A-type FHFs mediate resurgent currents through TTX-resistant voltage-gated sodium channels
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eLife assessment
This is an exciting and important study that constitutes a major advance in the molecular understanding of resurgent Na current. Reproducing resurgent current by expression of two proteins has never been done. Here, the authors have for the first time molecularly reconstituted Na channels that produce resurgent Na current. Not only do these experiments satisfactorily and convincingly address a long-standing question in the field, but they also open the door to molecular manipulation of this current, potentially of significant practical use given the proposed role of the current in several disorders and disease states, including pain. The work will be of interest to many neuroscientists.
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Abstract
Resurgent currents ( I NaR ) produced by voltage-gated sodium channels are required for many neurons to maintain high-frequency firing and contribute to neuronal hyperexcitability and disease pathophysiology. Here, we show, for the first time, that I NaR can be reconstituted in a heterologous system by coexpression of sodium channel α-subunits and A-type fibroblast growth factor homologous factors (FHFs). Specifically, A-type FHFs induces I NaR from Nav1.8, Nav1.9 tetrodotoxin (TTX)-resistant neuronal channels, and, to a lesser extent, neuronal Nav1.7 and cardiac Nav1.5 channels. Moreover, we identified the N-terminus of FHF as the critical molecule responsible for A-type FHFs-mediated I NaR . Among the FHFs, FHF4A is the most important isoform for mediating Nav1.8 and Nav1.9 I NaR . In nociceptive sensory neurons, FHF4A knockdown significantly reduces I NaR amplitude and the percentage of neurons that generate I NaR , substantially suppressing excitability. Thus, our work reveals a novel molecular mechanism underlying TTX-resistant I NaR generation and provides important potential targets for pain treatment.
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Author Response:
Reviewer #1 (Public Review):
This is an excellent paper with extensive data and important results. The authors present convincing data that resurgent sodium current from Nav1.8 and Nav1.9 channels is mediated, at least in part, by FHF proteins. [...] Altogether, the results in the paper make a major contribution to understanding the molecular events involved in generating resurgent current in Nav1.8 and Nav1.9 channels. The paper contains an impressive amount of data building on an equally impressive foundation of techniques developed in previous work and the results are clear and convincing.
We thank the reviewer for the positive comments.
There are some aspects of the presentation that could be improved. Line 74 "…and show for the first time that INaR can be reconstituted in heterologous systems by coexpressing …
Author Response:
Reviewer #1 (Public Review):
This is an excellent paper with extensive data and important results. The authors present convincing data that resurgent sodium current from Nav1.8 and Nav1.9 channels is mediated, at least in part, by FHF proteins. [...] Altogether, the results in the paper make a major contribution to understanding the molecular events involved in generating resurgent current in Nav1.8 and Nav1.9 channels. The paper contains an impressive amount of data building on an equally impressive foundation of techniques developed in previous work and the results are clear and convincing.
We thank the reviewer for the positive comments.
There are some aspects of the presentation that could be improved. Line 74 "…and show for the first time that INaR can be reconstituted in heterologous systems by coexpressing full-length A-type FHFs with VGSC α-subunits." It seems debatable whether the expression of Nav1.8 in ND7/23 cells constitutes truly "heterologous" expression. After all, ND7/23 cells are an immortalized DRG cell line. At the least, the authors need to explain why ND7/23 cells were used for the Nav1.8 expression and to acknowledge that ND7/23 cells may express proteins in addition to the transfected Nav1.8 and FHF that could be important for the generation of resurgent current. Did they ever attempt to express Nav1.8 and FHF4A in HEK or CHO cells? There should also be a reference to the literature (I suppose Lee et al PLoS One, 14:e0221156y, 2019) showing that ND7/23 cells do not express endogenous Nav1.8 currents.
We agree with the reviewer that ND7/23 cells are not the typical heterologous cell line. While ND7/23 cells are partly derived from rat DRG neurons, multiple reports have shown that they express Nav1.8. We now cite the paper from John et. Al, 2004 “Heterologous expression and functional analysis of rat Nav1.8 (SNS) voltage-gated sodium channels in the dorsal root ganglion neuroblastoma cell line ND7-23” along with Lee et al., 2019, which as the reviewer points out showed that these cells do not express Nav1.8 message but do express mouse Nav1.6 and Nav1.7. Based on these published studies, we believe that “heterologous expression” is appropriate, but we have clarified the use of the cell line in several places and We have attempted to transiently express Nav1.8 and FHF4A in Hek293 cells, but no Nav1.8 currents were elicited under whole-cell recording configuration. We have not attempted to express Nav1.8+FHF4A in CHO cells because the literature indicates that transient transfection of recombinant Nav1.8 in CHO cells has yielded no or low-level functional currents (Zhou et al., 2019; John et al., 2004). We now explicitly acknowledge state that “ND7/23 cells are derived from the fusion of rat DRG neurons with the N18Tg2 mouse neuroblastoma cell lines, and thus may express proteins in addition to the transfected VGSCs and FHF that could be important for the differential effects on resurgent currents and long-term inactivation that we observed with Nav1.8 and Nav1.6.” in the discussion.
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eLife assessment
This is an exciting and important study that constitutes a major advance in the molecular understanding of resurgent Na current. Reproducing resurgent current by expression of two proteins has never been done. Here, the authors have for the first time molecularly reconstituted Na channels that produce resurgent Na current. Not only do these experiments satisfactorily and convincingly address a long-standing question in the field, but they also open the door to molecular manipulation of this current, potentially of significant practical use given the proposed role of the current in several disorders and disease states, including pain. The work will be of interest to many neuroscientists.
