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

Link ER ion homeostasis maintained by an ER anion channel to ALS | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Biological Sciences - Article Link ER ion homeostasis maintained by an ER anion channel to ALS Yichang Jia, Liang Guo, Qionglei Mao, Ji He, Xiaoling LIU, Xuejiao Piao, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-536643/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract 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. Neurobiology of Disease Neurology Anion channel endoplasmic reticulum (ER) ion homeostasis ER stress ALS. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Full Text Due to technical limitations, full-text HTML conversion of this manuscript could not be completed. However, the manuscript can be downloaded and accessed as a PDF. Additional Declarations There is NO Competing Interest. Supplementary Files SupplementaryMovie1LinkCLCC1toALS.mp4 Extended Data Movie 1 | Early onset behavior phenotypes in the K298A/NM mouse. SupplementaryMovie2LinkCLCC1toALS.mp4 Extended Data Movie 2 | Increased penetrance of K298A allele. FiguresLinkCLCC1toALS0516.pdf Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-536643","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Biological Sciences - Article","associatedPublications":[],"authors":[{"id":27664881,"identity":"c270ce5b-ebff-438c-8b6b-459a60decda4","order_by":0,"name":"Yichang Jia","email":"data:image/png;base64,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","orcid":"","institution":"Tsinghua University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Yichang","middleName":"","lastName":"Jia","suffix":""},{"id":27664882,"identity":"a2b28273-2083-4f8d-8104-e826d7b7512f","order_by":1,"name":"Liang Guo","email":"","orcid":"","institution":"Tsinghua-Peking Joint Center for Life Sciences, Tsinghua University, Beijing, 100084, China.","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Liang","middleName":"","lastName":"Guo","suffix":""},{"id":27664883,"identity":"34b1cb16-a79e-47a9-8b95-0b6ae35d38d4","order_by":2,"name":"Qionglei Mao","email":"","orcid":"","institution":"Shanghai Institute of Materia Medica, Chinese Academy of Sciences.","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Qionglei","middleName":"","lastName":"Mao","suffix":""},{"id":27664884,"identity":"cb0949d4-80d8-4520-913b-9fa88551d26b","order_by":3,"name":"Ji He","email":"","orcid":"","institution":"Peking University Third Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ji","middleName":"","lastName":"He","suffix":""},{"id":27664885,"identity":"d2121377-ad82-4859-a70c-37d94d8e2a36","order_by":4,"name":"Xiaoling LIU","email":"","orcid":"","institution":"School of Pharmaceutical Sciences,Tsinghua University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xiaoling","middleName":"","lastName":"LIU","suffix":""},{"id":27664886,"identity":"b54c19e7-c638-42cb-82f1-d7bc3272f959","order_by":5,"name":"Xuejiao Piao","email":"","orcid":"","institution":"Tsinghua University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xuejiao","middleName":"","lastName":"Piao","suffix":""},{"id":27664887,"identity":"2c1428a3-86c0-47f7-a40d-13e537cd1db0","order_by":6,"name":"Li Luo","email":"","orcid":"","institution":"Tsinghua University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Li","middleName":"","lastName":"Luo","suffix":""},{"id":27664888,"identity":"69992550-130d-498b-a39c-d61dc238b349","order_by":7,"name":"Xiaoxu Hao","email":"","orcid":"","institution":"Zhejiang University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xiaoxu","middleName":"","lastName":"Hao","suffix":""},{"id":27664889,"identity":"5b24022a-0b39-4893-af24-f558afc1c230","order_by":8,"name":"Bailong Xiao","email":"","orcid":"https://orcid.org/0000-0002-2386-3322","institution":"State Key Laboratory of Membrane Biology, Tsinghua-Peking Joint Center for Life Sciences, Beijing Advanced Innovation Center for Structural Biology, IDG/McGovern Institute for Brain Research, School","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Bailong","middleName":"","lastName":"Xiao","suffix":""},{"id":27664890,"identity":"c9c12291-3ee7-4066-a465-3cb84a336166","order_by":9,"name":"Dongsheng Fan","email":"","orcid":"","institution":"Department of Neurology, Peking University Third hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Dongsheng","middleName":"","lastName":"Fan","suffix":""},{"id":27664891,"identity":"f7722650-a538-44c4-b81c-56c4cb061c7c","order_by":10,"name":"Zhaobing Gao","email":"","orcid":"","institution":"Shanghai Institute of Materia Medica, Chinese Academy of Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Zhaobing","middleName":"","lastName":"Gao","suffix":""}],"badges":[],"createdAt":"2021-05-18 00:11:04","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-536643/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-536643/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":9667985,"identity":"a76bf5b1-3069-4ad5-bd8c-75e0b3bb34ac","added_by":"auto","created_at":"2021-05-27 15:06:06","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":117105,"visible":true,"origin":"","legend":"ALS-associated CLCC1 mutations S263R and W267R reduce mutant CLCC1 expression and promote ER stress in vivo. a, The nonsynonymous (colored circle) and stopgain (red triangle) mutations of CLCC1 were identified in a Chinese sporadic ALS cohort. The potential damaging mutations are labeled in red. b, Validation of the potential diseasecausing mutations of CLCC1 by Sanger sequencing. Genomic DNA extracted from peripheral blood cells of individual ALS patients. The PCR products were subject to Sanger sequencing and the boundaries of adjacent exon and intron are marked. c, The Manhattan plot for an exome-wide rare variant burden analysis. The p value of CLCC1 (1.51×10-6, with OR = 5.72). d, A protein alignment of CLCC1 encompassing S263, W267, and neighboring residues. Note that S263 and W267 are located in a predicted alpha helix. e, ER stress and misfolded protein accumulation documented by Bip and ubiquitin (Ubi) staining in cerebella of compound heterozygous mice (S263R/NM and W267R/NM). NM, the NM2453 allele is an IAP (intracisternal A-particle) insertion in the intron 2 of Clcc1, which greatly reduces the expression of CLCC1 protein to ~10% of that in wildtype animals (PMID: 25698737). S263R/+ and wildtype (+/+) are negative for the phenotypes. f, Cerebellar expression of CLCC1 in the indicated genotypes. Both S263R and W267R lowered the expression of CLCC1. GAPDH, loading control. In e and f, +/+, 1.5 month; other genotypes, P35. Scale bar in E, 20 µm","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-536643/v1/4dcf32deb1d0bce76106500e.jpg"},{"id":9667986,"identity":"74e578ae-3562-4da6-8a84-607cac677d63","added_by":"auto","created_at":"2021-05-27 15:06:06","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":128174,"visible":true,"origin":"","legend":"CLCC1 is a pore-forming component of an ER anion channel and luminal Ca2+ inhibits the channel activity. a, Domain prediction of mouse CLCC1 (mCLCC1) that contains a signal peptide (SP) and three transmembrane segments (TMs). mCLCC1 is encoded by 10 exons (Ex2-11) (NM_145543.2). Note the N-terminal (12-200 a.a.) and Cterminal polypeptides (355-539 a. a.) used for generation of the N- and C-terminal antibodies, respectively. b, Naive 293FT cells were treated with disuccinimidyl suberate (DSS) at indicated concentrations. Cell lysates were separated by SDS-PAGE and blotted with CLCC1 C-terminal antibody (CLCC1-C). c, Chromatograph of His-tagged mCLCC1 expressed by an insect expression system and purified by Nickel column. Standard molecular weight markers are indicated by arrows. d, Purified mCLCC1 from (c) were incorporated into planar phospholipid bilayer and single channel currents were recorded in asymmetric KCl and NaCl solutions at indicated voltages (upper). C, closed state; O, open state. Current-voltage (I-V) relationships in asymmetric KCl and NaCl solutions (lower). e, Single channel currents recorded at 0 mV with 150 mM KCl in cis and 150 mM KBr, 75 mM K2SO4, or 150 mM KNO3 in trans (upper). I-V relationships under conditions (lower). f, Single channel activities (upper) and I-V relationships (lower) recorded from planar bilayers with purified human wildtype (hWT), S263R, and W267R mutant CLCC1, respectively. Solution, asymmetric KCl (In/Ex, 150/15 mM). In d, e, and f, values are presented as mean ± SEM (n ≥ 6). g, Topology of CLCC1 determined by microsome preparation. Microsomal vehicles prepared from mouse cerebellum were treated with trypsin alone, or trypsin together with Triton X-100. Protein lysates were then