β spectrin-dependent and domain specific mechanisms for Na⁺ channel clustering

Essential revisions:

The reviewers also found that there are several points that can be improved by including additional discussion.

1) The discussion of localization of β1 spectrin and AnkyrinR in a subset of neurons should note that both of these proteins are present predominantly in the cell body, both in wildtype and β4 spectrin null neurons. While some β1 staining can be detected in the β4 knockouts, this is minimal in comparison to the cell body.

We revised the text as follows: “Remarkably, in Nes-Cre;Sptbn4^F/F mice we found increased levels of AIS β1 spectrin in the majority of β1-high neurons (where somatic expression remained high; Figure 2D, arrow, E), but not at the AIS of β1-low neurons (Figure 2D, arrowheads, E).”

2) The Introduction is a single paragraph, and could be expanded.

We now include another introductory paragraph at the very beginning of the Introduction as follows:

“Clustered ion channels at axon initial segments (AIS) and nodes of Ranvier are essential for proper nervous system function. […] Using conditional knockout mice, we previously showed that loss of β4 spectrin from…”

3) The specialized function of β4 spectrin in recruiting CamKinase2 (missing in other β spectrins) should be mentioned. Similarly, discussion of specialized roles of giant AnkyrinG (ex-interactions with GABARAP, Nudel, EB proteins, and as a signaling platform) could help place the findings in perspective.

We revised the Discussion to include the suggested material as follows:

“β spectrins can also serve as a signaling platform. In cardiomyocytes, β4 spectrin regulates membrane excitability through coordinating CAMK2-mediated modification to Nav 1.5 channels for proper subcellular localization (Hund et al., 2010). […] For example, β4 spectrin-mediated signaling may influence downstream transcriptional regulation for axon regeneration after spectrin proteolysis by calpains.”

“AnkG’s giant exon participates in interactions with β4 spectrin (Jenkins et al., 2015), EB1/3 (Leterrier et al., 2011) and NDEL1 (Kuijpers et al., 2016). […] Giant AnkG also interacts with GABARAP to stabilize GABA receptors in the somatodendritic domain of neurons (Nelson et al., 2018).”

4) Inclusion of seizure data for individual β1 and β4 spectrin knockout animals will help to interpret results of the double knockout. If you already have these data, it would be appropriate to add them. Otherwise, you might consider to remove this data point.

We agree with the reviewers’ comment that seizure data for individual β1 and β4 conditional knockout mice would be very valuable. However, we did not perform EEG recordings from β1 conditional knockout mice since they had no phenotype whatsoever and brain morphology was completely normal. We did perform EEG recordings from 3 pairs of 6 month-old β4 conditional knockout mice (and control littermates). In these experiments we observed abnormal spike activity in both control and β4 conditional knockout mice, but seizures were not detected. In consultation with Dr. Jeffrey Noebels, who is an expert on mouse EEG recordings and who did this work, we decided not to include the β4 conditional knockout mice and only include the β1/β4 conditional knockout mice. We do not know how to explain the abnormal spike activity observed in the control mice. We concluded that new cohorts of conditional knockout and control mice would need to be prepared and the experiments performed again. This would require at least an additional 6 months. Because of the strong seizure phenotype in the β1/β4 conditional knockout mice, we strongly prefer to include these data found in Figure 4A-D.