Decision letter: Shank is a dose-dependent regulator of Cav1 calcium current and CREB target expression

Full text Figures and data Side by side Abstract Introduction Results Discussion Materials and methods References Decision letter Author response Article and author information Metrics Abstract Shank is a post-synaptic scaffolding protein that has many binding partners. Shank mutations and copy number variations (CNVs) are linked to several psychiatric disorders, and to synaptic and behavioral defects in mice. It is not known which Shank binding partners are responsible for these defects. Here we show that the C. elegans SHN-1/Shank binds L-type calcium channels and that increased and decreased shn-1 gene dosage alter L-channel current and activity-induced expression of a CRH-1/CREB transcriptional target (gem-4 Copine), which parallels the effects of human Shank copy number variations (CNVs) on Autism spectrum disorders and schizophrenia. These results suggest that an important function of Shank proteins is to regulate L-channel current and activity induced gene expression. https://doi.org/10.7554/eLife.18931.001 Introduction Shank is a synaptic scaffolding protein (containing SH3, Ankyrin, PDZ, proline-rich and SAM domains) (Grabrucker et al., 2011). Because Shank is highly enriched in the post-synaptic densities of excitatory synapses, prior studies have focused on the idea that Shank proteins regulate some aspect of synapse formation or function. Through its various domains, Shank proteins bind hundreds of other synaptic proteins (Lee et al., 2011; Sakai et al., 2011) thereby potentially altering diverse cellular functions. Shank proteins have been implicated in synaptic transmission, synapse formation, synaptic plasticity, and cytoskeletal remodeling (Jiang and Ehlers, 2013). Mammals have three Shank genes, each encoding multiple isoforms (Jiang and Ehlers, 2013). Several mouse Shank knockouts have been described but these mutants exhibit inconsistent (often contradictory) synaptic and behavioral defects (Jiang and Ehlers, 2013), most likely resulting from differences in which Shank isoforms are impacted by each mutation. The biochemical mechanism by which Shank mutations alter synaptic function and behavior has not been determined. In humans, Shank mutations and CNVs are linked to Autism Spectrum Disorders (ASD), schizophrenia, and mania (Durand et al., 2007; Peça et al., 2011). Haploinsufficiency for 22q13 (which spans the Shank3 locus) occurs in Phelan-McDermid syndrome (PMS), a syndromic form of ASD (Phelan and McDermid, 2012). PMS patients exhibit autistic behaviors accompanied by hypotonia, delayed speech, and intellectual disability (ID) (Bonaglia et al., 2011). Heterozygous inactivating Shank3 mutations are found in sporadic ASD and schizophrenia (Durand et al., 2007; Peça et al., 2011). These genetic studies suggest that decreased Shank3 function likely plays an important role in the pathophysiology of these psychiatric disorders. A parallel set of genetic studies suggest that increased Shank3 function also contributes to psychiatric diseases. 22q13 duplications spanning Shank3 are found in ASD, schizophrenia, ADHD, and bipolar disorder (Durand et al., 2007; Failla et al., 2007; Han et al., 2013). These 22q13 duplications involve multiple genes; consequently, the contribution of increased Shank3 to these psychiatric disorders was uncertain. To address this issue, a transgenic mouse model was developed that selectively over-expresses Shank3 (Han et al., 2013). This transgenic mouse exhibited hyperactive behavior and susceptibility to seizures. Taken together, these studies suggest that too little or too much Shank3 is associated with several psychiatric disorders. If Shank3 mutations and CNVs are causally associated with these psychiatric disorders, cellular and circuit phenotypes should also be sensitive to Shank3 copy number. Consistent with this idea, several defects have been reported in Shank3+/- heterozygotes, including: decreased mEPSC frequency and spine density (Zhou et al., 2016), decreased Ih current density (Yi et al., 2016), decreased TRPV1 current density (Han et al., 2016), increased tactile sensitivity (Orefice et al., 2016), and decreased post-synaptic Homer abundance (Wang et al., 2016). Increased Shank expression was associated with increased spine density in hippocampus and decreased inhibitory synapses (Han et al., 2013). In many cases (Han et al., 2013; Orefice et al., 2016; Wang et al., 2016; Zhou et al., 2016), it was not determined if these phenotypes are a cell autonomous consequence of altered Shank3 copy number. While these studies identify cellular