN subunits fused to GABAb receptor retention control sites at the endoplasmic reticulum. This trick allows to control the stoichiometry of the channels at the …

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   Referee #2

   Evidence, reproducibility and clarity

   The objective of this paper is to assess whether a single mutated subunit of GRIN can affect the function of various forms of NMDA receptors. In particular, this study investigates the functional consequences of a GRIN variant when assembled within tri-heteromers, containing 2 GluN1, 1 GluN2A and 1 GluN2B subunits, the major postnatal receptor type. For this purpose, the authors artificially forced the subunits to associate in predefined complexes, using chimeras of GRIN subunits fused to GABAb receptor retention control sites at the endoplasmic reticulum. This trick allows to control the stoichiometry of the channels at the membrane and thus to focus on the function of a single type of NMDA receptor. The take home message of the paper is that a single GluN2B‐variant, whether assembled with a GluN2B‐wt subunit to form mixed di‐heteromer or with a GluN2A‐wt‐subunit (tri‐heteromer), strongly impairs the receptor functioning, as reported by a decrease of the apparent glutamate affinity of the receptor.

   Altogether, this is a straightforward study of great interest for the GRIN community. However, the way the background and purpose of the study (title and abstract) are presented is a bit confusing for non-specialists and could be easily improved. Technical information, which is crucial to validate the conclusions drawn from data analysis, should be added to the article. Some additional experiments are suggested to consolidate the work. Finally, additional discussion points are strongly encouraged.

   Specific comments

   Abstract / Title:

   This work shows that a single GRIN variant impairs the function of various forms of NMDA receptors. Several sentences in the title and the abstract are confusing for a non-specialized audience. "Two extreme Loss‐of‐Function GRIN2B‐mutations are detrimental to triheteromeric NMDAR‐function, but rescued by pregnanolone‐sulfate." "Here, we have systematically examined how two de novo GRIN2B variants (G689C and G689S) affect the function of di‐ and tri‐heteromers." The number of variants tested is not of capital importance in the title, especially because one could believe that both are tested at the same time; similarly, when variants are named in the abstract, the fact that only 1 variant is studied at a time should be clarified (G689C OR G689S). Indeed, the problem is obvious to those familiar with GRIN disorders, but if this paper is to be published in a journal reaching a large audience rather than a specialized audience, the title of the paper should be modified.

   Introduction:

   "We find that the inclusion of a single GluN2B‐variant within mixed di‐ or tri‐heteromeric channels is sufficient to prompt a strong reduction in the receptors' glutamate affinity, but these reductions are not as drastic as in purely di‐heterometric receptors containing two copies of the variants. This observation is supported by the ability of a GluN2B‐selective potentiator (spermine) to potentiate mixed diheteromeric channels." Please, clarify the link between the two sentences. How do spermin potentiation of mixed diheteromeric channels supports the observation that the inclusion of a single GluN2B‐variant has less effect than the inclusion of two variants?

   Methods

   All this study is based on the use of a unique ER‐retention technique to limit expression of a desired receptor‐population at the membrane of cells. According to the ER system retention of GABAb receptor, used in this study, while C1/C1-fused subunits are retained in the ER, C2/C2 reach the cell surface and the association of C1/C2 in the ER enables cell-surface targeting of the heterodimer. However, GB2 does not contain any retention signal and can reach the cell surface in the absence of GB1, as a functionally inactive homo-dimer (doi: 10.1042/BJ20041435). If there is an experimental trick that prevents the addressing of C2/C2 to the cell membrane, it should be specified and explained. This is critically important for understanding which receptor populations the data are derived from: receptors containing C1/C2 fused subunits only as stated by the authors, or C1/C2 and C2/C2 fused subunits?

   Figures

   NMDA-receptor current amplitude should be normalized by the membrane expression of the receptors. A preliminary experiment should measure the effective cell surface expression of each of the subunits in the different transfection conditions.

   Fig.1a

   • The scheme should include C2-C2 complexes and mention whether these complexes are expressed at the cell surface (see previous and following comments).

