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

Figures

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  Figure 1.. SHN-1 regulates muscle AP firing patterns. (A) Representative traces of spontaneous muscle APs are shown for WT and shn-1(nu712 null) mutants. APs occur in bursts of ~10 APs/ burst. Plateau potentials (PPs), defined as transients lasting >150ms, are observed less frequently, often at the end of a burst. (B) APs become progressively longer during bursts. Successive APs taken from a representative burst are shown. (C) Repetitive depolarization to +30 mV leads to a progressive decrease in potassium currents. A representative recording from a WT animal is shown. This likely results from an accumulation of inactivated potassium channels during repetitive stimulation. (D–I) Mean PP rate (D), AP rate (E), AP width (F), RMP (G), AP amplitude (H), and input resistance (Rin, I) are compared in WT and shn-1 null mutants. All shn-1 data were obtained from shn-1(nu712) except for Rin (I), which were from shn-1(tm488). 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.

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  Figure 1—figure supplement 1.. SHN-1 is expressed in many tissues. Endogenous SHN-1 is broadly expressed, including in neurons, muscles, skin, and glia. A representative image of reconstituted fluorescence produced by shn-1(nu600 GFP11) and eft-3>GFP1-10 (left) and the corresponding bright field image (right) are shown. Pharyngeal muscles (Ph mm) are indicated in the bright field image. SHN-1(GFP11) expression in body muscles was not detected, consistent with the very low shn-1 mRNA levels reported in body muscles (Cao et al., 2017; Packer et al., 2019). Scale bar indicates 14 μm.

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  Figure 2.. SHN-1 acts in muscles to control AP duration. (A) A schematic of the shn-1 locus is shown. Open boxes indicate UTRs, black boxes indicate coding regions. Recombination sites mediating CRE induced deletions (LoxP) and inversions (FLEX) are indicated. The shn-1(nu697) allele allows CRE-induced shn-1 knockouts while shn-1(nu652) allows CRE-induced shn-1 rescue. In shn-1(nu652), an exon containing in frame stop codons was inserted into the second intron (in the ‘OFF’ orientation). This stop exon is bounded by FLEX sites. (B) Representative traces of spontaneous muscle APs are shown in shn-1(nu697) with and without muscle CRE expression. Mean PP rate (C) and AP width (D) are compared in the indicated shn-1 mutants without (-) and with CRE expression in muscles (m) or neurons (n). Sample sizes are as follows: shn-1(nu697) (17); shn-1(nu697) +muscle CRE (21); shn-1(nu697) +neuron CRE (15); shn-1(nu652) (18); shn-1(nu652) +muscle CRE (30); and shn-1(nu652) +neuron CRE (19). 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. Representative traces for genotypes in panels C and D…

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  Figure 2—figure supplement 1.. Representative traces for recordings summarized in Figure 2C and D. Representative traces of spontaneous muscle APs are shown for the indicated genotypes.

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  Figure 2—figure supplement 2.. Muscle AP defects in shn-1(null) and shn-1(muscle KO) are not significantly different. Mean PP rate and AP width are compared in the indicated shn-1 mutants. Sample sizes are as follows: shn-1(nu712 null) (21); shn-1(nu697) +muscle CRE (21). Error bars indicate SEM. No significant differences were observed.

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  Figure 3.. Mutations disrupting SHN-1 binding to EGL-19 increase AP duration. (A) A schematic illustrating the binding interaction between EGL-19’s c-terminus and SHN-1’s PDZ domain is shown. (B–C) Mean PP rate and AP width are compared in the indicated genotypes. Representative traces are shown in Figure 3—figure supplement 1. Mutations deleting the SHN-1 PDZ domain (nu542 ΔPDZ) or those deleting EGL-19’s c-terminal PDZ ligand (nu496 ΔVTTL) were edited into the endogenous genes using CRISPR. These mutations significantly increased AP width, compared to WT controls. Sample sizes are as follows: WT (41), shn-1(nu542) (22), and egl-19(nu496) (22). 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.

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  Figure 3—figure supplement 1.. Representative traces for recordings summarized in Figure 3B and C. Representative traces of spontaneous muscle APs are shown for the indicated genotypes.

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  Figure 3—figure supplement 2.. Muscle AP defects in shn-1(null) and shn-1(null); egl-19(ΔVTTL) double mutants are not significantly different. Mean PP rate and AP width are compared in the indicated genotypes. Sample sizes are as follows: shn-1(nu712 null) (21); shn-1(nu712); egl-19(ΔVTTL) (15). Error bars indicate SEM. No significant differences were observed.

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  Figure 4.. AP repolarization is mediated by SHK-1 and SLO channels. (A–D) Muscle voltage activated potassium currents are mediated by SHK-1 and SLO-2. 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 Figure 4—figure supplement 1) and mean AP widths (F) are shown. Alleles used in this figure were: shk-1(ok1581), slo-1(js379), and slo-2(nf100). Sample sizes are as follows: in panel B, WT (5), slo-2 (7), and shk-1 (5); in panel D, WT (10), slo-2 (5), shk-1 (5), shk-1;slo-2 (6); in panel F, WT (16), slo-1 (13), slo-2 (14), slo-1; slo-2 (8), shk-1 (7). Values that differ significantly from wild type controls are indicated (ns, not significant; *, p < 0.05; **, p < 0.01; ***, p <…

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  Figure 4—figure supplement 1.. Representative traces for recordings summarized in Figure 4E. Representative traces of spontaneous muscle APs are shown for the indicated genotypes.

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  Figure 5.. SLO-2 and SLO-1 function together in heteromeric channels. (A–B) IkhiCl was significantly decreased in slo-1(js379) and slo-2(nf100) single mutants but was not further decreased in slo-1; slo-2 double mutants. Representative traces (A) and mean current density (B) at +30 mV are shown. Sample sizes for panel B: WT (15), slo-1 (9), slo-2 (12), slo-1;slo-2 (6). (C–F) Expression of split GFP tagged SLO-2 (C–D) and SLO-1 (E–F) was analyzed in body muscles. CRISPR alleles were constructed adding 7 copies of GFP11 to the endogenous slo-1 and slo-2 genes (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—figure 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. Sample sizes are as follows: in panel D, WT (38) and slo-1 (23); in panel F, WT (34) and slo-2 (19). Values that differ significantly from wild type controls are indicated (ns, not significant; *, p < 0.05;…

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  Figure 5—figure supplement 1.. Analysis of GFP11 tagged slo-1 and slo-2 alleles. APs and potassium currents were analyzed in strains containing slo-1(nu678 GFP11) and slo-2(nu725 GFP11) together with the muscle>GFP1-10 transgene. Mean AP width (A), RMP (B), and IkhiCl current density (C) were not significantly different from WT controls. Sample sizes are as follows: in panels A and B, WT (25), slo-1(nu678) (15), and slo-2(nu725) (13); in panel C, WT (10), slo-1(nu678) (12), and slo-2(nu725) (6). Error bars indicate SEM.