1
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Mahapatra C, Kishore A, Gawad J, Al-Emam A, Kouzeiha RA, Rusho MA. Review of electrophysiological models to study membrane potential changes in breast cancer cell transformation and tumor progression. Front Physiol 2025; 16:1536165. [PMID: 40110186 PMCID: PMC11920174 DOI: 10.3389/fphys.2025.1536165] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/02/2024] [Accepted: 02/14/2025] [Indexed: 03/22/2025] Open
Abstract
The transformation of normal breast cells into cancerous cells is a complex process influenced by both genetic and microenvironmental factors. Recent studies highlight the significant role of membrane potential (Vm) alterations in this transformation. Cancer cells typically exhibit a depolarized resting membrane potential (RMP) compared to normal cells, which correlates with increased cellular activity and more aggressive cancer behavior. These RMP and Vm changes are associated with altered ion channel activity, altered calcium dynamics, mitochondrial dysfunction, modified gap junction communication, and disrupted signaling pathways. Such fluctuations in RMP and Vm influence key processes in cancer progression, including cell proliferation, migration, and invasion. Notably, more aggressive subtypes of breast cancer cells display more frequent and pronounced Vm fluctuations. Understanding the electrical properties of cancer cells provides new insights into their behavior and offers potential therapeutic targets, such as ion channels and Vm regulation. This review synthesizes current research on how various factors modulate membrane potential and proposes an electrophysiological model of breast cancer cells based on experimental and clinical data from the literature. These findings may pave the way for novel pharmacological targets for clinicians, researchers, and pharmacologists in treating breast cancer.
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Affiliation(s)
| | - Arnaw Kishore
- Microbiology and Immunology, Xavier University School of Medicine, Aruba, Netherlands
| | - Jineetkumar Gawad
- Department of Pharmaceutical Chemistry, VIVA Institute of Pharmacy, Virar, India
| | - Ahmed Al-Emam
- Department of Pathology, College of Medicine, King Khalid University, Asir, Saudi Arabia
| | - Riad Azzam Kouzeiha
- Faculty of Medical Sciences, Lebanese University, Hadath Campus, Beirut, Lebanon
| | - Maher Ali Rusho
- Department of Biomedical Engineering, University of Colorado Boulder, Boulder, CO, United States
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2
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Schrecker M, Son Y, Planells-Cases R, Kar S, Vorobeva V, Schulte U, Fakler B, Jentsch TJ, Hite RK. Structural basis of ClC-3 inhibition by TMEM9 and PI(3,5)P 2. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2025:2025.02.28.640562. [PMID: 40093093 PMCID: PMC11908120 DOI: 10.1101/2025.02.28.640562] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Subscribe] [Scholar Register] [Indexed: 03/19/2025]
Abstract
The trafficking and activity of endosomes relies on the exchange of chloride ions and protons by members of the CLC family of chloride channels and transporters, whose mutations are associated with numerous diseases. Despite their critical roles, the mechanisms by which CLC transporters are regulated are poorly understood. Here, we show that two related accessory β-subunits, TMEM9 and TMEM9B, directly interact with ClC-3, -4 and -5. Cryo-EM structures reveal that TMEM9 inhibits ClC-3 by sealing the cytosolic entrance to the Cl- ion pathway. Unexpectedly, we find that PI(3,5)P2 stabilizes the interaction between TMEM9 and ClC-3 and is required for proper regulation of ClC-3 by TMEM9. Collectively, our findings reveal that TMEM9 and PI(3,5)P2 collaborate to regulate endosomal ion homeostasis by modulating the activity of ClC-3.
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Affiliation(s)
- Marina Schrecker
- Structural Biology Program, Memorial Sloan Kettering Cancer Center; New York, NY, USA
| | - Yeeun Son
- Structural Biology Program, Memorial Sloan Kettering Cancer Center; New York, NY, USA
- BCMB Allied Program, Weill Cornell Graduate School; New York, NY, USA
| | - Rosa Planells-Cases
- Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP); Berlin, Germany
| | - Sumanta Kar
- Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP); Berlin, Germany
| | - Viktoriia Vorobeva
- Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP); Berlin, Germany
- Graduate program of the Free University; Berlin, Germany
| | - Uwe Schulte
- Institute of Physiology, Faculty of Medicine, University of Freiburg; Freiburg, Germany
- Logopharm GmbH; March-Buchheim, Germany
| | - Bernd Fakler
- Institute of Physiology, Faculty of Medicine, University of Freiburg; Freiburg, Germany
- Signalling Research Centers BIOSS and CIBSS; Freiburg, Germany
| | - Thomas J. Jentsch
- Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP); Berlin, Germany
- Neurocure Cluster of Excellence, Charité Universitätsmedizin; Berlin, Germany
| | - Richard K. Hite
- Structural Biology Program, Memorial Sloan Kettering Cancer Center; New York, NY, USA
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3
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Silverstein TP. Explaining neuronal membrane potentials: The Goldman equation vs. Lee's TELC hypothesis. Neuroscience 2025; 567:1-8. [PMID: 39755228 DOI: 10.1016/j.neuroscience.2024.12.065] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/20/2024] [Revised: 12/29/2024] [Accepted: 12/31/2024] [Indexed: 01/06/2025]
Abstract
In two recent papers (Curr Trends Neurol 17: 83-98, 2023; J Neurophysiol 124: 1029-1044, 2020), James Lee has argued that his Transmembrane Electrostatically-Localized Cations (TELC) hypothesis offers a model of neuron transmembrane potentials that is superior to Hodgkin-Huxley classic cable theory and the Goldman-Hodgkin-Katz (GHK) equation. Here we examine critically the arguments in these papers, finding key weaknesses and fallacies. We also examine closely the literature cited by Lee, and find (i) strong support for the GHK equation; (ii) published measurements that contradict TELC predictions; and (iii) no convincing support for the TELC hypothesis.
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4
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Jiang J, Ren R, Fang W, Miao J, Wen Z, Wang X, Xu J, Jin H. Lysosomal biogenesis and function in osteoclasts: a comprehensive review. Front Cell Dev Biol 2024; 12:1431566. [PMID: 39170917 PMCID: PMC11335558 DOI: 10.3389/fcell.2024.1431566] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/12/2024] [Accepted: 07/19/2024] [Indexed: 08/23/2024] Open
Abstract
Lysosomes serve as catabolic centers and signaling hubs in cells, regulating a multitude of cellular processes such as intracellular environment homeostasis, macromolecule degradation, intracellular vesicle trafficking and autophagy. Alterations in lysosomal level and function are crucial for cellular adaptation to external stimuli, with lysosome dysfunction being implicated in the pathogenesis of numerous diseases. Osteoclasts (OCs), as multinucleated cells responsible for bone resorption and maintaining bone homeostasis, have a complex relationship with lysosomes that is not fully understood. Dysregulated function of OCs can disrupt bone homeostasis leading to the development of various bone disorders. The regulation of OC differentiation and bone resorption for the treatment of bone disease have received considerable attention in recent years, yet the role and regulation of lysosomes in OCs, as well as the potential therapeutic implications of intervening in lysosomal biologic behavior for the treatment of bone diseases, remain relatively understudied. This review aims to elucidate the mechanisms involved in lysosomal biogenesis and to discuss the functions of lysosomes in OCs, specifically in relation to differentiation, bone resorption, and autophagy. Finally, we explore the potential therapeutic implication of targeting lysosomes in the treatment of bone metabolic disorders.
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Affiliation(s)
- Junchen Jiang
- Department of Orthopaedics, The Second Affiliated Hospital and Yuying Children’s Hospital of Wenzhou Medical University, Wenzhou, China
- The Second School of Medicine, Wenzhou Medical University, Wenzhou, China
| | - Rufeng Ren
- Department of Orthopaedics, The Second Affiliated Hospital and Yuying Children’s Hospital of Wenzhou Medical University, Wenzhou, China
- The Second School of Medicine, Wenzhou Medical University, Wenzhou, China
| | - Weiyuan Fang
- Department of Orthopaedics, The Second Affiliated Hospital and Yuying Children’s Hospital of Wenzhou Medical University, Wenzhou, China
- The Second School of Medicine, Wenzhou Medical University, Wenzhou, China
| | - Jiansen Miao
- Department of Orthopaedics, The Second Affiliated Hospital and Yuying Children’s Hospital of Wenzhou Medical University, Wenzhou, China
- The Second School of Medicine, Wenzhou Medical University, Wenzhou, China
| | - Zijun Wen
- Department of Orthopaedics, The Second Affiliated Hospital and Yuying Children’s Hospital of Wenzhou Medical University, Wenzhou, China
- The Second School of Medicine, Wenzhou Medical University, Wenzhou, China
| | - Xiangyang Wang
- Department of Orthopaedics, The Second Affiliated Hospital and Yuying Children’s Hospital of Wenzhou Medical University, Wenzhou, China
- The Second School of Medicine, Wenzhou Medical University, Wenzhou, China
| | - Jiake Xu
- Department of Orthopaedics, The Second Affiliated Hospital and Yuying Children’s Hospital of Wenzhou Medical University, Wenzhou, China
- Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, Shenzhen, China
| | - Haiming Jin
- Department of Orthopaedics, The Second Affiliated Hospital and Yuying Children’s Hospital of Wenzhou Medical University, Wenzhou, China
- The Second School of Medicine, Wenzhou Medical University, Wenzhou, China
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5
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Wan Y, Guo S, Zhen W, Xu L, Chen X, Liu F, Shen Y, Liu S, Hu L, Wang X, Ye F, Wang Q, Wen H, Yang F. Structural basis of adenine nucleotides regulation and neurodegenerative pathology in ClC-3 exchanger. Nat Commun 2024; 15:6654. [PMID: 39107281 PMCID: PMC11303396 DOI: 10.1038/s41467-024-50975-w] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/16/2023] [Accepted: 07/25/2024] [Indexed: 08/10/2024] Open
Abstract
The ClC-3 chloride/proton exchanger is both physiologically and pathologically critical, as it is potentiated by ATP to detect metabolic energy level and point mutations in ClC-3 lead to severe neurodegenerative diseases in human. However, why this exchanger is differentially modulated by ATP, ADP or AMP and how mutations caused gain-of-function remains largely unknow. Here we determine the high-resolution structures of dimeric wildtype ClC-3 in the apo state and in complex with ATP, ADP and AMP, and the disease-causing I607T mutant in the apo and ATP-bounded state by cryo-electron microscopy. In combination with patch-clamp recordings and molecular dynamic simulations, we reveal how the adenine nucleotides binds to ClC-3 and changes in ion occupancy between apo and ATP-bounded state. We further observe I607T mutation induced conformational changes and augments in current. Therefore, our study not only lays the structural basis of adenine nucleotides regulation in ClC-3, but also clearly indicates the target region for drug discovery against ClC-3 mediated neurodegenerative diseases.
