Jia Yichang of Tsinghua University School of Medicine/Tsinghua-Peking University Life Joint Center, Gao Zhaobing of Institute of Medicine of Chinese Academy of Sciences and Fan Dongsheng of Peking Uni

Although chloride ion is the most common anion in cells, relative to cations and related cation channel proteins, the physiological functions of chloride ion flux and related chloride channel proteins have not been paid attention to by the academic community for a long time.1989years, the pathogenic gene of fibrotic cystCFTRwas cloned, the gene encodes a protein belonging toABCtransporter family that encode a ligand-dependent chloride channel.1990ClC-0, the gene encodes a protein that gives torpedo skin cells the characteristic of firing. After a year,ClC-0in humansClC-1cloned, mutations in this gene cause myotonia. The cloning of these genes links the chloride flow across the cell membrane with various physiological phenomena and human diseases, opening a new chapter in the study of ion channels. Over the next three decades, multiple cell membrane-localized chloride channels were cloned, includingBestrophinfamily,TMEM16A/ B,LRRC8family,PACand so on, but the scientific community's understanding of organelles/endomembrane system are poorly understood.

The endoplasmic reticulum is a widespread inner membrane system in eukaryotic cells, and its main functions are the folding modification of nascent polypeptides, lipid synthesis and calcium storage. endoplasmic reticulum throughIP3R(inositol trisphosphate receptor) andRyR(ranneline receptor) channels release calcium ions, and throughSERCAa proton pump. The flow of calcium ions across the endoplasmic reticulum membrane will bring about a change in potential, requiring other ions to balance the charge. Disruptions of calcium homeostasis in the endoplasmic reticulum trigger an unfolded protein response (unfolded protein response,UPRs) causes a variety of diseases including neurodegenerative diseases. From1978years, using isolated sarcoplasmic reticulum and electrophysiological techniques, multiple research groups have recorded voltage-gated potassium currents and linearI-V(current-voltage) characteristic chloride ion flow, suggesting that there may be yin and cation channels in the sarcoplasmic network.2007years,trimeric intracellular cation channels(TRICs) is reported to be a potassium channel located in the endoplasmic reticulum, which can affect the calcium homeostasis of the endoplasmic reticulum.2001years, the use of expressionChloride Channel CLIC like 1(CLCC1)CHOcells (microsomes) component, the researcher recorded a completely new anion flow,CLCC1whether the pore formation of the endoplasmic reticulum chloride channel (into a hole) components are still in doubt; In addition, the anion channel located on the cell membrane is also folded and modified in the endoplasmic reticulum, so the anion flow detected by previous researchers in the microcapsule may also be derived from the channel protein located on the cell membrane.

2023year5month4day, 《cell researchpublished online."disruption of ER ion homeostasis maintained by the ER anion channel CLCC1 contributes to ALS-like pathology"/Tsinghua-Peking University Life Joint CenterJia Yichang, Gao Zhaobing of the Institute of Medicine of the Chinese Academy of Sciences and Fan Dongsheng Laboratory of the Third Hospital of Peking University confirmed for the first timeCLCC1is a pore-forming component of endoplasmic reticulum-localized chloride channels; from the full-length sequence,CLCC1andCLIC(chloride intracellular channel) protein sequences are not similar, and the team recommends that the protein sequences be based onCLCC1endoplasmic reticulum localization characteristics and electrophysiological data, the gene was renamedER anion channel 1 (ERAC1);CLCC1regulates the negative and positive homeostasis of the endoplasmic reticulum;CLCC1function disrupts the ion homeostasis of the endoplasmic reticulum, triggers endoplasmic reticulum stress and participates in amyotrophic lateral sclerosis (Amyotrophic Lateral Sclerosis, ALS) pathologyand pathogenicity.

CLCC1and any known ion channel, and only a signal peptide and three transmembrane domains are predicted. Immunofluorescence experiments and biochemical methods confirmedCLCC1in the endoplasmic reticulum. using purifiedCLCC1protein for single-channel electrophysiological detection, the research team confirmedCLCC1can independently mediate anion flow, but it is not permeable to sodium, potassium, calcium and other cations.CLCC1can becysteine350site (C350) by small moleculesMTSETandDIDSinhibition, whileC350Fdoes not affect the current but can relieve the inhibition of the above two drugs, suggesting thatC350site closeCLCC1the pore region of the chloride channel. Biochemical experiments revealedCLCC1topology, I .e., the conservativeNend and the second loop-forming region are on the lumen side of the endoplasmic reticulum. Electrophysiological experiments show thatNsideD25andD181is extremely important for calcium-inhibiting channel activity, this is consistent with the high concentration of calcium ion levels on the luminal side of the endoplasmic reticulum, suggesting a novel mechanism for the regulation of calcium-inhibited channel activity.

in order to studyCLCC1involved in regulating the ion homeostasis of the endoplasmic reticulum,the research team first prepared a ratio type of endoplasmic reticulum localization (ratio meter) chloride ion probe and potassium ion probe. They found,Knock lowCLCC1(knock-down efficiency reaches50%or more) leads to the concentration of chloride ions in the resting endoplasmic reticulum ([Cl^-]_ER) and potassium ion concentration(K⁺]_ER) elevated. The results of transmission electron microscopy showed that, unlike the slender endoplasmic reticulum structure in normal cells,CLCC1knockdown cells appear more short rod-shaped endoplasmic reticulum, and the average width of the endoplasmic reticulum will be significantly increased. Therefore,CLCC1maintains the concentration of chloride and potassium ions in the endoplasmic reticulum, as well as the endoplasmic reticulum osmolarity-related endoplasmic reticulum morphology.