deficits associated with Shank3 CNVs, it remains unclear which Shank binding partners and cellular functions are responsible for psychiatric traits, nor why these traits are sensitive to both increased and decreased Shank gene dosage. To further investigate how Shank proteins regulate nervous system development and function, we analyzed Shank function in an invertebrate genetic model. Here we show that C. elegans SHN-1 is a dose-sensitive regulator of Cav1 calcium current and CREB induced gene expression in C. elegans body muscles. Results The SHN-1 PDZ domain binds EGL-19/Cav1 channels 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 (Figure 1A). Many protein ligands have been identified for the Shank PDZ domain (Lee et al., 2011). Of these potential binding partners, we focused on EGL-19/Cav1 because human CACNA1C (which encodes a Cav1 α-subunit) is mutated in Timothy Syndrome (TS), a rare monogenic form of ASD (Splawski et al., 2005, 2004), and polymorphisms linked to CACNA1C are associated with multiple psychiatric disorders (Cross-Disorder Group of the Psychiatric Genomics Consortium, 2013). We confirmed that SHN-1's PDZ domain binds the EGL-19 carboxy-terminal motif (-VTTLCOOH) by both yeast two-hybrid and GST-pull down assays (Figure 1—figure supplement 1A and B). Thus, like their mammalian counterparts, SHN-1 binds to EGL-19/ Cav1 (Zhang et al., 2005). Figure 1 with 1 supplement see all Download asset Open asset SHN-1 promotes EGL-19/Cav1 channel function. (A) The protein domains found in SHN-1 and rat Shank3A are compared. SHN-1 lacks an SH3 domain but contains all other domains found in mammalian Shank proteins. Homology between the worm and mammalian protein is shown for each domain. (B–F) Voltage-activated Ca+2 currents were recorded from adult body wall muscles of the indicated genotypes at holding potentials of −60 to +40 mV. Averaged traces (B), mean current density as a function of holding potential (C), normalized conductance as a function of holding potential (D), mean current density at 10 mV (E), and mean deactivation time constants (F) are shown. shn-1 mutants had significantly decreased Ca+2 current-density and this defect was rescued by a single copy transgene expressing SHN-1 in body muscles (nuSi26) (D). No significant differences were observed for voltage-dependence of current activation and de-activation kinetics. The number of animals analyzed is indicated for each genotype. Values that differ significantly from wild type controls are indicated (***p<0.001). Error bars indicate SEM. Mean, standard errors, sample sizes, and p values for this figure are shown in Supplementary file 1. https://doi.org/10.7554/eLife.18931.002 EGL-19/Cav1 calcium currents are diminished in shn-1 mutants The impact of Shank binding on Cav1.3 function has been assessed by over-expression in Xenopus oocytes and in cultured neurons (Stanika et al., 2016; Zhang et al., 2005) but has not been tested in mutant or transgenic animals. Because shn-1 is expressed in muscles (Stefanakis et al., 2015), we assayed EGL-19/Cav1 channel function by recording voltage-activated calcium currents in body muscle, using salines containing potassium channel blockers (TEA and 4AP). In these conditions, the remaining voltage-activated inward current is entirely blocked by the EGL-19 antagonist nemadipine (Lainé et al., 2011). In shn-1(tm488) null mutants, calcium current density was significantly decreased (Figure 1B,C,E). Neither the voltage-dependence of calcium current activation (Figure 1D) nor the deactivation kinetics (Figure 1F) were altered in shn-1(tm488) null mutants. The shn-1 calcium current defect was rescued by a transgene restoring SHN-1 expression in body muscles (Figure 1E), confirming that SHN-1 has a cell autonomous effect on EGL-19/Cav1 currrent. To determine if SHN-1's effects on calcium currents were specific, we measured voltage-activated potassium currents in body muscles (Figure 1—figure supplement 1C). Neither the voltage-dependence nor the current density of fast and slow potassium currents were significantly altered in shn-1 mutants. Collectively, these results suggest that SHN-1 specifically regulates the expression or function of EGL-19/Cav1 channels. SHN-1 binding to EGL-19 increases Cav1 current density SHN-1 effects on calcium current density could result from direct binding of SHN-1 to EGL-19/Cav1 or indirectly via other SHN-1 binding partners. We did several experiments to distinguish between these possibilities. First, we utilized CRISPR to isolate two deletion alleles that alter the EGL-19 carboxy-terminus (nu495 and nu496) (Figure 2A). Both deletion mutants exhibited