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Affiliation(s)
- Yangzhuoqun Wan
- Department of Biophysics and Disease Center of the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang, China
- Liangzhu Laboratory, Zhejiang University Medical Center, Hangzhou, Zhejiang, China
| | - Shuangshuang Guo
- Department of Biophysics and Disease Center of the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang, China
- Liangzhu Laboratory, Zhejiang University Medical Center, Hangzhou, Zhejiang, China
| | - Wenxuan Zhen
- Department of Biophysics and Disease Center of the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang, China
- Liangzhu Laboratory, Zhejiang University Medical Center, Hangzhou, Zhejiang, China
| | - Lizhen Xu
- Department of Biophysics and Disease Center of the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang, China
- Liangzhu Laboratory, Zhejiang University Medical Center, Hangzhou, Zhejiang, China
| | - Xiaoying Chen
- Department of Biophysics and Disease Center of the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang, China
- Liangzhu Laboratory, Zhejiang University Medical Center, Hangzhou, Zhejiang, China
| | - Fangyue Liu
- Department of Neurobiology and Department of General Intensive Care Unit of the Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China
| | - Yi Shen
- Department of Neurobiology and Department of General Intensive Care Unit of the Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China
| | - Shuangshuang Liu
- Core Facilities, Zhejiang University School of Medicine, Hangzhou, China
| | - Lidan Hu
- The Children's Hospital, Zhejiang University School of Medicine, National Clinical Research Center for Child Health, Hangzhou, China
| | | | | | | | - Han Wen
- DP Technology, Beijing, China.
- Institute for Advanced Algorithms Research, Shanghai, China.
- State Key Laboratory of Medical Proteomics, Shanghai, China.
- AI for Science Institute, Beijing, China.
- National Key Laboratory of Lead Druggability Research, Beijing, China.
| | - Fan Yang
- Department of Biophysics and Disease Center of the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang, China.
- Liangzhu Laboratory, Zhejiang University Medical Center, Hangzhou, Zhejiang, China.
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6
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He H, Li X, Guzman GA, Bungert-Plümke S, Franzen A, Lin X, Zhu H, Peng G, Zhang H, Yu Y, Sun S, Huang Z, Zhai Q, Chen Z, Peng J, Guzman RE. Expanding the genetic and phenotypic relevance of CLCN4 variants in neurodevelopmental condition: 13 new patients. J Neurol 2024; 271:4933-4948. [PMID: 38758281 DOI: 10.1007/s00415-024-12383-4] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/24/2024] [Revised: 04/02/2024] [Accepted: 04/09/2024] [Indexed: 05/18/2024]
Abstract
OBJECTIVES CLCN4 variations have recently been identified as a genetic cause of X-linked neurodevelopmental disorders. This study aims to broaden the phenotypic spectrum of CLCN4-related condition and correlate it with functional consequences of CLCN4 variants. METHODS We described 13 individuals with CLCN4-related neurodevelopmental disorder. We analyzed the functional consequence of the unreported variants using heterologous expression, biochemistry, confocal fluorescent microscopy, patch-clamp electrophysiology, and minigene splicing assay. RESULTS We identified five novel (p.R41W, p.L348V, p.G480R, p.R603W, c.1576 + 5G > A) and three known (p.T203I, p.V275M, p.A555V) pathogenic CLCN4 variants in 13 Chinese patients. The p.V275M variant is found at high frequency and seen in four unrelated individuals. All had global developmental delay (GDD)/intellectual disability (ID). Seizures were present in eight individuals, and 62.5% of them developed refractory epilepsy. Five individuals without seizures showed moderate to severe GDD/ID. Developmental delay precedes seizure onset in most patients. The variants p.R41W, p.L348V, and p.R603W compromise the anion/exchange function of ClC-4. p.R41W partially impairs ClC-3/ClC-4 association. p.G480R reduces ClC-4 expression levels and impairs the heterodimerization with ClC-3. The c.1576 + 5G > A variant causes 22 bp deletion of exon 10. CONCLUSIONS We further define and broaden the clinical and mutational spectrum of CLCN4-related neurodevelopmental conditions. The p.V275M variant may be a potential hotspot CLCN4 variant in Chinese patients. The five novel variants cause loss of function of ClC-4. Transport dysfunction, protein instability, intracellular trafficking defect, or failure of ClC-4 to oligomerize may contribute to the pathophysiological events leading to CLCN4-related neurodevelopmental disorder.
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Affiliation(s)
- Hailan He
- Department of Pediatrics, Xiangya Hospital, Central South University, Changsha, China
| | - Xinyi Li
- Department of Pediatrics, Xiangya Hospital, Central South University, Changsha, China
| | - G A Guzman
- Institute of Biological Information Processing (IBI-7), Structural Biochemistry, Jülich Research Center, Jülich, Germany
| | - Stefanie Bungert-Plümke
- Institute of Biological Information Processing (IBI-1), Molecular and Cell Physiology, Jülich Research Center, Jülich, Germany
| | - Arne Franzen
- Institute of Biological Information Processing (IBI-1), Molecular and Cell Physiology, Jülich Research Center, Jülich, Germany
| | - XueQin Lin
- Department of Pediatrics, Xiangya Hospital, Central South University, Changsha, China
| | - Hongmin Zhu
- Department of Rehabilitation, Wuhan Children's Hospital, Tongji Medical College, Huazhong University of Science & Technology, Wuhan, China
| | - Guilan Peng
- Department of Neurology, Xiamen Maternal and Child Health Care Hospital, Xiamen, China
| | - Hongwei Zhang
- Epilepsy Center, Children's Hospital Affiliated to Shandong University, Jinan, China
| | - Yonglin Yu
- Department of Rehabilitation, Children's Hospital, Zhejiang University School of Medicine, Hangzhou, China
| | - Suzhen Sun
- Department of Pediatric Neurology, Hebei Children's Hospital, Hebei Medical University, Shijiazhuang, China
| | - Zhongqin Huang
- Department of Neurology, Xiamen Maternal and Child Health Care Hospital, Xiamen, China
| | - Qiongxiang Zhai
- Department of Pediatrics, Guangdong Provincial People's Hospital, Guangzhou, China
| | - Zheng Chen
- Department of Pediatrics, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, China
| | - Jing Peng
- Department of Pediatrics, Xiangya Hospital, Central South University, Changsha, China.
| | - Raul E Guzman
- Institute of Biological Information Processing (IBI-1), Molecular and Cell Physiology, Jülich Research Center, Jülich, Germany.
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7
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Tourkova IL, Larrouture QC, Liu S, Luo J, Shipman KE, Onwuka KM, Weisz OA, Riazanski V, Nelson DJ, MacDonald ML, Schlesinger PH, Blair HC. Chloride/proton antiporters ClC3 and ClC5 support bone formation in mice. Bone Rep 2024; 21:101763. [PMID: 38666049 PMCID: PMC11043850 DOI: 10.1016/j.bonr.2024.101763] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/09/2024] [Accepted: 04/12/2024] [Indexed: 04/28/2024] Open
Abstract
Acid transport is required for bone synthesis by osteoblasts. The osteoblast basolateral surface extrudes acid by Na+/H+ exchange, but apical proton uptake is undefined. We found high expression of the Cl-/H+ exchanger ClC3 at the bone apical surface. In mammals ClC3 functions in intracellular vesicular chloride transport, but when we found Cl- dependency of H+ transport in osteoblast membranes, we queried whether ClC3 Cl-/H+ exchange functions in bone formation. We used ClC3 knockout animals, and closely-related ClC5 knockout animals: In vitro studies suggested that both ClC3 and ClC5 might support bone formation. Genotypes were confirmed by total exon sequences. Expression of ClC3, and to a lesser extent of ClC5, at osteoblast apical membranes was demonstrated by fluorescent antibody labeling and electron microscopy with nanometer gold labeling. Animals with ClC3 or ClC5 knockouts were viable. In ClC3 or ClC5 knockouts, bone formation decreased ~40 % by calcein and xylenol orange labeling in vivo. In very sensitive micro-computed tomography, ClC5 knockout reduced bone relative to wild type, consistent with effects of ClC3 knockout, but varied with specific histological parameters. Regrettably, ClC5-ClC3 double knockouts are not viable, suggesting that ClC3 or ClC5 activity are essential to life. We conclude that ClC3 has a direct role in bone formation with overlapping but probably slightly smaller effects of ClC5. The mechanism in mineral formation might include ClC H+ uptake, in contrast to ClC3 and ClC5 function in cell vesicles or other organs.
