Nav1.2 and BK channel interaction shapes the action potential in the axon initial segment

  1. Luiza Filipis
  2. Laila Ananda Blömer
  3. Jérôme Montnach
  4. Gildas Loussouarn
  5. Michel De Waard
  6. Marco Canepari

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Abstract

In neocortical layer‐5 pyramidal neurons, the action potential (AP) is generated in the axon initial segment (AIS) when the membrane potential ( V m ) reaches the threshold for activation of the voltage‐gated Na + channels (VGNCs) Na v 1.2 and Na v 1.6. Yet, whereas these VGNCs are known to differ in spatial distribution along the AIS and in biophysical properties, our understanding of the functional differences between the two channels remains elusive. Here, using ultrafast Na + , V m and Ca 2+ imaging in combination with partial block of Na v 1.2 by the peptide G 1 G 4 ‐huwentoxin‐IV, we demonstrate an exclusive role of Na v 1.2 in shaping the generating AP. Precisely, we show that selective block of ∼30% of Na v 1.2 widens the AP in the distal part of the AIS and we demonstrate that this effect is due to a loss of activation of BK Ca 2+ ‐activated K + channels (CAKCs). Indeed, Ca 2+ influx via Na v 1.2 activates BK CAKCs, determining the amplitude and the early phase of repolarization of the AP in the AIS. By using control experiments using 4,9‐anhydrotetrodotoxin, a moderately selective inhibitor of Na v 1.6, we concluded that the Ca 2+ influx shaping the early phase of the AP is exclusive of Na v 1.2. Hence, we mimicked this result with a neuron model in which the role of the different ion channels tested reproduced the experimental evidence. The exclusive role of Na v 1.2 reported here is important for understanding the physiology and pathology of neuronal excitability. image

Key points

  • We optically analysed the action potential generated in the axon initial segment of mouse layer‐5 neocortical pyramidal neurons and its associated Na + and Ca 2+ currents using ultrafast imaging techniques.

  • We found that partial selective block of the voltage‐gated Na + channel Na v 1.2, produced by a recently developed peptide, widens the shape of the action potential in the distal part of the axon initial segment.

  • We demonstrate that this effect is due to a reduction of the Ca 2+ influx through Na v 1.2 that activates BK Ca 2+ ‐activated K + channels.

  • To validate our conclusions, we generated a neuron model that reproduces the ensemble of our experimental results.

  • The present results indicate a specific role of Na v 1.2 in the axon initial segment for shaping of the action potential during its generation.

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  1. Version published to 10.1113/jp283801
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    Reply to the reviewers

    We thank the two reviewers for the precious time devoted in the evaluation of our original manuscript and for the useful feedback that guided its revision. We addressed all points raised by the reviewers as detailed below. The changes in the revised manuscript are reported using green characters so that they can be more easily identified in the next evaluation. We would like to comment on the sentence of Reviewer 2 that

    “The bulk of this paper was either demonstrated or predicted by another paper Hanemaaijer et al. in 2020 using very similar methods.”

    We disagree with this statement and here is why. In Hanemaaijer et al. (2020) the authors demonstrate Nav1.2 permeability to calcium by experiments in HEK-293 cells expressing the channel. Then, they show that a calcium transient associated with the action potential (AP) in the AIS is mediated by sodium channels, but the pharmacology they used (TTX) could not distinguish between Nav1.2 and Nav1.6 (both channels being TTX-sensitive) and produced the blockade of the action potential. Thus, they inferred that at least part of the calcium transient associated with the AP is mediated by Nav1.2, but they could not exclude the involvement of Nav1.6. This is extremely important since we clearly demonstrate that it is only Nav1.2 that carries a calcium current and not Nav1.6. Regarding the interaction between Nav1.2 and BK channels, this possibility was only suggested in the Discussion on the basis of the channel distribution and its biophysical properties. However, here again, there was no formal demonstration. This seminal study is of course important nevertheless and hence was cited in our manuscript six times (reference [12]). However, this study did not reach formal conclusions. The reason why the authors could not further advance from this hypothesis relies on the lack of methods. In our study we fulfilled this limitation by adding two technical advances that were not available before. First, we achieved a selective partial block of Nav1.2 thanks to the newly optimized G1G4Huwentoxin-IV peptide. Second, we directly estimated the effect of Nav1.2 block on the sodium current underlying the AP generation using an imaging technique that we recently developed (Filipis & Canepari 2021, reference [15] in the manuscript). Thanks to these significant methodological differences with respect to Hanemaaijer et al. (2020), we unambiguously demonstrated a Nav1.2 calcium influx component associated with the AP and we assessed the question of the target of this calcium signal which turned out to be the BK channel. In line with that, we argue reasonably that when a research study reports interesting results and from there on suggests important working hypotheses that are not formally demonstrated, and then a second study demonstrates experimentally that these hypotheses are correct (thanks to innovative methodologies), then this second study is also of major importance and cannot be considered a confirmation of the first study.

