Non‐canonical endogenous expression of voltage‐gated sodium channel NaV1.7 subtype by the TE671 rhabdomyosarcoma cell line

Abstract The human TE671 cell line was originally used as a model of medulloblastoma but has since been reassigned as rhabdomyosarcoma. Despite the characterised endogenous expression of voltage‐sensitive sodium currents in these cells, the specific voltage‐gated sodium channel (VGSC) subtype underlying these currents remains unknown. To profile the VGSC subtype in undifferentiated TE671 cells, endpoint and quantitative reverse transcription–PCR (qRT‐PCR), western blot and whole‐cell patch clamp electrophysiology were performed. qRT‐PCR profiling revealed that expression of the SCN9A gene was ∼215‐fold greater than the SCN4A gene and over 400‐fold greater than any of the other VGSC genes, while western blot confirmed that the dominant SCN9A RNA was translated to a protein with a molecular mass of ∼250 kDa. Elicited sodium currents had a mean amplitude of 2.6 ± 0.7 nA with activation and fast inactivation V 50 values of −31.9 ± 1.1 and −69.6 ± 1.0 mV, respectively. The currents were completely and reversibly blocked by tetrodotoxin at concentrations greater than 100 nm (IC50 = 22.3 nm). They were also very susceptible to the NaV1.7 specific blockers Huwentoxin‐IV and Protoxin‐II with IC50 values of 14.6 nm and 0.8 nm, respectively, characteristic of those previously determined for NaV1.7. Combined, the results revealed the non‐canonical and highly dominant expression of NaV1.7 in the human TE671 rhabdomyosarcoma cell line. We show that the TE671 cell line is an easy to maintain and cost‐effective model for the study of NaV1.7, a major target for the development of analgesic drugs and more generally for the study of pain. Key points Undifferentiated TE671 cells produce a voltage‐sensitive sodium current when depolarised. The voltage‐gated sodium channel isoform expressed in undifferentiated TE671 cells was previously unknown. Through qRT‐PCR, western blot and toxin pharmacology, it is shown that undifferentiated TE671 cells dominantly (>99.5%) express the NaV1.7 isoform that is strongly associated with pain. The TE671 cell line is, therefore, a very easy to maintain and cost‐effective model to study NaV1.7‐targeting drugs.


21-Jul-2021 1st Editorial Decision
Dear Dr Mellor, Re: JP-RP-2021-282097 "Non-canonical endogenous expression of voltage-gated sodium channel NaV1.7 subtype by the TE671 rhabdomyosarcoma cell line" by Neville M Ngum, Muhammad Y A Aziz, Richard J Wall, Ian R Duce, and Ian R Mellor Thank you for submitting your manuscript to The Journal of Physiology. It has been assessed by a Reviewing Editor and by 2 expert Referees and I am pleased to tell you that it is considered to be acceptable for publication following satisfactory revision.
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The authors document the 'rogue' expression of voltage-gated sodium currents in the TE671 rhabdomyosarcoma skeletal muscle cell line. They present convincing data supporting the transcription of NaV1.7 mRNA and follow that up with pharmacological evidence that the expressed channels are primarily the NaV1.7 subtype. The short manuscript is well written, the results are presented in a clear and logical manner and the conclusions are supported by their data.

Referee #2:
Ngum et al examined voltage activated sodium currents in TE671 using molecular and physiological methods to determine the expressed subtypes. PCR and qPCR showed that the level of mRNA for Nav1.7 is much higher than all other subtypes. The VGS current was blocked with tetrodotoxin, Huwentoxin-IV and Protoxin-II. The authors suggest that this makes TE671 a useful system for the development of analgesic drugs.
In general, the manuscript is well written and flows very well. This applies to all sections which is commendable and appreciated. However, I have few points for the authors to address 1) In figure 1, it seems that the intensity of Nav1.7 bands is greater the higher the passage number is (relative to the GAPDH). Can the authors comments on this please? I suggest they add a figure comparing the level of Nav1.7 in different passages.
2) I am not sure how to interpret the fold difference between the expression of Nav1.7 and 1.4 in 1d in light of the lack of any product in 1a-c! In 1d there are differences between the levels for the channels that are "negative" in 1a-c. My questions is, are you saying the other subtypes are functionally expressed but 1.7 is expressed at a 200 fold higher level, OR are saying there is no evidence of biologically relevant expression of all subtypes except 1.7? Should a ratio of GAPDH of 0.0001 or lower be presented as Zero expression? A 200 fold difference sounds impressive but I feel it is not meaningful.
3) I expected an attempt to detect the 1.7 protein by either ICC or a western blot. I am not sure why this is not included in the molecular characterisation. There are good antibodies for 1.7 (e.g. from Neuromab) that can be used to support the qPCR data. 4) I wonder why one of the published small molecular inhibitors of Nav1.7 (e.g. PF-05089771) was not included in your study. Can you please comment on this?

