The study explores the modulation of kainate receptors GluK1-3 using the positive allosteric modulator BPAM344. Authors develop a calcium-sensitive fluorescence-based assay to screen for agonists, antagonists, and positive allosteric modulators (PAMs) of GluK1-K3 receptors. They used BPAM344 to enhance the receptor response and monitored intracellular free calcium levels using fluorescent probes Fluo-4-AM and Fluo-8-AM.
Stable cell lines expressing GluK1-K3 receptors were generated and validated using homologous competition binding with radiolabeled ligands. The potency of agonists (glutamate, kainate, and domoate) and antagonists (UBP310) was evaluated in the presence of BPAM344. The rank order of potency for GluK1 and GluK2 was domoate > kainate > glutamate, consistent with the effect of the known potentiator ConA. GluK3 showed higher potency for kainate than for glutamate, with no response to domoate.
The study also presents the biologically relevant dimeric structure of GluK3-LBD bound to kainate and BPAM344. They introduced an H573A mutation to improve dimer packing. The X-ray structures of GluK3-H523A-LBD were utilized to identify binding sites for sodium, chloride, and zinc ions. The authors resolved the density of chloride and zinc ions at the dimer interface. The sodium binding site was predicted through MD simulations and compared with GluK1 and GluK2.
Additionally, the study reports low-resolution structures of GluK3 determined by negative stain EM, showing a dimer of dimers arrangement of the LBDs, resembling the active state in 30% of the conformations, in the presence of BPAM344 and glutamate.

1. The authors claim ‘to establish robust assays at GluK1-3 that would allow testing of agonists, antagonists, and positive allosteric modulators using BPAM344 as a reference to diminish desensitization’. However, the number of ligands tested are very limited, for e.g. only one antagonist ‘UBP310’ was evaluated at GluK1.

2. In 2017, Larsen et al reported maximum potentiation from glutamate bound GluK3 receptors in presence of 100 µM of BPAM344 when compared to GluK1 and GluK2. Figure 2C does not replicate the same. No plausible explanation is provided for these two contradictory observations.

3. Also, BPAM344 is weakly potent at GluK3 (EC50 639 ± 29 µM) and still used to stabilize GluK3 receptor for capturing it in the active state with dimers of dimers conformation.

4. In electrophysiology, a single cell is patched to record the responses from the receptors expressed on the cell membrane. In calcium sensitive fluorescence assays, there are numerous cells with many receptors. The EC50 value determined from these experiments should technically be higher when compared to EC50 values generated from electrophysiology experiments right?

5. Overall, based on their calcium sensitive fluorescence experiments, BPAM344 could be used to potentiate responses from GluK1-K3 receptors to screen novel compounds under In vitro/ex vivo conditions only. The potential of BPAM344 cannot be replicated under In vivo conditions or for functional studies as it is not a selective KAR modulator and also shows weak potency for GluK3 receptors.

6. Reference for the adaptation of calcium fluorescence-based assay is not mentioned. Company details for Fluo-4-AM or Fluo-8-AM are not mentioned. Reference 36 is not found in the literature.

7. The biologically relevant dimeric structure of GluK3 LBD is an appreciable finding. However, the structural analysis could be done in depth in the results section to guide a reader through the figure.

8. ‘Kainate binds at the orthosteric binding site in all eight molecules, whereas two molecules of BPAM344 were found at each dimer interface in all four GluK3-H523ALBD dimers’ - Figures do not represent the same or is not clear. (Figure 6)

9. How is BPAM344 modulating the structure of GluK3-LBD is not discussed although the authors claim to have discussed this aspect in the introduction. (Page 7 Line6). Agreed, that the binding sites have been identified for BPAM344 for GluK3 LBD but what are the subtle changes it is bringing about in the structure for e.g., with respect to the S1 and S2 lobe distances could have been analysed. This structural information could later be translated to understand how BPAM344 binding is affecting channel activation at GluK3 receptors and helping in potentiation. Structural comparison could be carried out to highlight the overall changes induced by BPAM344.

Minor comments:
This sentence is confusing in the abstract ‘In the presence of 150 μM BPAM344,
domoate was found to ………. BPAM344 and 100 μM kainate.”. Please fix.

Please provide the reasoning behind the His523 mutation. How did the authors identify this specific residue, even though it may be clear from the dimeric structure.

The strain information for K3 LBD expression is not mentioned along with detailed purification protocol.
The conclusion of the study is very generalized and the limitations of the calcium sensitive fluorescence-based assay could have been discussed.
The introduction does not provide enough context to appreciate the study rationale.
Remove redundancies in the text between the Results and Discussion sections. Multiple instances results are just repeated again. E.g potency of BPAM344

Not clear why the full-length construct had P271H mutation in the NTD.

For negative stain EM, the resolution achieved is in the range of 18-20 Å and hence this sentence does not make sense and should be removed “Resolutions of 6.7 Å were calculated using the FSC gold standard for both volumes.”

The authors have addressed all the queries raised by this reviewer to satisfaction. I don't have any more concerns.