   Fig.1b and c

   • Current from cells transfected with GluN2B‐wt‐C1 and GluN2B‐wt‐C2 should be compared to current expressed in cells expressing untagged receptor subunits: GluN2B‐wt
   • Current from cells transfected with GluN2B‐wt‐C1 alone should be shown as well (although expected to be retained in the ER) (as performed for GluN2A‐wt‐C1 GluN2B‐wt‐C1 in suppl Fig. 1a)
   • How could you explain the null current from cells transfected with GluN2B‐wt‐C2 alone (Fig.1b middle, and 1c)? since GB2 does not contain any retention signal and can reach the cell surface in the absence of GB1, GluN2B‐wt‐C2 is supposed to reach the cell surface. This is a very important point to clarify (I am probably missing a technical detail) because if the sub-unit tagged with C2 does reach the cell surface, then all the results and conclusions drawn from the C1-C2 conditions are wrong and could be attributed to a mix of complexes containing either C1-C2 or C2-C2. My following comments are based on the assumption that only receptors containing C1-C2 tagged subunits reach the membrane (as assumed by the authors and suggested in Figure 1b middle), but explanations should absolutely be provided to convince the reader.

   Fig. 4a and 5a

   • Please, keep the current scale constant between all current illustrations within the same figure (4a and 5a). Indeed, not only the Spermin- or SP- induced potentiation is an important data (which is presently quantified on the histograms of fig. 4b and 5b) but also knowing whether the amount of current recorded in cells expressing one mutant subunit in presence of SP (for example GluN2A‐wt‐C1 GluN2B‐G689S‐C2) is comparable to the current recorded in wt receptor-expressing cells (GluN2A‐wt‐C1 GluN2B‐wt‐C2) in absence of SP would be an excellent added value for the paper. A special figure could quantify this rescue effect of SP, measuring and comparing the mean currents recorded in these conditions (one current illustration is not sufficient given variations between similar samples). By the way 5mM glutamate might be an excessive concentration. At 1mM, the expected synaptic concentration of glutamate following action potential, according to figures 3 and Suppl1 the response of the mutated receptor is much lower than that of the WT which is already almost maximal. In these conditions, SP-induced potentiation by a factor of two of GluN2A‐wt‐C1 GluN2B‐G689S‐C2 current could be equivalent to control currents recorded in GluN2A‐wt‐C1 GluN2B‐wt‐C2 cells.

   Fig. 6

   • Figure 6 is not convincing because cultured hippocampal neurons do express endogenous NMDA receptors. To what extent the recording currents are affected by endogenous, non-mutated GluN2B subunits? Western Blots showing an extinction of endogenous subunits expression when transfected tagged subunits are competitively expressed would be required.
   • Fig.6b "PE-S" on the graph should be replaced by "PS"

   Discussion

   The authors are surprised by the fact (Fig.2) that 1 variant reduces the apparent glutamate affinity of the receptor, but not as much as 2 variants, despite the fact that "NMDARs opening requires all four subunits to be liganded (i.e., occupied by a ligand) which implies that the least affine subunit should have dominated the final affinity of the receptor". I agree that the difference is noticeable, however the glutamate affinity for receptors containing 1 variant is much closer to that of receptors containing 2 variants than that of wild-type receptors. Hence, the results obtained do not seem so surprising and could result, as rightly explained by the authors from a possible cooperativity between the subunits.

   On the other hand, the data in Figure 6 are much more difficult to interpret and reconcile with the nature of the expressed receptor subunits (which this time is not controlled) nor their association within the same receptor. However, this aspect, which is essential to the understanding of the consequences of 1 variant on neuronal signalling, is not discussed: Whatever the stoichiometry of the complexes in the heterozygous disease, the mutated and wild type GluN2B subunits coexist in the same cell: Either within the same di-heteromeric complexes GluN2B-wt + GluN2B-mutant, or in separate complexes but nevertheless expressed in the same cell, in di heteromeric (GluN2B-wt + GluN2B-wt and GluN2B-mutant + GluN2B-mutant); or tri-heteromeric (GluN2A-wt + GluN2B-wt and GluN2A-wt + GluN2B-mutant) complexes. Assuming that half of the complexes remain wild-type, e.g. (GluN2A-wt + GluN2B-wt and GluN2A-wt + GluN2B-mutant) we would expect (Fig. 6) a small decrease in NMDA current (carried only by the half that expresses the mutated subunit, and whose function is not zero but only decreased by about 20% in response to 5mM Glutamate, Fig. 3b). The same reasoning applies to the di-heteromeric conditions (GluN2B-wt + GluN2B-wt; GluN2B-mutant + GluN2B-mutant), here again the decrease observed Fig. 6b is difficult to reconcile with the responses measured Fig. 2b. In other words, how to explain a 50% decrease of the currents, instead of the 10% expected by the previous reasoning. In this experiment we do not know which subunits are expressed, their proportions, nor how they are associated in functional complexes, which makes the interpretation of the data impossible. The only explanation, far-fetched, for 50 % decrease would be that the complexes were to contain all (or the vast majority) 1 wild-type subunits associated with 1 variant, then a homogeneous 50% reduction in current could be expected. But this extreme condition could only be possible in the case of di-heteromers, which is unlikely the case in Fig.6 as GluN2A currents are measured in presence of Ifenprodil. To conclude 1) the comparison of the currents in transfected and non-transfected neurons does not make sense in figure 6b which is not convincing because we do not know the nature of the currents actually measured. A comparison in controlled condition would make more sense (as I suggested in the criticism of figures 4, 5). 2) The reality of the combinations of expression and association between subunits within different complexes expressed in the same cell must be considered and taken into account in the interpretation of the data. Undoubtedly, the means of restoring the NMDA current will be different depending on the presence of mutated subunits in all functional channels or not.

