BK channel density is regulated by endoplasmic reticulum associated degradation and influenced by the SKN-1A/NRF1 transcription factor

Abstract

Ion channels are present at specific levels within subcellular compartments of excitable cells. The regulation of ion channel trafficking and targeting is an effective way to control cell excitability. The BK channel is a calcium-activated potassium channel that serves as a negative feedback mechanism at presynaptic axon terminals and sites of muscle excitation. The C. elegans BK channel ortholog, SLO-1, requires an endoplasmic reticulum (ER) membrane protein for efficient anterograde transport to these locations. Here, we found that, in the absence of this ER membrane protein, SLO-1 channels that are seemingly normally folded and expressed at physiological levels undergo SEL-11/HRD1-mediated ER-associated degradation (ERAD). This SLO-1 degradation is also indirectly regulated by a SKN-1A/NRF1-mediated transcriptional mechanism that controls proteasome levels. Therefore, our data indicate that SLO-1 channel density is regulated by the competitive balance between the efficiency of ER trafficking machinery and the capacity of ERAD.

Fig 1. SEL-11/HRD1 is required for normal degradation of SLO-1.

(A) and (B) Representative images and quantification of SLO-1 in the dorsal cord and body wall muscle (white asterisk indicates dorsal cord). Data are means ± SEM; ****P < 0.0001, One-way ANOVA, Tukey’s post hoc test. (scale bar = 10 μm). (C) Western blot analysis shows that total SLO-1 levels are reduced in erg-28 mutants and partially restored in sel-11 erg-28 double mutants. SLO-1 intensity was normalized to that of the α-tubulin. N2 does not express GFP and is a negative control for western blotting with anti-GFP antibody.

Fig 2. A sel-11 mutation reverses the reduced SLO-1 functions observed in the absence of ERG-28.

(A) Locomotory speed was measured in mutants with slo-1(ky399gf) backgrounds. erg-28 slo-1(ky399gf) mutant animals move faster than slo-1(ky399gf). sel-11 mutation depresses locomotory speed in erg-28 slo-1(ky399gf) mutant animals. Data are means ± SEM; ***P < 0.001, One-way ANOVA, Tukey’s post hoc test. (B) sel-11 mutation increases aldicarb resistance in erg-28 slo-1(ky399gf) animals. Aldicarb-induced paralysis was analyzed using Kaplan-Meier survival analysis. (C) Electrophysiology reveals that a sel-11 mutation depresses evoked amplitude in erg-28 slo-1(ky399gf) mutants. Data are means ± SEM; ****P < 0.0001, One-way ANOVA, Tukey’s post hoc test.

Fig 3. A forward genetic screen reveals sel-11 as a critical gene for SLO-1 degradation.

(A) Representative images of SLO-1 levels in slo-1(cim105) and slo-1(cim113gf) nerve rings. (scale bar = 10 μm). SLO-1 levels of the dorsal cords and body wall muscles were similar between cim105 and cim113gf. Data are means ± SEM; NS, paired two-tailed t-test. (B) Phenotypic changes and the associated SLO-1 levels. (C) Schematic of the EMS forward genetic screen. (D) Representative image of the nerve ring of cim54, a mutant isolated from the screen. (E) sel-11(cim54) mutation abolished the thrash rate recovery by the erg-28 mutation. cimEx107, a transgene expressing a fosmid with the sel-11 gene, restored the thrash rates in sel-11(cim54) erg-28 slo-1(cim113gf) mutants. Data are means ± SEM; ****P < 0.0001, One-way ANOVA, Tukey’s post hoc test. (F) Western blot analysis shows that total SLO-1 levels were reduced in erg-28 animals and restored in sel-11(cim54) erg-28 slo-1(cim113gf) mutants. The extrachromosomal cimEx107 transgene reduced SLO-1 levels toward erg-28 slo-1(cim113gf) mutants. N2 does not express GFP and is a negative control for Western blotting with anti-GFP antibody. (G) Model of SEL-11, based on a previously described report [42], as a multi-pass transmembrane ER protein with a Really Interesting New Gene (RING) finger domain (purple) and proline-rich regions (teal). The arrow indicates the predicted proline to serine point mutation at position 371, the beginning of the proline-rich regions.The line indicates the predicted complex substitution in the sel-11(tm1743) allele.

Fig 4. SEL-1/HRD3, a member of the SEL-11 ubiquitin ligase complex, targets SLO-1 channels for degradation.

(A) and (B) Representative images and quantification of SLO-1 at the dorsal cord and body wall muscle. (scale bar = 10 μm). Data are means ± SEM; ***P < 0.001; **P < 0.01, One-way ANOVA, Tukey’s post hoc test.

Fig 5. The aspartic protease DDI-1 functions in the SEL-11-dependent ERAD of SLO-1.

(A) Representative images and quantification of SLO-1 at the dorsal cord and body wall muscle. ddi-1 null mutation and a mutation in the aspartic protease domain of ddi-1 elevate SLO-1 channels to similar levels. (B) Representative images and quantification of SLO-1 at the dorsal cord and body wall muscle. ddi-1, sel-11, and ddi-1;sel-11 restore SLO-1 channels to similar levels. Data are means ± SEM; ****P < 0.0001, One-way ANOVA, Tukey’s post hoc test. (scale bar = 10 μm).

Fig 6. SKN-1A mediates SLO-1 degradation independently of the SEL-11 ERAD pathway.

Representative images and quantification of SLO-1 at the dorsal cord and body wall muscle. (A) skn-1a mutation elevates SLO-1 levels in erg-28 mutants. Data are means ± SEM; ****P < 0.0001, One-way ANOVA, Tukey’s post hoc test. (B) cimEx108, a transgene expressing skn-1a[cut, 4ND] under pan-neuronal promoter rgef-1 and muscle-specific promoter myo-3, dampened SLO-1 levels in ddi-1;sel-11 erg-28 mutants dampened SLO-1 density at the dorsal cord and body wall muscle. Data are means ± SEM; ***P < 0.001; **P < 0.01 (paired two-tailed t-test). (C) cimSi4, a single-copy transgene of skn-1a(cut, 4ND) under the rgef-1 promoter, constitutively induces the transcriptional rpt-3p::gfp reporter (mgIs72) expression, specifically in neurons. The bottom panel demonstrates rpt-3p::gfp expression when treated with 40 μM bortezomib. (D) cimSi4 partially dampened SLO-1 levels in sel-11 erg-28 mutants at the dorsal cord, but the remaining SLO-1 levels are still higher than in erg-28 mutant animals. Data are means ± SEM; ****P < 0.0001, One-way ANOVA, Tukey’s post hoc test. (scale bar = 10 μm).