JP-RP-2021-282097 "Non-canonical endogenous expression of voltage-gated sodium channel NaV1.7 subtype by the TE671 rhabdomyosarcoma cell line" by Neville M Ngum, Muhammad Y A Aziz, Richard J Wall, Ian R Duce, and Ian R Mellor
We should like to thank the editors and the referees for taking the time to review our manuscript and for their constructive comments on it. Please find our responses below.

EDITOR COMMENTS
Reviewing Editor: Comments for Authors to ensure the paper complies with the Statistics Policy: In preparing your revision please refer to the Journal's 'Statistics Policy' guidelines: If n<30, all data points must be plotted in the figure in a way that reveals their range and distribution. A bar graph with data points overlaid, a box and whisker plot or a violin plot (the latter two also preferably with data points included) are acceptable formats. SD should be used instead of SEM; all 'n' values must be clearly stated in main text, figures and their legends. The exact p values should be provided for statistical tests. A Statistical Summary document must be provided with the revised manuscript (see Statistics Policy for a template).
Response: We have added individual data points to the bar graph in Figure 1D. We have ensured that all mean values quoted in the text are accompanied by SD and n, and that P has been given for any statistical comparisons. A statistical summary has been completed and submitted.
Comments to the Author: The manuscript reports robust expression of nociceptive-neuron-specific voltage gated Na+ channel subunit Nav1.7 in the TE671 rhabdomyosarcoma cell line. This is an important finding as Nav1.7 is a verified drug target for pain and cellular models for pharmacological screenings against Nav1.7 are highly sought after. The PCR and pharmacology data are convincing and the manuscript is well-written. The reviewers are broadly supportive of the study, however, reviewer #2 identified several aspects in which the study should be improved. These include verifying Nav1.7 protein expression as well as testing small-molecule Nav1.7 inhibitors. Indeed, as the functional expression of Nav1.7 in TE671 cells is the main finding of this study, these additional tests are necessary. The reviewer also asked to clarify the PCR data.

Response:
It would be impossible to conduct the two additional sets of experiments that have been suggested within a response time of only four weeks and it would even be challenging to obtain the reagents in the current climate. However, both of these suggestions were considered during our study and we felt that neither of these provided additional weight to the case. We have given our reasons for this in our response to Referee #2 below.

REFEREE COMMENTS
Referee #1: The authors document the 'rogue' expression of voltage-gated sodium currents in the TE671 rhabdomyosarcoma skeletal muscle cell line. They present convincing data supporting the transcription of NaV1.7 mRNA and follow that up with pharmacological evidence that the expressed channels are primarily the NaV1.7 subtype. The short manuscript is well written, the results are presented in a clear and logical manner and the conclusions are supported by their data.

Response:
There are no points to respond to but we thank Referee #1 for their positive comments about our manuscript.