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Affiliation(s)
- Irina L. Tourkova
- Department of Pathology, University of Pittsburgh, Pittsburgh, PA, USA
- Research Service, VA Healthcare System, Pittsburgh, PA, USA
| | | | - Silvia Liu
- Department of Pathology, University of Pittsburgh, Pittsburgh, PA, USA
| | - Jianhua Luo
- Department of Pathology, University of Pittsburgh, Pittsburgh, PA, USA
| | - Katherine E. Shipman
- Renal Electrolyte Division, Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA
| | - Kelechi M. Onwuka
- Department of Pathology, University of Pittsburgh, Pittsburgh, PA, USA
| | - Ora A. Weisz
- Renal Electrolyte Division, Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA
| | - Vladimir Riazanski
- Dept of Neurobiology, Pharmacology & Physiology, University of Chicago, Chicago, IL, USA
| | - Deborah J. Nelson
- Dept of Neurobiology, Pharmacology & Physiology, University of Chicago, Chicago, IL, USA
| | - Matthew L. MacDonald
- Department of Psychiatry, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA
| | | | - Harry C. Blair
- Department of Pathology, University of Pittsburgh, Pittsburgh, PA, USA
- Research Service, VA Healthcare System, Pittsburgh, PA, USA
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8
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Alam I, Hardman SL, Gerard-O'Riley RL, Acton D, Parker RS, Hong JM, Bruzzaniti A, Econs MJ. Effect of Roflumilast, a Selective PDE4 Inhibitor, on Bone Phenotypes in ADO2 Mice. Calcif Tissue Int 2024; 114:419-429. [PMID: 38300304 DOI: 10.1007/s00223-023-01180-2] [Citation(s) in RCA: 1] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/29/2023] [Accepted: 12/25/2023] [Indexed: 02/02/2024]
Abstract
Autosomal Dominant Osteopetrosis type II (ADO2) is a rare bone disease of impaired osteoclastic bone resorption that usually results from heterozygous missense mutations in the chloride channel 7 (CLCN7) gene. We previously created mouse models of ADO2 (p.G213R) with one of the most common mutations (G215R) as found in humans and demonstrated that this mutation in mice phenocopies the human disease of ADO2. Previous studies have shown that roflumilast (RF), a selective phosphodiesterase 4 (PDE4) inhibitor that regulates the cAMP pathway, can increase osteoclast activity. We also observed that RF increased bone resorption in both wild-type and ADO2 heterozygous osteoclasts in vitro, suggesting it might rescue bone phenotypes in ADO2 mice. To test this hypothesis, we administered RF-treated diets (0, 20 and 100 mg/kg) to 8-week-old ADO2 mice for 6 months. We evaluated bone mineral density and bone micro-architecture using longitudinal in-vivo DXA and micro-CT at baseline, and 6-, 12-, 18-, and 24-week post-baseline time points. Additionally, we analyzed serum bone biomarkers (CTX, TRAP, and P1NP) at baseline, 12-, and 24-week post-baseline. Our findings revealed that RF treatment did not improve aBMD (whole body, femur, and spine) and trabecular BV/TV (distal femur) in ADO2 mice compared to the control group treated with a normal diet. Furthermore, we did not observe any significant changes in serum levels of bone biomarkers due to RF treatment in these mice. Overall, our results indicate that RF does not rescue the osteopetrotic bone phenotypes in ADO2 heterozygous mice.
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Affiliation(s)
- Imranul Alam
- Division of Endocrinology, Department of Medicine, Indiana University School of Medicine, Indianapolis, IN, 46202, USA.
| | - Sara L Hardman
- Division of Endocrinology, Department of Medicine, Indiana University School of Medicine, Indianapolis, IN, 46202, USA
| | - Rita L Gerard-O'Riley
- Division of Endocrinology, Department of Medicine, Indiana University School of Medicine, Indianapolis, IN, 46202, USA
| | - Dena Acton
- Division of Endocrinology, Department of Medicine, Indiana University School of Medicine, Indianapolis, IN, 46202, USA
| | - Reginald S Parker
- Division of Endocrinology, Department of Medicine, Indiana University School of Medicine, Indianapolis, IN, 46202, USA
| | - Jung Min Hong
- Biomedical Sciences and Comprehensive Care, Indiana University School of Dentistry, Indianapolis, IN, 46202, USA
| | - Angela Bruzzaniti
- Biomedical Sciences and Comprehensive Care, Indiana University School of Dentistry, Indianapolis, IN, 46202, USA
| | - Michael J Econs
- Division of Endocrinology, Department of Medicine, Indiana University School of Medicine, Indianapolis, IN, 46202, USA
- Medical and Molecular Genetics, Indiana University School of Medicine, 1120 West Michigan St, CL459, Indianapolis, IN, 46202, USA
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9
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Hong JM, Gerard-O'Riley RL, Acton D, Alam I, Econs MJ, Bruzzaniti A. The PDE4 Inhibitors Roflumilast and Rolipram Rescue ADO2 Osteoclast Resorption Dysfunction. Calcif Tissue Int 2024; 114:430-443. [PMID: 38483547 PMCID: PMC11239147 DOI: 10.1007/s00223-024-01191-7] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/04/2023] [Accepted: 01/29/2024] [Indexed: 03/22/2024]
Abstract
Autosomal Dominant Osteopetrosis type II (ADO2) is a rare bone disease of impaired osteoclastic bone resorption caused by heterozygous missense mutations in the chloride channel 7 (CLCN7). Adenylate cyclase, which catalyzes the formation of cAMP, is critical for lysosomal acidification in osteoclasts. We found reduced cAMP levels in ADO2 osteoclasts compared to wild-type (WT) osteoclasts, leading us to examine whether regulating cAMP would improve ADO2 osteoclast activity. Although forskolin, a known activator of adenylate cyclase and cAMP levels, negatively affected osteoclast number, it led to an overall increase in ADO2 and WT osteoclast resorption activity in vitro. Next, we examined cAMP hydrolysis by the phosphodiesterase 4 (PDE4) proteins in ADO2 versus WT osteoclasts. QPCR analysis revealed higher expression of the three major PDE4 subtypes (4a, 4b, 4d) in ADO2 osteoclasts compared in WT, consistent with reduced cAMP levels in ADO2 osteoclasts. In addition, we found that the PDE4 antagonists, rolipram and roflumilast, stimulated ADO2 and WT osteoclast formation in a dose-dependent manner. Importantly, roflumilast and rolipram displayed a concentration-dependent increase in osteoclast resorption activity which was greater in ADO2 than WT osteoclasts. Moreover, treatment with roflumilast rescued cAMP levels in ADO2 OCLs. The key findings from our studies demonstrate that osteoclasts from ADO2 mice exhibit reduced cAMP levels and PDE4 inhibition rescues cAMP levels and ADO2 osteoclast activity dysfunction in vitro. The mechanism of action of PDE4 inhibitors and their ability to reduce the high bone mass of ADO2 mice in vivo are currently under investigation. Importantly, these studies advance the understanding of the mechanisms underlying the ADO2 osteoclast dysfunction which is critical for the development of therapeutic approaches to treat clinically affected ADO2 patients.
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Affiliation(s)
- Jung Min Hong
- Department of Biomedical Sciences and Comprehensive Care, Indiana University School of Dentistry, 1121 West Michigan Street, DS266, Indianapolis, IN, 46202, USA
| | - Rita L Gerard-O'Riley
- Department of Medicine, Indiana University School of Medicine, Indianapolis, IN, 46202, USA
| | - Dena Acton
- Department of Medicine, Indiana University School of Medicine, Indianapolis, IN, 46202, USA
| | - Imranul Alam
- Department of Medicine, Indiana University School of Medicine, Indianapolis, IN, 46202, USA
| | - Michael J Econs
- Department of Medicine, Indiana University School of Medicine, Indianapolis, IN, 46202, USA
- Department of Medical and Molecular Genetics, Indiana University School of Medicine, Indianapolis, IN, 46202, USA
| | - Angela Bruzzaniti
- Department of Biomedical Sciences and Comprehensive Care, Indiana University School of Dentistry, 1121 West Michigan Street, DS266, Indianapolis, IN, 46202, USA.
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10
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Kostritskaia Y, Klüssendorf M, Pan YE, Hassani Nia F, Kostova S, Stauber T. Physiological Functions of the Volume-Regulated Anion Channel VRAC/LRRC8 and the Proton-Activated Chloride Channel ASOR/TMEM206. Handb Exp Pharmacol 2024; 283:181-218. [PMID: 37468723 DOI: 10.1007/164_2023_673] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 07/21/2023]
Abstract
Volume-regulated anion channels (VRACs) and the acid-sensitive outwardly rectifying anion channel (ASOR) mediate flux of chloride and small organic anions. Although known for a long time, they were only recently identified at the molecular level. VRACs are heteromers consisting of LRRC8 proteins A to E. Combining the essential LRRC8A with different LRRC8 paralogues changes key properties of VRAC such as conductance or substrate selectivity, which is how VRACs are involved in multiple physiological functions including regulatory volume decrease, cell proliferation and migration, cell death, purinergic signalling, fat and glucose metabolism, insulin signalling, and spermiogenesis. VRACs are also involved in pathological conditions, such as the neurotoxic release of glutamate and aspartate. Certain VRACs are also permeable to larger, organic anions, including antibiotics and anti-cancer drugs, making them an interesting therapeutic target. ASOR, also named proton-activated chloride channel (PAC), is formed by TMEM206 homotrimers on the plasma membrane and on endosomal compartments where it mediates chloride flux in response to extracytosolic acidification and plays a role in the shrinking and maturation of macropinosomes. ASOR has been shown to underlie neuronal swelling which causes cell death after stroke as well as promoting the metastasis of certain cancers, making them intriguing therapeutic targets as well.
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Affiliation(s)
- Yulia Kostritskaia
- Institute for Molecular Medicine, MSH Medical School Hamburg, Hamburg, Germany
| | - Malte Klüssendorf
- Institute for Molecular Medicine, MSH Medical School Hamburg, Hamburg, Germany
| | - Yingzhou Edward Pan
- Institute for Molecular Medicine, MSH Medical School Hamburg, Hamburg, Germany
| | - Fatemeh Hassani Nia
- Institute for Molecular Medicine, MSH Medical School Hamburg, Hamburg, Germany
| | - Simona Kostova
- Institute for Molecular Medicine, MSH Medical School Hamburg, Hamburg, Germany
| | - Tobias Stauber
- Institute for Molecular Medicine, MSH Medical School Hamburg, Hamburg, Germany.
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11
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Ergen PH, Shorter S, Ntziachristos V, Ovsepian SV. Neurotoxin-Derived Optical Probes for Biological and Medical Imaging. Mol Imaging Biol 2023; 25:799-814. [PMID: 37468801 PMCID: PMC10598172 DOI: 10.1007/s11307-023-01838-1] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/01/2023] [Revised: 07/04/2023] [Accepted: 07/05/2023] [Indexed: 07/21/2023]
Abstract
The superb specificity and potency of biological toxins targeting various ion channels and receptors are of major interest for the delivery of therapeutics to distinct cell types and subcellular compartments. Fused with reporter proteins or labelled with fluorophores and nanocomposites, animal toxins and their detoxified variants also offer expanding opportunities for visualisation of a range of molecular processes and functions in preclinical models, as well as clinical studies. This article presents state-of-the-art optical probes derived from neurotoxins targeting ion channels, with discussions of their applications in basic and translational biomedical research. It describes the design and production of probes and reviews their applications with advantages and limitations, with prospects for future improvements. Given the advances in imaging tools and expanding research areas benefiting from the use of optical probes, described here resources should assist the discovery process and facilitate high-precision interrogation and therapeutic interventions.
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Affiliation(s)
- Pinar Helin Ergen
- Faculty of Engineering and Science, University of Greenwich London, Chatham Maritime, Kent, ME4 4TB, United Kingdom
| | - Susan Shorter
- Faculty of Engineering and Science, University of Greenwich London, Chatham Maritime, Kent, ME4 4TB, United Kingdom
| | - Vasilis Ntziachristos
- Chair of Biological Imaging at the Central Institute for Translational Cancer Research (TranslaTUM), School of Medicine, Technical University of Munich, 81675, Munich, Germany
- Institute of Biological and Medical Imaging, Helmholtz Zentrum München (GmbH), 85764, Neuherberg, Germany
- Munich Institute of Robotics and Machine Intelligence (MIRMI), Technical University of Munich, 80992, Munich, Germany
- DZHK (German Centre for Cardiovascular Research), partner site Munich Heart Alliance, Munich, Germany
| | - Saak Victor Ovsepian
- Faculty of Engineering and Science, University of Greenwich London, Chatham Maritime, Kent, ME4 4TB, United Kingdom.
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12
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Li S, Zhang W, Liang P, Zhu M, Zheng B, Zhou W, Wang C, Zhao X. Novel variants in the CLCN4 gene associated with syndromic X-linked intellectual disability. Front Neurol 2023; 14:1096969. [PMID: 37789889 PMCID: PMC10542403 DOI: 10.3389/fneur.2023.1096969] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/13/2022] [Accepted: 08/15/2023] [Indexed: 10/05/2023] Open
Abstract
Objective The dysfunction of the CLCN4 gene can lead to X-linked intellectual disability and Raynaud-Claes syndrome (MRXSRC), characterized by severe cognitive impairment and mental disorders. This study aimed to investigate the genetic defects and clinical features of Chinese children with CLCN4 variants and explore the effect of mutant ClC-4 on the protein expression level and subcellular localization through in vitro experiments. Methods A total of 401 children with intellectual disabilities were screened for genetic variability using whole-exome sequencing (WES). Clinical data, including age, sex, perinatal conditions, and environmental exposure, were collected. Cognitive, verbal, motor, and social behavioral abilities were evaluated. Candidate variants were verified using Sanger sequencing, and their pathogenicity and conservation were analyzed using in silico prediction tools. Protein expression and localization of mutant ClC-4 were measured using Western blotting (WB) and immunofluorescence microscopy. The impact of a splice site variant was assessed with a minigene assay. Results Exome analysis identified five rare CLCN4 variants in six unrelated patients with intellectual disabilities, including two recurrent heterozygous de novo missense variants (p.D89N and p.A555V) in three female patients, and two hemizygous missense variants (p.N141S and p.R694Q) and a splicing variant (c.1390-12T > G) that are maternally inherited in three male patients. The p.N141S variant and the splicing variant c.1390-12(T > G were novel, while p.R694Q was identified in two asymptomatic heterozygous female patients. The six children with CLCN4 variants exhibited a neurodevelopmental spectrum disease characterized by intellectual disability (ID), delayed speech, autism spectrum disorders (ASD), microcephaly, hypertonia, and abnormal imaging findings. The minigene splicing result indicated that the c.1390-12T > G did not affect the splicing of CLCN4 mRNA. In vitro experiments showed that the mutant protein level and localization of mutant protein are similar to the wild type. Conclusion The study identified six probands with CLCN4 gene variants associated with X-linked ID. It expanded the gene and phenotype spectrum of CLCN4 variants. The bioinformatic analysis supported the pathogenicity of CLCN4 variants. However, these CLCN4 gene variants did not affect the ClC-4 expression levels and protein location, consistent with previous studies. Further investigations are necessary to investigate the pathogenetic mechanism.
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Affiliation(s)
- Sinan Li
- Department of Rehabilitation, Children's Hospital of Nanjing Medical University, Nanjing, China
| | - Wenxin Zhang
- Department of Rehabilitation, Children's Hospital of Nanjing Medical University, Nanjing, China
| | - Piao Liang
- Department of Rehabilitation, Children's Hospital of Nanjing Medical University, Nanjing, China
| | - Min Zhu
- Department of Rehabilitation, Children's Hospital of Nanjing Medical University, Nanjing, China
| | - Bixia Zheng
- Nanjing Key Laboratory of Pediatrics, Children's Hospital of Nanjing Medical University, Nanjing, China
| | - Wei Zhou
- Nanjing Key Laboratory of Pediatrics, Children's Hospital of Nanjing Medical University, Nanjing, China
| | - Chunli Wang
- Nanjing Key Laboratory of Pediatrics, Children's Hospital of Nanjing Medical University, Nanjing, China
| | - Xiaoke Zhao
- Department of Rehabilitation, Children's Hospital of Nanjing Medical University, Nanjing, China
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Wang C, Li J, Liu W, Li S, Zhang Y, Jin Y, Cui J. Comprehensive analysis and experimental validation reveal elevated CLCN4 is a promising biomarker in endometrial cancer. Aging (Albany NY) 2023; 15:8744-8769. [PMID: 37671947 PMCID: PMC10522378 DOI: 10.18632/aging.204994] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/13/2023] [Accepted: 07/25/2023] [Indexed: 09/07/2023]
Abstract
Several studies have reported the role of CLCN4 in tumor progression. However, its mechanism remains to be thoroughly studied. The objective of this study was to explore the potential pathogenic role of CLCN4 in endometrial carcinoma (UCEC) with a better understanding of the pathological mechanisms involved. The potential roles of CLCN4 in different tumors were explored based on The Cancer Genome Atlas (TCGA), the expression difference, mutation, survival, pathological stage, Immunity subtypes, Immune infiltration, tumor microenvironment (TME), tumor mutation burden (TMB), microsatellite instability (MSI), mismatch repair (MMR) related to CLCN4 were analyzed. Then, the expression, prognosis, mutation, and functional enrichment of CLCN4 in UCEC were analyzed. Immunohistochemical experiment was used to verify the expression of CLCN4 in endometrial cancer tissues and normal tissues. In vitro, we knocked down of CLCN4 in HEC-1-A cells and performed CCK8, WB, RT-PCR, wound-healing, transwell assays to further validation of the molecular function. Results revealed that high expression of CLCN4 was observed in 20 cancer types of TCGA. CLCN4 expression correlates with poor survival in MESO, BLCA, THCA, especially UCEC tumors. CLCN4 expression was significantly associated with CD4+ T-cell infiltration, especially CD4+ Th1-cell. Immunohistochemical experiment reveals that CLCN4 is high expressed in endometrial tumors, in vitro experiment reveals that knockdown of CLCN4 inhibits the cells proliferation, migration and invasion. Our study is the first to offer a comprehensive understanding of the oncogenic roles of CLCN4 on different tumors. CLCN4 may become a potential biomarker in UCEC.
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Affiliation(s)
- Chenyang Wang
- Department of Gynecology, The Second Affiliated Hospital of Zhengzhou University, Zhengzhou 450000, China
- Academy of Medical Sciences, Zhengzhou University, Zhengzhou 450000, China
| | - Jing Li
- Department of Gynecology, Qingdao Hospital, University of Health and Rehabilitation Sciences (Qingdao Municipal Hospital), Qingdao, Shandong 266000, China
| | - Weina Liu
- Department of Gynecology, The Second Affiliated Hospital of Zhengzhou University, Zhengzhou 450000, China
- Academy of Medical Sciences, Zhengzhou University, Zhengzhou 450000, China
| | - Shiya Li
- Department of Gynecology, The Second Affiliated Hospital of Zhengzhou University, Zhengzhou 450000, China
- Academy of Medical Sciences, Zhengzhou University, Zhengzhou 450000, China
| | - Yi Zhang
- Department of Gynecology, The University of Auckland, Grafton, Auckland 1023, New Zealand
| | - Yanbin Jin
- Department of Gynecology, Hainan Affiliated Hospital of Hainan Medical University (Hainan General Hospital), Haikou 570311, China
| | - Jinquan Cui
- Department of Gynecology, The Second Affiliated Hospital of Zhengzhou University, Zhengzhou 450000, China
- Academy of Medical Sciences, Zhengzhou University, Zhengzhou 450000, China
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14
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Kettritz R, Loffing J. Potassium homeostasis - Physiology and pharmacology in a clinical context. Pharmacol Ther 2023; 249:108489. [PMID: 37454737 DOI: 10.1016/j.pharmthera.2023.108489] [Citation(s) in RCA: 14] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/15/2023] [Revised: 07/03/2023] [Accepted: 07/06/2023] [Indexed: 07/18/2023]
Abstract
Membrane voltage controls the function of excitable cells and is mainly a consequence of the ratio between the extra- and intracellular potassium concentration. Potassium homeostasis is safeguarded by balancing the extra-/intracellular distribution and systemic elimination of potassium to the dietary potassium intake. These processes adjust the plasma potassium concentration between 3.5 and 4.5 mmol/L. Several genetic and acquired diseases but also pharmacological interventions cause dyskalemias that are associated with increased morbidity and mortality. The thresholds at which serum K+ not only associates but also causes increased mortality are hotly debated. We discuss physiologic, pathophysiologic, and pharmacologic aspects of potassium regulation and provide informative case vignettes. Our aim is to help clinicians, epidemiologists, and pharmacologists to understand the complexity of the potassium homeostasis in health and disease and to initiate appropriate treatment strategies in dyskalemic patients.
