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

Mutations altering the scaffolding protein Shank are linked to several psychiatric disorders, and to synaptic and behavioral defects in mice. Among its many binding partners, Shank directly binds CaV1 voltage activated calcium channels. Here we show that the C. elegans SHN-1/Shank promotes CaV1 coupling to calcium activated potassium channels. Mutations inactivating SHN-1, and those preventing SHN-1 binding to EGL-19/CaV1 all increase action potential durations in body muscles. Action potential repolarization is mediated by two classes of potassium channels: SHK-1/KCNA and SLO-1 and SLO-2 BK channels. BK channels are calcium-dependent, and their activation requires tight coupling to EGL-19/CaV1 channels. SHN-1’s effects on AP duration are mediated by changes in BK channels. In shn-1 mutants, SLO-2 currents and channel clustering are significantly decreased in both body muscles and neurons. Finally, increased and decreased shn-1 gene copy number produce similar changes in AP width and SLO-2 current. Collectively, these results suggest that an important function of Shank is to promote nanodomain coupling of BK with CaV1.


Summary:
Mutations altering the scaffolding protein Shank are linked to several psychiatric disorders, and to synaptic and behavioral defects in mice. Among its many binding partners, Shank directly binds CaV1 voltage activated calcium channels. Here we show that the C. elegans SHN-1/Shank promotes CaV1 coupling to calcium activated potassium channels. Mutations inactivating SHN-1, and those preventing SHN-1 binding to EGL-19/CaV1 all increase action potential durations in body muscles. Action potential repolarization is mediated by two classes of potassium channels: SHK-1/KCNA and SLO-1 and SLO-2 BK channels. BK channels are calcium-dependent, and their activation requires tight coupling to EGL-19/CaV1 channels. SHN-1's effects on AP duration are mediated by changes in BK channels.
In shn-1 mutants, SLO-2 currents and channel clustering are significantly decreased in both body muscles and neurons. Finally, increased and decreased shn-1 gene copy number produce similar changes in AP width and SLO-2 current. Collectively, these results suggest that an important function of Shank is to promote nanodomain coupling of BK with CaV1.
synaptic densities of excitatory synapses; consequently, most studies have focused on the idea that Shank proteins regulate some aspect of synapse formation or function. Through its various domains, Shank proteins bind many proteins (Lee et al., 2011;Sakai et al., 2011), thereby potentially altering diverse cellular functions. Shank proteins have been implicated in activity induced gene transcription (Perfitt et al., 2020;Pym et al., 2017), synaptic transmission (Zhou et al., 2016), synapse maturation (Harris et al., 2016), synaptic homeostasis (Tatavarty et al., 2020), cytoskeletal remodeling (Lilja et al., 2017), and sleep (Ingiosi et al., 2019). Each of these defects could contribute to the neurodevelopmental and cognitive deficits observed in ASD and schizophrenia.
Several recent studies suggest that an important function of Shank is to regulate the subcellular localization of ion channels. Shank mutations decrease the synaptic localization of NMDA and AMPA type glutamate receptors (Peca et al., 2011;Won et al., 2012). Other studies show that Shank proteins promote delivery of several ion channels to the plasma membrane, including HCN channels (Yi et al., 2016;Zhu et al., 2018), TRPV channels (Han et al., 2016), and CaV1 voltage activated calcium channels (Pym et al., 2017;Wang et al., 2017). Of these potential binding partners, we focus on CaV1 because human CACNA1C (which encodes a CaV1 a-subunit) is mutated in Timothy Syndrome (TS), a rare monogenic form of ASD (Splawski et al., 2005;Splawski et al., 2004), and polymorphisms linked to CACNA1C are associated with multiple psychiatric disorders (Cross-Disorder Group of the Psychiatric Genomics, 2013). For this reason, we asked how Shank regulates the coupling of CaV1 channels to their downstream effectors.
C. elegans has a single Shank gene, shn-1. The SHN-1 protein lacks an SH3 domain but has all other domains found in mammalian Shank proteins. Mammalian Shank proteins directly bind CaV1 channels through both the SH3 and PDZ domains (Zhang et al., 2005). We previously showed that the SHN-1 PDZ domain directly binds to a carboxy-terminal ligand in EGL-19/CaV1 (Pym et al., 2017). CaV1 channels are tightly coupled to multiple downstream calcium activated pathways. C. elegans and mouse Shank proteins have been shown to promote CaV1 mediated activation of the transcription factor CREB (Perfitt et al., 2020;Pym et al., 2017).
Here we test the idea that SHN-1 regulates CaV1 coupling to a second effector, calcium activated potassium currents (which are mediated by BK channels). BK channels are activated by both membrane depolarization and by cytoplasmic calcium. At resting cytoplasmic calcium levels (~100 nM), BK channels have extremely low open probability. Following depolarization, cytoplasmic calcium rises thereby activating BK channels. BK channels bind calcium with a K d ranging from 1-10 µM (Contreras et al., 2013); consequently, efficient BK channel activation requires tight spatial coupling to voltage activated calcium (CaV) channels. BK channels associate with all classes of CaV channels (Berkefeld et al., 2006). The co-clustering of BK and CaV channels allows rapid activation of hyperpolarizing potassium currents following depolarization. BK channels decrease action potential (AP) durations, promote rapid after hyperpolarizations, decrease the duration of calcium entry, and limit secretion of neurotransmitters and hormones in neurons and muscles (Adams et al., 1982;Edgerton and Reinhart, 2003;Petersen and Maruyama, 1984;Storm, 1987). Thus, BK channels have profound effects on circuit activity.
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 (Fig. 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 (>150 ms), which are hereafter designated plateau potentials (PPs). PPs often occur at the end of an AP burst (Fig. 1A).
In shn-1 null mutants, PP frequency and AP widths were significantly increased, AP amplitudes were decreased, resting membrane potential (RMP) was depolarized, while AP frequency and input resistance were unaltered ( Fig. 1D-I). Similar increases in AP widths and PP frequency 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 GFP 11 ) ( Fig. 1 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 ( Fig. 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) ( Fig. 2B-D).
By contrast, shn-1 knockout in neurons had no effect on PP rate or AP widths . 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). Collectively, these results suggest that SHN-1 acts in body muscles to control AP duration.