decreased calcium current density (similar to the defect observed in shn-1 null mutants) (Figure 2B–C). The egl-19(nu496) mutation had no effect on the voltage-dependence of calcium current activation nor on deactivation kinetics (Figure 2—figure supplement 1A and B). The egl-19(nu496) and shn-1 null mutations did not have additive effects on calcium current density in double mutants, as would be predicted if SHN-1's effects on calcium current require direct binding to EGL-19's carboxy-terminus (Figure 2D and E). To further examine the functional impact of the SHN-1 PDZ interaction with EGL-19, we analyzed shn-1(ok1241) mutants, which have an in-frame deletion spanning exons encoding the PDZ domain and part of the proline-rich domain (Figure 1—figure supplement 1D). The shn-1(ok1241) mutants exhibited a decrease in calcium current density similar to those observed in shn-1 null and the egl-19 carboxy-terminal deletion mutants and had no effect on voltage-dependence of current activation nor on deactivation kinetics (Figure 2—figure supplement 1C–F). Collectively, these results suggest that SHN-1 binding to EGL-19's carboxy-terminus promotes the expression or function of L-type calcium channels. Figure 2 with 1 supplement see all Download asset Open asset SHN-1 binding to EGL-19's carboxy-terminus promotes the expression or function of L-type calcium channels. (A) Predicted c-terminal sequences of mutant EGL-19 proteins are shown. egl-19(nu496) is a 22 bp deletion and egl-19(nu495) is a 5 bp deletion, both resulting in frame shifts that delete the carboxy-terminal PDZ ligand of EGL-19 (-VTTLCOOH). Residues in blue represent the PDZ ligand. Residues in red represent those introduced by the frame shift mutations. (B–E) Voltage-activated Ca+2 currents were recorded from adult body wall muscles of the indicated genotypes at holding potentials of −60 to +40 mV. Representative traces (B), mean current density at 0 mV (C, E), and mean current density as a function of holding potential (D) are shown. The egl-19(nu496) and shn-1(tm488) single mutants had similar decreases in Ca+2 current-density, and additive defects were not observed in the double mutant. The number of animals analyzed is indicated for each genotype. Values that differ significantly from wild type controls are indicated (***p<0.001). Error bars indicate SEM. Mean, standard errors, sample sizes, and p values for this figure are shown in Supplementary file 1. https://doi.org/10.7554/eLife.18931.004 SHN-1 promotes trafficking of EGL-19 channels to the cell surface SHN-1 effects on calcium current could result from a change in EGL-19 delivery to the cell surface. We performed two further experiments to test this idea. First, we measured the gating currents of voltage-activated channels in body muscles (Figure 3A–B). The activation of Cav channels is mediated by depolarization-induced movements of positively charged residues in membrane-spanning S4 helices, which are termed gating charges. When there is no net calcium current (by holding the muscle membrane at the reversal potential), gating charge movement can be measured as a small voltage-activated current. The magnitude of gating currents can be used as a measure of Cav channel surface abundance (Fu et al., 2011; Hulme et al., 2006). The total voltage activated gating charge in body muscles was significantly reduced in shn-1 null mutants (Figure 3B). This shn-1 mutant defect in gating charge is unlikely to result from decreased surface delivery of other voltage-activated channels because voltage-activated potassium currents were unaltered in shn-1 mutants (Figure 1—figure supplement 1C). Thus, analysis of gating currents suggests that the decreased calcium current exhibited in shn-1 mutants arises from decreased trafficking of EGL-19/Cav1 channels to the cell surface. Figure 3 Download asset Open asset SHN-1 promotes EGL-19/Cav1 delivery to the cell surface. (A–B) Voltage-activating gating currents were significantly decreased in shn-1 null mutants. Averaged trace of gating current in wild type adult body muscles (A) and mean gating charge (normalized to capacitance) (B) are shown. (C–G) Surface delivery of the Terrier fusion protein is significantly reduced in shn-1 null mutants. (C) A schematic illustrating the structure of the Terrier fusion protein is shown. (D) A schematic illustrating the imaged region (left) and representative images of Terrier pHluorin fluorescence in the nerve ring are shown. Mean pHluorin puncta intensity (E), pHluorin puncta area (F), and total pHluorin puncta fluorescence (G) are shown. Regions of interest utilized to quantify Terrier fluorescence are indicated (D). The number of animals analyzed is indicated for each genotype. Values that differ significantly from wild type controls are indicated (**p<0.01, *p<0.05). Error bars indicate SEM. https://doi.org/10.7554/eLife.18931.006 To further investigate SHN-1's effects on EGL-19 trafficking, we designed a fluorescent reporter construct containing a trans-membrane domain fused to EGL-19's cytoplasmic tail domain (504 amino acids), which includes the carboxy-terminal PDZ ligand (Figure 3C). This chimeric protein (designated Terrier to indicate the presence of EGL-19's cytoplasmic tail) was expressed in body muscles. The Terrier protein contains tagRFP in the cytoplasmic domain, and pHluorin (a pH-sensitive GFP) in the ectodomain (Figure 3C). Thus, red fluorescence reports total Terrier protein while green fluorescence reports surface Terrier molecules. When expressed in body muscles, Terrier exhibits a punctate green and red fluorescence in the nerve ring, where body muscles receive synaptic input (Figure 3C–D). In shn-1 null mutants, the intensity and size of green Terrier puncta were significantly decreased (Figure 3D–G), indicating decreased surface Terrier protein in the nerve ring. Thus, decreased calcium current density (20%) in shn-1 mutants was mirrored by similar decreases in gating charge (26%) and total surface Terrier fluorescence (36%). Collectively, these results suggest that the decreased calcium current in shn-1 null mutants arises from decreased delivery of EGL-19/Cav1 channels to the cell surface. gem-4 is an activity-induced CRH-1/CREB target expressed in muscles Increased cytoplasmic calcium activates expression of a large number genes, hereafter designated activity-induced gene expression. Although Cav1 channels account for a small fraction of bulk calcium entry in neurons, Cav1 channels account for the majority of activity-induced gene expression (Ma et al., 2012). This privileged ability of Cav1 channels to activate gene expression is thought to be mediated by direct physical coupling of Cav1 channels to the calcium sensors responsible for activating CREB (Deisseroth et al., 1996; Wheeler et al., 2008). Because shn-1 mutations alter EGL-19/Cav1 current, we hypothesized that SHN-1 may also play a role in activity-induced gene expression. To test this idea, we first identified activity-induced muscle genes. We analyzed gene expression following depolarization of body muscles with a nicotinic acetylcholine (ACh) agonist (levamisole, Lev). Lev-induced genes were identified using the Affymetrix C. elegans gene chip. This analysis identified 427 genes whose expression was significantly increased following muscle depolarization (>2 fold change, FDR p<0.05) (Figure 4, Supplementary file 2). 67% (287/427) of Lev-induced genes contain binding sites for the myogenic transcription factor HLH-1 (<5 kb from the transcriptional start site, TSS) in chromatin-immunoprecipitation experiments (http://www.modencode.org), suggesting that these genes are expressed in body muscles. Of the HLH-1 binding genes, 81% (233/287) contain predicted CREB binding sites (<5 kb from the TSS). These results suggest that C. elegans body muscles (like other excitable cells) have a large number of activity-induced genes, many of which are potential CREB transcriptional targets. Using this Lev-induced gene list, we devised a simple reporter assay for CREB induced gene expression. For this purpose, we focused on the gem-4/Copine gene, which was the top hit from our analysis of Lev-induced genes (induced ~30 fold) (Figure 4). The gem-4 promoter contains multiple CRH-1/CREB binding sites, implying that it could be a direct CRH-1 transcriptional target. Quantitative RT-PCR confirmed that Lev treatment increased gem-4 mRNA levels 8-fold (Figure 5A). We designed a transcriptional reporter (Figure 5B) that compares expression of the gem-4 promoter (expressing NLS-GFP) with a control promoter unaffected by depolarization (the myo-3 promoter, expressing NLS-mCherry) in individual muscle cells. Using this reporter, we found that Lev treatment increased gem-4 expression 8–12-fold (Figure 5C–D) while myo-3 expression was unaltered (Figure 5—figure supplement 1A). Lev-induction of the gem-4 reporter was eliminated by mutations inactivating a Lev receptor subunit (UNC-29), indicating that gem-4 induction was not mediated by an off target effect of Lev (Figure 5C). Lev-induction of gem-4 was also blocked in mutants lacking CRH-1/CREB and this defect was rescued by a transgene expressing CRH-1 in body muscles (Figure 5D). These results identify gem-4 as a CRH-1/CREB target expressed in body muscles. Figure 4 Download asset Open asset