EGL-19/CaV1 channels mediate the primary depolarizing current during body muscle APs (Jospin et al., 2002; Liu et al., 2011). Because SHN-1 directly binds EGL-19 (Pym et al., 2017), we asked if SHN-1 regulates muscle AP firing patterns. In WT animals, body muscles exhibit a pattern of spontaneous AP bursts (~10 APs/burst; burst frequency 0.1 Hz) (Figure 1A). Within a burst, APs became progressively wider (Figure 1B). A similar pattern of progressive AP broadening during burst firing has been reported for many neurons (Geiger and Jonas, 2000; Jackson et al., 1991). Outward potassium currents were progressively decreased during repetitive depolarization (Figure 1C), suggesting that progressive AP broadening most likely results from accumulation of inactivated potassium channels during bursts, as seen in other cell types (Geiger and Jonas, 2000; Kole et al., 2007). Occasionally, WT muscles also exhibited prolonged depolarizations ( > 150ms), which are hereafter designated plateau potentials (PPs). PPs often occur at the end of an AP burst (Figure 1A).

In shn-1 null mutants, PP rate and AP widths were significantly increased, AP amplitudes were decreased, resting membrane potential (RMP) was depolarized, while AP frequency and input resistance were unaltered (Figure 1D–I). Similar increases in AP widths and PP rate were observed in three, independently derived shn-1 null mutants (nu712, nu652, and tm488) (Table 1). Single-cell RNA sequencing studies suggest that SHN-1 is expressed in muscles, neurons, glia, and epithelial cells (Cao et al., 2017; Packer et al., 2019), consistent with the broad expression of split GFP tagged shn-1(nu600 GFP₁₁) (Figure 1—figure supplement 1). To determine if SHN-1 functions in body muscles to control AP duration, we edited the endogenous shn-1 locus to construct alleles that are either inactivated (nu697) or rescued (nu652) by the CRE recombinase (Figure 2A). Using these alleles, we found that AP widths and PP frequency were increased in shn-1(muscle Knockout, KO) and that this defect was eliminated in shn-1(muscle rescue) (Figure 2B–D). By contrast, shn-1(neuron KO) and shn-1(neuron rescue) had no effect on PP rate or AP widths (Figure 2C–D). Because CRE expression in muscles produced opposite changes in AP firing patterns in strains containing the shn-1 nu697 and nu652 alleles, these results are unlikely to be caused by toxicity associated with CRE expression (Speed et al., 2019). The PP rate and AP width defects observed in shn-1(muscle KO) were not significantly different from those in shn-1(null) (Figure 2—figure supplement 2). Collectively, these results suggest that SHN-1 acts in body muscles to control AP duration.

Because SHN-1 has multiple binding partners, we sought to confirm that prolonged APs result from decreased SHN-1 binding to EGL-19. To address this question, we recorded APs in strains containing mutations that disrupt this interaction (Figure 3A). PP frequency was significantly increased by a deletion removing the EGL-19 carboxy-terminal PDZ ligand [egl-19(nu496 ΔVTTL)] (Figure 3B). AP widths were significantly increased by a deletion removing the SHN-1 PDZ domain [shn-1(nu542 ΔPDZ)] and by the egl-19(nu496 ΔVTTL) mutation (Figure 3C). 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—figure supplement 2). Taken together, these results support the idea that SHN-1 binding to EGL-19/CaV1 accelerates AP repolarization.