    Rebuttal to Reviewer 1

    However, statistical analysis should be performed with non-parametric tests such as Mann-Whitney or Wilcoxon tests and not t-test because of the small samples (t-test can be used only for large samples).

    We strongly agree that a “two-population” t-test, being parametric, is not adequate when comparing two or more small sets of independent samples. However, in this study, we never compared sets of independent samples, but we compared two sets of correlated samples where the first sample is the measurement in control condition and the second sample is the same measurement after addition of the channel blocker. Thus, for this type of datasets, we used a “paired” t-test. While the standard Mann-Whitney or Wilcoxon are not appropriate for sets of correlated samples, we followed the reviewer recommendation to perform a non-parametric test and we applied the “Wilcoxon ranked sign test” (non-parametric equivalent to the paired t-test). The results of this additional test were consistent with the paired t-test. As reported in the manuscript, all data and metadata used in this study will be available in the public repository Zenodo (doi: 10.5281/zenodo.5835995) after the manuscript will be accepted by a journal. Thus, while in the manuscript we only report p

    1. abstract, line 5, "...by a recent peptide,...". I guess the authors mean "...by a peptide recently identified..."

    We replaced with “recently modified peptide” since the wild-type Huwentoxin-IV was identified some time ago.

    Rebuttal to Reviewer 2

    It would be fair to point out somewhere in the introduction or discussion that the role of Nav1.2 and its interaction with BK channels was already predicted by Hanemaaijer et al. (2020).

    We pointed out this prediction in the Discussion of the revised manuscript. It must be said that the important work of Hanemaaijer et al. (2020) (reference [12]) is cited six times in the manuscript. As stated above, the interaction between Nav1.2 and BK channels was only suggested in Hanemaaijer et al. (2020) and not demonstrated.

    I would recommend citing Huang & Rasband (2018) for a relatively up to date review of channels in the AIS.

    This review is now cited in the Discussion of the revised manuscript (reference [38]).

    The authors should state whether the L5 cells are L5a or L5b (or whether they didn't distinguish).

    Although L5a and L5b could be in principle recognised also by their morphology, no attempt was done to distinguish between two functionally different groups of cells. This is now stated in the Introduction of the revised manuscript.

    The choice of colors in the figures made it hard to see which line was which. The authors use blue vs light blue and green vs light green, which were indistinguishable on my screen and in print. The orange vs yellow figs could just be made out.

    In control conditions, we use green for sodium, red for voltage and blue for calcium, which also makes it easy to follow in the simulations. We followed the reviewer’s recommendation and used always grey traces for all traces after addition of the channel blocker. It should be easier now in the revised manuscript to visually discriminate the traces.

    Are the plots on the RHS of Fig. 2b averages?

    The plots are from the cell in panel a. This is now stated in the figure legend of the revised manuscript.

    I don't understand the sentence starting, "This signal translated in a substantial delay..."

    We replaced this sentence, in the revised manuscript, with “In our experiments, the peak of the Ca2+ current in the distal axon preceded both the peak of the somatic AP and the peak of the Ca2+ in the proximal part of the AIS”. We thank the reviewer for finding this ambiguous sentence.

    I also didn't understand the point being made in the sentence: "The evident anticipation of the AP peak, also with respect to the somatic AP peak, suggests that Ca2+ influx associated with the AP is mainly mediated by VGNCs in the distal axon, whereas it is mainly mediated by VGCCs in the proximal axon." The block of calcium with VGCC-blockers is convincing in itself but this argument based on timing isn't convincing. Isn't the point that VGCCs are to be found closer to the soma, despite the fact that Nav1.2 is also found closer to the soma, so that the presence of Ca-conducting Na+ channels near the cell body could be masked?

    We erased this sentence in the revised manuscript.

    I think it would make more sense to write, "the effects produced by 1 µM IK CAKC inhibitor tram-34 were not significant".

    We replaced “variable” with “not significant” in the revised manuscript.

    There is a mismatch in Fig. S5 between the 400 nM, 800 nM, 1600 nM labelling in the figure and the "40 nM, 80 nM or 160 nm" in the legend.

    We thank the reviewer for having found this mismatch. The values are those in the figure labels, so we corrected the values in the figure legend in the revised manuscript.

    In Fig. S7c-d, the authors write "... the widening of the distal axonal AP was observed in 4 cells, whereas the AP waveforms did not change in 3 cells". I think this is not appropriate if the results are statistically insignificant. It implies that the authors know somehow that the outliers are not just noise.

    We changed this inappropriate sentence in the revised manuscript.

