Control of AMPA receptor activity by the extracellular loops of auxiliary proteins

Thank you for submitting your article "Control of AMPA receptor activity by the extracellular loops of auxiliary proteins" for consideration by eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by Kenton Swartz as the Reviewing Editor and Gary Westbrook as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Lonnie Wollmuth (Reviewer #1); Hiro Furukawa (Reviewer #2).

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Summary:

A series of recent and exciting publications have explored structural and mechanistic interactions between AMPA receptors and TARPs, a family of accessory subunits that modulate gating of the receptor. In the present manuscript, the authors use chimeras as well as site specific mutations to identify interactions of the extracellular regions of gamma2 and gamma8, L1 loop and L2 loop, with AMPA receptor subunits. They find that L1 loop, which interacts predominantly with LBD, modulates desensitization properties. On the other hand, L2 loop, which presumably interacts with the LBD-TMD linkers, has much more notable effects on desensitization and superactivation. The authors are also able to generate what they call kinetic null constructs – constructs that show no effect on gating – for either the extracellular loops of gamma2 or gamma8 as well as for LBD-TMD linkers. This is a particularly interesting discovery. Overall, the manuscript is well-written and for the most part data are presented clearly. The results and conclusions are extremely interesting. There was concern with the model because it wasn't clear how it was generated and it has not been validated as far as we could see. The following are essential revisions that the authors should address.

Essential revisions:

1) The value and validity of the model presented in Figure 1 is unclear. The authors need to expand on the description of how the model was generated and describe what kind of constraints were added to the loop region. The fact that there is no density in cryo-EM may indicate that the loop is disordered or has too many conformations to be tracked. We think that the quality of the manuscript, which is otherwise excellent, is diminished by this casual web-based model and the extent to which the authors build hypotheses around it. The authors should also try to validate the model, perhaps by calculating rmsd for bond length and bond angle using programs such as Procheck and Molprobity, keeping in mind that this analysis only tests the geometrical parameters of created models and cannot validate the existence of the loop conformations that authors show in their manuscript. We also request that the authors revise the manuscript to diminish the extent to which the model is used to frame hypotheses and perhaps simply state that the "loop is long enough to perhaps transiently interact with AMPAR LBD."

2) The experiments shown in Figure 1B need to be better introduced and documented by the authors. It is very difficult to know what the authors did here experimentally. Did the authors detect bound LBD tagged with poly histidine tag? It needs some explanations in the figure legend or in the Materials and methods section. The authors also need to justify what they think this assay can tell us. How strong or specific must the interactions be to be detected here? There are multiple controls in these experiments, and the authors should walk the reader through them and explain how they constrain their results.