To investigate how SHN-1 controls AP duration, we first asked which potassium channels promote repolarization following APs. Prior studies showed that voltage-activated potassium currents in body muscles are mediated by SHK-1/KCNA and BK channels (Gao and Zhen, 2011; Liu et al., 2011). SHK-1 channel function can be assessed in recordings using an internal solution containing low chloride levels (hereafter Ik_loCl). Ik_loCl was nearly eliminated in shk-1 single mutants (Figure 4A–B). BK channel function can be assayed in recordings utilizing internal solutions with high chloride levels (hereafter Ik_hiCl), which activates SLO-2 channels (Yuan et al., 2000). Ik_hiCl was ~50% reduced in single mutants lacking either SLO-2 or SHK-1 and was eliminated in slo-2; shk-1 double mutants (Figure 4C–D). These results suggest that SHK-1/KCNA and SLO-2/BK are the primary channels promoting AP repolarization. Consistent with this idea, AP duration was significantly increased in mutants lacking SHK-1/KCNA (Figure 4E–F), as previously reported (Gao and Zhen, 2011; Liu et al., 2011).

Contradictory results have been reported for AP firing patterns in slo-1 and slo-2 BK mutants (Gao and Zhen, 2011; Liu et al., 2011). These studies used intracellular solutions that alter BK channel function. In (Liu et al., 2011), an intracellular solution containing high chloride levels was used, thereby exaggerating SLO-2’s contribution to AP repolarization (Yuan et al., 2000). In (Gao and Zhen, 2011), an intracellular solution containing a fast calcium chelator (BAPTA) was used, which inhibits BK activation thereby minimizing their impact on APs. We re-investigated the effect of SLO channels on APs using intracellular solutions with low chloride and a slow calcium chelator (EGTA), finding that AP durations were increased to a similar extent in slo-1 and slo-2 single mutants (Figure 4E–F). Taken together, these results confirm that SHK-1/KCNA and SLO/BK are the primary channels promoting AP repolarization in body muscles.

SLO-1 and SLO-2 subunits are co-expressed in muscles and could potentially co-assemble to form heteromeric channels. To determine if channels containing both SLO-1 and SLO-2 regulate AP repolarization, we analyzed AP widths in slo-1; slo-2 double mutants. AP widths in slo-1; slo-2 double mutants were not significantly different from those found in the single mutants (Figure 4F). Because slo-1 and slo-2 mutations did not have additive effects on AP widths, these results support the idea that heteromeric SLO-1/2 channels mediate rapid repolarization of muscle APs.

We did several experiments to further test the idea that SLO-1 and SLO-2 function together in heteromeric channels. First, we recorded voltage-activated potassium current in body muscles and found that Ik_hiCl was modestly reduced in slo-1 mutants, was dramatically reduced in slo-2 mutants, and was not further reduced in slo-1; slo-2 double mutants (Figure 5A–B). These results suggest that Ik_hiCl is mediated by heteromeric channels (containing both SLO-1 and SLO-2 subunits) and by SLO-2 homomers.

As a final test of this idea, we asked if subcellular localization of SLO-1 and SLO-2 subunits requires expression of both subunits. For this analysis, endogenous SLO-1 and SLO-2 subunits were labeled with split GFP. Using CRISPR, we introduced the eleventh β-strand of GFP (GFP₁₁) into the endogenous slo-1 and slo-2 genes and visualized their expression by expressing GFP_1-10 in body muscles. Strains containing the GFP₁₁ tagged alleles exhibited wild-type AP widths, RMP, and Ik_hiCl currents, indicating that the tag did not interfere with SLO channel function (Figure 5—figure supplement 1). Using these alleles, we find that SLO-2 puncta intensity was significantly reduced in slo-1 null mutants, indicating that channels containing SLO-2 subunits require SLO-1 for their trafficking (Figure 5C–D). By contrast, SLO-1 puncta intensity was unaffected in slo-2 mutants, suggesting that BK channels lacking SLO-2 were trafficked normally (Figure 5E–F). Collectively, these results suggest that rapid muscle repolarization following APs is mediated by SLO-1/2 heteromeric channels and by SLO-2 homomers. Two prior studies also suggested that SLO subunits form heteromeric channels when heterologously expressed in Xenopus oocytes. SLO-1 currents were inhibited by a dominant-negative SLO-2 construct (Yuan et al., 2000). Similarly, mammalian SLO2 subunits (KCNT1 and 2) co-assemble to form heteromeric channels (Chen et al., 2009). Our results suggest that endogenously expressed SLO subunits also form heteromeric channels in native tissues.

