Probing the ionotropic activity of the orphan glutamate delta 2 receptor with genetically-engineered photopharmacology

  1. Damien Lemoine
  2. Sarah Mondoloni
  3. Jérôme Tange
  4. Bertrand Lambolez
  5. Philippe Faure
  6. Antoine Taly
  7. Ludovic Tricoire
  8. Alexandre Mourot

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Abstract

Glutamate delta (GluD) receptors belong to the ionotropic glutamate receptor family, yet whether they actually form functional and physiologically-relevant ion channels in neurons remains a debated question. Here we used a chemo-genetic approach to engineer specific and photo-reversible pharmacology in the orphan GluD2 receptor. We incorporated a cysteine mutation in the cavity located above the putative ion channel pore, for site-specific conjugation with a photoswitchable ligand. We first showed that, in the constitutively-open GluD2 Lurcher mutant, current could be rapidly and reversibly decreased with light. We then transposed the cysteine mutation to the native receptor, to demonstrate with absolute pharmacological specificity that metabotropic glutamate receptor signaling opens the GluD2 ion channel in heterologous expression system. Our results assess the functional relevance of GluD2 ion channel and introduce an optogenetic tool that will provide a novel and powerful means for probing GluD2 ionotropic contribution to neuronal physiology.

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  1. eLife

    ###Reviewer #3:

    Unlike other ionotropic glutamate receptors, GluD2 is not gated by glutamate. No specific or high-affinity chemical modulators that induce channel activity exist for this receptor--as such, it’s role as a functional channel has been questioned. To address this challenge, the authors have utilized a previously characterized photoswitchable tethered ligand (PTL) called MAGu to target a very non-specific blocker (pentamidine) to a new ion channel target (the GluD2 receptor). This approach (using this exact PTL) has been used to target knock-in cysteine mutants of the GABAA receptor in mouse brain slices and in vivo in an awake, behaving mouse. Based on this precedent, it is not unreasonable to believe that this tool could similarly be used for the GluD2 receptor (which would be a significant advance in the field for understanding the physiological role of this protein in disease), although the authors only characterized MAGu response against GluD2 in heterologous cell culture within this manuscript. Because the GluD2 receptor is not ligand-activated in the traditional sense, the authors have exploited a previously characterized constitutively open point mutant (L654T) as a background to test different photoactivatable GluD2 cysteine mutants and have nicely demonstrated a reversible current block response in the presence of purple (380 nm = "cis-" = channel "on") and green (535 nm = "trans-" = channel "off") light. The authors have numerous publications and experience in the photopharmacology of ion channels, and the characterization data here look solid.

    That said, there are a few questions that should potentially be addressed:

    1. How does MAGu work on the cysteine-engineered receptor that would presumably be used for future in vivo studies? Because the GluD2-I677C point mutant (lacking the L654T background) does not show current, the authors use the known effect of mGlu1 receptor agonism as a readout of GluD2-I677C activity in response to light and only see a 23% decrease in mGlu1 current - is this very small effect physiologically significant or to be expected? It seems like MAGu might be a useful tool to modulate GluD2 in Lurcher mice (which harbor the L654T mutation), but it is hard to know what the probe efficacy and usefulness is for evaluating the physiology of the WT GluD2 receptor in the absence of a way to measure a direct functional effect on the channel. How else might this be addressed?

    2. PTLs have been shown to generate a high local concentration of ligand to accelerate pharmacological response (and in this case, provide some level of specificity for a very non-specific, greasy cation), but it is hard to rationalize "absolute" pharmacological specificity claimed by the authors (line 35, 211). At the mid-micromolar concentrations required to elicit response, it seems unlikely that MAGu will not react with any other extracellular cysteines present in cells. Further, the guanidinium group by itself will certainly not direct the maleimide reactivity towards GluD2 over any other cation channel or electronegative protein surface. The language of this claim should be modified in the absence of other types of specificity assays.

