Fig. 5: ALS-associated CLCC1 mutations S263R and W267R impair CLCC1 channel functions and promote ER stress in vivo. | Cell Research

Fig. 5: ALS-associated CLCC1 mutations S263R and W267R impair CLCC1 channel functions and promote ER stress in vivo.

From: Disruption of ER ion homeostasis maintained by an ER anion channel CLCC1 contributes to ALS-like pathologies

Fig. 5

a The nonsynonymous (colored circle) and stop-gain (red triangle) mutations of CLCC1 were identified in a Chinese sporadic ALS cohort. The potential damaging mutations are labeled in red. b The Manhattan plot for an exome-wide rare variant burden analysis. The P value of CLCC1 = 1.51 × 10–6, with OR = 5.72. c Protein sequence alignment of CLCC1 encompassing S263, W267, and neighboring residues. S263 and W267 are located in a predicted α-helix. d, e Single channel activities (d) and I-V relationships (e) recorded from planar phospholipid bilayers in the presence of PIP2 after the incorporation of purified hWT CLCC1 and its S263R, and W267R mutants, respectively. f, g Data summary of slope conductance (f) and channel open probability (Po) at 0 mV (g) in the absence and presence of PIP2. h Measurement of [Cl–]ER in 293FT cells expressing WT or ALS-associated mutant CLCC1 by RaMorideER. [Cl–]ER was reflected by the ratio of YFP/DsRed fluorescent signals. Cells expressing WT or ALS-associated mutant CLCC1 were sorted by an engineered near-infrared fluorescent protein, miRFP670S,70 by FACS. i ATP-induced internal Ca2+ release was impaired by S263R and W267R mutants. Human WT (hWT) and S263 and W267 mutant CLCC1 were expressed in 293FT cells loaded with Fura-2. j ER stress and misfolded protein accumulation documented by Bip and ubiquitin (Ubi) staining in cerebella of compound heterozygous mice (S263R/NM and W267R/NM). S263R/+and WT (+/+) are negative for the phenotypes. k, l A diagram for tunicamycin challenge (upper, k). Animals with indicated genotypes were treated with one dose of tunicamycin (3 mg/kg body weight), an ER stress inducer. ER stress and accumulation of misfolded proteins were documented in W267R/W267R brains but not in that of WT (+/+). Data summary shown in (lower, k) and representative images shown in l. Values are presented as mean ± SD. N.S., no significant difference; ***P < 0.001, by one-way ANOVA. In f and g, n = 4–20; in h, n = 4 and in i, n = 5, > 50 cells per experiment; in k, 3 animals/genotypes, at least 4 slides/animal. In j and l, animal age, P30–P45; scale bar, 20 µm.

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