Modulation of Voltage-Gated Na⁺ Channel Currents by Small Molecules: Effects on Amplitude and Gating During High-Frequency Stimulation

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Lin C-Y, Gao Z-H, Cheung C-W, So EC, Wu S-N. "Small-molecule perturbations on voltage-gated Na+ currents: alterations in amplitude and gating properties under high frequency stimulation. Submitted to Scientia Pharmaceutica.

Abstract:

This section reads as an abstract for a primary research article. Statements about the effects of drugs are presented here as if there are novel results from experiments not previously published. After stating these putative experimental results, the authors claim that "Our investigations show that when cells are exposed to small-molecule modulators, these compounds can perturb INa, under high frequency stimulation condition".

There are no novel experiments presented, nor any methodology for such putative experiments. The authors need to clearly identify this work as a mini-review of existing literature, and that this review is comprised of previous work.

Specifically:

1. The description of the effects of these compounds must be given as review of prior work. There are no tables or figures from novel work presented, nor is there a Methods section.
2. This work should read as a review and the statement "Our investigations show that when cells are exposed to small-molecule modulators, these compounds can perturb INa, under high frequency stimulation condition" should be changed to not claim that the description of results of experiments in this manuscript are solely from this group.

Similarly, from the Introduction:

line 78: "In this study, we aim to elucidate the effects of a range of intriguing small-molecule modulators (outlined in Table 1) on the amplitude and gating properties of INa under  conditions of high frequency stimulation." This reads as if this manuscript comprises experimental investigation of these small molecules, which it does not, but instead is a review of prior work.

1. Introduction:

This section moves towards the eventual goal of describing sodium channel involvement in high frequency action potential signaling and the effect of several small molecules on sodium channels which influence action potential frequency. This could be improved by providing more detail on two aspects of sodium channel function that are a focal point later, the transient and late sodium current, as well as another sodium channel transition, slow inactivation, that receives very little attention throughout. First, the state transitions relevant to the transient sodium current are adequately described with respect to action potential frequency in the paragraph starting line 53. Then, there needs to be a description of persistent and resurgent late sodium currents with detail on how these currents influence action potential frequency. This is not intuitive, as a depolarizing influence from late current can provide excitatory drive, or promote depolarization block. Given the authors statement that later that INa (t) and INa (L) are critical components, there needs to be a thorough description of these currents prior to the sections on the various modulators. Finally, the phenomenon of slow inactivation and its relevance to action potential frequency  is not well addressed, see below:

paragraph starting line 60: This section needs to be re-worked. The use-dependent inhibition of sodium current amplitude as reflecting a decreased availability is written as if this phenomenon has not been investigated from a mechanistic standpoint. Use-dependent inhibition is typically one means to demonstrate the impact of slow inactivation on sodium channels. There is a body of literature on the proposed mechanism of action of slow inactivation, a complex process which may comprise several states of slow inactivation. Thus the overall mechanism of slow  inactivation remains an active area of research, yet both the mechanism involving voltage sensor transition and pore module conformational changes have been studied extensively in prokaryotic or eukaryotic sodium channels. In addition, the pharmacology of slow inactivation has received considerable attention, since slow inactivation is an important aspect of sodium channelopathies. To that end, one of the small molecules emphasized in this report, lacosamide, has been characterized with its primary impact as an anti-epileptic drug, by targeting slow inactivation. The review would be improved by the addition of an examination of this target (slow inactivation) of small molecule perturbation under high frequency conditions.

 

Table 1  lists references that are not included in the References section, and those that are included (i.e. 19) should be checked for their appropriate citation for the specific pharmaceutical in this Table.

2. Effects of small-molecule modulators on INa during high-frequency stimulation (20 or 40 Hz)

2.1. The first sentence does not have a predicate.

line 93 "included" should be "include"

line 102 "exhibited" should be "exhibit". There is no reference to the effect of CBZ on (presumably steady-state) fast inactivation, or slowed recovery.

line 107: The authors should state the well described, differential impact of INa (t) and INa (L) and make a distinction between persistent late current and resurgent late current.  These currents are especially well studied in DRG neurons and this may be relevant to CBZ action on neuropathic pain. Lines 107 to 112 are vague and without references of any specific studies of INa.

2.2

line 126 What is the means of determining of EC50 for GV-58; in other words what is measured for the effects of GV-58 transient and late sodium currents to be used to create a dose response curve? Is there a comparison of absolute magnitude of effect or just the dose response? The term "2,6 uM" should have a period not comma.

line 132 This is speculative and there is no reference to support the notion that slow inactivation requires channels to open prior to reaching this state, nor that recovery from slow inactivation open-state.

2.3

Extensive work has been done to determine the primary action of this modulator is on slow inactivation. Loss of function with lacosamide is ascribed to enhanced slow inactivation. A distinction between this effect and that of lacosamide actions on fast gating is not presented.

2.4

Are there any studies to examine the effect  of MGB on NaV channels in heterologous expression to more clearly address the direct versus indirect (CaV channel) influence on sodium current?

2.5

See above.  In studies of native cells such as neurons, or others such as neuroblastoma cells, one drawback is that the effect of a modulator may be on several distinct channels or other proteins, and dissection of these effects (with other modulators) is difficult. Overexpression of a channel, use of heterologous expression in cells lacking prominent channel expression (i.e. oocytes) has its own drawbacks but does offer a  reductionist approach. Has such an approach been used to study phenobarbital?

Comments on the Quality of English Language

Overall the English is fine. However, it is recommended  that the authors carefully proof this manuscript for errors in tense. Use of present versus past tense matters in statements that should read as relating to previous investigations, for example

Author Response

Lin C-Y, Gao Z-H, Cheung C-W, So EC, Wu S-N. "Small-molecule perturbations on voltage-gated Na+ currents: alterations in amplitude and gating properties under high frequency stimulation. Submitted to Scientia Pharmaceutica.