    Minor Comments:

    1. Provide description of the step-by-step protocol for Fig. 2C (or label "washout" of pentamidine)

    2. Why does normalized current plateau at 80% for 535 nm (Fig. 4B)?

    3. There is a current biorxiv paper reporting the GluD2 structure. https://www.biorxiv.org/content/10.1101/2020.01.10.902072v2.full.pdf If this is published during the course of this review, it would be interesting for the authors to comment on how this compares to their homology model and if it makes sense with respect to their mutagenesis experiments.

  2. eLife

    ###Reviewer #2:

    The present manuscript investigates the development of a photo-activatable pore blocker to block the glutamate receptor delta receptor (GluD) ion channel as a potential tool to study this receptor in vitro and in vivo. GluD shares structural homology to other members of the family and plays key roles in synapse formation and signaling. However, in contrast to other members of the family, it does not have a clear ionotropic function - complicating defining how it contributes to synaptic function in vivo. Many labs have studied GluD and have provided key insights into its function and role. Still, the availability of new a tool to study and clarify its function has high potential.

    The manuscript lays out quite well, with some minor quibbles (see below), the issues. Proper controls are carried out to define the specificity of the action of the photo-switchable MAGu and how it can alter membrane currents and how it might work. The potential for a photo-switchable pore blocker to study the role of the ion channel in GluD is extremely high. I do have some concerns about signal-to-noise, since the pore block by trans-MAGu is only a fraction of total presumed current through GluD. In addition, how to introduce a specific cysteine in vivo will not be trivial. Still, overall this is an important manuscript that introduces an interesting strategy to study and further clarify GluD in the brain.

    1. Abstract/Introduction. It would be helpful to define early and explicitly what the photoswitchable functional strategy is - that it is working via a pore block mechanism. In the Abstract, for example, instead of calling it '...a photoswitchable ligand.' how about just '...a photoswitchable pore blocker." Once I realized the general functional strategy (at the beginning of description of results, where it was explicitly stated), everything became clearer. The functional strategy, that you are generating a photoswitchable pore blocker, should also be explicitly stated in the Introduction, where right now it is touched on but not explicitly stated.

    2. Figure 2C. The extent of block for photoswitching is being quantified relative to that for pentamidine, which is reasonable. However, for pentamidine, what is the concentration used for the experiments? Where is it at on the concentration-block curve for pentamidine? Presumably, if a complete block the leak current should go to zero and hence the efficacy of the photoswitching blocker would be less (e.g., Figure 4B). Please clarify.

    3. Figure 4A. Would be nice to see difference currents and perhaps to contrast to what is shown in Figure 2A. This would clarify the 'voltage-independence' of action for those unfamiliar.

    4. Figure 4D. Not clear how the 'ion channel' or red/green pore was generated? Is this from the structure or from some modeling? Please add details. This is an interesting figure but it is also somewhat speculative, I think, but needs more details to understand its basis. One question is what is driving the positioning of the trans MAGu? Is it being fixed? And what is driving the change in the coloration - presumed pore blocking by trans MAGu?

    Minor Comments:

    1. Figure 1. Minor point. Technically, there is no transmembrane segment 2 (TM2) in iGluRs. M2 is a pore loop, like the P loop in K+ channels, and enters and exits on the same side of the membrane - and does not span the membrane (and hence not a transmembrane segment). Simple solution would be to just rename TM2 to M2 leaving TM1, TM3, and TM4 as is and just noting somewhere in Figure legend that M2 is a non-membrane spanning pore loop.

    2. Figure 2D. Minor point. Although I understand the intent of figure, it is Very hard to discern what is being shown. Might be helpful to remove the 'red' subunit?

  3. eLife

    ###Reviewer #1:

    The study by Lemoine and colleagues demonstrates a novel chemogenetic tool to probe ion channel function of GluD2 in HEK cells. By introducing cysteine mutations and engineering a photoswitchable ligand, ionic current carried by constitutively-open GluD2 mutant channels was reversibly decreased by light. Further, GluD2 current produced by activation of mGluRs was partially reduced by light. This tool has the potential to be very powerful to advance the understanding of GluD2 channel function in neurons.