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Affiliation(s)
- Ralph Kettritz
- Department of Nephrology and Medical Intensive Care, Charité - Universitätsmedizin Berlin, Freie Universität Berlin and Humboldt-Universität zu Berlin, Berlin, Germany; Experimental and Clinical Research Center, Max Delbrück Center for Molecular Medicine in the Helmholtz Association and Charité Universitätsmedizin Berlin, Germany.
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15
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Kang JS, Zhou X, Liu YT, Wang K, Zhou ZH. Theoretical framework and experimental solution for the air-water interface adsorption problem in cryoEM. BIOPHYSICS REPORTS 2023; 9:215-229. [PMID: 38516618 PMCID: PMC10951471 DOI: 10.52601/bpr.2023.230008] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/07/2023] [Accepted: 10/31/2023] [Indexed: 03/23/2024] Open
Abstract
As cryogenic electron microscopy (cryoEM) gains traction in the structural biology community as a method of choice for determining atomic structures of biological complexes, it has been increasingly recognized that many complexes that behave well under conventional negative-stain electron microscopy tend to have preferential orientation, aggregate or simply mysteriously "disappear" on cryoEM grids. However, the reasons for such misbehavior are not well understood, which limits systematic approaches to solving the problem. Here, we have developed a theoretical formulation that explains these observations. Our formulation predicts that all particles migrate to the air-water interface (AWI) to lower the total potential surface energy-rationalizing the use of surfactant, which is a direct solution to reduce the surface tension of the aqueous solution. By performing cryogenic electron tomography (cryoET) on the widely-tested sample, GroEL, we demonstrate that, in a standard buffer solution, nearly all particles migrate to the AWI. Gradually reducing the surface tension by introducing surfactants decreased the percentage of particles exposed to the surface. By conducting single-particle cryoEM, we confirm that suitable surfactants do not damage the biological complex, thus suggesting that they might provide a practical, simple, and general solution to the problem for high-resolution cryoEM. Applying this solution to a real-world AWI adsorption problem involving a more challenging membrane protein, namely, the ClC-1 channel, has resulted in its near-atomic structure determination using cryoEM.
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Affiliation(s)
- Joon S. Kang
- Department of Microbiology, Immunology & Molecular Genetics, University of California, Los Angeles (UCLA), Los Angeles, CA 90095, USA
- Molecular Biology Institute, UCLA, Los Angeles, CA 90095, USA
| | - Xueting Zhou
- Department of Microbiology, Immunology & Molecular Genetics, University of California, Los Angeles (UCLA), Los Angeles, CA 90095, USA
| | - Yun-Tao Liu
- Department of Microbiology, Immunology & Molecular Genetics, University of California, Los Angeles (UCLA), Los Angeles, CA 90095, USA
- California NanoSystems Institute, UCLA, Los Angeles, CA 90095, USA
| | - Kaituo Wang
- Department of Microbiology, Immunology & Molecular Genetics, University of California, Los Angeles (UCLA), Los Angeles, CA 90095, USA
- California NanoSystems Institute, UCLA, Los Angeles, CA 90095, USA
| | - Z. Hong Zhou
- Department of Microbiology, Immunology & Molecular Genetics, University of California, Los Angeles (UCLA), Los Angeles, CA 90095, USA
- Molecular Biology Institute, UCLA, Los Angeles, CA 90095, USA
- California NanoSystems Institute, UCLA, Los Angeles, CA 90095, USA
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16
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Coppola MA, Tettey-Matey A, Imbrici P, Gavazzo P, Liantonio A, Pusch M. Biophysical Aspects of Neurodegenerative and Neurodevelopmental Disorders Involving Endo-/Lysosomal CLC Cl -/H + Antiporters. Life (Basel) 2023; 13:1317. [PMID: 37374100 DOI: 10.3390/life13061317] [Citation(s) in RCA: 5] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/22/2023] [Revised: 05/30/2023] [Accepted: 05/31/2023] [Indexed: 06/29/2023] Open
Abstract
Endosomes and lysosomes are intracellular vesicular organelles with important roles in cell functions such as protein homeostasis, clearance of extracellular material, and autophagy. Endolysosomes are characterized by an acidic luminal pH that is critical for proper function. Five members of the gene family of voltage-gated ChLoride Channels (CLC proteins) are localized to endolysosomal membranes, carrying out anion/proton exchange activity and thereby regulating pH and chloride concentration. Mutations in these vesicular CLCs cause global developmental delay, intellectual disability, various psychiatric conditions, lysosomal storage diseases, and neurodegeneration, resulting in severe pathologies or even death. Currently, there is no cure for any of these diseases. Here, we review the various diseases in which these proteins are involved and discuss the peculiar biophysical properties of the WT transporter and how these properties are altered in specific neurodegenerative and neurodevelopmental disorders.
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Affiliation(s)
- Maria Antonietta Coppola
- Istituto di Biofisica, Consiglio Nazionale delle Ricerche, 16149 Genova, Italy
- Department of Pharmacy-Drug Sciences, University of Bari "Aldo Moro", 70125 Bari, Italy
| | | | - Paola Imbrici
- Department of Pharmacy-Drug Sciences, University of Bari "Aldo Moro", 70125 Bari, Italy
| | - Paola Gavazzo
- Istituto di Biofisica, Consiglio Nazionale delle Ricerche, 16149 Genova, Italy
| | - Antonella Liantonio
- Department of Pharmacy-Drug Sciences, University of Bari "Aldo Moro", 70125 Bari, Italy
| | - Michael Pusch
- Istituto di Biofisica, Consiglio Nazionale delle Ricerche, 16149 Genova, Italy
- RAISE Ecosystem, 16149 Genova, Italy
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17
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Schmollinger S, Chen S, Merchant SS. Quantitative elemental imaging in eukaryotic algae. Metallomics 2023; 15:mfad025. [PMID: 37186252 PMCID: PMC10209819 DOI: 10.1093/mtomcs/mfad025] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/30/2022] [Accepted: 03/03/2023] [Indexed: 05/17/2023]
Abstract
All organisms, fundamentally, are made from the same raw material, namely the elements of the periodic table. Biochemical diversity is achieved by how these elements are utilized, for what purpose, and in which physical location. Determining elemental distributions, especially those of trace elements that facilitate metabolism as cofactors in the active centers of essential enzymes, can determine the state of metabolism, the nutritional status, or the developmental stage of an organism. Photosynthetic eukaryotes, especially algae, are excellent subjects for quantitative analysis of elemental distribution. These microbes utilize unique metabolic pathways that require various trace nutrients at their core to enable their operation. Photosynthetic microbes also have important environmental roles as primary producers in habitats with limited nutrient supplies or toxin contaminations. Accordingly, photosynthetic eukaryotes are of great interest for biotechnological exploitation, carbon sequestration, and bioremediation, with many of the applications involving various trace elements and consequently affecting their quota and intracellular distribution. A number of diverse applications were developed for elemental imaging, allowing subcellular resolution, with X-ray fluorescence microscopy (XFM, XRF) being at the forefront, enabling quantitative descriptions of intact cells in a non-destructive method. This Tutorial Review summarizes the workflow of a quantitative, single-cell elemental distribution analysis of a eukaryotic alga using XFM.
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Affiliation(s)
- Stefan Schmollinger
- California Institute for Quantitative Biosciences (QB3), University of California, Berkeley, CA 94720, USA
- Departments of Molecular and Cell Biology and Plant and Microbial Biology, University of California, Berkeley, CA 94720, USA
| | - Si Chen
- X-ray Science Division, Argonne National Laboratory, Lemont, IL 60439, USA
| | - Sabeeha S Merchant
- California Institute for Quantitative Biosciences (QB3), University of California, Berkeley, CA 94720, USA
- Departments of Molecular and Cell Biology and Plant and Microbial Biology, University of California, Berkeley, CA 94720, USA
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18
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Yan P, Ke B, Fang X. Ion channels as a therapeutic target for renal fibrosis. Front Physiol 2022; 13:1019028. [PMID: 36277193 PMCID: PMC9581181 DOI: 10.3389/fphys.2022.1019028] [Citation(s) in RCA: 7] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/14/2022] [Accepted: 09/20/2022] [Indexed: 11/13/2022] Open
Abstract
Renal ion channel transport and electrolyte disturbances play an important role in the process of functional impairment and fibrosis in the kidney. It is well known that there are limited effective drugs for the treatment of renal fibrosis, and since a large number of ion channels are involved in the renal fibrosis process, understanding the mechanisms of ion channel transport and the complex network of signaling cascades between them is essential to identify potential therapeutic approaches to slow down renal fibrosis. This review summarizes the current work of ion channels in renal fibrosis. We pay close attention to the effect of cystic fibrosis transmembrane conductance regulator (CFTR), transmembrane Member 16A (TMEM16A) and other Cl− channel mediated signaling pathways and ion concentrations on fibrosis, as well as the various complex mechanisms for the action of Ca2+ handling channels including Ca2+-release-activated Ca2+ channel (CRAC), purinergic receptor, and transient receptor potential (TRP) channels. Furthermore, we also focus on the contribution of Na+ transport such as epithelial sodium channel (ENaC), Na+, K+-ATPase, Na+-H+ exchangers, and K+ channels like Ca2+-activated K+ channels, voltage-dependent K+ channel, ATP-sensitive K+ channels on renal fibrosis. Proposed potential therapeutic approaches through further dissection of these mechanisms may provide new therapeutic opportunities to reduce the burden of chronic kidney disease.