Referee #2:
Ngum et al examined voltage activated sodium currents in TE671 using molecular and physiological methods to determine the expressed subtypes. PCR and qPCR showed that the level of mRNA for Nav1.7 is much higher than all other subtypes. The VGS current was blocked with tetrodotoxin, Huwentoxin-IV and Protoxin-II. The authors suggest that this makes TE671 a useful system for the development of analgesic drugs.
In general, the manuscript is well written and flows very well. This applies to all sections which is commendable and appreciated. However, I have few points for the authors to address 1) In figure 1, it seems that the intensity of Nav1.7 bands is greater the higher the passage number is (relative to the GAPDH). Can the authors comments on this please? I suggest they add a figure comparing the level of Nav1.7 in different passages.
Response: We have added a comment to the main text on page 9 (highlighted in red) noting that "There was also an apparent increase in band intensity with increasing passage number", however, because the qRT-PCR was only conducted for passage 10 we are unable to precisely quantify the apparent increase in expression.
2) I am not sure how to interpret the fold difference between the expression of Nav1.7 and 1.4 in 1d in light of the lack of any product in 1a-c! In 1d there are differences between the levels for the channels that are "negative" in 1a-c. My questions is, are you saying the other subtypes are functionally expressed but 1.7 is expressed at a 200 fold higher level, OR are saying there is no evidence of biologically relevant expression of all subtypes except 1.7? Should a ratio of GAPDH of 0.0001 or lower be presented as Zero expression? A 200 fold difference sounds impressive but I feel it is not meaningful.
The data presented in Figure 1D are derived from the qRT-PCR experiments that provide much more sensitive detection of RNA levels than visualising the PCR products on the gels. Perhaps the log scale of the plot in Figure 1D may have been misleading and showing up levels and differences that were effectively negligible and, therefore, not visible on the gels. Therefore, we have changed the Y-axis in this plot back to a linear scale. The >200 fold difference quoted in the text is based on the qRT-PCR data comparing Na V 1.7 to Na V 1.4, which is the next highest in terms of expression. In answer to the referee's question, we are saying that Na V 1.7 is expressed at a > 200 fold higher level than Na V 1.4 but also it may be true that the very low level of Na V 1.4 expression (and lower for others) is not biologically relevant. On page 9 we have clarified the text a little and added "In effect, the only biologically relevant level of RNA is of that coding for Na V 1.7" (highlighted in red). We hope this clarifies the situation.
3) I expected an attempt to detect the 1.7 protein by either ICC or a western blot. I am not sure why this is not included in the molecular characterisation. There are good antibodies for 1.7 (e.g. from Neuromab) that can be used to support the qPCR data.
Response: Interestingly, we considered this. However, we felt that ICC or Western blot would only be meaningful if done with the full set of Nav antibodies and, in any case, this would only show that we have Na V 1.7 protein; we have gone one step further and show functional membrane protein with clear Na V 1.7 pharmacology. This is well supported by the clear dominance of Na V 1.7 RNA. So we didn't feel that this further experiment added anything useful to the study. 4) I wonder why one of the published small molecular inhibitors of Nav1.7 (e.g. PF-05089771) was not included in your study. Can you please comment on this?
Response: We compiled a list of candidate inhibitors with some level of Na V 1.7 selectivity for the pharmacological study. PF-05089771 was considered as one these candidates but rejected on the grounds that it is not as selective as ProTx-II or HWTX-IV; only about 10x over Na V 1.2 and 1.6, and there is limited data for Na V 1.8 and 1.9, some of which suggests that it is selective for Na V 1.8 also.

02-Sep-2021 1st Revision -Editorial Decision
Dear Dr Mellor, Re: JP-RP-2021-282097R1 "Non-canonical endogenous expression of voltage-gated sodium channel NaV1.7 subtype by the TE671 rhabdomyosarcoma cell line" by Neville M Ngum, Muhammad Y A Aziz, Richard J Wall, Ian R Duce, and Ian R Mellor Thank you for submitting your manuscript to The Journal of Physiology. It has been assessed by a Reviewing Editor and by 1 Referees and the reports are copied below.
Please let your co-authors know of the following editorial decision as quickly as possible.
As you will see, in its current form, the manuscript is not acceptable for publication in The Journal of Physiology. In comments to me, the Reviewing Editor expressed interest in the potential of this study, but much work still needs to be done (and this may include new experiments) in order to satisfactorily address the concerns raised in the reports.
In view of this interest, I would like to offer you the opportunity to carry out all of the changes requested in full, and to resubmit a new manuscript using the "Submit Special Case Resubmission for JP-RP-2021-282097R1..." on your homepage.
We cannot, of course, guarantee ultimate acceptance at this stage as the revisions required are substantial. However, we encourage you to consider the requested changes and resubmit your work to us if you are able to complete or address all changes.
A new manuscript would be renumbered and redated, but the original referees would be consulted wherever possible. An additional referee's opinion could be sought, if the Reviewing Editor felt it necessary. A full response to each of the reports should be uploaded with a new version.
I hope that the points raised in the reports will be helpful to you. Thank you for your revision. In moving a manuscript from provisional acceptance to final acceptance the Editors take particular note of whether specific requests have been completed. In this case two relatively straightforward additional experiments were requested; without this information the manuscript is of lesser impact and would not likely reach our criterion for acceptance. If the additional experiments will take more time, then we are happy to provide extra time (in this case we would technically reject this version but invite you to re-submit, as a special case resubmission.