SHN-1’s impact on AP duration could be mediated by changes in either SHK-1 or SLO channels. To determine if SHN-1 acts through SLO channels, we asked if slo-2 mutations block SHN-1’s effects on AP widths. 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). These results suggest that SHN-1 controls AP duration by regulating SLO-2 channels.

To confirm that SHN-1 regulates SLO-2 channels, we analyzed potassium currents in shn-1 mutants. A shn-1 null mutation had no effect on Ik_loCl currents, indicating that SHK-1 function was unaffected (Figure 6B–C). By contrast, Ik_hiCl was ~30% reduced in shn-1 null mutants, ~ 50% reduced in slo-2 mutants, and was not further reduced in shn-1; slo-2 double mutants (Figure 6D–E). Lack of additivity in shn-1; slo-2 double mutants suggests that the SHN-1-sensitive potassium current was mediated by SLO-2. A similar decrease in Ik_hiCl current was observed in shn-1(nu542 ΔPDZ) mutants (Figure 6E). Ik_hiCl current was unaltered in egl-19(nu496 ΔVTTL) mutants, implying that SHN-1 binding to EGL-19’s carboxy terminus is not required for SLO-2 current (Figure 6E). Thus, shn-1 inactivation decreased SLO-1/2 BK current but had little or no effect on SHK-1 KCNA current; consequently, SHN-1 regulates AP widths by promoting activation of SLO-1/2 channels.

BK channels bind calcium with affinities ranging from 1 to 10 μM (Contreras et al., 2013). As a result of this calcium dependence, BK channels have very low open probability at resting cytoplasmic calcium levels (~100 nM) and efficient BK activation typically requires close spatial coupling to calcium channels (Barrett et al., 1982). We next asked if body muscle BK channels are functionally coupled to EGL-19/CaV1 channels. Consistent with this idea, Ik_hiCl current was significantly decreased by nemadipine, an EGL-19/CaV1 antagonist (Figure 7A–B; Kwok et al., 2008). The inhibitory effect of nemadipine on Ik_hiCl was eliminated in slo-2 mutants (Figure 7A–B), suggesting that the nemadipine-sensitive potassium current was mediated by SLO-2.

Is EGL-19 coupling to SLO-2 mediated by microdomain signaling? To test this idea, we compared Ik_hiCl and AP widths recorded with intracellular solutions containing fast (BAPTA) and slow (EGTA) calcium chelators (Figure 7C–E). We found that Ik_hiCl recorded with BAPTA was significantly smaller than that recorded with EGTA (Figure 7C). Similarly, AP widths recorded with BAPTA were significantly longer than those recorded with EGTA (Figure 7D–E). The effect of BAPTA on Ik_hiCl and AP widths was eliminated in slo-2 mutants (Figure 7C and E), suggesting that the BAPTA sensitive potassium current is mediated by SLO-2. BAPTA’s effect on Ik_hiCl and AP widths was reduced but not eliminated in shn-1 mutants (Figure 7C and E), consistent with the partial loss of SLO-2 current in these mutants (Figure 6D–E). Taken together, these results suggest that SHN-1 promotes SLO-2 microdomain coupling to EGL-19/CaV1.