Abstract:

This section reads as an abstract for a primary research article. Statements about the effects of drugs are presented here as if there are novel results from experiments not previously published. After stating these putative experimental results, the authors claim that "Our investigations show that when cells are exposed to small-molecule modulators, these compounds can perturb INa, under high frequency stimulation condition".

Ans: We thank the reviewer for this insightful comment. We acknowledge that the language in the indicated section may give the impression that we are reporting novel experimental findings. Our intention, however, was to summarize findings from previously published literature in a way that highlights the broader implications of small-molecule modulators on I_Na under high-frequency stimulation. To clarify this, we have revised the sentence in question to.  “Previous studies have shown that exposure to certain small-molecule modulators can perturb I_Na during high-frequency stimulation, influencing the extent of cumulative inhibition and electrical excitability in excitable cells.” (lines 36-38).

        This revision makes it clear that we are referring to findings reported in the literature, not presenting new experimental data. We have also reviewed the surrounding text to ensure that no other statements unintentionally imply novel findings.

There are no novel experiments presented, nor any methodology for such putative experiments. The authors need to clearly identify this work as a mini-review of existing literature, and that this review is comprised of previous work.

Ans: We appreciate the reviewer’s careful reading and constructive feedback. We agree that the current manuscript may have unintentionally implied the presence of original experimental data. To clarify the nature of this review article, we have made revisions throughout the manuscript to explicitly state that this work is a review synthesizing finding from previously published studies.  Specifically, we have added the following clarifying sentence to the Introduction. That is, “This review synthesizes existing research on the amplitude and gating characteristics of I_Na, as well as the cumulative inhibition of I_Na during high-frequency stimulation, with particular emphasis on the modulatory effects of various small molecules, as summarized in Table 1.” (lines 94-97).

Specifically:

1. The description of the effects of these compounds must be given as review of prior work. There are no tables or figures from novel work presented, nor is there a Methods section.

Ans: We thank the reviewer for this important observation. We agree that the descriptions of compound effects must be framed within the context of previously published research. To ensure this is clear to readers, we have revised the manuscript to consistently reflect that all data and interpretations are drawn from previously published studies. Specifically, we have: (1) explicitly identified the article as a review in both the abstract and introduction; (2) avoided any language that might suggest original experimentation or novel data; (3) confirmed that no methods section is included, as this work does not present original experiments; and (4) clarified that Table 1 summarizes findings from the existing literature, and do not originate from novel analyses or experiments. We appreciate the reviewer’s guidance in helping us maintain transparency and precision regarding the nature of this work.

 

1. This work should read as a review and the statement "Our investigations show that when cells are exposed to small-molecule modulators, these compounds can perturb INa, under high frequency stimulation condition" should be changed to not claim that the description of results of experiments in this manuscript are solely from this group.

Ans: We appreciate the reviewer’s thoughtful feedback. We agree that the original phrasing could be misinterpreted as implying that the described findings result from our own experimental investigations. As this manuscript is strictly a review of previously published work, we have revised the statement to ensure that the information is derived from prior studies and not from original research conducted by our group. The revised sentence in the revised manuscript now reads: “Previous investigations have shown that exposure to small-molecule modulators can perturb I_Na under conditions of high-frequency stimulation.” (lines 36-38).

 

Similarly, from the Introduction:

line 78: "In this study, we aim to elucidate the effects of a range of intriguing small-molecule modulators (outlined in Table 1) on the amplitude and gating properties of INa under conditions of high frequency stimulation." This reads as if this manuscript comprises experimental investigation of these small molecules, which it does not, but instead is a review of prior work.

Ans: We appreciate the reviewer’s observation and agree that the original phrasing may give the misleading impression that this manuscript presents novel experimental work. As this article is a review, its aim is to synthesize and contextualize findings from previously published studies. To avoid any confusion, we have revised the sentence. That is, “This review synthesizes existing research on the amplitude and gating characteristics of INa, as well as the cumulative inhibition of I_Na during high-frequency stimulation, with particular emphasis on the modulatory effects of various small molecules, as summarized in Table 1.” (lines 94-97).

1. Introduction:

This section moves towards the eventual goal of describing sodium channel involvement in high frequency action potential signaling and the effect of several small molecules on sodium channels which influence action potential frequency. This could be improved by providing more detail on two aspects of sodium channel function that are a focal point later, the transient and late sodium current, as well as another sodium channel transition, slow inactivation, that receives very little attention throughout. First, the state transitions relevant to the transient sodium current are adequately described with respect to action potential frequency in the paragraph starting line 53. Then, there needs to be a description of persistent and resurgent late sodium currents with detail on how these currents influence action potential frequency. This is not intuitive, as a depolarizing influence from late current can provide excitatory drive, or promote depolarization block. Given the authors statement that later that INa (t) and INa (L) are critical components, there needs to be a thorough description of these currents prior to the sections on the various modulators. Finally, the phenomenon of slow inactivation and its relevance to action potential frequency  is not well addressed, see below:

Ans: We thank the reviewer for the detailed and insightful comments, which have helped us substantially improve the clarity and depth of our manuscript. Hence, an additional paragraph was included in the revised manuscript. That is, “The transient Na⁺ current (I_Na(T)) represents the fast-activating and rapidly inactivating component responsible for the upstroke of the action potential.  In addition to this primary current, Na_V channels give rise to late Na⁺ currents (I_Na(L)), which include both persistent and resurgent components. The persistent sodium current (I_Na(P)) consists of a small, non-inactivating inward current that can lower firing thresholds and promote repetitive firing, while the resurgent Na⁺ current emerges during repolarization and is thought to support high-frequency firing, particularly in specialized neuronal populations. Moreover, slow inactivation, a distinct gating process that develops over prolonged depolarizations, serves as a protective mechanism by reducing Na_V channel availability during sustained activity and modulating responsiveness to high-frequency input. Together, these current components and gating transitions are critical in defining the cellular response to high-frequency stimulation and are thus essential targets for pharmacological modulation “ (lines 60-72). These additions strengthen the physiological context for our discussion of pharmacological modulators and address the reviewer’s concern that the manuscript lacked sufficient background before transitioning into compound-specific effects.

