Aminomethanesulfonic acid illuminates the boundary between full and partial agonists of the pentameric glycine receptor
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Evaluation Summary:
Ivica et al. provide both functional and structural characterization of a relatively unstudied glycine receptor agonist that is structurally in between a full and partial agonist. The combination of cryogenic electron microscopy and electrophysiological approaches allows for complementary structural and functional investigations into the criteria that determine ligand efficacy at the glycine receptor. This manuscript will be of interest to both biophysical and pharmacological investigations of ligand-gated ion channels.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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Abstract
To clarify the determinants of agonist efficacy in pentameric ligand-gated ion channels, we examined a new compound, aminomethanesulfonic acid (AMS), a molecule intermediate in structure between glycine and taurine. Despite wide availability, to date there are no reports of AMS action on glycine receptors, perhaps because AMS is unstable at physiological pH. Here, we show that at pH 5, AMS is an efficacious agonist, eliciting in zebrafish α1 glycine receptors a maximum single-channel open probability of 0.85, much greater than that of β-alanine (0.54) or taurine (0.12), and second only to that of glycine itself (0.96). Thermodynamic cycle analysis of the efficacy of these closely related agonists shows supra-additive interaction between changes in the length of the agonist molecule and the size of the anionic moiety. Single particle cryo-electron microscopy structures of AMS-bound glycine receptors show that the AMS-bound agonist pocket is as compact as with glycine, and three-dimensional classification demonstrates that the channel populates the open and the desensitized states, like glycine, but not the closed intermediate state associated with the weaker partial agonists, β-alanine and taurine. Because AMS is on the cusp between full and partial agonists, it provides a new tool to help us understand agonist action in the pentameric superfamily of ligand-gated ion channels.
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Author Response
Reviewer #2 (Public Review):
Ivica et al., conducted a series of electrophysiological and cryo-electron microscopy studies to investigate what differentiates partial agonist versus full agonist effects at the glycine receptor, a member of the cys-loop receptor superfamily. To this end, they used aminomethanesulfonic acid (AMS), a novel partial agonist that possesses efficacy intermediate between the high efficacy agonist glycine and the partial agonists beta-alanine and taurine. AMS was shown to possess a maximal channel open probability of 0.85, compared to 0.96 for glycine and only 0.12 for taurine. Cryo-EM structures of glycine receptors that had bound glycine, AMS, or taurine differed, with only glycine and AMS yielding a compact conformation that differed from that seen after taurine binding. This is thought to …
Author Response
Reviewer #2 (Public Review):
Ivica et al., conducted a series of electrophysiological and cryo-electron microscopy studies to investigate what differentiates partial agonist versus full agonist effects at the glycine receptor, a member of the cys-loop receptor superfamily. To this end, they used aminomethanesulfonic acid (AMS), a novel partial agonist that possesses efficacy intermediate between the high efficacy agonist glycine and the partial agonists beta-alanine and taurine. AMS was shown to possess a maximal channel open probability of 0.85, compared to 0.96 for glycine and only 0.12 for taurine. Cryo-EM structures of glycine receptors that had bound glycine, AMS, or taurine differed, with only glycine and AMS yielding a compact conformation that differed from that seen after taurine binding. This is thought to be partially responsible for the different efficacies of these ligands. This study was performed meticulously, with compelling evidence provided supporting the authors' primary hypothesis.
The authors should consider defining what they mean by glycine being a "full agonist". In previous publications, they have argued that, since efficacy is a ratio of rates of transitions among different states of receptors, what anyone currently defines as a full agonist is in reality just the highest efficacy ligand discovered to date. There isn't any problem with the use of the term "full agonist" per se, since it is a concise way of comparing the high efficacy of glycine versus other ligands at the GlyR, but the reader would be served by having this clarified.
This is correct. We have changed “full agonist” into “highly efficacious” wherever possible (all tracked) and added in the introduction that we shall refer to glycine as a full agonist (Line 54). In the interest of conciseness, we have tried to keep this as light-handed as possible. A complete explanation of what a full agonist is would have to include various caveats such as the fact that it depends on the receptor isoform and that glycine itself becomes partial in loss-of-function hyperekplexia mutants.
Is there a qualitative rather than just a quantitative difference between high and low efficacy agonists at glycine receptors, in that only low efficacy compounds can interact with the loop B serine 174 residue and only high efficacy ligands yield compact binding pockets? In other words, should ligand efficacy be considered a continuum at the GlyR, or should it be considered more quantal in nature, with different agents occupying discrete categories? Explicitly addressing this issue would likely be of interest to the reader.
The reviewer raises a good point. In this receptor, differences in the size of the binding pockets with agonists of different efficacy was relatively small (see our reply to point 1 of reviewer #1). The numbers have been added to the text on lines 239 and following.