If BK channels are functionally coupled to EGL-19/CaV1, these channels should be co-localized. Endogenous SLO-2 channels (tagged with GFP₁₁) were distributed in a punctate pattern on the muscle surface. A subset of the SLO-2 puncta co-localized with EGL-19/CaV1 channels (tagged with Cherry₁₁), suggesting that EGL-19 nanocomplexes are heterogeneous (Figure 7F). SLO-2 puncta intensity was significantly reduced in shn-1 null mutants (Figure 7F–G), consistent with the decreased SLO-2 current observed in these mutants. By contrast, SLO-2 puncta intensity was unaltered in shn-1(nu542 ΔPDZ) and egl-19(nu496 ΔVTTL) mutants (Figure 7G), in which SHN-1 binding to EGL-19’s c-terminus is disrupted (Pym et al., 2017). 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—figure 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(null) mutants (Figure 7H and Figure 7—figure supplement 2C-D), suggesting that SHN-1 does not broadly regulate co-localization of ion channels at ER-plasma membrane junctional contacts. Collectively, these results suggest that SHN-1 stabilizes SLO-2 clusters in the plasma membrane and promotes activation of heteromeric SLO-1/2 channels by nearby calcium channels.

Deletion and duplication of human shank genes are both associated with ASD, schizophrenia, and mania (Bonaglia et al., 2006; Durand et al., 2007; Failla et al., 2007; Gauthier et al., 2010; Han et al., 2013). These results suggest that Shank phenotypes relevant to psychiatric disorders should exhibit a similar sensitivity to Shank copy number. For this reason, we analyzed the effect of shn-1 gene dosage on Ik_hiCl and AP duration (Figure 8). We analyzed animals with 1 (nu712/+ heterozygotes), 2 (WT), and 4 (WT +2 single copy shn-1 transgenes) copies of shn-1. Compared to wild-type controls, AP duration was significantly increased (Figure 8A and B) while muscle Ik_hiCl was significantly decreased (Figure 8C and D) in animals containing 1 and 4 copies of shn-1. Thus, increased and decreased shn-1 gene dosage produced similar defects in AP duration and SLO-2 current.

Thus far, our results suggest that SHN-1 promotes EGL-19 to SLO-2 coupling in muscles. We next asked if SHN-1 also promotes coupling in motor neurons. To test this idea, we analyzed Ik_hiCl in cholinergic motor neurons and found that it was significantly reduced in shn-1 null mutants (Figure 9A–C). The slo-2 and shn-1 mutations did not have additive effects on Ik_hiCl in double mutants, suggesting that the shn-1 mutation selectively decreases SLO-2 current in motor neurons (Figure 9A–C). Consistent with decreased SLO-2 currents, we observed a corresponding decrease in axonal SLO-2(nu725 GFP₁₁) puncta fluorescence in shn-1 mutant motor neurons (Figure 9D–E). Thus, our results suggest that SHN-1 promotes EGL-19/CaV1 to SLO-2 coupling in both body muscles and cholinergic motor neurons.

Several recent studies suggest that an important function of Shank proteins is to regulate ion channel density and localization. Mutations inactivating Shank have been shown to decrease AMPA and NMDA receptor abundance and post-synaptic currents (Peça et al., 2011; Won et al., 2012), HCN channels (Yi et al., 2016; Zhu et al., 2018), TRPV channels (Han et al., 2016), and voltage-activated CaV1 calcium channels (Pym et al., 2017; Wang et al., 2017). Here, we show that Shank also regulates BK channel densities in C. elegans muscles and motor neurons. Collectively, these studies suggest that Shank proteins have the capacity to control localization of many ion channels, thereby shaping neuron and muscle excitability.

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., 1999; Storm, 1987). In pre-synaptic terminals, BK channels limit the duration of calcium influx during APs, thereby decreasing neurotransmitter release (Griguoli et al., 2016; Yazejian et al., 2000). In muscles, BK channels regulate AP firing patterns, calcium influx during APs, and muscle contraction (Dopico et al., 2018; Latorre et al., 2017). Thus, Shank mutations could broadly alter neuron and muscle function via changes in CaV-BK coupling. It will be very interesting to determine if this new function for Shank is conserved in other animals, including humans.