 

paragraph starting line 60: This section needs to be re-worked. The use-dependent inhibition of sodium current amplitude as reflecting a decreased availability is written as if this phenomenon has not been investigated from a mechanistic standpoint. Use-dependent inhibition is typically one means to demonstrate the impact of slow inactivation on sodium channels. There is a body of literature on the proposed mechanism of action of slow inactivation, a complex process which may comprise several states of slow inactivation. Thus the overall mechanism of slow  inactivation remains an active area of research, yet both the mechanism involving voltage sensor transition and pore module conformational changes have been studied extensively in prokaryotic or eukaryotic sodium channels. In addition, the pharmacology of slow inactivation has received considerable attention, since slow inactivation is an important aspect of sodium channelopathies. To that end, one of the small molecules emphasized in this report, lacosamide, has been characterized with its primary impact as an anti-epileptic drug, by targeting slow inactivation. The review would be improved by the addition of an examination of this target (slow inactivation) of small molecule perturbation under high frequency conditions.

Ans: We thank the reviewer for their expert insight and for highlighting this important oversight. We agree that the original phrasing did not adequately acknowledge the extensive body of mechanistic research surrounding use-dependent inhibition and its strong linkage to slow inactivation of voltage-gated sodium channels. To address this, we have significantly revised the relevant section (starting at line 60) to reflect the current understanding of slow inactivation as a multistate process influenced by both voltage sensor dynamics and conformational changes in the pore module, supported by evidence from both prokaryotic and eukaryotic sodium channel studies (lines 75-79).

In addition, we highlight the importance of this mechanism in pharmacology, particularly in the case of lacosamide, which we have revised to clearly state as a well-characterized antiepileptic agent that selectively enhances slow inactivation of sodium channels. (lines 226-234).

To better support this point, we have added a reference (ref #5) to representative studies on the structural and functional aspects of slow inactivation and expanded our review of small-molecule modulators that target this mechanism under high-frequency stimulation conditions. We believe these revisions significantly improve the depth and clarity of our manuscript and appreciate the reviewer’s guidance in helping us highlight this crucial aspect of sodium channel function and pharmacology.

 

Table 1  lists references that are not included in the References section, and those that are included (i.e. 19) should be checked for their appropriate citation for the specific pharmaceutical in this Table.

Ans: We thank the reviewer for carefully reviewing the reference list and identifying inconsistencies related to Table 1. We have thoroughly reviewed the table and cross-checked all citations to ensure accuracy and completeness. Specifically, we have added any missing references cited in Table 1 to the References section. We have also verified that all references already included (e.g., Reference #7) appropriately support the information provided in the table regarding the pharmacological properties of the respective small molecules. We have also revised the style within Table 1, where necessary, to ensure proper alignment with the reference numbers and formatting used throughout the manuscript.

We appreciate the reviewer’s attention to detail in helping us improve the accuracy and scholarly integrity of the manuscript.

 

1. Effects of small-molecule modulators on INa during high-frequency stimulation (20 or 40 Hz)

Ans: High-frequency stimulation at 20 or 40 Hz used to study effects of small-molecule modulators on I_Na is generally acceptable, although 20 Hz is usually sufficient. However, when the frequency exceeds 50 Hz, mammalian neurons are more prone to damage, and the resistance between the electrode and the cell membrane may also decrease.

2.1. The first sentence does not have a predicate.

Ans: The sentence was changed to “Carbamazepine (CBZ) is an aromatic anticonvulsant commonly used to treat seizures and neuropathic pain, such as trigeminal neuralgia” (lines 108-109).

line 93 "included" should be "include"

Ans: As pointed out by the reviewer, “included” was hence replaced with “include” (line 113).

line 102 "exhibited" should be "exhibit". There is no reference to the effect of CBZ on (presumably steady-state) fast inactivation, or slowed recovery.

Ans: The sentence was changed to “CBZ’s effect on I_Na(T) inactivation, triggered by pulse-train depolarizing stimuli, was reported to exhibit an excessive enhancement.  The recovery of I_Na(T) inactivation during varying interpulse intervals was slowed in the CBZ presence [7].” (lines 138-140).

line 107: The authors should state the well described, differential impact of INa (t) and INa (L) and make a distinction between persistent late current and resurgent late current.  These currents are especially well studied in DRG neurons and this may be relevant to CBZ action on neuropathic pain. Lines 107 to 112 are vague and without references of any specific studies of INa.

Ans: We thank the reviewer for this valuable comment and agree that the distinctions between I_Na(T), persistent I_Na(L), and resurgent I_Na(L) currents should be more clearly articulated. In response, we have revised the section beginning at line 107 to explicitly describe the differential roles of these Na⁺ current components in shaping neuronal excitability, particularly under high-frequency stimulation.

We now clarify that: I_Na(T) is responsible for the rapid depolarization phase of the action potential. Persistent I_Na(L) contributes to subthreshold depolarization and repetitive firing. Resurgent I_Na(L) supports high-frequency firing by enabling rapid recovery from inactivation.

We have also emphasized that these current components are especially well characterized in dorsal root ganglion (DRG) neurons, where their dysregulation is implicated in neuropathic pain. This is particularly relevant to the mechanism of action of carbamazepine (CBZ), which has been shown to modulate Na_V channel activity in DRG neurons and is widely used in the treatment of neuropathic pain.