endoplasmic reticulum-localized ion channels can be mediated by ion hedge (counter ion) mechanismto balance the endoplasmic reticulumcalcium ions (endocalcium release). ByIP3R,RyRandTRICspotassium current to simultaneously balance the changes in potential and osmotic pressure caused by internal calcium release.CLCC1-mediated chloride flow can be2Ca^{2 +}+ Cl^-= 3K⁺way to perfectly balance the changes in potential and osmotic pressure caused by the release of calcium. The team foundCLCC1knockdown of cells not only significantlygroundreduced the magnitude of calcium release and the rate of release, but also weakened the cytoplasmic calcium shock, suggesting thatCLCC1is what the industry has long been looking.2Ca^{2 +}+ Cl^-= 3K⁺. in primary cardiomyocytes,CLCC1loss of function also impairs caffeine-stimulatedRyRchannel-mediated calcium release. The above experiments show that,CLCC1to endoplasmic reticulum/sarcoplasmic reticulum calcium release are not limited to specific calcium channels. Using a low-affinity endoplasmic reticulum-localized calcium probe, the team found that knockdownCLCC1level ~80%and not ~50%significantly reduced the endoplasmic reticulum calcium ion concentration ([Ca^{2 +}]_ER), promptCLCC1dose-dependent effects[Ca^{2 +}]_ER, and this effect is likely dueCLCC1caused by increased volume of the endoplasmic reticulum due to loss of function.

the research team further discovered that phosphatidylinositol【PI(4,5)P2] can increaseCLCC1can also increase its open probability.PI(4,5)P2rightCLCC1activity depends onCLCC1the secondinto a ringregion (K298).K298Amutant protein has channel activity, but completelyis lost.PIP2rightCLCC1conductance and open probabilitypromotion. induced expression in cellsK298AmutatedCLCC1impairs endocalcium release and subsequent calcium shock.K298Amutant knock-in not only increases endoplasmic reticulum stress and endoplasmic reticulum swelling in mouse cerebellum, but alsoinspinal cordChat +the number of motor neurons. The above results show that,CLCC1channel activity can disrupt endoplasmic reticulum ion homeostasis and damage endoplasmic reticulum morphology, and ultimately lead to neuronal death.

the research team in ChinaALSqueue (670patients and1910controls) were foundCLCC18rare mutations includeW267RandS263R. among them,S263Rwas found in two unrelated patients. It is worth mentioning that,W267andS263is relatively conservative and exists in the samealphahelices, both mutated to lysine, suggestingALSW267RandS263RmutationrightCLCC1function may be similar. Through a series of experiments such as single channel electrophysiology, calcium imaging, pathological detection of mutant knock-in mice,them.confirmedS263RandW267Ris a loss-of-function mutation. Low-dose intraperitoneal injection of tunicamycin (tunicamycin, a drug that induces endoplasmic reticulum stress) can be used inCLCC1mutation in the brain of mice caused the endoplasmic reticulum stress of neurons, while the same concentration of tunicamycin could not cause endoplasmic reticulum stress in the brain of normal mice, indicating thatCLCC1ALSmutations may cause disease by impairing a patient's ability to cope with stress. InK298A,S263RandW267Rthree mutant knock-in mouse strains, the mutatedCLCC1protein will undergo ubiquitination-dependent degradation, suggesting thatALSmutatedCLCC1cannot be efficiently assembledCLCC1channel multimers. in the spinal cordChat +Motor NeuronsClcc1,causedmassive motor neuron death, motor neuron endoplasmic reticulum stress,andALScharacteristic pathologies, includingTDP-43protein nucleation andTDP-43ubiquitination aggregation. The above results for the first time willCLCC1is relatedALSlinks and tipsCLCC1may be a newALSpathogenic gene.

the study was the first to confirmCLCC1is the pore-forming component of the endoplasmic reticulum-localized chloride channel;CLCC1may be a long-sought anion channel component of the endoplasmic reticulum.2Ca^{2 +}+ Cl^-= 3K⁺way; through cooperation with clinicians, it is proposed for the first time thatCLCC1is a newALSdisease-causing genes, and through researchCLCC1's lack of function is proposedALSpathogenesis, includingCLCC1loss of function activates the classicATF4andATF6ratherIRE1/XBP1access.

endoplasmic reticulum localizationCLCC1/ERAC1regulates endoplasmic reticulum ion homeostasis

Tsinghua University Academy of Sciences/Tsinghua-Peking UniversityJoint Center for Life Sciences, Dr. Mao Qionglei, Shanghai Institute of Medicine, Chinese Academy of Sciences, and Dr. He He, Peking University Third Hospital are the parallel papers. Professor Jia Yichang,Gao Zhaobing Researcher,Professor Fan Dongsheng is the co-author of the correspondence. School of Pharmacy, Tsinghua University/Tsinghua-Peking UniversityLife Science Joint Center Xiao Bailong,Dr. Liu Xiaoling, School of Pharmacy, Tsinghua University, Dr. Park Xuejiao, School of Medicine, Tsinghua Universityand, Dr. Qiang Song,Luo Li, Yu Hanzhi, an undergraduate student of the Tsinghua University Academy of Sciences, and Hao Xiaoxu of Shanghai Medicine of the Chinese Academy of Sciences have all made important contributions. This work was awarded by the Joint Center for Life Sciences, the Young Thousand Program,IDG/McGovern Institute, National Natural Science FoundationandBeijing Municipal Science and Technology Commission and other support.

Original Link:https://doi.org/10.1038/s41422-023-00798-z