   Referees cross-commenting

   The referees' comments are highly relevant. In particular, referee 3's comment 1 seems very interesting because it may help to better understand the discrepancy in the results observed in neurons, i.e. a 50% decrease in the currents induced by the expression of the mutant and wild type subunits in the same cells, whereas theoretically one would expect a 10% decrease of this current (cf. referee 2's 2nd comment in the discussion section). This comment 1 of referee 3 indeed stresses the fact that the control (non-transfected neurons) to which the heterozygous condition is compared is not the correct control, which should rather be neurons transfected with wild type receptor subunits. More generally, this comment underlines the importance of monitoring the effective membrane expression of the different subunits in each of the experimental conditions in order to be able to compare conditions and draw conclusions.

   Significance

   The novelty of the study, is to evaluate the consequences of a single mutated subunit within NMDA receptors affected by GRIN variant, to mimic the heterozygous condition of GRIN encephalopathies, this is of potential value for the field and the interest could also be extended to other genetic diseases (at least the experimental way to study the functioning of only one desired stoichiometric configuration).

   The strength of this paper is precisely to isolate technically and to study the functioning of a desired stoichiometric configuration only. The main limitation of the paper is the interpretation that the authors make of their data in a physio-pathological context

   This work could be of interest for general audience, providing the title and summary are slightly modified My area of expertise could not be closer to the topic of the article: Glutamate receptors; GRIN; molecular tinkering, cell culture, electrophysiology, receptor stoichiometry...

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   Referee #1

   Evidence, reproducibility and clarity

   Summary:

   Kelner and Berlin present their research findings pertaining to the effect of GRIN2B variants that modify NMDA receptor function and pharmacology. While these mutations were published previously, the new manuscript provides a more thorough investigation into the effects that these variants pose when incorporated into heteromeric complexes with either wildtype GluN2B or GluN2A - NMDA receptors containing only a single mutated GluN2B subunits is more relevant to the disease cases because the associated patients are heterozygous for the variant. The authors achieved selective expression of receptor heteromeric complexes by …

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   Referee #1

   Evidence, reproducibility and clarity

   Summary:

   Kelner and Berlin present their research findings pertaining to the effect of GRIN2B variants that modify NMDA receptor function and pharmacology. While these mutations were published previously, the new manuscript provides a more thorough investigation into the effects that these variants pose when incorporated into heteromeric complexes with either wildtype GluN2B or GluN2A - NMDA receptors containing only a single mutated GluN2B subunits is more relevant to the disease cases because the associated patients are heterozygous for the variant. The authors achieved selective expression of receptor heteromeric complexes by utilising an established trafficking control system. The authors found that while a single variant subunit in the receptor complex is largely dominant in its effect on reducing glutamate potency of the NMDA receptor, it 's effect on receptor pharmacology varied. Unlike diheteromeric receptors containing mutated subunits, polyamine spermine potentiated GluN1/2B (but not GluN1/2A/2B) receptors that contained a single mutated GluN2B. In contrast, the neurosteroid, pregnenolone-sulfate (PS), was effective at potentiating the NMDA receptor currents (to varying degrees) regardless of the subunit composition. The potentiation of NMDA receptor currents by PS was also observed in neurons overexpressing the variants.

   The techniques used in this study were appropriate to address the objectives and the overall effects are large, and generally convincing. I like the way the results are presented, although have a few (easily addressable) comments.