We appreciate the reviewer’s suggestion, which has helped us improve the clarity, specificity, and scientific rigor of this portion of the manuscript.

2.2

line 126 What is the means of determining of EC50 for GV-58; in other words what is measured for the effects of GV-58 transient and late sodium currents to be used to create a dose response curve? Is there a comparison of absolute magnitude of effect or just the dose response? The term "2,6 uM" should have a period not comma.

Ans: We thank the reviewer for this important observation. In the study, the EC₅₀ values for GV-58 were determined by measuring its concentration-dependent effects on both transient (peak) and late components of I_Na.in GH₃ pituitary cells. Specifically, whole-cell patch-clamp recordings were used to quantify the amplitude of I_Na in response to depolarizing voltage steps across a range of GV-58 concentrations. The resulting concentration-response relationships were fitted using nonlinear regression to estimate EC₅₀ values, which were found to be 8.9 mM for peak I_Na and 2.6 mM for late I_Na. These values reflect the potency of GV-58 in enhancing each current component, rather than a direct comparison of their absolute magnitudes. We have corrected the typographical error in the manuscript by replacing “2,6 mM” with “2.6 mM”, as indicated in lines 162-163).

line 132 This is speculative and there is no reference to support the notion that slow inactivation requires channels to open prior to reaching this state, nor that recovery from slow inactivation open-state.

Ans: As advised by the reviewer, because of being speculative, the sentence “The post-spike I_Na during rapid repetitive stimuli is linked to the prominent slow inactivation in GH₃ cells, suggesting such slow inactivation develops from open channels, and, if recovery from slow inactivation occurs through the open state(s) (conformation(s)), it would be accompanied by a small residual steady current.” appearing in the original manuscript, has been removed from the revised manuscript.

2.3

Extensive work has been done to determine the primary action of this modulator is on slow inactivation. Loss of function with lacosamide is ascribed to enhanced slow inactivation. A distinction between this effect and that of lacosamide actions on fast gating is not presented.

Ans: Thanks for the valuable comments provided by the reviewer. In response, an additional paragraph has been included in the revised manuscript. That is, “Previous electrophysiological studies have demonstrated that LCS selectively enhances the slow inactivation of NaV channels, distinguishing it mechanistically from traditional antiepileptic drugs that primarily affect fast inactivation.  This enhancement stabilizes hyperexcitable neuronal membranes and reduces repetitive firing without significantly altering fast gating kinetics.  While LCS’s primary mechanism is well characterized, a detailed comparative analysis of its effects on fast versus slow inactivation has not been comprehensively presented.  Therefore, further investigations—particularly using voltage-clamp protocols designed to isolate fast inactivation parameters—would be valuable to delineate any subtle modulatory effect on fast gating.” (lines 226-234).

2.4

Are there any studies to examine the effect  of MGB on NaV channels in heterologous expression to more clearly address the direct versus indirect (CaV channel) influence on sodium current?

Ans: Thanks for the valuable comments by the reviewer. We thank the reviewer for this important point. To our knowledge, no studies have yet investigated the direct effect of mirogabalin (MGB) on Na_V channels in heterologous expression systems such as HEK293 cells or Xenopus oocytes. The present findings in native cells suggest modulation of Na⁺ currents; however, we acknowledge that this may be an indirect consequence of mirogabalin’s action on Ca_V channels via the α2δ-1 subunit. Future studies using heterologous expression of Na_V channels, with or without co-expression of α2δ-1, will be essential to clarify whether mirogabalin exerts a direct modulatory effect on Na_V channel function. Hence, an additional paragraph was included in the revised manuscript (lines 288-294).

2.5

See above.  In studies of native cells such as neurons, or others such as neuroblastoma cells, one drawback is that the effect of a modulator may be on several distinct channels or other proteins, and dissection of these effects (with other modulators) is difficult. Overexpression of a channel, use of heterologous expression in cells lacking prominent channel expression (i.e. oocytes) has its own drawbacks but does offer a  reductionist approach. Has such an approach been used to study phenobarbital?

Ans: We appreciate the reviewer’s thoughtful observation regarding the complexity of interpreting modulator effect in native cells due to the presence of multiple ion channels and interacting proteins. Indeed, heterologous expression systems such as Xenopus oocytes or HEK293 cells offer a reductionist approach that can help isolate the effects of a compound on specific ion channel subtype.

        To our knowledge, phenobarbital has not been extensively studied using heterologous expression systems specifically to dissect its direct effects on individual volage-gated ion channels. Most investigators have focused on its broader pharmacological actions, including enhancement of GABA_A receptor activity and modulation of neuronal excitability in native or mixed- cell environments. However, studies have explored phenobarbital’s molecular mechanisms through gene induction pathways, such as its interaction with nuclear receptors like the constitutive androstane receptor and pregnane X receptor (Men and Wang, Drug Metab Dispos 2023;51(2):210-218), which have been examined in heterologous systems to understand transcriptional regulation.

        We agree that applying heterologous expression systems to study phenobarbital’s direct effects on specific ion channels—such as Na_V channels—would provide valuable mechanistic insights and help disentangle its multi-target actions. We have added a brief discussion of this point in the revised manuscript, as indicated in lines 352-358).

 

Comments on the Quality of English Language

Overall the English is fine. However, it is recommended  that the authors carefully proof this manuscript for errors in tense. Use of present versus past tense matters in statements that should read as relating to previous investigations, for example

Ans: We thank the reviewer for the careful reading and thoughtful suggestion regarding tense usage. We agree that the distinction between present and past tense is important, particularly when referencing prior investigations versus the current study. In response, we have conducted a thorough review of the manuscript and have revised the text where necessary to ensure appropriate and consistent use of tense. Specifically, we now use the past tense when discussing findings from previous studies, and the present tense when referring to accepted facts or current interpretations based on our results. These changes are intended to improve clarity and maintain consistency throughout the revised manuscript.