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Genetic spectrum and founder effect of non-dystrophic myotonia: a Japanese case series study. J Neurol 2022; 269:6406-6415. [PMID: 35907044 DOI: 10.1007/s00415-022-11305-6] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/25/2022] [Revised: 07/20/2022] [Accepted: 07/21/2022] [Indexed: 10/16/2022]
Abstract
Non-dystrophic myotonias (NDM) are rare skeletal muscle channelopathies, mainly linked to two voltage-gated ion channel genes, CLCN1 and SCN4A. The aim of this study is to identify the clinical and genetic features of patients with NDM in Japan. We collected a Japanese nationwide case series of patients with clinical diagnosis of NDM (1999-2021). Among 71 out of 88 pedigrees, using Sanger and next-generation sequencing targeting both CLCN1 and SCN4A genes, variants classified as pathogenic/likely pathogenic/unknown significance were detected from CLCN1 (31 probands), SCN4A (36 probands), or both genes (4 probands), and 11 of them were novel. Pedigrees carrying mono-allelic CLCN1 variants were more commonly seen than that with bi-allelic/double variants (24:7). Compared to patients with CLCN1 variants, patients harboring SCN4A variants showed younger onset age (5.64 ± 4.70 years vs. 9.23 ± 5.21 years), fewer warm-up phenomenon, but more paramyotonia, hyperCKemia, transient muscle weakness, and cold-induced myotonia. Haplotype analysis verified founder effects of the hot spot variants in both CLCN1 (p.T539A) and SCN4A (p.T1313M). This study reveals variants in CLCN1 and SCN4A from 80.7% of our case series, extending genetic spectrum of NDM, and would further our understanding of clinical similarity/diversity between CLCN1- and SCN4A-related NDM, as well as the genetic racial differences.
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Anoctamin 1 controls bone resorption by coupling Cl - channel activation with RANKL-RANK signaling transduction. Nat Commun 2022; 13:2899. [PMID: 35610255 PMCID: PMC9130328 DOI: 10.1038/s41467-022-30625-9] [Citation(s) in RCA: 22] [Impact Index Per Article: 7.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/21/2021] [Accepted: 05/05/2022] [Indexed: 12/18/2022] Open
Abstract
Osteoclast over-activation leads to bone loss and chloride homeostasis is fundamental importance for osteoclast function. The calcium-activated chloride channel Anoctamin 1 (also known as TMEM16A) is an important chloride channel involved in many physiological processes. However, its role in osteoclast remains unresolved. Here, we identified the existence of Anoctamin 1 in osteoclast and show that its expression positively correlates with osteoclast activity. Osteoclast-specific Anoctamin 1 knockout mice exhibit increased bone mass and decreased bone resorption. Mechanistically, Anoctamin 1 deletion increases intracellular Cl- concentration, decreases H+ secretion and reduces bone resorption. Notably, Anoctamin 1 physically interacts with RANK and this interaction is dependent upon Anoctamin 1 channel activity, jointly promoting RANKL-induced downstream signaling pathways. Anoctamin 1 protein levels are substantially increased in osteoporosis patients and this closely correlates with osteoclast activity. Finally, Anoctamin 1 deletion significantly alleviates ovariectomy induced osteoporosis. These results collectively establish Anoctamin 1 as an essential regulator in osteoclast function and suggest a potential therapeutic target for osteoporosis.
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The Role of the Lysosomal Cl−/H+ Antiporter ClC-7 in Osteopetrosis and Neurodegeneration. Cells 2022; 11:cells11030366. [PMID: 35159175 PMCID: PMC8833911 DOI: 10.3390/cells11030366] [Citation(s) in RCA: 7] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/01/2021] [Revised: 01/16/2022] [Accepted: 01/19/2022] [Indexed: 12/04/2022] Open
Abstract
CLC proteins comprise Cl− channels and anion/H+ antiporters involved in several fundamental physiological processes. ClC-7 is a lysosomal Cl−/H+ antiporter that together with its beta subunit Ostm1 has a critical role in the ionic homeostasis of lysosomes and of the osteoclasts’ resorption lacuna, although the specific underlying mechanism has so far remained elusive. Mutations in ClC-7 cause osteopetrosis, but also a form of lysosomal storage disease and neurodegeneration. Interestingly, both loss-of- and gain-of-function mutations of ClC-7 can be pathogenic, but the mechanistic implications of this finding are still unclear. This review will focus on the recent advances in our understanding of the biophysical properties of ClC-7 and of its role in human diseases with a focus on osteopetrosis and neurodegeneration.
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22
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Recent advance in dual-functional luminescent probes for reactive species and common biological ions. Anal Bioanal Chem 2022; 414:5087-5103. [DOI: 10.1007/s00216-021-03792-9] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/09/2021] [Indexed: 01/17/2023]
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Luo Y, Liu X, Li X, Zhong W, Lin J, Chen Q. Identification and validation of a signature involving voltage-gated chloride ion channel genes for prediction of prostate cancer recurrence. Front Endocrinol (Lausanne) 2022; 13:1001634. [PMID: 36246902 PMCID: PMC9561150 DOI: 10.3389/fendo.2022.1001634] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/23/2022] [Accepted: 09/06/2022] [Indexed: 11/26/2022] Open
Abstract
Voltage-gated chloride ion channels (CLCs) are transmembrane proteins that maintain chloride ion homeostasis in various cells. Accumulating studies indicated CLCs were related to cell growth, proliferation, and cell cycle. Nevertheless, the role of CLCs in prostate cancer (PCa) has not been systematically profiled. The purpose of this study was to investigate the expression profiles and biofunctions of CLCs genes, and construct a novel risk signature to predict biochemical recurrence (BCR) of PCa patients. We identified five differentially expressed CLCs genes in our cohort and then constructed a signature composed of CLCN2 and CLCN6 through Lasso-Cox regression analysis in the training cohort from the Cancer Genome Atlas (TCGA). The testing and entire cohorts from TCGA and the GSE21034 from the Gene Expression Omnibus (GEO) were used as internal and independent external validation datasets. This signature could divide PCa patients into the high and low risk groups with different prognoses, was apparently correlated with clinical features, and was an independent excellent prognostic indicator. Enrichment analysis indicated our signature was primarily concentrated in cellular process and metabolic process. The expression patterns of CLCN2 and CLCN6 were detected in our own cohort based immunohistochemistry staining, and we found CLCN2 and CLCN6 were highly expressed in PCa tissues compared with benign tissues and positively associated with higher Gleason score and shorter BCR-free time. Functional experiments revealed that CLCN2 and CLCN6 downregulation inhibited cell proliferation, colony formation, invasion, and migration, but prolonged cell cycle and promoted apoptosis. Furthermore, Seahorse assay showed that silencing CLCN2 or CLCN6 exerted potential inhibitory effects on energy metabolism in PCa. Collectively, our signature could provide a novel and robust strategy for the prognostic evaluation and improve treatment decision making for PCa patients.
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Affiliation(s)
- Yong Luo
- Department of Urology, The Second People’s Hospital of Foshan, Affiliated Foshan Hospital of Southern Medical University, Foshan, China
| | - Xiaopeng Liu
- Department of Science and Teaching, The Second People’s Hospital of Foshan, Affiliated Foshan Hospital of Southern Medical University, Foshan, China
| | - Xiaoxiao Li
- Department of Nursing Administration, the Second People’s Hospital of Foshan, Affiliated Foshan Hospital of Southern Medical University, Foshan, China
| | - Weide Zhong
- Department of Urology, The Second People’s Hospital of Foshan, Affiliated Foshan Hospital of Southern Medical University, Foshan, China
- Department of Urology, Guangdong Key Laboratory of Clinical Molecular Medicine and Diagnostics, Guangzhou First People’s Hospital, School of Medicine, South China University of Technology, Guangzhou, China
- Urology Key Laboratory of Guangdong Province, The First Affiliated Hospital of Guangzhou Medical University, Guangzhou Medical University, Guangzhou, China
- Guangdong Provincial Institute of Nephrology, Nanfang Hospital, Southern Medical University, Guangzhou, China
- State Key Laboratory of Quality Research in Chinese Medicines, Macau University of Science and Technology, Macau, Macau SAR, China
- *Correspondence: Qingbiao Chen, ; Jingbo Lin, ; Weide Zhong,
| | - Jingbo Lin
- Department of Urology, The Second People’s Hospital of Foshan, Affiliated Foshan Hospital of Southern Medical University, Foshan, China
- *Correspondence: Qingbiao Chen, ; Jingbo Lin, ; Weide Zhong,
| | - Qingbiao Chen
- Department of Urology, The Second People’s Hospital of Foshan, Affiliated Foshan Hospital of Southern Medical University, Foshan, China
- *Correspondence: Qingbiao Chen, ; Jingbo Lin, ; Weide Zhong,
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24
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Wilczyński B, Dąbrowska A, Saczko J, Kulbacka J. The Role of Chloride Channels in the Multidrug Resistance. MEMBRANES 2021; 12:38. [PMID: 35054564 PMCID: PMC8781147 DOI: 10.3390/membranes12010038] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 11/30/2021] [Accepted: 12/23/2021] [Indexed: 12/19/2022]
Abstract
Nowadays, one of medicine's main and most challenging aims is finding effective ways to treat cancer. Unfortunately, although there are numerous anti-cancerous drugs, such as cisplatin, more and more cancerous cells create drug resistance. Thus, it is equally important to find new medicines and research the drug resistance phenomenon and possibilities to avoid this mechanism. Ion channels, including chloride channels, play an important role in the drug resistance phenomenon. Our article focuses on the chloride channels, especially the volume-regulated channels (VRAC) and CLC chloride channels family. VRAC induces multidrug resistance (MDR) by causing apoptosis connected with apoptotic volume decrease (AVD) and VRAC are responsible for the transport of anti-cancerous drugs such as cisplatin. VRACs are a group of heterogenic complexes made from leucine-rich repetition with 8A (LRRC8A) and a subunit LRRC8B-E responsible for the properties. There are probably other subunits, which can create those channels, for example, TTYH1 and TTYH2. It is also known that the ClC family is involved in creating MDR in mainly two mechanisms-by changing the cell metabolism or acidification of the cell. The most researched chloride channel from this family is the CLC-3 channel. However, other channels are playing an important role in inducing MDR as well. In this paper, we review the role of chloride channels in MDR and establish the role of the channels in the MDR phenomenon.