   Major comments:

   • When incrementally adding drugs (e.g. traces in figures 5 and 6), it doesn't always appear like the response has plateaued before changing the solutions/drugs. Therefore, I am curious to what extent the effects observed are underestimated.
   • Also, in relation to figure 6, to what extent does agonist application cause desensitization here? Looking at traces in Figure 6b it appears that there is some desensitization and it isn't clear to what extent this persists during the solution changes. Could the authors conduct/show the controls where NMDA alone (for 50-60s), or NMDA followed by PE-S (without ifenprodil).
   • Finally, figure 5 shows the effect of the neurosteroid (and ifenprodil) on NMDA-evoked currents in neurons overexpressing the GluN2B variants in neurons. However, there currents probably reflect a mixture of extrasynaptic and synaptic receptors. To what extent are synaptic NMDA receptors affected by the variants?

   Minor comments:

   • Looking at the fits in the graph of Figure 2b it appears that the slope on the concentration response curves is less steep for the mixed 2B-diheteromeric NMDA receptors. How much are the Hill coefficients changing and can this be interpreted to provide more mechanistic insight? Wouldn't it make sense to include the Hill coefficients in Table 1?
   • The authors illustrate the changes in potency by the shift in the concentration response curves, but is there any change in efficacy? A simple way to illustrate this would be also present a simple graph showing the maximum current amplitudes (i.e. to 10 mM glutamate) for each of the receptor complexes.
   • The authors characterize the 'apparent' affinity (or potency) of the receptor using concentration-response curves, but numerous points in the manuscript refer to changes in affinity. None of the experiments shown directly measure affinity (which would require ligand-binding assays) and so the use of the word affinity is inaccurate/misleading. I suggest the authors replace the instances of the word 'affinity' with 'potency'.
   • In the third line of the abstract, the authors wrote, 'for which there are no treatments' in relation to GRINopathies. My understanding is that there are symptomatic treatments but that there are no disease-modifying treatments.
   • The authors have interchangeably used the terms NMDAR or GluNRs throughout the manuscript. I suggest sticking to one of these terms. I would suggest NMDARs since this is less likely to be misread as a a specific NMDA receptor subunit..
   • Typos: 
1) Results paragraph 2 sentence one: 'We thereby produced GluN2B-wt, GluN2B-G689C and GluN2B-G689S subunits tagged with C1 or C2, co-expressed these along with the GluN1a-wt subunits in...'
2) Results paragraph 2: '...but these were mainly noticeable when oocytes are were exposed to high (saturating) glutamate concentrations...'
3) Last sentence in the second to last paragraph of the results section entitled 'Mixed di-and tri-heteromeric channels...': 'This , PS may serve to rescue...'
4) Last sentence in last paragraph of the results section entitled 'Mixed di-and tri-heteromeric channels...': 'Despite the latter, we found no evidence for any direct effect of three different physiologically relevant concentrations of the drug on di- or tri-heteromeric receptors'
   • Figures 1e, 2b, 3b: it would be helpful to add a legend to the graph so that the curves can be interpreted without having to read through the figure legend.
   • The bar graphs in Figure 6 show individual data points but those in figures 4b and 5b don't. Can the authors please add the data points to these graph.
   • It would be helpful to reviewers that future manuscripts by the authors include page numbers and line numbers

   Referees cross-commenting

   Reviewers 2 and 3 highlight an important issue concerning figure 6 and the extent to which the overexpressed variants subunits can compete and assemble with endogenous NMDA receptors (unlike the system where the surface expression of specific receptor complexes is controlled). Indeed in the recent paper by the same authors, the two variants differed in their surface expression (in HEK cells), with G689C expressing particularly poorly. With reference to the second minor comment of Reviewer 1, the maximum current amplitudes would of course need to be normalized to cell surface expression of the receptor to gain any insight into efficacy.

   Significance

   This study emphasizes the complex pattern of effects that variants can have on glutamate receptor function and pharmacology, especially considering the context of receptor subunit composition. The authors have followed up their previous findings on the same mutants (Kellner et al, 2021, Elife), but used a trafficking control system here to characterize properties of mutated receptor complexes that are most likely to exist in neurons. The authors show that the defective currents mediated by NMDA receptors containing a loss-of-function GluN2B variant can be enhanced by neurosteroids (and in the case of GluN1/2B receptors, polyamines also). Development and approval of neurosteroids for the clinic would be required for the findings to translate to a therapy for patients. Readers should also be aware that neurosteroids act on other receptors too (e.g. GABA receptors), which could complicate the outcome. The expertise of the reviewer is in glutamate receptors and synaptic transmission.

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   Dec 13, 2022