Reviewer 2 Report

Comments and Suggestions for Authors

Lin et al. review how various small molecules (carbamazepine, GV-58, lacosamide, mirogabalin (MGB) and phenobarbital (PHB)) affect voltage-gated Na⁺-channel currents (I_Nas) evoked by a high-frequency depolarized pulse.  I_Na in response to the pulse is schematically shown in Fig. 1, and the chemical structures of the molecules are given in Table 1.  Figure 2 gives a schematic diagram showing that MGB inhibits not only I_Na but also voltage-gated Ca²⁺-channel currents, resulting in alleviating neuropathic pain.  Moreover, it is schematically shown in Fig. 3 that PHB suppresses multiple ion currents induced by high-frequency depolarized pulse while enhancing GABA_A-receptor channels, leading to sedative/hypnotic, anesthetic and anticonvulsant effects.  Figure 4 demonstrates that inhibition by their drugs of I_Nas evoked by high-frequency stimulation results in modulating stimulus-secretion coupling, membrane excitability and action potential firing.  There are known to be countless small molecule modulators, so it is unclear why the chemicals shown in Table 1 were selected.  So many mistakes are noted throughout the manuscript.  Some of points, which should be addressed and may serve to amend this manuscript, are as follows:

 

1. Title: this should be revised, because Figs. 2 and 3 also show the effects of the drugs on channel currents other than I_Na.
2. Line 23: not “.. current ..” but “.. channel current ..”? Please amend this point.
3. Line 28: not “were” but “was”.
4. Line 38: is there any need to emphasize “in vitro or in vivo” here? Please reply to this question.
5. Keywords: “persistent Na⁺ current” seems to be inappropriate as keywords, because this type of I_Na appears only three times in the text (see lines 173, 182 and 184). Please reply to this question.
6. Line 48: is “are” OK? Please check English.
7. Lines 51 and 52: “heart” is contained in ”muscles”. Please amend this point.
8. Line 57: in “.. of in ..” OK? Please check English.
9. Table 1: it is unclear what the various colored atoms in the chemical structures shown in this Table represent. Please make this point clear.  “stimu    lation” should be amended.
10. Line 88: not “.. useful ..” but “.., is useful ..”? Please check English.
11. Line 91: not “, myotonia” but “and myotonia”? Please check English.
12. Lines 95 and 96: please explain shortly how transient I_Na (I_Na(T)) component differs from late one (I_Na(L)). What is the difference between the mechanisms behind these two current components?  Please reply to this question.
13. Line 97: please spell out “IC₅₀”.
14. Line 98: not “18μM” but “18 μM”. Please put a space between value and unit throughout this manuscript.
15. Line 99: “window Na⁺ current “ should be shortly explained.
16. Line 105: it is not necessary to repeatedly define “Nav” (please see line 44). Please amend this point.
17. Lines 124 and 125: it is not necessary to repeatedly define “I_Na(T)” and “I_Na(L)” (please see lines 95 and 96). Please amend this point.
18. Line 126: please spell out “EC₅₀”.
19. Line 127: not “2,6” but “2.6”.
20. Line 148: please remove the leftmost character (a.).
21. Line 173: “persistent Na⁺ current” should be shortly explained.
22. Line 185: not “figure-eight” but “figure-of-eight” (please see line 183)? Please check this point.
23. Line 192: it is not necessary to repeatedly define “Cav” (please see line 116). Please amend this point.
24. Line 212: “tefluthrin” should be explained here, not in line 252.
25. Line 242: not “depress” but “depresses”? Not “slow” but “slows”?  Please check English.
26. Line 247: not “suppress” but “suppresses”? Please check English.
27. Line 249: not “GABA receptors” but “GABA_A receptors”? Please check this point.
28. Line 251: it is not necessary to repeatedly define “Tef” (please see line 212). Please amend this point.
29. Line 262: please specify “x” in “KCNQx”.
30. Lines 266 and 267: “hyperpolarize the after-potential” is scientifically not right. Please amend this point.
31. 3: “γ-aminobutyric acid receptor” should be “γ-aminobutyric acid_A receptor”.
32. Line 285: “(GABA)” should be “(GABA)_A”.
33. The effects of MGB and PHB on ion channels and the resulting physiological phenomena have been illustrated in Figs. 2 and 3, respectively, which have not been done with other substances (carbamazepine, GV-58 and lacosamide). This point should be amended.
34. The neuropathic pain alleviation produced by MGB (see Fig. 2) and the PHB-induced sedative/hypnotic, anesthetic and anticonvulsant effects (see Fig. 3) are not reflected in Fig. 4, which summarizes the results of the main text. Please amend this point.
35. Abbreviations: “I_Na(P)” is not in this list. Please confirm that all the abbreviations used in the text are in this list.
36. There are likely many more scientific and English language errors than those mentioned above. This manuscript should be checked very carefully.

Comments on the Quality of English Language

English language should be amended.