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Affiliation(s)
- Bartosz Wilczyński
- Faculty of Medicine, Wroclaw Medical University, L. Pasteura 1, 50-367 Wroclaw, Poland; (B.W.); (A.D.)
| | - Alicja Dąbrowska
- Faculty of Medicine, Wroclaw Medical University, L. Pasteura 1, 50-367 Wroclaw, Poland; (B.W.); (A.D.)
| | - Jolanta Saczko
- Department of Molecular and Cellular Biology, Faculty of Pharmacy, Wroclaw Medical University, Borowska 211A, 50-556 Wroclaw, Poland;
| | - Julita Kulbacka
- Department of Molecular and Cellular Biology, Faculty of Pharmacy, Wroclaw Medical University, Borowska 211A, 50-556 Wroclaw, Poland;
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25
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Wang Y, Zeng W, Lin B, Yao Y, Li C, Hu W, Wu H, Huang J, Zhang M, Xue T, Ren D, Qu L, Cang C. CLN7 is an organellar chloride channel regulating lysosomal function. SCIENCE ADVANCES 2021; 7:eabj9608. [PMID: 34910516 PMCID: PMC8673761 DOI: 10.1126/sciadv.abj9608] [Citation(s) in RCA: 23] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/14/2023]
Abstract
Neuronal ceroid lipofuscinoses (NCLs) are a group of autosomal recessive lysosomal storage diseases. One variant form of late-infantile NCL (vLINCL) is caused by mutations of a lysosomal membrane protein CLN7, the function of which has remained unknown. Here, we identified CLN7 as a novel endolysosomal chloride channel. Overexpression of CLN7 increases endolysosomal chloride currents and enlarges endolysosomes through a Ca2+/calmodulin-dependent way. Human CLN7 and its yeast homolog exhibit characteristics of chloride channels and are sensitive to chloride channel blockers. Moreover, CLN7 regulates lysosomal chloride conductance, luminal pH, and lysosomal membrane potential and promotes the release of lysosomal Ca2+ through transient receptor potential mucolipin 1 (TRPML1). Knocking out CLN7 causes pathological features that are similar to those of patients with vLINCL, including retinal degeneration and autofluorescent lipofuscin. The pathogenic mutations in CLN7 lead to a decrease in chloride permeability, suggesting that reconstitution of lysosomal Cl− homeostasis may be an effective strategy for the treatment of vLINCL.
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Affiliation(s)
- Yayu Wang
- Hefei National Laboratory for Physical Sciences at Microscale, the CAS Key Laboratory of Innate Immunity and Chronic Disease, Neurodegenerative Disorder Research Center, School of Basic Medical Sciences, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, Anhui 230026, China
| | - Wenping Zeng
- Hefei National Laboratory for Physical Sciences at Microscale, the CAS Key Laboratory of Innate Immunity and Chronic Disease, Neurodegenerative Disorder Research Center, School of Basic Medical Sciences, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, Anhui 230026, China
| | - Bingqian Lin
- Hefei National Laboratory for Physical Sciences at Microscale, the CAS Key Laboratory of Innate Immunity and Chronic Disease, Neurodegenerative Disorder Research Center, School of Basic Medical Sciences, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, Anhui 230026, China
| | - Yichuan Yao
- CAS Key Laboratory of Brain Function and Disease, School of Life Sciences, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, Anhui 230026, China
| | - Canjun Li
- Hefei National Laboratory for Physical Sciences at Microscale, the CAS Key Laboratory of Innate Immunity and Chronic Disease, Neurodegenerative Disorder Research Center, School of Basic Medical Sciences, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, Anhui 230026, China
| | - Wenqi Hu
- Hefei National Laboratory for Physical Sciences at Microscale, the CAS Key Laboratory of Innate Immunity and Chronic Disease, Neurodegenerative Disorder Research Center, School of Basic Medical Sciences, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, Anhui 230026, China
| | - Haotian Wu
- Hefei National Laboratory for Physical Sciences at Microscale, the CAS Key Laboratory of Innate Immunity and Chronic Disease, Neurodegenerative Disorder Research Center, School of Basic Medical Sciences, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, Anhui 230026, China
| | - Jiamin Huang
- Hefei National Laboratory for Physical Sciences at Microscale, the CAS Key Laboratory of Innate Immunity and Chronic Disease, Neurodegenerative Disorder Research Center, School of Basic Medical Sciences, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, Anhui 230026, China
| | - Mei Zhang
- CAS Key Laboratory of Brain Function and Disease, School of Life Sciences, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, Anhui 230026, China
| | - Tian Xue
- Hefei National Laboratory for Physical Sciences at Microscale, the CAS Key Laboratory of Innate Immunity and Chronic Disease, Neurodegenerative Disorder Research Center, School of Basic Medical Sciences, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, Anhui 230026, China
- CAS Key Laboratory of Brain Function and Disease, School of Life Sciences, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, Anhui 230026, China
| | - Dejian Ren
- Department of Biology, University of Pennsylvania, Philadelphia, PA 19104, USA
| | - Lili Qu
- Hefei National Laboratory for Physical Sciences at Microscale, the CAS Key Laboratory of Innate Immunity and Chronic Disease, Neurodegenerative Disorder Research Center, School of Basic Medical Sciences, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, Anhui 230026, China
- Corresponding author. (L.Q.); (C.C.)
| | - Chunlei Cang
- Hefei National Laboratory for Physical Sciences at Microscale, the CAS Key Laboratory of Innate Immunity and Chronic Disease, Neurodegenerative Disorder Research Center, School of Basic Medical Sciences, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, Anhui 230026, China
- Corresponding author. (L.Q.); (C.C.)
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Abstract
Chloride transport across cell membranes is broadly involved in epithelial fluid transport, cell volume and pH regulation, muscle contraction, membrane excitability, and organellar acidification. The human genome encodes at least 53 chloride-transporting proteins with expression in cell plasma or intracellular membranes, which include chloride channels, exchangers, and cotransporters, some having broad anion specificity. Loss-of-function mutations in chloride transporters cause a wide variety of human diseases, including cystic fibrosis, secretory diarrhea, kidney stones, salt-wasting nephropathy, myotonia, osteopetrosis, hearing loss, and goiter. Although impactful advances have been made in the past decade in drug treatment of cystic fibrosis using small molecule modulators of the defective cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel, other chloride channels and solute carrier proteins (SLCs) represent relatively underexplored target classes for drug discovery. New opportunities have emerged for the development of chloride transport modulators as potential therapeutics for secretory diarrheas, constipation, dry eye disorders, kidney stones, polycystic kidney disease, hypertension, and osteoporosis. Approaches to chloride transport-targeted drug discovery are reviewed herein, with focus on chloride channel and exchanger classes in which recent preclinical advances have been made in the identification of small molecule modulators and in proof of concept testing in experimental animal models.
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Affiliation(s)
- Alan S Verkman
- Department of Medicine, University of California, San Francisco, California.,Department of Physiology, University of California, San Francisco, California
| | - Luis J V Galietta
- Telethon Institute of Genetics and Medicine, Pozzuoli, Italy.,Department of Translational Medical Sciences, University of Naples Federico II, Naples, Italy
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27
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Ritter M, Bresgen N, Kerschbaum HH. From Pinocytosis to Methuosis-Fluid Consumption as a Risk Factor for Cell Death. Front Cell Dev Biol 2021; 9:651982. [PMID: 34249909 PMCID: PMC8261248 DOI: 10.3389/fcell.2021.651982] [Citation(s) in RCA: 30] [Impact Index Per Article: 7.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/11/2021] [Accepted: 04/29/2021] [Indexed: 12/11/2022] Open
Abstract
The volumes of a cell [cell volume (CV)] and its organelles are adjusted by osmoregulatory processes. During pinocytosis, extracellular fluid volume equivalent to its CV is incorporated within an hour and membrane area equivalent to the cell's surface within 30 min. Since neither fluid uptake nor membrane consumption leads to swelling or shrinkage, cells must be equipped with potent volume regulatory mechanisms. Normally, cells respond to outwardly or inwardly directed osmotic gradients by a volume decrease and increase, respectively, i.e., they shrink or swell but then try to recover their CV. However, when a cell death (CD) pathway is triggered, CV persistently decreases in isotonic conditions in apoptosis and it increases in necrosis. One type of CD associated with cell swelling is due to a dysfunctional pinocytosis. Methuosis, a non-apoptotic CD phenotype, occurs when cells accumulate too much fluid by macropinocytosis. In contrast to functional pinocytosis, in methuosis, macropinosomes neither recycle nor fuse with lysosomes but with each other to form giant vacuoles, which finally cause rupture of the plasma membrane (PM). Understanding methuosis longs for the understanding of the ionic mechanisms of cell volume regulation (CVR) and vesicular volume regulation (VVR). In nascent macropinosomes, ion channels and transporters are derived from the PM. Along trafficking from the PM to the perinuclear area, the equipment of channels and transporters of the vesicle membrane changes by retrieval, addition, and recycling from and back to the PM, causing profound changes in vesicular ion concentrations, acidification, and-most importantly-shrinkage of the macropinosome, which is indispensable for its proper targeting and cargo processing. In this review, we discuss ion and water transport mechanisms with respect to CVR and VVR and with special emphasis on pinocytosis and methuosis. We describe various aspects of the complex mutual interplay between extracellular and intracellular ions and ion gradients, the PM and vesicular membrane, phosphoinositides, monomeric G proteins and their targets, as well as the submembranous cytoskeleton. Our aim is to highlight important cellular mechanisms, components, and processes that may lead to methuotic CD upon their derangement.