We did ask ourselves whether the interactions of the partial agonists with loop B could also make parts of the receptor more rigid, and thus reduce efficacy in a quantal fashion, but this is highly speculative at the moment. Answering this question will require the availability of more structures showing the receptor bound to other agonists, and possibly MD.
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Evaluation Summary:
Ivica et al. provide both functional and structural characterization of a relatively unstudied glycine receptor agonist that is structurally in between a full and partial agonist. The combination of cryogenic electron microscopy and electrophysiological approaches allows for complementary structural and functional investigations into the criteria that determine ligand efficacy at the glycine receptor. This manuscript will be of interest to both biophysical and pharmacological investigations of ligand-gated ion channels.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
Ivica et al. provide both functional and structural characterization of a relatively unstudied glycine receptor agonist AMS. They show that at pH 5 where AMS does not readily degrade, that AMS is slightly less efficacious than the full agonist glycine but more efficacious than partial agonists beta-alanine and taurine. Consistent with AMS acting as a nearly full agonist, cryo-EM structures of AMS-bound glycine receptors adopted primarily an open pore conformation similar to glycine-bound channels, whereas AMS-bound channels were not observed in a closed pore conformation as previously observed for partial agonist-bound channels (Yu et al. 2021). This work lends support to the authors' prior conclusions of the actions of full vs. partial agonists at these receptors, but also provides valuable functional and …
Reviewer #1 (Public Review):
Ivica et al. provide both functional and structural characterization of a relatively unstudied glycine receptor agonist AMS. They show that at pH 5 where AMS does not readily degrade, that AMS is slightly less efficacious than the full agonist glycine but more efficacious than partial agonists beta-alanine and taurine. Consistent with AMS acting as a nearly full agonist, cryo-EM structures of AMS-bound glycine receptors adopted primarily an open pore conformation similar to glycine-bound channels, whereas AMS-bound channels were not observed in a closed pore conformation as previously observed for partial agonist-bound channels (Yu et al. 2021). This work lends support to the authors' prior conclusions of the actions of full vs. partial agonists at these receptors, but also provides valuable functional and structural data for a ligand that is structurally in between the full agonist glycine and the partial agonist taurine. The authors further show that the energetic effect of swapping between carboxylate and sulfonate charged groups on the ligand differs depending on the ligand's length. Together, these data will be of interest to both biophysical and pharmacological investigations of ligand-gated ion channels. My only concern is in the interpretation of some rather small structural differences.
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Reviewer #2 (Public Review):
Ivica et al., conducted a series of electrophysiological and cryo-electron microscopy studies to investigate what differentiates partial agonist versus full agonist effects at the glycine receptor, a member of the cys-loop receptor superfamily. To this end, they used aminomethanesulfonic acid (AMS), a novel partial agonist that possesses efficacy intermediate between the high efficacy agonist glycine and the partial agonists beta-alanine and taurine. AMS was shown to possess a maximal channel open probability of 0.85, compared to 0.96 for glycine and only 0.12 for taurine. Cryo-EM structures of glycine receptors that had bound glycine, AMS, or taurine differed, with only glycine and AMS yielding a compact conformation that differed from that seen after taurine binding. This is thought to be partially …
Reviewer #2 (Public Review):
Ivica et al., conducted a series of electrophysiological and cryo-electron microscopy studies to investigate what differentiates partial agonist versus full agonist effects at the glycine receptor, a member of the cys-loop receptor superfamily. To this end, they used aminomethanesulfonic acid (AMS), a novel partial agonist that possesses efficacy intermediate between the high efficacy agonist glycine and the partial agonists beta-alanine and taurine. AMS was shown to possess a maximal channel open probability of 0.85, compared to 0.96 for glycine and only 0.12 for taurine. Cryo-EM structures of glycine receptors that had bound glycine, AMS, or taurine differed, with only glycine and AMS yielding a compact conformation that differed from that seen after taurine binding. This is thought to be partially responsible for the different efficacies of these ligands. This study was performed meticulously, with compelling evidence provided supporting the authors' primary hypothesis.
The authors should consider defining what they mean by glycine being a "full agonist". In previous publications, they have argued that, since efficacy is a ratio of rates of transitions among different states of receptors, what anyone currently defines as a full agonist is in reality just the highest efficacy ligand discovered to date. There isn't any problem with the use of the term "full agonist" per se, since it is a concise way of comparing the high efficacy of glycine versus other ligands at the GlyR, but the reader would be served by having this clarified.
Is there a qualitative rather than just a quantitative difference between high and low efficacy agonists at glycine receptors, in that only low efficacy compounds can interact with the loop B serine 174 residue and only high efficacy ligands yield compact binding pockets? In other words, should ligand efficacy be considered a continuum at the GlyR, or should it be considered more quantal in nature, with different agents occupying discrete categories? Explicitly addressing this issue would likely be of interest to the reader.
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