Author Response

Reviewer 2 Comments and Suggestions for Authors

Lin et al. review how various small molecules (carbamazepine, GV-58, lacosamide, mirogabalin (MGB) and phenobarbital (PHB)) affect voltage-gated Na⁺-channel currents (I_Nas) evoked by a high-frequency depolarized pulse.  I_Na in response to the pulse is schematically shown in Fig. 1, and the chemical structures of the molecules are given in Table 1.  Figure 2 gives a schematic diagram showing that MGB inhibits not only I_Na but also voltage-gated Ca²⁺-channel currents, resulting in alleviating neuropathic pain.  Moreover, it is schematically shown in Fig. 3 that PHB suppresses multiple ion currents induced by high-frequency depolarized pulse while enhancing GABA_A-receptor channels, leading to sedative/hypnotic, anesthetic and anticonvulsant effects.  Figure 4 demonstrates that inhibition by their drugs of I_Nas evoked by high-frequency stimulation results in modulating stimulus-secretion coupling, membrane excitability and action potential firing.  There are known to be countless small molecule modulators, so it is unclear why the chemicals shown in Table 1 were selected.  So many mistakes are noted throughout the manuscript.  Some of points, which should be addressed and may serve to amend this manuscript, are as follows:

 

Ans: We sincerely thank the reviewer for the thorough and constructive comments. We appreciate the opportunity to clarify and improve our manuscript. Below is our detailed response to the points raised:

• Selection of small molecules (Table 1): We acknowledge the reviewer’s concern regarding the selection of small molecules. Our choice was based on two key criteria: (a) compounds that are known or clinically used to influence excitability in neural or muscular tissues, and (b) agents for which prior literature supports an interaction with Na_V channels, especially under conditions mimicking high-frequency stimulation. These molecules were selected to illustrate representative mechanisms through which modulation of I_Na may affect excitability and stimulus-secretion coupling. We have clarified this selection rationale in the revised manuscript. Of note, Table 1 was redone.
• Figure legends and schematic representations: We appreciate the reviewer’s comments on the schematic figures. We have modified Figure 4 to better explain their scientific context and visual meaning. Additional textual clarifications have also been added to the revised manuscript to ensure the message conveyed in each schematic is fully supported by experimental findings.
• MGB and PHB mechanisms (Figures 2 and 3): In response to the reviewer’s comment, we revised the descriptions of MGB and PHB actions to more accurately reflect their effects on ionic current and receptor (i.e., GABA_A) activity. Specifically, we emphasized that MGB inhibits both I_Na and I_Ca in a frequency-dependent manner, and that PHB exerts dual actions—suppressing depolarization-induced ionic currents while enhancing GABA_A-mediated inhibition. Corresponding references have been shown to substantiate these mechanisms.
• I_Na inhibition and functional outcomes (Figure 4): We revised the description of Figure 4 to clarify how high-frequency stimulation and cumulative I_Na inhibition influence physiological processes such as membrane excitability, stimulus-secretion coupling, and AP firing. These connections are grounded in previous electrophysiological studies and our own observation, which are now more clearly articulated in the revised figure legend and discussion.
• General clarifications and language revisions: We apologize for any oversight in language and technical phrasing throughout the manuscript. A comprehensive revision has been performed to correct typographical and grammatical issues and to improve scientific clarity. We hope these changes address the reviewer’s concerns regarding errors and improve the overall readability and precision of the text.

 

The following reply corresponds to the sequence in which the reviewer’s questions were presented.

 

1. Title: this should be revised, because Figs. 2 and 3 also show the effects of the drugs on channel currents other than I_Na.

Ans: Thank you for the reviewer’s valuable suggestions. Although some of the compounds discussed in this study may also affect other currents besides I_Na, our primary focus is on the amplitude and gating kinetics of I_Na, particularly under conditions of high-frequency stimulation. Nevertheless, we agree with the reviewer’s perspective and have accordingly revised the title of the revised manuscript to “Modulation of voltage-gated Na⁺ currents by small molecules: effects on amplitude and gating during high-frequency stimulation” (indicated in the red color).

1. Line 23: not “.. current ..” but “.. channel current ..”? Please amend this point.

Ans: Apologies—we did not find any specific error regarding the term “current”. Nevertheless, we have revised the sentence as follows for clarity: Cumulative inhibition of voltage-gated Na⁺ current (I_Na) induced by high-frequency depolarization helps regulate electrical activity in excitable cells. A variety of small molecules that modulate the amplitude and/or gating kinetics of I_Na influence the degree of their inhibition. (line 23-27).

1. Line 28: not “were” but “was”.

Ans: Thanks for bringing our attention. “were” was replaced with “was” (line 30).

1. Line 38: is there any need to emphasize “in vitro or in vivo” here? Please reply to this question.

Ans: As pointed out by the reviewer, the sentence was optimally changed to “This ionic mechanism plays a crucial role in modulating membrane excitability thereby, supporting the validity of these findings (lines 38-39).

1. Keywords: “persistent Na⁺ current” seems to be inappropriate as keywords, because this type of I_Na appears only three times in the text (see lines 173, 182 and 184). Please reply to this question.

Ans: As advised by the reviewer, “persistent Na⁺ current” was removed from Keywords. However, “current kinetics” was included in the Keywords section (lines 40-41).

1. Line 48: is “are” OK? Please check English.

Ans: As advised by the reviewer, the sentences were appropriately changed to “Furthermore, the b subunit of Na_V channels serve as auxiliary proteins that regulate the function and expression of the b subunits, which form the ion-conducting pore for Na⁺ ions. These b subunits are essential for the proper operation of Na_V channels and play a critical role in the generation and propagation of action potentials (APs) in excitable tissues, including nerves as well as skeletal and cardiac muscles.” (lines 46-51).

1. "Lines 51 and 52: “heart” is contained in ”muscles”. Please amend this point.

Ans: Thanks for bringing our attention. The sentence was changed to “….., including nerves as well as skeletal and cardiac muscles. (lines 50-51).

1. Line 57: in “.. of in ..” OK? Please check English.

Ans: Thanks for bringing our attention. The sentence was change to “The I_Na amplitude, once triggered, propels the cell membrane into depolarization through a positive feedback cycle, initiating the AP upstroke in excitable cells.”. (lines 55-57).

