Monitoring TRPC7 Conformational Changes by BRET Following GPCR Activation

Round 1

Reviewer 1 Report

In this manuscript, the authors demonstrate TRPC7 conformational change to use BRET signal with GPCR activation. They showed that the receptor stimulation by agonists induced the conformation change of TRPC7 in HEK cells and rat cardiac fibroblast where GFP10-TRPC7-RLucII was heterologously expressed. Although this reviewer realizes the meaning of experiments performed, this manuscript includes a couple of weakness. In particular, due to the narrow range of signals and the low signal to noise ratio, all data only show a trend but not statistical difference. Therefore, the authors should add this point to discussion as a weak point of the present experiments.

 

1. All data only show a trend but not statistical difference. The authors should explain this point in Discussion as a weak point of the present experiments.

 

2. The legend of Fig.2; 'at least seven', This is not adequate. The authors should show exact n numbers in each experiment.

Author Response

We thank reviewer 1 for his overall positive appreciation of our study.
1. All data only show a trend but not statistical difference. The authors should explain this point in Discussion as a weak point of the present experiments.
We have now performed a statistical analysis of the BRET results. Accordingly we have updated figures 2, 3, 4, 5, 6 and supplementary figure 2, where the data points showing statistical significance are now clearly identified. The figure legends have also been updated. We believe that the data and conclusions are strengthened by this analysis.
2. The legend of Fig.2; 'at least seven', This is not adequate. The authors should show exact n numbers in each experiment.
This is a valid point and we now clearly state the number of experiments performed in all figure legends.

Reviewer 2 Report

The authors develop a BRET-based conformational changes of TRPC7 upon GqPCR stimulation in overexpressing cell system. The S/N ratio of this BRET assay seems nice, and the specificity of GqPCR-stimulated TRPC7 activation is well reflected in this assay system. However, the authors need some substantial experiments to Emphasize the specificity and selectivity of this assay system.

 

Major comments.

1. As the TRPC7 is a diacylglycerol-activated channel, it is more interesting to show whether GqPCR-independent TRPC7 activation by the treatment with cell-permeable diacylglycerol analogue, such as OAG and SAG, also reduces BRET ratio. If not, they need to discuss about the discrepancy of the result.
2. It is important to exclude a possibility that the TRPC7-mediated cation influx affects the BRET ratio. Does the treatment with inhibitor for TRPC7-mediated cation influx have any impact on GqPCR-stimulated BRET changes?
3. TRPC7 may form the heterotetramer with TRPC3 or TRPC6. Is the GqPCR-stimulated BRET changes conserved in TRPC3/6/7-coexpressing HEK293 cells? This information will be helpful for better understanding the structure-function relationship of TRPC channels.

 

Author Response

We have modified the introduction to address the reviewer’s concern. We now highlight how a BRET-based approach has been successfully used by other groups to characterize other TRP channels and have added the appropriate references in the introduction (page 2, lines 72-74). This has allowed us to better argue for the pertinence of our approach towards TRPC7.
We have also included some new experimental data to strengthen the research design of our study to better support our conclusions (see below).
1. As the TRPC7 is a diacylglycerol-activated channel, it is more interesting to show whether GqPCR-independent TRPC7 activation by the treatment with cell-permeable diacylglycerol analogue, such as OAG and SAG, also reduces BRET ratio. If not, they need to discuss about the discrepancy of the result.
We acknowledge that this is a limitation of our study. We have not been able to reproducibly show a decrease in BRET signal of the TRPC7 biosensor following a direct stimulation by either cell permeable OAG or SAG. This could, for instance, indicate that a crucial component of the GPCR signalling upstream of TRPC7 is missing in order to detect responsiveness of the biosensor. We note that other BRET based studies of TRPC3 have also not shown GRCR independent activation using a cell-permeable DAG analogue (see Liu et al, ref 18).
2. It is important to exclude a possibility that the TRPC7-mediated cation influx affects the BRET ratio. Does the treatment with inhibitor for TRPC7-mediated cation influx have any impact on GqPCR-stimulated BRET changes?
We agree this is an important question and to address this concern, we performed 2 sets of experiments. First, we show that the TRPC7 biosensor remains responsive in the absence of extracellular calcium (Figure S2 b). Second, we used SKF96365, a non-selective calcium entry blocker acting on TRPC channels, to show that the response of the TRPC7 biosensor was unaltered following stimulation (Figure S2 c). Together, these data indicate that cation influx has little impact on GqPCR-stimulated BRET changes (page 3, lines 137-139).
3. TRPC7 may form the heterotetramer with TRPC3 or TRPC6. Is the GqPCR-stimulated BRET changes conserved in TRPC3/6/7-coexpressing HEK293 cells? This information will be helpful for better understanding the structure-function relationship of TRPC channels.
The reviewer correctly mentions that TRPC channels associate as homo or heterotetramers. To address his question, we now show in figure 3 the effect of overexpressing untagged TRPC3 monomers on the GFP10-TRPC7-RLucII BRET signal following GqPCR stimulation (Figures 3b, 3d). As observed for untagged TRPC7, overexpressing untagged TRPC3 also lowered the BRET signal, but only partially. This reveals an intermolecular interaction between TRPC7 and TRPC3, in addition to intermolecular interactions between individual TRPC7 subunits (page 6 lines 175-184).

Round 2

Reviewer 2 Report

The authors sufficiently responded to the reviewer's concerns.