Analysis of mRNA abundance following muscle depolarization. mRNA abundance in Lev (200 µM, 1 hr) versus mock treated synchronized L4 larvae is plotted. Fold change (x-axis) is plotted against the statistical significance (y-axis) for each probeset. Fold changes are shown in log2 scale. Adjusted P values are shown in - log10 scale. Genes with increased (red dots) and decreased (green dots) expression are indicated (>2 Fold-change, FDR p<0.05). Probe sets corresponding to gem-4 and cex-1 are indicated. All genes that are differentially expressed following Lev treatment are listed in Supplementary file 2. https://doi.org/10.7554/eLife.18931.007 Figure 5 with 1 supplement see all Download asset Open asset gem-4 Copine expression in body muscle is induced by depolarization. Induction of gem-4 expression was analyzed by qPCR (A) and using a transcriptional reporter (B–H). (A) The abundance of gem-4 mRNA (assessed by qPCR) was increased following 1 hr levamisole (Lev) exposure. The number of biological replicates is indicated. (B) A schematic diagram of the gem-4 reporter construct (left) and representative images of muscle nuclei (right) before and after a 20 min Lev exposure, and 2 hr recovery. (C–D) The mean fold induction of the gem-4 reporter (Pgem-4) after Lev treatment is shown. Lev-induced gem-4 expression was abolished in mutants lacking UNC-29, an essential subunit of the Lev receptor (C) and in mutants lacking the transcription factor CRH-1 (D). The crh-1 mutant defect in gem-4 induction was rescued by a transgene expressing CRH-1 in body muscles (D). (E) Expression of the gem-4 reporter was measured following photo-stimulation of transgenic animals that express ChR2 in cholinergic motorneurons. Expression of gem-4 was significantly increased by 2, 5, 10, and 20 Hz photo-stimulation (for 20 min). Photo-evoked gem-4 expression was not observed when animals were not cultured with ATR. (F–G) The fold induction of the gem-4 reporter following Lev exposure was significantly reduced in shn-1 null mutants (F) but not in shn-1(ok1241) mutants, which lack the PDZ domain (G). Lev-induced gem-4 expression was significantly increased in egl-19(nu496) mutants, which lack the carboxy-terminal PDZ ligand (H). (I–J) Lev induction of the cex-1 reporter is significantly reduced in shn-1 mutants. (I) Schematics of the cex-1 and myo-3 reporters are shown. (J) Expression of the cex-1 reporter (normalized to myo-3 expression in the same nucleus) was significantly increased by Lev treatment. The Lev-induced expression of the cex-1 reporter was significantly reduced in shn-1 mutants. The number of animals analyzed is indicated for each genotype. Values that differ significantly from wild type controls are indicated (***p<0.001; **p<0.01; *p<0.05). Error bars indicate SEM. https://doi.org/10.7554/eLife.18931.008 Lev treatment is a non-physiological stimulus that produces prolonged muscle depolarization. To determine if synaptic activity induces the gem-4 reporter, we evoked excitatory synaptic input to muscles by photo-stimulating transgenic animals expressing channel rhodopsin in cholinergic motor neurons (Figure 5E). In patch clamp recordings of body muscles, spontaneous action potentials are observed at ~1 Hz (Gao and Zhen, 2011). Therefore, to mimic a realistic pattern of synaptic input, we photo-stimulated motor neurons (25 ms light pulse) at 1–20 Hz for 20 min. The gem-4 reporter was significantly induced following 2, 5, 10, and 20 Hz photo-stimulation (Figure 5E). Induction was not observed at lower frequencies (0.5 and 1 Hz) nor when animals were cultured without all trans-retinal (ATR) (Figure 5E). Photo-induction of gem-4 expression was eliminated in unc-13 mutants (Figure 5—figure supplement 1B), which have dramatically reduced synaptic vesicle exocytosis (Richmond et al., 1999). By contrast, unc-13 mutations did not prevent Lev-induced gem-4 expression (Figure 5—figure supplement 1C). Similar levels of gem-4 induction were produced by 20 Hz photo-stimulation and Lev treatment (Figure 5—figure supplement 1B–C). Thus, the gem-4 reporter was induced by both synaptic and Lev-evoked muscle depolarization. Expression of a mouse gem-4 paralog (N-copine) in hippocampal neurons is also induced by high frequency stimulation of acute brain slices (Nakayama et al., 1998); consequently, activity-induced copine expression is observed in both muscles and neurons and is conserved across phylogeny. SHN-1 is required for gem-4 induction Using this gem-4 reporter construct, we next asked if SHN-1 is required for CRH-1/CREB-induced gene expression. Lev-induced gem-4 expression was significantly decreased in shn-1(tm488) null