1. Table 1: it is unclear what the various colored atoms in the chemical structures shown in this Table represent. Please make this point clear.  “stimu    lation” should be amended.

Ans: Thanks for bringing our attention. Table 1 was redone. The text in Table 1 was appropriately changed (Lines 101-103).

1. Line 88: not “.. useful ..” but “.., is useful ..”? Please check English.

Ans: Thanks for bringing our attention. Hence, the sentence was changed to “Carbamazepine (CBZ) is an aromatic anticonvulsant commonly used to treat seizures and neuropathic pain, such as trigeminal neuralgia” (lines 108-109).

1. Line 91: not “, myotonia” but “and myotonia”? Please check English.

Ans: As pointed out by the reviewer, the sentence has been properly changed to “It is also used as an adjunctive treatment for schizophrenia and myotonia, and serves as a second-line treatment option for bipolar disorder.” (lines 110-112).

1. Lines 95 and 96: please explain shortly how transient I_Na (I_Na(T)) component differs from late one (I_Na(L)). What is the difference between the mechanisms behind these two current components?  Please reply to this question.

Ans: As advised by the reviewer, several additional sentences were included in the revised manuscript. That is, “I_Na(T) is the rapid, short-lived influx of Na⁺ that occurs right at the beginning of an action potential. It activates and then quickly inactivates within a few milliseconds.  I_Na(L) is a much smaller but persistent current that continues flowing during the plateau phase of the action potential.  It results from a small portion of Na_V channels either failing to inactivate completely or reopening intermittently.  Mechanistically, the key difference is that I_Na(T) is driven by fast activation and rapid inactivation of Na_V channels, whereas I_Na(L) arises from slow or incomplete inactivation, or stochastic reopening of these same channels.” (lines 116-123).

1. Line 97: please spell out “IC₅₀”.

Ans: IC₅₀ (half maximal inhibitory concentration) is the concentration of a compound that is required to reduce a specific biological or biochemical function by 50% under defined conditions. (lines 129-131).

1. Line 98: not “18μM” but “18 μM”. Please put a space between value and unit throughout this manuscript.

Ans: Goof! “18mM” was replaced with “18 mM”. (line 129).

1. Line 99: “window Na⁺ current “ should be shortly explained.

Ans: Window Na⁺ current refers to a small, steady, non-inactivating component of sodium current that occurs when the activation and inactivation curves of voltage-gated sodium channels (Na_V channels) overlap within a certain voltage range. The sentence was included in the revised manuscript.

1. Line 105: it is not necessary to repeatedly define “Nav” (please see line 44). Please amend this point.

Ans: As noted by the reviewer, “voltage-gated Na⁺..” was hence removed from text in the revised manuscript (line 107).

1. Lines 124 and 125: it is not necessary to repeatedly define “I_Na(T)” and “I_Na(L)” (please see lines 95 and 96). Please amend this point.

Ans: The sentence was accordingly to “….. the differential stimulation by GV-58 of I_Na(T) and I_Na(L) may participate in the regulation of electrical activities of excitable cells….” (lines 160-162).

1. Line 126: please spell out “EC₅₀”.

Ans: EC₅₀ (half maximal effective concentration) is a pharmacological term that refers to the concentration of a compound (or ligand) that produces 50% of its maximum possible effect.  The sentence was included in the revised manuscript (lines 163-165).

 

1. Line 127: not “2,6” but “2.6”.

Ans: “2,6” was replaced with “2.6” (indicated in line 162).

1. Line 148: please remove the leftmost character (a.).

Ans: Goof! “a” was removed from the text of the removed manuscript.

1. Line 173: “persistent Na⁺ current” should be shortly explained.

Ans: Persistent sodium current (I_Na(P)) refers to a non-inactivating or slowly inactivating component of the voltage-gated sodium current (I_Na) that persists after the initial fast transient current has decayed. The sentence was included in the revised manuscript (lines 208-209).

1. Line 185: not “figure-eight” but “figure-of-eight” (please see line 183)? Please check this point.

Ans: Thanks! “figure-eight” was hence replaced with “figure-of-eight” (line 222).

1. Line 192: it is not necessary to repeatedly define “Cav” (please see line 116). Please amend this point.

Ans: As noted by the reviewer, the sentence was changed to “MGB, an orally administered gabapentinoid, was reported to act as a ligand for the a2d-1 subunit of Ca_V channels.”. (lines 237-238).

1. Line 212: “tefluthrin” should be explained here, not in line 252.

Ans: As advised by the reviewer, the sentence was moved up to lines 259-260 in the revised manuscript.

1. Line 242: not “depress” but “depresses”? Not “slow” but “slows”?  Please check English.

Ans: Thanks for bringing our attention. The sentence was hence changed to “PHB is a barbiturate that depresses the central nervous system, slowing down brain activities and producing both anticonvulsant and hypnotic effects.: (lines 288-289).

1. Line 247: not “suppress” but “suppresses”? Please check English.

Ans: Goof! We made a mistake. “suppress” was hence replaced with “suppresses” (in line 293 of the revised manuscript).

1. Line 249: not “GABA receptors” but “GABA_A receptors”? Please check this point.

Ans: GABA receptors were replaced by GABAA receptors (indicated in line 295).

1. Line 251: it is not necessary to repeatedly define “Tef” (please see line 212). Please amend this point.

Ans: Thanks! As indicated by the reviewer, “Tefluthrin” was hence removed from the revised manuscript. (indicated in line 297).

29. Line 262: please specify “x” in “KCNQx”.

Ans: In this context, KCNQx primarily refers to KCNQ2, KCNQ3, and KCNQ5. KCNQ1 is predominantly expressed in the heart, while KCNQ4 is mainly found in the inner ear and auditory system. (indicated in lines 308-310 of the revised manuscript).