AP repolarization is controlled by SHK-1 KCNA and SLO-1/2 BK channels
To investigate how SHN-1 controls AP duration, we first asked which potassium channels promote repolarization following APs. Prior studies showed that voltageactivated potassium currents in body muscles are mediated by SHK-1/KCNA and BK channels Liu et al., 2011). SHK-1 channel function can be assessed in recordings using an internal solution containing low chloride levels (hereafter Ik loCl ).
Contradictory results have been reported for AP firing patterns in slo-1 and slo-2 BK mutants 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 , 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 ( Fig. 4E-F). Taken together these results confirm that SHK-1 and SLO are the primary channels promoting AP repolarization in body muscles.

SLO-1 and SLO-2 function together to promote AP repolarization
SLO-1 and SLO-2 subunits are co-expressed in muscles and could potentially coassemble 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 (Fig. 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 ( Fig. 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 labelled with split GFP. Using CRISPR, we introduced the eleventh b-strand of GFP (GFP 11 ) into the endogenous slo-1 and slo-2 genes and visualized their expression by expressing GFP 1-10 in body muscles. Strains containing the GFP 11 tagged alleles exhibited wild type Ik hiCl currents, AP widths, and RMP, indicating that the tag did not interfere with SLO channel function ( Fig. 5 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 ( Fig. 5C-D). By contrast, SLO-1 puncta intensity was unaffected in slo-2 mutants, suggesting that BK channels lacking SLO-2 were trafficked normally ( Fig. 5E-F). Collectively, these results suggest that rapid muscle repolarization following APs is mediated by SLO-1/2 heteromeric channels. Two prior studies also suggested that SLO subunits form heteromeric channels when heterologously expressed in Xenopus oocytes.

