ERG-28 controls BK channel trafficking in the ER to regulate synaptic function and alcohol response in C. elegans
- Rosalind Franklin University of Medicine and Science, United States
- Lake Forest College, United States
- Cincinnati Children’s Hospital Research Foundation, United States
- University of Illinois at Chicago, United States
Figures
Figure 1 with 3 supplements
Loss-of-function mutation in erg-28 suppresses phenotypic defects of slo-1 gain-of-function mutant.
(A) Mutation in erg-28 suppresses sluggish locomotion of slo-1(ky399gf) mutant. Average speed of animals with indicated genotypes on NGM agar plates without food. slo-1(gf) and erg-28 alleles are ky3…
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Figure 1—source data 1
erg-28 mutation suppresses phenotipic defects of slo-1(gf) mutant.
Raw data and statistics for the speed measurement (A) and egg laying assay (C).
- https://doi.org/10.7554/eLife.24733.003
Figure 1—figure supplement 1
Identification of cim16 mutation in erg-28 gene.
(A) Genetic mapping and fosmid rescue were used to identify the genetic lesion of cim16. Genetic mapping was performed using single nucleotide polymorphisms (SNPs) present between Bristol N2 and …
Figure 1—figure supplement 2
Both gk697770 and tm6168 alleles of erg-28 suppress the sluggish movement of slo-1(ky399) gain-of-function mutant.
Speed measurement was performed as in Figure 1A. Error bars represent S.E.M. (standard error of the mean). n = 10. One way ANOVA with Tukey’s post-hoc analysis.
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Figure 1—figure supplement 2—source data 1
Two other alleles of erg-28 suppress the locomotory defect of slo-1(gf) mutant.
Raw data and statistics for the speed measurement.
- https://doi.org/10.7554/eLife.24733.006
Figure 1—figure supplement 3
The human homolog C14orf1 (herg-28) can partially replace C. elegans erg-28.
Speed measurement was performed as in Figure 1A. C14orf1(herg-28) was expressed under the control of the pan-neuronal promoter in erg-28(cim16) slo-1(ky399gf) double mutant animals. The speeds of …
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Figure 1—figure supplement 3—source data 1
Functional conservation of human homolog with C. elegans erg-28.
Raw data and statistics for the speed measurement.
- https://doi.org/10.7554/eLife.24733.008
Figure 2
ERG-28 is required for SLO-1 function in synaptic transmission.
(A) erg-28(gk697770) mutation suppresses aldicarb resistance of slo-1(ky399gf) mutant and is aldicarb hypersensitive. Aldicarb-induced paralysis was analyzed using Kaplan-Meier survival analysis. …
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Figure 2—source data 1
erg-28 mutation suppresses the synaptic transmission defect of slo-1(gf) mutant.
Raw data and statistics for the aldicarb assay (A) and the electrophysiology (B-E).
- https://doi.org/10.7554/eLife.24733.010
Figure 3 with 1 supplement
ERG-28 is an endoplasmic reticulum resident protein expressed in muscles and neurons.
(A) erg-28 is a part of an operon with C14C10.5. 2.5 kb upstream of C14C10.5 gene including 30 bp of coding region of C14C10.5 was fused with GFP (cimEx54[erg-28p::gfp, ttx-3p::mRFP]). Expression …
Figure 3—figure supplement 1
GFP::ERG-28 co-localizes with the ER marker (A), but not with the Golgi marker (B) in muscle cells.
Muscle cells with a low expression level of GFP::ERG-28 were imaged using the z-scanning acquisition (0.2 micron intervals) from the muscle plasma membrane (top) to the muscle interior/muscle belly …
Figure 4 with 2 supplements
ERG-28 is required for the normal expression level of SLO-1 at synaptic terminals and muscle excitation sites.