18-Aug-2021
Senior Editor: Comments for Authors to ensure the paper complies with the Statistics Policy: Dose response curves need raw data points.
Comments to the Author: I agree with the Editor's assessment that this MS needs to show the additional data in order proceed to publication. We are happy to give you more time to do this.

EDITOR COMMENTS
Reviewing Editor: Thank you for your revision. In moving a manuscript from provisional acceptance to final acceptance the Editors take particular note of whether specific requests have been completed. In this case two relatively straightforward additional experiments were requested; without this information the manuscript is of lesser impact and would not likely reach our criterion for acceptance. If the additional experiments will take more time, then we are happy to provide extra time (in this case we would technically reject this version but invite you to re-submit, as a special case resubmission.

Senior Editor:
Comments for Authors to ensure the paper complies with the Statistics Policy: Dose response curves need raw data points.
Comments to the Author: I agree with the Editor's assessment that this MS needs to show the additional data in order proceed to publication. We are happy to give you more time to do this.

RESPONSE:
We thank you for the provision of more time to complete further experimental work and the opportunity to resubmit this. We have now performed a Western Blot according to the original request of Referee #2 and have added this to the manuscript (text and new Figure 2). An additional author has been added, who performed this work. We have also added raw data points to the dose-response curves in Figures 4-6 as requested. A highlighted copy of the manuscript has been provided indicating all of the additions. I am clear now on the justification for using the protein blockers, please include this in the manuscript. RESPONSE: We added a note in our methods (section 2.5) justifying the selection of the peptide spider toxins over the small molecule inhibitors like PF-05089771 on the grounds of their much superior and well documented selectivity for Na V 1.7. Please could authors include: * Author contributions * Acknowledgements

RESPONSE:
We have now included the author contributions within the manuscript file. We have no further acknowledgements beyond the funding bodies already given.

22-Mar-2022 2nd Revision -Editorial Decision
Dear Dr Mellor, Re: JP-RP-2022-283055X "Non-canonical endogenous expression of voltage-gated sodium channel NaV1.7 subtype by the TE671 rhabdomyosarcoma cell line" by Neville M Ngum, Muhammad Y A Aziz, Muhammad Liaque Latif, Richard J Wall, Ian R Duce, and Ian R Mellor Thank you for submitting your revised Research Article to The Journal of Physiology. It has been assessed by the original Reviewing Editor and Referees and has been well received. Some final revisions have been requested.
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NEW POLICY: In order to improve the transparency of its peer review process The Journal of Physiology publishes online as supporting information the peer review history of all articles accepted for publication. Readers will have access to decision letters, including all Editors' comments and referee reports, for each version of the manuscript and any author responses to peer review comments. Referees can decide whether or not they wish to be named on the peer review history document.
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-Statistics Summary Document completed appropriately upon revision ----------------EDITOR COMMENTS Reviewing Editor: Comments for Authors to ensure the paper complies with the Statistics Policy: It seems that Figure 3 does not include primary data and it also uses S.E.M. rather than SD, which is against journal's policy.
If the Statistical Summary Document has errors please describe what is incorrect? : Several panels of Fig. 3C report mean data but only the data for panel 3C is included in the statistical summary.
Comments to the Author: The revision adequately addressed reviewer's concerns. There are still few minor inconsistencies with regards to data presentation and statistics (i.e. Fig. 3 may not be in full compliance with the journal style).
Senior Editor: