Shank promotes action potential repolarization by recruiting BK channels to calcium microdomains

Essential revisions:

Major Concerns:

1. Figures 2C and D: using floxed (nu697) or flexed (nu652) shn-1 alleles, they show that expressing CRE in muscles enhances PP rate and AP width, while expressing CRE in neurons has no effect on PP rate and a moderate, but not significant effect on AP width. Could it be that shn-1 affects muscle AP through a function in both tissues? This may be addressed by expressing CRE simultaneously in neurons and muscle to test if PP rate or AP width are further enhanced/rescued. Also, it appears that the authors omitted the control expressing CRE in neurons of nu652, which will strengthen the conclusions.

As requested, we now analyze AP firing patterns following neuronal CRE expression in nu652 (Figure 2). These data are described in the revised text, as follows:

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“By contrast, shn-1(neuron KO) and shn-1(neuron rescue) had no effect on PP rate or AP widths (Figure 2C-D).”

2. Figure 3 could be strengthened by including analysis of ∆VTTL; ∆PDZ double mutants. The findings will support that these two domains indeed interact with each other, as opposed to the idea that each domain mediates a different task, each incrementally affecting AP repolarization.

To address this concern, we now analyze ΔVTTL; shn-1(null) double mutants. Because the shn-1 null has a stronger SLO channel defect than the ΔPDZ mutant, we thought it would be better to analyze this double mutant. These new results are described as follows:

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“Furthermore, the shn-1(nu712 null) and egl-19(nu496 ΔVTTL) mutations did not have additive effects on PP rate and AP widths in double mutants (Figure 3, supplement 2).”

3. Figure 5A-B implies potassium currents are mediated by 2 sources, one channel composed of SLO-1/SLO-2 and another homomeric SLO-2. The finding in Figure 5C-D that SLO-2 signals are reduced in the absence of SLO-1 implies that the remaining GFP signals may represent SLO-2 homomers. If SLO-1 is exclusively heterodimer with SLO-2, how do the authors explain the observation that SLO-1 signals are unchanged in slo-2 mutants? Based on the data, it remains possible that functioning SLO-1 containing channels are not required for repolarization and instead, SLO-1 is only required for SLO-2 trafficking or localization. The statement that "..these results suggest that rapid muscle repolarization following Aps is mediated by SLO1/2 heteromeric channels." The authors should acknowledge that SLO-2 homomers may also support rapid repolarization.

The text was revised as recommended:

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“Collectively, these results suggest that rapid muscle repolarization following APs is mediated by SLO-1/2 heteromeric channels and by SLO-2 homomers.”

4. Figure 6D-E: The authors suggest that SHN-1 stabilizes SLO to nearby EGL-19 channels to link Ca to K currents. However, they also observe that ∆VTTL does not affect SLO-2 currents. While the authors address this contradiction in their discussion, a test of the AP phenotypes in egl-19∆VTTL; slo-2 double mutants compared to the singles (related to point 2 about the Figure 3 analysis) would help rule out any SLO-2 independent role for the egl-19 VTTL domain.

The requested experiment was included in the original submission. These results are described in the text as follows:

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“Consistent with this idea, AP widths in slo-2 single mutants were not significantly different from those in slo-2 double mutants containing shn-1(nu712 null), shn-1(nu542 ΔPDZ), or egl-19(nu496 ΔVTTL) mutations (Figure 6A).”

5. Table 2 is a list of alleles used or generated. The authors should also include a comprehensive list of strains used in this study.

A complete list of strains utilized is now provided in the Key Resource Table.

6. Figures 2 and 3. The only data in the manuscript that are less than completely compelling are the assessments of PP rate where a subset of recordings seem to be affected and the changes are generally less than 1/10th of a Hz. The effect seems to reach high levels of significance due to the fact that the rate is near zero in controls (as well as a large number of the mutant recordings). There is a disruption, but the relevance here to muscle and animal function is a bit hard to understand. This should be clarified in the text and, perhaps, the authors could consider other aspects of the data that could be quantified – charge comes to mind.

We agree that identifying a physiological significance for the muscle plateau potentials (PPs) would be interesting. Experimentally, this would require a mutation (or drug) that blocks SHK-1/KCNA inactivation (thereby reducing PP rate). Given that we lack such a mutant (or drug), we have no data examining this issue; consequently, we would rather not speculate about this. Nonetheless, we believe that readers will find it helpful if the text comments on PPs and SHN-1’s impact on them because PPs are a salient feature of our AP recordings.

7. Action potentials. These are slow, calcium-driven, muscle action potentials. The most appropriate reference point is probably cardiac muscle. The implicit assertion that there is direct relevance to sodium-driven action potentials in the CNS should be revised and broadened to non-neuronal muscle. This need not diminish the relevance of the work – in fact it may broaden the relevance.

We agree that CaV-BK coupling is unlikely to alter sodium-driven APs; however, we cannot find anywhere in the text where this possibility is suggested (or implied). If the reviewer can point out a specific comment that should be revised, we will be happy to do so. Instead, we list several potential effects of altered CaV-BK coupling on neuron and muscle physiology:

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“Shank regulation of BK channels could have broad effects on neuron and muscle function. In neurons, BK channels are functionally coupled to CaV channels in the soma and dendrites, thereby regulating AP firing patterns and somatodendritic calcium transients Golding et al., 1999Storm, 1987(; ). In pre-synaptic terminals, BK channels limit the duration of calcium influx during APs, thereby decreasing neurotransmitter release Griguoli et al., 2016Yazejian et al., 2000(; ). In muscles, BK channels regulate AP firing patterns, calcium influx during APs, and muscle contraction Dopico et al., 2018Latorre et al., 2017(; ). Thus, Shank mutations could broadly alter neuron and muscle function via changes in CaV-BK coupling.”

8. The emphasis on nano-domain organization is problematic. As I understand, the sites of protein co-localization represent large micro-domains within the muscle t-tubule system. Obviously, nano-domains are relevant in mammalian neurons to couple calcium entry to BK channel function in both time and space during the sub-millisecond kinetics of the sodium action potential. The tight temporal coupling demands physical coupling. Work at the frog NMJ, imaging calcium domains, argues that the size of calcium domains at sites of entry are approx. 1.5µm and this size is not influenced significantly by the presence of EGTA. Given the much slower temporal dynamics of the C. elegans muscle action potential, and the large size of the protein micro-domains, I do not fully understand the argument for nano-domain coupling. This should probably be revised.

We thank the reviewers for this comment. As recommended, we replaced nano-domain with micro-domain throughout the text (including the title).

9. The muscle sites seem to be coincident with T-tubule domains. Can the authors provide evidence of other resident markers that do, or do not change? This will provide more general relevance and give a sense of whether there is broad-based disorganization of these sites versus specific effects on the channels focused on in the text.

As suggested, we now analyze SHN-1’s impact on three additional junctional markers (SLO-1, EGL-19, and UNC-68). These results are described as follows:

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“Next, we asked if inactivating SHN-1 alters the localization of other muscle ion channels. SLO-1 puncta intensity was unaltered in shn-1 null mutants, indicating that BK channels lacking SLO-2 were trafficked normally (Figure 7 supplement 2A-B). In body muscles, EGL-19/CaV1 channels are extensively co-localized with calcium channels in the endoplasmic reticulum (ER), UNC-68/Ryanodine Receptors (RYR) Piggott et al., 2021(). However, the puncta intensity of endogenous EGL-19(Cherry₁₁) and UNC-68(GFP₁₁)/RYR in body muscles were unaltered in shn-1(nu712 null) mutants (Figure 7H and Figure 7 supplement 2C-D), suggesting that SHN-1 does not broadly regulate co-localization of ion channels at ER-plasma membrane junctional contacts.”

10. The authors imply that shank, CaV1, and the Slo's form a functional complex in the muscle membrane, but the data are somewhat indirect. It would be useful if the authors could spell this out better so the reader can assess the strength of this inference. Is there direct evidence (pull-downs, etc) for such a 3-way complex?

The text was revised as suggested:

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“Although SHN-1 binds EGL-19, SHN-1 may not directly link EGL-19 to SLO/BK channels. Instead, SHN-1 may link EGL-19 to other proteins required for SLO channel localization, e.g. components of the dystrophin complex Kim et al., 2009Sancar et al., 2011(; ).”