(A) SLO-1::GFP expression is reduced in erg-28(gk697770) and erg-28(cim16) mutants. N.R: nerve ring. Scale bar: 10 μm. (B and C) SLO-1 expression is restored by expression of ERG-28 in muscle cells …
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Figure 4—source data 1
SLO-1 expression in the muscle (B) and neuron (C) is reduced in erg-28 mutant.
Raw data and statistics for the fluorescence measurement.
- https://doi.org/10.7554/eLife.24733.015
Figure 4—figure supplement 1
Introduction of GFP and fRFP (FusionRed) into the slo-1 (A) and elks-1 (B) genomic loci using CRISPR/Cas9 technology.
sgRNA sequences are underlined and PAM sequences are shown in red.
Figure 4—figure supplement 2
Introduction of GFP at the end of the genomic slo-1 coding sequence does not interfere with head bending angle (A and B) or normal locomotory speed (C).
The data are presented as the mean ± S.E.M. ns, not significant (p>0.05); one-way ANOVA with Dunnett’s post hoc analysis. (D) SLO-1::GFP is expressed as distinct puncta in neurons and muscles. d.c. …
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Figure 4—figure supplement 2—source data 1
The C-terminal GFP fusion does not interfere with normal function of SLO-1.
Raw data and statistics for the head angle (A) and the speed (C) measurement.
- https://doi.org/10.7554/eLife.24733.018
Figure 5 with 1 supplement
SLO-1 is degraded by proteasome in the absence of ERG-28.
(A) Proteasome inhibition with bortezomib (BZ) partially restores SLO-1 expression at the presynaptic terminals in erg-28(gk69777) mutant. Scale bar: 10 μm. Right panel: SLO-1 average pixel …
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Figure 5—source data 1
Proteasome inhibition and ddi-1 mutation partially restore SLO-1 expression in erg-28 mutant.
Raw data and statistics for the fluorescence measurement.
- https://doi.org/10.7554/eLife.24733.020
Figure 5—figure supplement 1
Co-localization of SLO-1 and ERG-28 in the ventral cord.
ERG-28 and SLO-1 occasionally co-localize together. Scale bar: 10 μm.
Figure 6 with 1 supplement
erg-28 mutation confers the resistance to the intoxicating effect of ethanol and ddi-1 mutation reverses the ethanol-resistant behavior.
(A) The locomotory speed was measured in the presence of ethanol and its values are divided by the average speed of untreated animals. Error bars represent S.E.M. **p=0.0033, ****p<0.0001, one way …
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Figure 6—source data 1
ddi-1 mutation reverses alcohol-resistant locomotion of erg-28 mutant.
Raw data and statistics for the speed measurement.
- https://doi.org/10.7554/eLife.24733.023
Figure 6—figure supplement 1
A model for ERG-28 function.
ERG-28 as a chaperone for the SLO-1 channel complex in the ER. In the absence of ERG-28, SLO-1 is not efficiently delivered to the synaptic terminals, and is highly susceptible to DDI-1 and …
Videos
Video 1
ERG-28 is mobile.
mCherry::ERG-28 was expressed using erg-28 promoter, cimIs39[erg-28p::mCherry::erg-28, odr-1p::gfp]. Time lapse image. A movie recorded for 14 s.
Video 2
A movie for wild-type, erg-28(gk697770) and slo-1(eg142) in the presence of intoxicating dose of ethanol.
https://doi.org/10.7554/eLife.24733.025
Video 3
A movie for wild-type, erg-28(gk697770) and ddi-1(ok1468);erg-28(gk697770) in the presence of intoxicating dose of ethanol.
https://doi.org/10.7554/eLife.24733.026
Video 4
A movie for wild-type, slo-1(eg142) and ddi-1(ok1468) in the presence of intoxicating dose of ethanol.
https://doi.org/10.7554/eLife.24733.027Additional files
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Supplementary file 1
Lists of plasmid constructs and strains used in this study.
- https://doi.org/10.7554/eLife.24733.028