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Reviewer #1 (Public Review):
This is an excellent paper with extensive data and important results. The authors present convincing data that resurgent sodium current from Nav1.8 and Nav1.9 channels is mediated, at least in part, by FHF proteins. They show that expression of FHF4A, and to a lesser extent, FHF1A, FHF2A, and FHF3A, can induce resurgent Na current in Nav1.8 channels expressed in ND7/23 cells and Nav1.9/beta1/beta2 channels expressed in HEK cells. They further show that shRNA knock-down of FHF4 can substantially reduce (though not eliminate) native Nav1.8-mediated resurgent current in DRG neurons, that this reduces repetitive firing of the neurons, and that repetitive firing can be restored by a peptide derived from the N-terminal of FHF4A. An additional very interesting result is the identification of an inner-pore residue …
Reviewer #1 (Public Review):
This is an excellent paper with extensive data and important results. The authors present convincing data that resurgent sodium current from Nav1.8 and Nav1.9 channels is mediated, at least in part, by FHF proteins. They show that expression of FHF4A, and to a lesser extent, FHF1A, FHF2A, and FHF3A, can induce resurgent Na current in Nav1.8 channels expressed in ND7/23 cells and Nav1.9/beta1/beta2 channels expressed in HEK cells. They further show that shRNA knock-down of FHF4 can substantially reduce (though not eliminate) native Nav1.8-mediated resurgent current in DRG neurons, that this reduces repetitive firing of the neurons, and that repetitive firing can be restored by a peptide derived from the N-terminal of FHF4A. An additional very interesting result is the identification of an inner-pore residue that differs between Nav1.9 and Nav1.5 and when mutated in Nav1.5 almost eliminates the ability of the Navβ4 peptide to produce resurgent current without affecting resurgent current induced by FHF2A, suggesting different intrapore binding sites for the Navβ4 peptide and FHFs. Altogether, the results in the paper make a major contribution to understanding the molecular events involved in generating resurgent current in Nav1.8 and Nav1.9 channels. The paper contains an impressive amount of data building on an equally impressive foundation of techniques developed in previous work and the results are clear and convincing.
There are some aspects of the presentation that could be improved.
Line 74 "...and show for the first time that INaR can be reconstituted in heterologous systems by coexpressing full-length A-type FHFs with VGSC α-subunits."
It seems debatable whether the expression of Nav1.8 in ND7/23 cells constitutes truly "heterologous" expression. After all, ND7/23 cells are an immortalized DRG cell line. At the least, the authors need to explain why ND7/23 cells were used for the Nav1.8 expression and to acknowledge that ND7/23 cells may express proteins in addition to the transfected Nav1.8 and FHF that could be important for the generation of resurgent current. Did they ever attempt to express Nav1.8 and FHF4A in HEK or CHO cells? There should also be a reference to the literature (I suppose Lee et al PLoS One, 14:e0221156y, 2019) showing that ND7/23 cells do not express endogenous Nav1.8 currents.
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Reviewer #2 (Public Review):
This is an excellent report describing a new functionality for FHF sodium channel-binding proteins. Certain combinations of FHF and sodium channel isoforms enable the generation of resurgent current. Specifically, TTX-resistant sodium channels (Nav1.8 and Nav1.9) in conjunction with A-type FHFs result in a resurgent current. The mechanism involves an open-channel block analogous to the way A-type FHFs induce long-term inactivation of TTX-sensitive sodium channels. The researchers also employ FHF4 knockdown to show that endogenous A-type FHF underlies resurgent current in DRG neurons and enhanced excitability.
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Reviewer #3 (Public Review):
This is an extremely exciting and important study that constitutes a major (and long-awaited) advance in the molecular understanding of resurgent Na current. Reproducing resurgent current by expression of two proteins has never been done before: All studies that have tried to work their way toward the molecular mechanism have relied on peptides from candidate blocking proteins which mimic resurgent current, and co-expression of those candidate proteins with Na channels has been unsuccessful. Here, the authors have accomplished three things:
(1) They have for the first time successfully molecularly reconstituted Na channels that produce resurgent Na current by expressing the FHF4A protein with NaV1.8 or 1.9.
(2) They have also reconstituted resurgent currents by coexpressing FHF2A with NaV1.5 or 1.7.
(3) They …Reviewer #3 (Public Review):
This is an extremely exciting and important study that constitutes a major (and long-awaited) advance in the molecular understanding of resurgent Na current. Reproducing resurgent current by expression of two proteins has never been done before: All studies that have tried to work their way toward the molecular mechanism have relied on peptides from candidate blocking proteins which mimic resurgent current, and co-expression of those candidate proteins with Na channels has been unsuccessful. Here, the authors have accomplished three things:
(1) They have for the first time successfully molecularly reconstituted Na channels that produce resurgent Na current by expressing the FHF4A protein with NaV1.8 or 1.9.
(2) They have also reconstituted resurgent currents by coexpressing FHF2A with NaV1.5 or 1.7.
(3) They have identified a key residue that regulates the sensitivity of NaV alpha subunits to resurgent-current mimicking open-channel block by the NaVbeta4 peptide.Not only do these experiments satisfactorily and convincingly address a long-standing question (and give an idea of the wealth of molecular combinations that may generate resurgent current), they also open the door to molecular manipulation of this current, which will be highly informative and potentially of significant practical use, given the proposed role of the current in several disorders and disease states, including pain (as shown by these authors). The work is convincingly done and clearly presented.
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