SHN-1 controls AP duration through BK channels
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 DPDZ), or egl-19(nu496 DVTTL) mutations ( Fig. 6A). These results suggest that SHN-1 controls AP duration by regulating SLO-2 channels.

SHN-1 promotes nanodomain coupling of SLO-2 with EGL-19/CaV1 channels
BK channels bind calcium with affinities ranging from 1-10 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 ( Fig. 7A-B) (Kwok et al., 2008). The inhibitory effect of nemadipine on Ik hiCl was eliminated in slo-2 mutants ( Fig. 7A-B), suggesting that the nemadipine sensitive potassium current was mediated by SLO-2.
Is EGL-19 coupling to SLO-2 mediated by nanodomain signaling? To test this idea, we compared Ik hiCl and AP widths recorded with intracellular solutions containing fast (BAPTA) and slow (EGTA) calcium chelators ( Fig. 7C-E). We found that Ik hiCl recorded with BAPTA was significantly smaller than that recorded with EGTA ( Fig. 7C).

EGL-19 to SLO-2 coupling is sensitive to shn-1 gene dose
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 (Fig. 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, muscle Ik hiCl was significantly decreased while AP duration was significantly increased in animals containing 1 and 4 copies of shn-1 (Fig. 8). Thus, increased and decreased shn-1 gene dosage produced similar defects in AP duration and SLO-2 current.

SHN-1 regulates BK channel activation and neurotransmitter release in motor neurons
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 ( Fig. 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 ( Fig. 9A-C). Consistent with decreased SLO-2 currents, we observed a corresponding decrease in axonal SLO-2(nu725 GFP 11 ) puncta fluorescence in shn-1 mutant motor neurons ( Fig. 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.

Discussion:
Our results lead to six principal conclusions. First, we show that SHN-1 acts cell autonomously in muscles to promote rapid repolarization of APs. Second, heteromeric BK channels containing both SLO-1 and SLO-2 subunits promote AP repolarization.
Third, SHN-1 limits AP duration by promoting BK channel activation. Fourth, shn-1 mutants have decreased SLO-2 channel clustering and decreased SLO-2 currents. Fifth, increased and decreased SHN-1 gene dosage produce similar defects in AP durations and SLO-2 currents. And sixth, SHN-1 also promotes SLO-2 activation in motor neurons.
Below we discuss the significance of these findings.
Shank as a regulator of ion channel density. 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 (Peca 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.

Implications for understanding neurodevelopmental disorders. Shank3
deletions and duplications both confer risk for ASD and schizophrenia (Bonaglia et al., 2006;Durand et al., 2007;Failla et al., 2007;Gauthier et al., 2010;Han et al., 2013). It is currently unclear how opposite changes in Shank3 levels produce similar psychiatric phenotypes. Different (potentially opposite) biochemical defects arising from decreased and increased Shank dosage could produce similar 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 similar 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) 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.

Experimental Procedures:
Strains: 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. 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

CRISPR alleles
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).
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 564bp 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) (Schnutgen 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).

Fluorescence imaging
Confocal imaging was performed using a Nikon 60x objective (NA 1.45) on a Nikon AR1 confocal microscope. 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). Muscles just anterior to the vulva were imaged. For quantitation of florescence intensities, puncta were analyzed using Fiji.

Electrophysiology
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  2, 323 mOsm). The voltage-clamp protocol consisted of -60mV for 50ms, -90mV for 50 ms, test voltage (from -60mV to +60mV) 150 ms. The repetitive stimulus protocol was -20mV for 50ms, +30mV 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 5µM nemadipine to the pipette solution. Patch clamp recording of IkhiCl in ACh motor neurons was done using solutions described above for the muscle recordings. ACh neurons were identified for patching by expression of unc-17 VAChT>GFP.

Statistical methods
For normally distributed data, significant differences were assessed with unpaired t tests (for 2 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 (2 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.