BK channel activation requires tight coupling to CaV channels (Berkefeld et al., 2006). Our results suggest that SHN-1 promotes CaV1-BK microdomain coupling. APs were prolonged in shn-1 (null), shn-1(ΔPDZ), and egl-19(ΔVTTL) mutants and in all cases these defects were eliminated in double mutants lacking SLO-2. Interestingly, although all impair SLO-2 mediated AP repolarization, these mutations had distinct effects on SLO-2 channels. SLO-2 currents were reduced in shn-1 (null) and shn-1(ΔPDZ) mutants but were unaffected in egl-19(ΔVTTL) mutants. SLO-2 puncta intensity was decreased in shn-1(null) mutants but was unaffected in shn-1(ΔPDZ) and egl-19(ΔVTTL) mutants. These differences suggest that these mutants comprise an allelic series for EGL-19 to SLO-2 coupling defects in the following hierarchy shn-1 (null) >shn-1(ΔPDZ) > egl-19(ΔVTTL) mutants. Based on these results, we propose that multiple protein interactions progressively tighten CaV-BK coupling. Specifically, we propose that: (1) multiple SHN-1 domains act together to promote SLO-2 coupling to EGL-19, accounting for the distinct phenotypes observed in shn-1(null) and shn-1(ΔPDZ) mutants; (2) SHN-1 promotes formation (or stability) of SLO-2 clusters in the plasma membrane, as indicated by decreased SLO-2 puncta intensity in shn-1(null) mutants; (3) beyond this trafficking function, SHN-1’s PDZ domain tightens SLO-2 coupling to nearby calcium channels, accounting for the smaller SLO-2 current but unaltered SLO-2 puncta intensity in shn-1(ΔPDZ) mutants; (4) SHN-1 PDZ binding to EGL-19’s c-terminus promotes rapid SLO-2 activation during APs, accounting for the increased AP width but unaltered SLO-2 current and puncta intensity in egl-19(ΔVTTL) mutants; and (5) SHN-1’s PDZ must bind multiple proteins (not just EGL-19) to promote SLO-2 activation, accounting for the different phenotypes found in in shn-1(ΔPDZ) and egl-19(ΔVTTL) mutants. Multivalent interactions between scaffolds and their client proteins may represent a general mechanism for promoting microdomain signaling. 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, for example components of the dystrophin complex (Kim et al., 2009; Sancar et al., 2011).

Several studies suggest that Shank3 deletions and duplications are both linked to ASD and schizophrenia, suggesting that opposite changes in Shank3 levels produce similar or overlapping psychiatric phenotypes (Bonaglia et al., 2006; Durand et al., 2007; Failla et al., 2007; Gauthier et al., 2010; Han et al., 2013). It remains possible that more detailed analysis will reveal phenotypic differences between gain and loss of human Shank3. In either case, it is currently unclear how opposite changes in Shank3 levels produce psychiatric phenotypes. Different (potentially opposite) biochemical defects arising from decreased and increased Shank dosage could produce psychiatric traits, perhaps by circuit level mechanisms (Antoine et al., 2019; Peixoto et al., 2016). For example, Shank duplications and hemizygosity could act in different cells or circuits to produce psychiatric traits. Our results provide support for a second possibility. We find that increased and decreased shn-1 gene dosage produce similar cell autonomous CaV1-BK coupling defects. Two prior studies suggest that bidirectional changes in Shank produce similar defects in Wnt signaling and CaV1 current density (Harris et al., 2016; Pym et al., 2017). Collectively, these results suggest that some biochemical functions of Shank exhibit this unusual pattern of dose sensitivity and consequently could contribute to the pathophysiology of human Shankopathies (i.e. Shank3 mutations, CNVs, or PMS).