    Major:

    1. The introduction and abstract are rather general and antiquated, to the disservice of the readers. It may be time to move away from the notion that the ion channel function of GluD is debated. The authors have published many elegant studies demonstrating ion channel function. By appearances of the literature, the interpretation of these studies are not contested. In addition to pharmacology, ion channel function of GluD has been demonstrated using selective genetic strategies (e.g. Ady et al., 2013; Benamer et al., 2018; Gantz et al., 2020). To this end, lines 28-29, 51, 55, & 73-75 should be changed. It does not seem fitting to state "direct evidence for ionotropic activity of GluD in neuronal setting [sic] is lacking" provided the studies referenced above. Broadly, the readers would benefit from restructuring of the introduction and abstract to state the specific issue addressed by the present study (i.e. the lack of specific antagonists/pore blockers to study GluD without affecting other iGluRs) and highlight the potential application of the ligand.

    2. This photoswitchable ligand MAGu has great potential to probe GluD channel function in neurons, although the present study stops short of demonstrating its utility in neurons. Lines 211-212 state that the WT receptor is insensitive to MAGu, but it is not clear where those data are presented. It would be beneficial to show the magnitude of the DHPG-induced current in WT GluD2-expressing cells before and after addition of MAGu to address the possibility that MAGu affects the current irrespective of trans- or cis- conformation. It is also not clear how MAGu will be selective for site-specific conjugation when introduced in a neuronal setting. Is it expected MAGu will react with any available cysteine? It would be helpful to discuss possible limitations going forward towards use in neurons.

    3. The data show convincingly that 380 nm light unblocks MAGu-induced GluD2 block by darkness or 535 nm light. But it is not clear how trans-MAGu affected leak current from GluD2 Lurcher mutant channels. In Figure 2C I677C, there is still substantial leak in 535 nm. The quantification in Figure 2C (% photoswitching) shows the % of I-Blockphoto over I-Blockpenta, but the arrows in the right-hand trace, it would appear I-Blockphoto is actually the current unblocked. It would be helpful to quantify the amount of leak current blocked by trans-MAGu. Additional discussion as the structural basis for incomplete block may also be helpful.

    Minor:

    1. Recommendation to include model system in the title ("in expression systems" or "in HEK cells", vel sim)
  4. eLife

    ##Preprint Review

    This preprint was reviewed using eLife’s Preprint Review service, which provides public peer reviews of manuscripts posted on bioRxiv for the benefit of the authors, readers, potential readers, and others interested in our assessment of the work. This review applies only to version 1 of the manuscript.

    This manuscript was assessed by three reviewers. After the completion of their reviews, the editor and reviewers discussed the paper and arrived at the following consensus review. For transparency, the individual reviews are also presented.

    ###Summary:

    Unlike other ionotropic glutamate receptors, GluD2 is not gated by glutamate. No specific or high-affinity chemical modulators that induce channel activity exist for this receptor. To address this challenge, the authors used a previously characterized photoswitchable tethered ligand (PTL) called MAGu to target a very non-specific blocker (pentamidine) to a new ion channel target (the GluD2 receptor). This approach (using this exact PTL) has been used to target knock-in cysteine mutants of the GABAA receptor in mouse brain slices and in vivo in an awake, behaving mouse. Based on this precedent, it is not unreasonable to believe that this tool could similarly be used for the GluD2 receptor, which would be a significant advance in the field for understanding the physiological role of this protein in disease.

    The original reviews, below, reflect the reviewers’ initial enthusiasm for the potential of the approach to study GluD2 channels. In the discussion, all reviewers agreed that the issue of signal-to-noise is critical and that additional experiments are essential to demonstrate that the MAGu response will be sufficient for physiological studies in vivo.

  5. Version published to 10.1101/2020.05.14.093419 on bioRxiv