separated by SDS-PAGE and probed with CLCC1 N- and C-terminal antibodies. As a control, Bip, an ER lumen resident, was protected from trypsinization. h, Application of 2 mM MTSET in trans but not cis side blocked mCLCC1 channel activity. We defined cis side as the chamber we applied purified CLCC1 proteins (upper). The C350F mCLCC1 mutant was resistant to MTSET (n ≥ 6). i, Cysteine residues of CLCC1, with C350 highlighted. j, An alignment of predicted TM3 of CLCC1 across different species. C350 is labeled with an asterisk. Note the corresponding residue of Homo sapiens and Mus musculus C350 is phenylalanine in Xenopus. k, Application of 10 mM CaCl2 in cis but not trans reduced mCLCC1 channel activity. The inhibitory effect of Ca2+ was partially prevented by EDTA (10mM). l, Statistical analysis of normalized relative open probability (Po). Relative Po, Po-post/Po-pre, Po after CaCl2 or EDTA treatment divided by that before the treatment. Values are presented as mean ± SEM (n = 6).","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-536643/v1/4a52b5ab071aa23e79164130.jpg"},{"id":9667650,"identity":"c5f10682-81c9-4e85-8b00-089f4b34da08","added_by":"auto","created_at":"2021-05-27 15:00:06","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":124674,"visible":true,"origin":"","legend":"Dysfunction of CLCC1 impairs steady state [Cl- ]ER and leads to ER swelling. a, Ratiometric Cl- sensor (RaMorideER) for ER [Cl- ] ([Cl- ]ER) measurement. A signal peptide (SP) was tagged to the N-terminus of a previously reported Cl- sensor (PMID: 24901231), which was then fused to a monomeric DsRed as an internal control for the probe expression level and an ER retention signal (KDEL) at the C-terminal end. The resulting ratiometric Clsensor (RaMorideER) for ER [Cl- ] ([Cl- ]ER) measurement (upper). Cell expressing wildtype (WT) or ALS-associated mutant CLCC1 was monitored by the expression of an engineered near-infrared fluorescent protein, miRFP670S (PMID: 23770755). IRES (Internal Ribosome Entry Sites) sequences were employed to ensure the co-expression (lower). b, ER localization of RaMorideER, showing a prominent overlap of the DsRed fluorescence with Calnexin immunostaining signals. c and d, 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 miRFP670S. Representative FACS (Fluorescence Activated Cell Sorting) plots (c) and the summary data (d) are shown. e, Knockdown of CLCC1 in 293FT cells infected with lentiviral H3 and H4 shRNAs, measured by western blot (left), FACS (middle), and RNA-seq (right). Ctrl., MOCK control; Scra., scrambled shRNA; H3 and H4, shRNAs specific for CLCC1. f, Steady state [Cl- ]ER measured by RaMorideER in 293FT cells infected with the indicated shRNAs. g and h, Transmission electron microscopy (TEM) images of 293FT cells infected with the indicated shRNAs. Ribosome-bound rough ER was marked by red arrows. ER width was calculated and the summary data are shown in (h). In d, f, and h, values are presented as mean ± SEM from at least three independent experiments or biological replicates; N.S., no significant difference, *p\u003c0.05, **p\u003c0.01, ***p​","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-536643/v1/52a6a8075560099cd32e06fe.jpg"},{"id":9667800,"identity":"f0b1e4af-dd08-4737-86e4-a86372dc0cf7","added_by":"auto","created_at":"2021-05-27 15:03:06","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":122963,"visible":true,"origin":"","legend":"Dysfunction of CLCC1 impairs internal Ca2+ release and depletion of CLCC1 dosage-dependently reduces [Ca2+]ER. a-c, 293FT cells infected with the indicated shRNAs were loaded with Fura-2 and stimulated with ATP in a calcium-free medium (gray rectangle). Representative Ca2+ release traces averaged from at least 50 cells (a). The knockdown of CLCC1 reduced the amplitude (b) but increased the time-to-peak (c) of ATPinduced Ca2+ release. d and e, Full-length WT mCLCC1 but not the Δ2nd loop mutant CLCC1 restored the ATP-induced Ca2+ release damaged by H3 shRNA knockdown. The data summary shown in (e). f, ATP-induced internal Ca2+ release was impaired by S263R and W267R mutants. Human wildtype (hWT) and S263 and W267 mutant CLCC1 were expressed in 293FT cells. g-i, ER Ca2+ content was estimated by CPA-induced cytosolic Ca2+ rise in the calcium-free medium (gray rectangle) in 293FT cells infected with the indicated shRNAs. Shown are representative traces of CPA-induced calcium leak averaged from at least 50 cells (g) and summary data for the amplitude (h) and time-to-half peak (i) of CPA-induced cytosolic Ca2+ rise. j and k, Steady state [Ca2+]ER in 293FT cells infected with the indicated shRNAs was measured by fluorescent signals of ER-GCaMP6-210 a previously reported low affinity Ca2+ probe (PMID: 28162809) by FACS. Baseline, 1 mM EGTA + 10 µM ionomycin; Steady, normal medium containing 2 mM Ca2+; Max, 10 mM Ca2+ + 10 µM ionomycin. The summary data (k) were from three independent experiments. ΔFsteady = (Fsteady-Fbaseline); ΔFmax = (Fmax-Fbaseline). Values are presented as mean ± SEM. In b, c, e, f, h, and i, n \u003e 150 cells pooled from three independent experiments. N.S., no significant difference, *p\u003c0.05, **p\u003c0.01, ***p​","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-536643/v1/9e8793a90efc677a09195d31.jpg"},{"id":9667987,"identity":"ebd608a8-6b0c-4905-b8e4-693d7b6acf74","added_by":"auto","created_at":"2021-05-27 15:06:06","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":169654,"visible":true,"origin":"","legend":"Mutation of K298 a PIP2-sensing residue for CLCC1 channel activity impairs ER Ca2+ release and promotes ER swelling and motor neuron degeneration. a and b, Single channel activities recorded after incorporating the purified wildtype (WT), K298A, and K298E mutant mCLCC1 into the planar phospholipid bilayer. In b, the phospholipid bilayer contained 2% PIP2. c, An alignment of sequences encompassing the 2nd loop of CLCC1 among different species. d and e, I-V relationships in the absence (d) and presence (e) of PIP2 for WT mCLCC1 and its K298A and K298E mutants recorded from planar phospholipid bilayer in the asymmetric KCl solutions. f, Summary data of slope conductance (upper) and channel open probability (Po) at 0 mV (lower) in the asymmetric KCl solutions. g, A lentiviral inducible system was used to express wildtype mCLCC1 (WT) and its K298A mutant (K298A) in 293FT cells. ATP-induced Ca2+ release was measured in the calcium-free culture medium in Fura-2 loaded cells with (+Dox) or without (-Dox) induction. h and i, Summary data of amplitude (h) and time-to-peak (i) of ATP-induced Ca2+ release under conditions shown in (g). j, Cerebellar expression of CLCC1 in the indicated genotypes. GAPDH, loading control. k, ER stress and misfolded protein accumulation documented by Bip and ubiquitin (Ubi) staining, respectively, in cerebella of NM2453 homozygotes (NM/NM) and K298A and NM2453 compound heterozygotes (K298A/NM). P, Purkinje cells. Wildtype (+/+), negative control; NM2453 homozygotes (NM/NM), positive control. l and m, Ubiquitinpositive inclusions in ChAT-positive motor neurons in lumbar 4–5 spinal cords of K298A/NM mice. Representative images (l) and quantification of number of ChAT-positive motor neurons in the ventral horn (m) are shown. n and o, TEM images of cerebellar granule neurons from wildtype (+/+) and K298A/NM mice (n). Red arrows indicate ribosome-bound rough ER. Summary data are shown in (o). Mouse age: j and k, 1.5 month; l and m, +/+, 10 months, K298A/NM, 14 months; n and o, 3 months. Quantification: d, e, and f, n = 4-20; h and i, more than 150 cells from three independent experiments; m and o, 14-18 slides (m) and more than 25 granule cells (o) per mouse from three individual animals for each genotype. Scale bar, k, 20 µm; l, 10 µm; n, 50 nm. Values are presented as mean ± SEM; N.S., no significant difference; *p\u003c0.05; ***p\u003c0.001, by t-test or one-way ANOVA.","description":"","filename":"58.jpg","url":"https://assets-eu.researchsquare.com/files/rs-536643/v1/004198b9986f997b9a0917e9.jpg"},{"id":9667647,"identity":"1888970d-75bf-41d1-b734-d7780ce6b5ae","added_by":"auto","created_at":"2021-05-27 15:00:06","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":134288,"visible":true,"origin":"","legend":"Increased penetrance of K298A allele and cell-autonomous effect of Clcc1 loss-of-function in motor neuron loss. a, Percent of phenotype onset in wildtype (+/+) and K298A/+ animals. The early onset phenotypes of K298A/+ animals (K298A/+*, 12/182, onset time: postnatal 90.9 ± 5.5 days) include body weight loss, hindlimb weakness, trunk shaking, tail flagging, abnormal gaits, and ataxia (also see Movie S2). b, A K298A/+ mutant mouse displaying early onset phenotype (K298A/+*) together with a wildtype and a normal K298A/+ mouse. c, Curved spine shown in K298A/+* but not in wildtype and K298A/+ mice. d, Expression levels of CLCC1 in various tissues in wildtype, K298A/+, and K298A/+* mice. Summary data in Extended Data Fig. 13. e, Construction of Clcc1 conditional knockout mouse. f-j, Knockout of Clcc1 in ChAT-positive motor neurons (ChAT-Cre;Clcc1fl/fl) leads to ER stress (f and g), TDP-43 pathology (h), early death (i), and motor neuron loss (j). ChAT-Cre;Clcc1fl/+ served as a negative control. In j, 14-18 slides per mouse from lumbar 4–5 spinal cords of three individual animals for each genotype were analyzed. Mouse age: b-d, 10 months; f, g, h, and j, P20-25. Scale bar in f-h, 10 µm.","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-536643/v1/88caebc8eb7d24381bfd4370.jpg"},{"id":13636804,"identity":"9c74f058-5199-44dc-ad8e-5c0072beb8a7","added_by":"auto","created_at":"2021-09-17 08:43:25","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":958070,"visible":true,"origin":"","legend":"Article File","description":"","filename":"ManuscriptLinkCLCC1toALS0516.pdf","url":"https://assets-eu.researchsquare.com/files/rs-536643/v1_covered.pdf"},{"id":9667990,"identity":"95c1e615-7c71-48ee-8dbe-7381b5b36989","added_by":"auto","created_at":"2021-05-27 15:06:14","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":953994,"visible":true,"origin":"","legend":"Article File","description":"","filename":"ManuscriptLinkCLCC1toALS0516.pdf","url":"https://assets-eu.researchsquare.com/files/rs-536643/v1_covered.pdf"},{"id":9667642,"identity":"9a773d33-58b4-468f-ba89-0b364e3269f1","added_by":"auto","created_at":"2021-05-27 15:00:06","extension":"mp4","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":5126853,"visible":true,"origin":"","legend":"Extended Data Movie 1 | Early onset behavior phenotypes in the K298A/NM mouse.","description":"","filename":"SupplementaryMovie1LinkCLCC1toALS.mp4","url":"https://assets-eu.researchsquare.com/files/rs-536643/v1/918c289f743c2411eff9c8ac.mp4"},{"id":9667804,"identity":"f60de977-fea9-4bec-adf7-e7008308c8d7","added_by":"auto","created_at":"2021-05-27 15:03:06","extension":"mp4","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":5889222,"visible":true,"origin":"","legend":"Extended Data Movie 2 | Increased penetrance of K298A allele.","description":"","filename":"SupplementaryMovie2LinkCLCC1toALS.mp4","url":"https://assets-eu.researchsquare.com/files/rs-536643/v1/f82469aa176d6a1bfca87e08.mp4"},{"id":9667805,"identity":"ae305c45-0180-4c62-b518-d4afa3cabf79","added_by":"auto","created_at":"2021-05-27 15:03:06","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":18236767,"visible":true,"origin":"","legend":"","description":"","filename":"FiguresLinkCLCC1toALS0516.pdf","url":"https://assets-eu.researchsquare.com/files/rs-536643/v1/24a13342daacd1a758a43a01.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Link ER ion homeostasis maintained by an ER anion channel to ALS","fulltext":[{"header":"Full Text","content":"\u003cp\u003eDue to technical limitations, full-text HTML conversion of this manuscript could not be completed. However, the manuscript can be downloaded and accessed as a PDF.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Anion channel, endoplasmic reticulum (ER), ion homeostasis, ER stress, ALS.","lastPublishedDoi":"10.21203/rs.3.rs-536643/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-536643/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"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.","manuscriptTitle":"Link ER ion homeostasis maintained by an ER anion channel to ALS","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2021-05-27 15:00:04","doi":"10.21203/rs.3.rs-536643/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"64fa2115-2838-4029-8f98-62133893bce8","owner":[],"postedDate":"May 27th, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":4622112,"name":"Neurobiology of Disease"},{"id":4622113,"name":"Neurology"}],"tags":[],"updatedAt":"2022-03-29T20:25:41+00:00","versionOfRecord":[],"versionCreatedAt":"2021-05-27 15:00:04","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-536643","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-536643","identity":"rs-536643","version":["v1"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}