The role of human Shank in CaV1-BK coupling has not been tested. Nonetheless, it seems plausible that this new physiological function could contribute to neuropsychiatric or co-morbid phenotypes associated with human Shankopathies. Consistent with this idea, PMS and human KCNMA1/BK mutations are associated with several shared phenotypes including: autism, developmental delay, intellectual disability, hypotonia, seizures, and gastrointestinal defects (i.e. vomiting, constipation, or diarrhea) (Bailey et al., 2019; Laumonnier et al., 2006; Phelan and McDermid, 2012; Soorya et al., 2013; Witmer et al., 2019). Moreover, BK channels are regulated by two high confidence ASD genes (UBE3A and hnRNP U). BK channels are degraded by the ubiquitin ligase UBE3A (Sun et al., 2019), mutations in which cause Angelman’s syndrome. The RNA binding protein hnRNP U promotes translation of slo-2 mRNA (Liu et al., 2018). Collectively, these results support the idea that disrupted CaV1-BK channel coupling could play an important role in shankopathies and that BK channels may represent an important therapeutic target for treating these disorders.

Strain maintenance and genetic manipulation were performed as described (Brenner, 1974). Animals were cultivated at room temperature (~22 °C) on agar nematode growth media seeded with OP50 bacteria. Alleles used in this study are described in Table 2 and are identified in each figure legend. All strains utilized are listed in the Key Resources Table. Transgenic animals were prepared by microinjection, and integrated transgenes were isolated following UV irradiation, as described (Dittman and Kaplan, 2006). Single copy transgenes were isolated by the MoSCI and miniMoS techniques (Frøkjaer-Jensen et al., 2008; Frøkjær-Jensen et al., 2014).

Animals with different shn-1 copy numbers were constructed as follows: 0 copies, shn-1(nu712) homozygotes; one copy, unc-17::gfp (LX929) males were crossed with shn-1(nu712) homozygotes and gfp-expressing hermaphrodites were analyzed; two copies, WT were analyzed; four copies, WT animals homozygous for the single copy transgene expressing SHN-1A in body muscles (nuSi26). The nuSi26 transgene was described in our earlier study (Pym et al., 2017).

CRISPR alleles were isolated as described (Arribere et al., 2014). Briefly, we used unc-58 as a co-CRISPR selection to identify edited animals. Animals were injected with two guide RNAs (gRNAs) and two repair templates, one introducing an unc-58 gain of function mutation and a second modifying a gene of interest. Progeny exhibiting the unc-58(gf) uncoordinated phenotype were screened for successful editing of the second locus by PCR. Split GFP and split sfCherry constructs are described in Feng et al., 2017. MiniMOS inserts in which Pmyo-3 drives expression of either GFP_1-10 (nuSi144) or sfCherry_1-10 SL2 GFP_1-10 (nuSi458) were created.

Tissue specific shn-1 knockout was performed by introducing LoxP sites into intron 1 and the 3’UTR of the endogenous locus, in shn-1(nu697), and expressing CRE in muscles (pat-10 promoter) or neurons (sbt-1 promoter). Tissue-specific shn-1 rescue was performed by introducing a stop cassette into intron 2 of shn-1 using CRISPR, creating the shn-1(nu652) allele. The stop cassette consists of a synthetic exon (containing a consensus splice acceptor sequence and stop codons in all reading frames) followed by a 3’ UTR and transcriptional terminator taken from the flp-28 gene (the 564 bp sequence just 3’ to the flp-28 stop codon). The stop cassette is flanked by FLEX sites (which are modified loxP sites that mediate CRE induced inversions) (Schnütgen and Ghyselinck, 2007). In this manner, orientation of the stop cassette within the shn-1 locus is controlled by CRE expression. Expression of shn-1 is reduced when the stop cassette is in the OFF configuration (i.e. the same orientation as shn-1) but is unaffected in the ON configuration (opposite orientation). The endogenous flp-28 gene is located in an intron of W07E11.1 (in the opposite orientation). Consequently, we reasoned that the flp-28 transcriptional terminator would interfere with shn-1 expression in an orientation selective manner. A similar strategy was previously described for conditional gene knockouts in Drosophila (Fisher et al., 2017).