Acknowledgements
We thank the following for strains, advice, reagents, and comments on the manuscript: C. elegans stock center, S. Mitani, and members of the Kaplan lab. This work was supported by an NIH research grant to J.K. (NS32196).     Voltage activated potassium currents were recorded using pipette solutions containing low (IkloCl, A-B) and high (IkhiCl, C-D) chloride concentrations. Representative traces (A,C) and mean current density (B,D) at +30 mV are shown. IkloCl is mediated by SHK-1 whereas SHK-1 and SLO-2 equally contribute to IkhiCl. (E-F) AP durations are significantly increased in mutants lacking SHK-1, SLO-1, and SLO-2 channels. The AP widths observed in slo-1; slo-2 double mutants were not significantly different from those found in slo-2 single mutants. Representative traces (E) and mean AP widths (F) are shown. Alleles used in this figure were: shk-1(ok1581), slo-1(js379), and slo-2(nf100).
Values that differ significantly from wild type controls are indicated (ns, not significant; *, p <0.05; **, p <0.01; ***, p <0.001). Error bars indicate SEM.  (Table 2) and fluorescence was reconstituted by expressing GFP1-10 in body muscles. Controls showing that the GFP11 tags had no effect on AP width, RMP, and potassium currents are shown in Figure 5 supplement 1. Representative images (C and E) and mean puncta intensity (D and F) are shown. SLO-2 puncta intensity was significantly decreased in slo-1(js379) mutants. SLO-1 puncta intensity was unaltered in slo-2(nf100) mutants. Values that differ significantly from wild type controls are indicated (ns, not significant; *, p <0.05; **, p <0.01; ***, p <0.001). Error bars indicate SEM. Scale bar indicates 4 µm.   activation is functionally coupled to EGL-19. IkhiCl was significantly reduced by nemadapine . This inhibitory effect of nemadopine on IkhiCl was eliminated in slo-2(nf100) mutants, indicating that the nemadapine sensitive current is mediated by SLO-2. IkhiCl currents were recorded from adult body wall muscles of the indicated genotypes at holding potentials of -60 to +60 mV. Representative IkhiCl traces (A) and mean current density as a function of membrane potential (B) are shown. (C) SLO-2 activation requires nanodomain coupling to EGL-19. IkhiCl currents recorded in BAPTA are significantly smaller than those in EGTA. The inhibitory effect of BAPTA was reduced in shn-1(nu712 null) mutants and was eliminated in slo-2(nf100) mutants, indicating that the BAPTA sensitive current is mediated by SLO-2. The ratio of IkhiCl current density at +30 mV recorded in BAPTA to the mean current density recorded in EGTA is plotted for the indicated genotypes. (D-E) AP repolarization is mediated by nanodomain activation of SLO-2. AP widths recorded in solutions containing BAPTA are wider than those recorded in EGTA. The effect of BAPTA on AP widths was reduced in shn-1(nu712 null) mutants and was eliminated in slo-2(nf100) mutants, indicating that BAPTA's effect is mediated by SLO-2. Representative traces of WT muscle APs recorded in EGTA and BAPTA are shown (D). The ratio of AP widths recorded in BAPTA to the mean AP widths recorded in EGTA is plotted for the indicated genotypes (E). (F-G) SLO-2(nu725 GFP11) is partially co-localized with EGL-19(nu722 Cherry11) in body muscles. GFP and Cherry fluorescence were reconstituted with GFP1-10 and Cherry1-10 expressed in body muscles. SLO-2 puncta intensity was significantly reduced in shn-1(nu712 null) mutants but was unaffected in shn-1(nu542 DPDZ) and egl-19(nu496 DVTTL) mutants. Representative images (F) and mean SLO-2 puncta intensity (G) are shown. Values that differ significantly from wild type controls are indicated (ns, not significant; *, p <0.05; **, p <0.01; ***, p <0.001). Error bars indicate SEM. Scale bar indicates 4 µm.