mutants (Figure 5F). To determine if SHN-1 controls expression of other activity-induced genes, we developed a transcriptional reporter for a second Lev-induced gene (cex-1) 4 and Expression of the cex-1 reporter in body muscles was dramatically induced following Lev treatment (Figure Because cex-1 expression in muscles could not be we were to measure the of the cex-1 reporter following Lev treatment. with the gem-4 reporter, we found that Lev-induced cex-1 expression in muscles was dramatically reduced in shn-1 null mutants (Figure Taken together, these results the idea that SHN-1 promotes activity-induced gene expression in body muscles. we asked if gem-4 induction binding of SHN-1's PDZ domain to EGL-19's EGL-19's PDZ ligand egl-19(nu496) significantly increased gem-4 induction while SHN-1's PDZ domain shn-1(ok1241) had no effect on gem-4 induction (Figure These results indicate that SHN-1 regulates EGL-19/Cav1 current and CRH-1/CREB activation by the PDZ binding to EGL-19 while the current and gem-4 induction are sensitive to shn-1 gene and of human genes are both associated with ASD, schizophrenia, and mania (Bonaglia et al., et al., 2007; Failla et al., 2007; et al., Han et al., 2013). If Shank CNVs are causally associated with these psychiatric disorders, cellular phenotypes should be sensitive to Shank copy number. To test this idea, we analyzed the effect of shn-1 gene dosage on calcium currents and Lev-induced gem-4 expression (Figure We analyzed animals with 0 1 2 and 4 single copy shn-1 of shn-1. to wild type muscle calcium current density was significantly increased in animals containing 1 and 4 of shn-1 and was significantly decreased in animals containing 0 of shn-1 (Figure The kinetics of calcium current deactivation were not significantly altered by changes in shn-1 dosage (Figure when to wild type gem-4 induction was significantly diminished in animals containing and 4 shn-1 (Figure Thus, L-type calcium current density and gem-4 induction were both sensitive to shn-1 copy number. increased and decreased shn-1 gene dosage produced similar defects in calcium current and gem-4 Figure Download asset Open asset shn-1 gene dosage regulates calcium current density and gem-4 The effect of shn-1 gene dosage on calcium current density (A–B) and gem-4 induction (C) was The following genotypes were 0 of shn-1 1 copy of shn-1 2 of shn-1 and 4 of shn-1 in (A–B) Ca+2 current was sensitive to changes in shn-1 gene with decreased shn-1 and increased and 4 shn-1 current density observed in the indicated Mean current density as a function of holding potential mean current density at 10 mV (B), and mean current deactivation time constants (C) are shown. (D) Lev-induced gem-4 expression was significantly reduced in animals with and 4 of shn-1. The number of animals analyzed is indicated for each genotype. Values that differ significantly from wild type controls are indicated (***p<0.001; Error bars indicate SEM. Mean, standard errors, sample sizes, and p values for are shown in Supplementary file 1. is not sensitive to shn-1 gene dosage To determine SHN-1's role in synaptic transmission, we recorded excitatory post-synaptic currents from body muscles. were significantly in shn-1 null mutants (Figure The of evoked in wild type and shn-1 mutants were indicating that the kinetics of evoked was unaltered (Figure The and of spontaneous were unaltered in shn-1 mutants (Figure supplement The change in evoked with unaltered mEPSC a change in cholinergic The shn-1 null defect was rescued by expressing SHN-1 in body muscles, implying that SHN-1 functions (Figure The evoked (Figure and (Figure supplement observed in animals with 2, and 4 shn-1 were not significantly indicating that synaptic is not sensitive to shn-1 copy number. Similar results were reported in where in the was in but this effect was not observed in et al., 2016). Figure with 1 supplement see all Download asset Open asset at the is not sensitive to shn-1 gene dosage. were recorded from adult body wall muscles. Representative traces of evoked shn-1 and evoked (normalized to (B), and mean evoked charge (C) are shown. charge was significantly increased in shn-1 and this defect was rescued by a single copy transgene (nuSi26) that SHN-1 expression in body muscles Averaged normalized shn-1 and evoked have and indicating that and kinetics were unaltered charge (D) and (E) did not differ significantly in animals containing 2, and 4 shn-1 The number of animals analyzed is indicated for each genotype. Values

Topic: Cellular transport and secretion
Subfield: Cell Biology
Field: Biochemistry, Genetics and Molecular Biology
Domain: Life Sciences