Worms were immobilized on 10% agarose pads with 0.3 µl of 0.1 µm diameter polystyrene microspheres (Polysciences 00876–15, 2.5% w/v suspension). Body muscles just anterior to the vulva were imaged. Images were taken with a Nikon A1R confocal, using a 60 X/1.49 NA oil objective, with Nyquist sampling. Image volumes spanning the muscle surface were collected (~10 planes/volume, 0.15 μm between planes, and 0.06 μm /pixel). Maximum intensity projections for each volume were auto-thresholded, and puncta were identified as round fluorescent objects (area >0.1 μm²), using analysis of particles. Mean fluorescent intensity in each punctum was analyzed in the raw images. All image analysis was done using FIJI.

Whole-cell patch-clamp measurements were performed using a Axopatch 200B amplifier with pClamp 10 software (Molecular Devices). The data were sampled at 10 kHz and filtered at 5 kHz. All recordings were performed at room temperature (~19°C–21°C).

Muscle AP recordings- The bath solution contained (in mM): NaCl 140, KCl 5, CaCl₂ 5, MgCl₂ 5, dextrose 11 and HEPES 5 (pH 7.2, 320 mOsm). The pipette solution contained (in mM): Kgluconate 120, KOH 20, Tris 5, CaCl₂ 0.25, MgCl₂ 4, sucrose 36, EGTA 5 (or BAPTA 5), and Na₂ATP 4 (pH 7.2, 323 mOsm). Spontaneous APs were recorded in current-clamp without current injection. Cell resistance (R_in) was measured following a 10 pA pulse injection. AP traces were analyzed in Matlab. APs were defined as depolarizations lasting <150ms. PPs were defined as depolarizations lasting >150ms.

K⁺ current recordings - The bath solution contained (in mM): NaCl 140, KCl 5, CaCl₂ 5, MgCl₂ 5, dextrose 11 and HEPES 5 (pH 7.2, 320 mOsm). For Ik_loCl recordings, the pipette solution contained (in mM): Kgluconate 120, KOH 20, Tris 5, CaCl₂ 0.25, MgCl₂ 4, sucrose 36, EGTA 5, and Na₂ATP 4 (pH 7.2, 323 mOsm). For Ik_hiCl recordings, the pipette solution contained (in mM): KCl 120, KOH 20, Tris 5, CaCl₂ 0.25, MgCl₂ 4, sucrose 36, EGTA 5 (or BAPTA 5), and Na₂ATP 4 (pH 7.2, 323 mOsm). The voltage-clamp protocol consisted of –60 mV for 50ms, –90 mV for 50ms, test voltage (from –60 mV to +60 mV) 150ms. The repetitive stimulus protocol was –20 mV for 50ms, + 30 mV for 50ms, which was repeated 20 times. In figures, we show outward currents evoked at +30 mV, which corresponds to the peak amplitude of muscle APs. In some recordings, EGL-19 channels were blocked by adding 5 μM nemadipine to the pipette solution. Patch clamp recording of Ik_hiCl in ACh motor neurons was done using solutions described above for the muscle recordings. ACh neurons were identified for patching by expression of an unc-17 transcriptional reporter (Punc-17::GFP).

For normally distributed data, significant differences were assessed with unpaired t tests (for two groups) or one way ANOVA with post-hoc Dunn’s multiple comparisons test (for >2 groups). For non-normal data, differences were assessed by Mann-Whitney (two groups) or Kruskal-Wallis test with post-hoc Dunn’s multiple comparisons test ( > 2 groups). Data graphing and statistics were performed in GraphPad Prism 9. No statistical method was used to select sample sizes. Data shown in each figure represent contemporaneous measurements from mutant and control animals over a period of 1–2 weeks. For electrophysiology, data points represent mean values for individual neuron or muscle recordings (which were considered biological replicates). For imaging studies, data points represent mean puncta fluorescence values in individual animals (which were considered biological replicates). All data obtained in each experiment were analyzed, without any exclusions.