1. Lines 266 and 267: “hyperpolarize the after-potential” is scientifically not right. Please amend this point.

Ans: In this context, "after-potential" refers to the membrane potential following long-lasting high-frequency stimulation. During such stimulation, I_K(M) may accumulate progressively. As a result, when the high-frequency stimulation ceases, the enhanced I_K(M) leads to a more hyperpolarized after-potential. This membrane hyperpolarization facilitates faster recovery of Na_V channels from inactivation, thereby increasing their availability. Hence, additional sentences were included in lines 316-320 in the revised manuscript.

1. 3: “γ-aminobutyric acid receptor” should be “γ-aminobutyric acid_A receptor”.

Ans: Thanks! The sentence was changed to “The drug stimulates g-aminobutyric acid type A (GABA_A) receptors and concurrently exerts inhibitory effects on various ion currents across the cell membrane.” (lines 337-339).

1. Line 285: “(GABA)” should be “(GABA)_A”.

Ans: Thanks! GABA was replaced with GABA_A (line 337) in the revised manuscript.

1. The effects of MGB and PHB on ion channels and the resulting physiological phenomena have been illustrated in Figs. 2 and 3, respectively, which have not been done with other substances (carbamazepine, GV-58 and lacosamide). This point should be amended.

Ans: We thank the reviewer for the helpful reminder. To address the reviewer’s concern, Table 1 has been replaced to include carbamazepine, GV-58, lacosamide, mirogabalin, and phenobarbital. The corresponding references have also been provided.

 

1. The neuropathic pain alleviation produced by MGB (see Fig. 2) and the PHB-induced sedative/hypnotic, anesthetic and anticonvulsant effects (see Fig. 3) are not reflected in Fig. 4, which summarizes the results of the main text. Please amend this point.

Ans: We thank the reviewer for the helpful reminder. Accordingly, we have added the symbols * and # in Figure 4, and an additional sentence has been included in the figure legend as follows: ^*Mirogabalin alleviates neuropathic pain, whereas ^#phenobarbital exerts sedative, hypnotic, anesthetic, and anticonvulsant effects.” (indicated in lines 374-375 of the revised manuscript).

 

1. Abbreviations: “I_Na(P)” is not in this list. Please confirm that all the abbreviations used in the text are in this list.

Ans: Thank you for the reviewer’s reminder. The abbreviation “I_Na(P)” has been included in the list of abbreviations in the revised manuscript.

 

1. There are likely many more scientific and English language errors than those mentioned above. This manuscript should be checked very carefully.

Ans: Thank you for the reviewer’s comments. All Scientific and language-related errors have been extensively corrected in the revised manuscript.

 

Comments on the Quality of English Language

English language should be amended.

Submission Date

03 June 2025

Date of this review

26 Jun 2025 09:10:30

 

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

This revised manuscript has been largely amended.  There remain to be several minor points that may serve to amend this manuscript, as follows:

 

1. Lines 2, 23, 40, 54 and 86: if “voltage-gated” is stated, not “currents” but “channel currents”?
2. Line 116: not “I_Na((T) ” “I_Na(T) ”.
3. Lines 134 and 135: not “voltage-gated sodium channels (Na_V channels)” but “Na_V channels”?
4. Line 168: not “[9]” but “, [9]”?
5. Line 183: not “(R)” but “(2R)”?
6. Line 291: not “CaV” but “Cav”.
7. References do not appear to be presented in a uniform manner, as noted from comparison of the titles of Refs. 10 and 11. Please amend this point.

Author Response

1. Lines 2, 23, 40, 54 and 86: if “voltage-gated” is stated, not “currents” but “channel currents”?

Ans: Thank you for bringing this to our attention. As advised by the reviewer, we replaced “voltage-gated Na⁺ currents” with “voltage-gated Na⁺ channel currents”, as indicated by the reviewer at the specified line number in the revised manuscript.

1. Line 116: not “INa((T) ” “INa(T) ”.

Ans: Thank you to the reviewer for pointing this out. “I_Na((T))” has been corrected to “I_Na(T)”.

1. Lines 134 and 135: not “voltage-gated sodium channels (NaV channels)” but “NaV channels”?

Ans: As advised by the reviewer, the sentence was hence changed to “…when the activation and inactivation curves of Na_V channels overlap within a certain voltage range.” (lines 135-136).

1. Line 168: not “[9]” but “, [9]”?

Ans: As pointed out by the reviewer, the sentence was changed to “.. and further addition of ranolazine, an inhibitor of I_Na(L) [9],…..

1. Line 183: not “(R)” but “(2R)”?

Ans: Thank you for bringing this to our attention. The IUPAC name was changed to “(2R)-2-acetamido-N-benzyl-3-methoxypropanamide)”.

1. Line 291: not “CaV” but “Cav”.

Ans: “CaV” was hence replaced with “Ca_V”.^.

1. References do not appear to be presented in a uniform manner, as noted from comparison of the titles of Refs. 10 and 11. Please amend this point.

Ans: As pointed out by the reviewer, references 10 and 11 in this version of the revised manuscript were hence changed to the following”

10. Wu N., Hsiao C.F. & Chandler S.H. Membrane Resonance and Subthreshold Membrane Oscillations in Mesencephalic V Neurons: Participants in Burst Generation. J Neurosci. 2001; 21(11):3729-3739.
11. Wu P.M., Lin Y.C., Chiang C.W., Cho H.Y., Chuang T.H., Yu M.C., Wu S.N. & Tu Y.F. Effective Modulation by Lacosamide on Cumulative Inhibition of I(Na) during High-Frequency Stimulation and Recovery of I(Na) Block during Conditioning Pulse Train. Int J Mol Sci. 2022; 23(19):11966.