Link ER ion homeostasis maintained by an ER anion channel to ALS


 Although anion channel activities have been demonstrated in sarcoplasmic reticulum/endoplasmic reticulum (SR/ER), their molecular identities and functions remain unclear. Here, we link rare variants of CLCC1 (Chloride Channel CLIC Like 1) to ALS (amyotrophic lateral sclerosis). We demonstrate that CLCC1 is a pore-forming component of an ER anion channel and that ALS-associated mutations impair the channel activity. CLCC1 unitary conductance is inhibited by luminal Ca2+ but facilitated by phosphatidylinositol 4,5-bisphosphate (PIP2). We identified a conserved lysine 298 (K298) in CLCC1 intraluminal loop as the critical PIP2-sensing residue. CLCC1 maintains steady-state [Cl-]ER and morphology and regulates ER Ca2+ homeostasis including steady-state [Ca2+]ER and efficiency of internal Ca2+ release. ALS-associated mutant CLCC1 increase steady-state [Cl-]ER and impair ER Ca2+ homeostasis. Phenotypic comparisons of multiple Clcc1 mutant alleles, including ALS-associated mutations, reveal a CLCC1 dosage-dependence in severity of disease phenotypes in vivo. Conditional knockout of Clcc1 cell-autonomously causes motor neuron loss and ER stress, misfolded protein accumulation, and characteristic ALS pathologies in the spinal cord. Thus, we argue that disruption of ER ion homeostasis maintained by CLCC1 underlies etiology of neurodegenerative diseases.

shown to lead to ER stress and neurodegeneration in mice 18 . A burden analysis 113 23 was further carried out and revealed that CLCC1 is associated with ALS (p = 114 1.51×10 -6 , with OR = 5.72), reaching suggestive significance (Fig. 1c).

ALS mutations S263R and W267R reduce CLCC1 expression and promote
116 ER stress and protein misfolding in vivo 117 Homozygous knockout of Clcc1 in mice is lethal, indicating Clcc1 is essential 118 (Extended Data Fig. 2a and Supplementary Table 2). Evolutionarily, CLCC1 119 Link CLCC1 to ALS. 6 orthologues appear in vertebrate bot not invertebrate (Fig. 1d). Human and 120 mouse CLCC1 share 73% identity, but S263 and W267 are conserved between 121 the two species. To examine the biological consequence of S263R and W267R 122 in vivo, we generated S263R and W267R knock-in mouse lines (Extended Data 123 Fig. 2b and 2c). Mice heterozygous for S263R and W267R were viable and fertile, 124 and no obvious ER stress and protein misfolding was disclosed in S263R  Table 2). Therefore, our data support the 139 Link CLCC1 to ALS. 7 notion that ALS-associated S263R and W267R mutations are potential disease-140 causing.

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CLCC1 forms homomultimer in the ER membrane 142 Based on its primary sequence, CLCC1 shares little sequence similarity with any 143 known ion channel and is predicted to contain three transmembrane segments 144 (TMs) and an N-terminal signal peptide (Fig. 2a). We generated antibodies 145 against the N-and C-termini of CLCC1 (Extended Data Fig. 3a). Using the C- 146 terminal antibody, we confirm that as suggested by a previous report 18 CLCC1 is 147 predominantly ER-localized, as demonstrated by its co-localization with 148 CALNEXIN, an ER resident protein (Extended Data Fig. 3b).

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To understand how CLCC1 functions in the ER, we treated human 293FT cells 150 with disuccinimidyl suberate (DSS), a crosslinker with a spacer length of 11.4 Å.

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The C-terminal antibody detected high molecular weight complexes in a DSS 152 dosage-dependent manner from whole cell lysate. From the complex sizes, we 153 speculated that CLCC1 forms homomultimers (Fig. 2b) amplitude, but also the rate (as reflected by the increase in time-to-peak), of ATP-283 induced Ca 2+ release ( Fig. 4b and 4c). Although the two shRNAs had different 284 CLCC1 knockdown efficiencies, they impaired the ATP-induced Ca 2+ amplitude 285 and rate to a similar extent. Interestingly, both ALS-associated S263R and W267R 296 mutations located in the 2 nd loop also significantly affected the internal Ca 2+ release 297 relative to wildtype hCLCC1 (hWT) (Fig. 4f)  consecutive row of six conserved residues-VPPTKA in the 2 nd loop, which is 338 required for CLCC1 facilitation of internal Ca 2+ release ( Fig. 4d and 4e). In 339 addition, K298 is downstream of two proline residues, which usually present strong 340 conformational rigidity, and lies at the beginning of a predicted alpha-helix.
We expressed and purified K298A mutant mCLCC1 and incorporated it into 342 the lipid bilayer in the absence of PIP2, the mutant protein exhibited single channel 343 activity with a slope conductance of 31.8 ± 0.7 pS, slightly lower than that of 344 wildtype mCLCC1 (39.9 ± 1.0 pS) (Fig. 5a, 5d, and 5f). The Po at 0 mV did not 345 differ from that of wildtype mCLCC1 ( Fig. 5a and 5f). Next, we mutated K298 to 346 the negatively charged residue glutamate (K298E). Like K298A, K298E also has 347 little effect on the channel activity in absence of PIP2 (Fig. 5f). However, unlike 348 wildtype mCLCC1 responsible to PIP2 ( Fig. 5b and 5e), both K298A and K298E 349 mutants abolished the responses, in terms of conductance and Po ( Fig. 5e and 5f).

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Therefore, we conclude that PIP2 facilitates CLCC1 channel activity and a 351 conserved K298 in the 2 nd loop is responsible for the facilitation.

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In comparison to the K298A mutant protein in the lipid bilayer assay, proteins 353 with the ALS patient mutations showed slope conductance that were significantly 354 lower than that of hWT in presence of PIP2 (Extended Data Fig. 10a-10c).

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As knockdown of CLCC1 impairs ER ion homeostasis and leads to ER swelling 411 (Fig. 3), we next asked whether dysfunction of CLCC1 impairs ER morphology in 412 vivo. To this end, we examined the cerebella from wildtype and K298A/NM mice 413 by TEM. We observed that instead of ribosome-bound and tubule-like ER 414 morphologies observed in wildtype cerebellar granule neurons, the mutant 415 neurons harbored enlarged, stubby, and less ribosome-bound ER (Fig. 5n).

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was also evidenced by upregulation of ubiquitin in these Clcc1 conditional KO 441 neurons ( Fig. 6f and 6g). Compared to nucleus-localized TDP-43 in ChAT-

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The generation and purification of CLCC1 polyclonal antibodies.