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    Referee #2

    Evidence, reproducibility and clarity

    Filipis et al. present a study of the underlying mechanisms for the generation of action potentials in the axon initial segment (AIS) of layer 5 somatosensory pyramidal neurons. The experiments employed three different imaging approaches for following sodium, voltage and calcium changes at two different locations in the axon near the cell body. This was combined with pharmacology for comparing the contribution of two different types of sodium channels: Nav1.6 and Nav1.2, the latter having a concomitant Ca2+ conductance. They confirm the previously established findings in terms of the location of Nav1.6 lying more distally than Nav1.2 on the AIS, and show that while Nav1.6 contributes mostly to the initiation of the AP, Nav1.2 plays an important role in controlling the shape of the AP via Ca2+ activation of BK (potassium) channels. The study represents a careful attempt to investigate a difficult subject owing to the small size of the axon initial segment and the difficulty of disentangling the different channels and influences. I find the paper well carried out and well presented and a useful contribution to understanding the firing properties of these important neurons.

    I only have a few minor points:

    1. It would be fair to point out somewhere in the introduction or discussion that the role of Nav1.2 and its interaction with BK channels was already predicted by Hanemaaijer et al. (2020).
    2. I would recommend citing Huang & Rasband (2018) for a relatively up to date review of channels in the AIS.
    3. The authors should state whether the L5 cells are L5a or L5b (or whether they didn't distinguish).
    4. The choice of colors in the figures made it hard to see which line was which. The authors use blue vs light blue and green vs light green, which were indistinguishable on my screen and in print. The orange vs yellow figs could just be made out.
    5. Are the plots on the RHS of Fig. 2b averages?
    6. I don't understand the sentence starting, "This signal translated in a substantial delay..."
    7. I also didn't understand the point being made in the sentence: "The evident anticipation of the AP peak, also with respect to the somatic AP peak, suggests that Ca2+ influx associated with the AP is mainly mediated by VGNCs in the distal axon, whereas it is mainly mediated by VGCCs in the proximal axon." The block of calcium with VGCC-blockers is convincing in itself but this argument based on timing isn't convincing. Isn't the point that VGCCs are to be found closer to the soma, despite the fact that Nav1.2 is also found closer to the soma, so that the presence of Ca-conducting Na+ channels near the cell body could be masked?
    8. I think it would make more sense to write, "the effects produced by 1 µM IK CAKC inhibitor tram-34 were not significant".
    9. There is a mismatch in Fig. S5 between the 400 nM, 800 nM, 1600 nM labelling in the figure and the "40 nM, 80 nM or 160 nm" in the legend.
    10. In Fig. S7c-d, the authors write "... the widening of the distal axonal AP was observed in 4 cells, whereas the AP waveforms did not change in 3 cells". I think this is not appropriate if the results are statistically insignificant. It implies that the authors know somehow that the outliers are not just noise.

    Hanemaaijer, N. A., Popovic, M. A., Wilders, X., Grasman, S., Arocas, O. P., & Kole, M. H. (2020). Ca2+ entry through NaV channels generates submillisecond axonal Ca2+ signaling. Elife, 9, e54566. Huang, C. Y. M., & Rasband, M. N. (2018). Axon initial segments: structure, function, and disease. Annals of the New York Academy of Sciences, 1420(1), 46-61.

    Significance

    • The results are useful and important (but perhaps not major).
    • The bulk of this paper was either demonstrated or predicted by another paper Hanemaaijer et al. in 2020 using very similar methods.
    • The audience for this paper will be a relatively specialized group focusing on biophysical explanations for axonal excitability and/or modellers.
    • My expertise is a specialization on the electrical properties of these neurons (L5 pyramidal neurons).
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    Referee #1

    Evidence, reproducibility and clarity

    This paper investigates the mechanisms of BK potassium channels activation in the axon initial segment (AIS) of cortical neurons. Using whole-cell patch-clamp recording, voltage imaging of the AIS, pharmacological tools, use of peptides and computer simulations, the authors show that calcium influx via Nav1.2 channels activates BK channels, thus shaping the action potential waveform.

    The manuscript is well written and the data are clear. The conclusions are convincing as they are in agreement with a recent study showing that Nav1.2 are permeable to calcium ions (Hanemaaijer et al., eLife 2020). The results and methods are presented in a way that they can be reproduced. However, statistical analysis should be performed with non-parametric tests such as Mann-Whitney or Wilcoxon tests and not t-test because of the small samples (t-test can be used only for large samples).

    Minor points:

    abstract, line 5, "...by a recent peptide,...". I guess the authors mean "...by a peptide recently identified..."

    Significance

    The study is interesting as it provides a target (activation of BK channels) for the Nav1.2-mediated calcium influx. Audience: specialized journal in neurobiology.

    My field of expertise is the cellular neurophysiology (axon, ion channels, synapse and plasticity). I have sufficient expertise to evaluate all the aspects of this paper.

  5. Version published to 10.1101/2022.04.12.488116v1 on bioRxiv