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Affiliation(s)
- Markus Ritter
- Center for Physiology, Pathophysiology and Biophysics, Institute for Physiology and Pathophysiology, Paracelsus Medical University, Salzburg, Austria
- Institute for Physiology and Pathophysiology, Paracelsus Medical University, Nuremberg, Germany
- Gastein Research Institute, Paracelsus Medical University, Salzburg, Austria
- Ludwig Boltzmann Institute for Arthritis und Rehabilitation, Salzburg, Austria
- Kathmandu University School of Medical Sciences, Dhulikhel, Nepal
| | - Nikolaus Bresgen
- Department of Biosciences, University of Salzburg, Salzburg, Austria
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28
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Ji M, Duan X, Han X, Sun J, Zhang D. Exogenous transforming growth factor-β1 prevents the inflow of fluoride to ameleoblasts through regulation of voltage-gated chloride channels 5 and 7. Exp Ther Med 2021; 21:615. [PMID: 33936272 PMCID: PMC8082615 DOI: 10.3892/etm.2021.10047] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/22/2020] [Accepted: 03/15/2021] [Indexed: 02/07/2023] Open
Abstract
Dental fluorosis is a global issue. Although there are multiple causes of dental fluorosis, the precise mechanism remains controversial. Previous studies have demonstrated that extracellular fluoride may promote an accumulation of fluoride ions in ameloblasts, which may induce oxidative and endoplasmic reticulum stresses, leading to dental fluorosis. However, the exact process by which fluoride ions enter cells has not been determined. In the present study, intracellular fluoride concentration was determined using a newly developed specific fluorescent probe called probe 1. Under high extracellular fluoride concentrations, the fluorescence intensity of the ameloblasts increased, however, exogenous transforming growth factor-β1 (TGF-β1) was able to inhibit the increase. Furthermore, changes in the expression of the voltage-gated chloride channels 5 and 7 (ClC5 and ClC-7), which are responsible for the transport of fluoride were investigated. The results indicated that fluoride reduced the expression of endogenous TGF-β1 and increased the expression of ClC-5 and ClC-7. Additionally, exogenous TGF-β1 reduced the expression of ClC-5 and ClC-7. The results of the present study indicate that exogenous TGF-β1 may prevent accumulation of fluoride in ameloblasts through the regulation of ClC-5 and ClC-7 under high extracellular fluoride concentrations.
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Affiliation(s)
- Mei Ji
- Department of Stomatology, Shandong Provincial Hospital Affiliated to Shandong University, Jinan, Shandong 250021, P.R. China
| | - Xuejing Duan
- Department of Stomatology, Shandong Provincial Hospital Affiliated to Shandong University, Jinan, Shandong 250021, P.R. China
| | - Xiaohui Han
- Department of Stomatology, Shandong Provincial Hospital Affiliated to Shandong University, Jinan, Shandong 250021, P.R. China
| | - Jing Sun
- Department of Stomatology, Shandong Provincial Hospital Affiliated to Shandong University, Jinan, Shandong 250021, P.R. China
| | - Dongsheng Zhang
- Department of Stomatology, Shandong Provincial Hospital Affiliated to Shandong University, Jinan, Shandong 250021, P.R. China
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29
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Bose S, He H, Stauber T. Neurodegeneration Upon Dysfunction of Endosomal/Lysosomal CLC Chloride Transporters. Front Cell Dev Biol 2021; 9:639231. [PMID: 33708769 PMCID: PMC7940362 DOI: 10.3389/fcell.2021.639231] [Citation(s) in RCA: 24] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/08/2020] [Accepted: 02/03/2021] [Indexed: 12/15/2022] Open
Abstract
The regulation of luminal ion concentrations is critical for the function of, and transport between intracellular organelles. The importance of the acidic pH in the compartments of the endosomal-lysosomal pathway has been well-known for decades. Besides the V-ATPase, which pumps protons into their lumen, a variety of ion transporters and channels is involved in the regulation of the organelles' complex ion homeostasis. Amongst these are the intracellular members of the CLC family, ClC-3 through ClC-7. They localize to distinct but overlapping compartments of the endosomal-lysosomal pathway, partially with tissue-specific expression. Functioning as 2Cl−/H+ exchangers, they can support the vesicular acidification and accumulate luminal Cl−. Mutations in the encoding genes in patients and mouse models underlie severe phenotypes including kidney stones with CLCN5 and osteopetrosis or hypopigmentation with CLCN7. Dysfunction of those intracellular CLCs that are expressed in neurons lead to neuronal defects. Loss of endosomal ClC-3, which heteromerizes with ClC-4, results in neurodegeneration. Mutations in ClC-4 are associated with epileptic encephalopathy and intellectual disability. Mice lacking the late endosomal ClC-6 develop a lysosomal storage disease with reduced pain sensitivity. Human gene variants have been associated with epilepsy, and a gain-of-function mutation causes early-onset neurodegeneration. Dysfunction of the lysosomal ClC-7 leads to a lysosomal storage disease and neurodegeneration in mice and humans. Reduced luminal chloride, as well as altered calcium regulation, has been associated with lysosomal storage diseases in general. This review discusses the properties of endosomal and lysosomal Cl−/H+ exchange by CLCs and how various alterations of ion transport by CLCs impact organellar ion homeostasis and function in neurodegenerative disorders.
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Affiliation(s)
- Shroddha Bose
- Institute for Chemistry and Biochemistry, Freie Universität Berlin, Berlin, Germany
| | - Hailan He
- Institute for Chemistry and Biochemistry, Freie Universität Berlin, Berlin, Germany.,Department of Pediatrics, Xiangya Hospital, Central South University, Changsha, China
| | - Tobias Stauber
- Institute for Chemistry and Biochemistry, Freie Universität Berlin, Berlin, Germany.,Department of Human Medicine and Institute for Molecular Medicine, MSH Medical School Hamburg, Hamburg, Germany
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30
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Park C, Sakurai Y, Sato H, Kanda S, Iino Y, Kunitomo H. Roles of the ClC chloride channel CLH-1 in food-associated salt chemotaxis behavior of C. elegans. eLife 2021; 10:e55701. [PMID: 33492228 PMCID: PMC7834019 DOI: 10.7554/elife.55701] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/03/2020] [Accepted: 01/04/2021] [Indexed: 01/19/2023] Open
Abstract
The ability of animals to process dynamic sensory information facilitates foraging in an ever-changing environment. However, molecular and neural mechanisms underlying such ability remain elusive. The ClC anion channels/transporters play a pivotal role in cellular ion homeostasis across all phyla. Here, we find a ClC chloride channel is involved in salt concentration chemotaxis of Caenorhabditis elegans. Genetic screening identified two altered-function mutations of clh-1 that disrupt experience-dependent salt chemotaxis. Using genetically encoded fluorescent sensors, we demonstrate that CLH-1 contributes to regulation of intracellular anion and calcium dynamics of salt-sensing neuron, ASER. The mutant CLH-1 reduced responsiveness of ASER to salt stimuli in terms of both temporal resolution and intensity, which disrupted navigation strategies for approaching preferred salt concentrations. Furthermore, other ClC genes appeared to act redundantly in salt chemotaxis. These findings provide insights into the regulatory mechanism of neuronal responsivity by ClCs that contribute to modulation of navigation behavior.
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Affiliation(s)
- Chanhyun Park
- Department of Biological Sciences, School of Science, The University of TokyoTokyoJapan
| | - Yuki Sakurai
- Department of Biological Sciences, School of Science, The University of TokyoTokyoJapan
| | - Hirofumi Sato
- Department of Biological Sciences, School of Science, The University of TokyoTokyoJapan
| | - Shinji Kanda
- Department of Biological Sciences, School of Science, The University of TokyoTokyoJapan
- Laboratory of Physiology, Atmosphere and Ocean Research Institute, The University of TokyoChibaJapan
| | - Yuichi Iino
- Department of Biological Sciences, School of Science, The University of TokyoTokyoJapan
| | - Hirofumi Kunitomo
- Department of Biological Sciences, School of Science, The University of TokyoTokyoJapan
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31
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Westman J, Grinstein S. Determinants of Phagosomal pH During Host-Pathogen Interactions. Front Cell Dev Biol 2021. [PMID: 33505976 DOI: 10.3389/fcell.2020.624958/bibtex] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022] Open
Abstract
The ability of phagosomes to halt microbial growth is intimately linked to their ability to acidify their luminal pH. Establishment and maintenance of an acidic lumen requires precise co-ordination of H+ pumping and counter-ion permeation to offset the countervailing H+ leakage. Despite the best efforts of professional phagocytes, however, a number of specialized pathogens survive and even replicate inside phagosomes. In such instances, pathogens target the pH-regulatory machinery of the host cell in an effort to survive inside or escape from phagosomes. This review aims to describe how phagosomal pH is regulated during phagocytosis, why it varies in different types of professional phagocytes and the strategies developed by prototypical intracellular pathogens to manipulate phagosomal pH to survive, replicate, and eventually escape from the phagocyte.
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Affiliation(s)
- Johannes Westman
- Program in Cell Biology, The Hospital for Sick Children, Toronto, ON, Canada
| | - Sergio Grinstein
- Program in Cell Biology, The Hospital for Sick Children, Toronto, ON, Canada.,Department of Biochemistry, University of Toronto, Toronto, ON, Canada
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32
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Westman J, Grinstein S. Determinants of Phagosomal pH During Host-Pathogen Interactions. Front Cell Dev Biol 2021; 8:624958. [PMID: 33505976 PMCID: PMC7829662 DOI: 10.3389/fcell.2020.624958] [Citation(s) in RCA: 33] [Impact Index Per Article: 8.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/01/2020] [Accepted: 12/15/2020] [Indexed: 12/15/2022] Open
Abstract
The ability of phagosomes to halt microbial growth is intimately linked to their ability to acidify their luminal pH. Establishment and maintenance of an acidic lumen requires precise co-ordination of H+ pumping and counter-ion permeation to offset the countervailing H+ leakage. Despite the best efforts of professional phagocytes, however, a number of specialized pathogens survive and even replicate inside phagosomes. In such instances, pathogens target the pH-regulatory machinery of the host cell in an effort to survive inside or escape from phagosomes. This review aims to describe how phagosomal pH is regulated during phagocytosis, why it varies in different types of professional phagocytes and the strategies developed by prototypical intracellular pathogens to manipulate phagosomal pH to survive, replicate, and eventually escape from the phagocyte.
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Affiliation(s)
- Johannes Westman
- Program in Cell Biology, The Hospital for Sick Children, Toronto, ON, Canada
| | - Sergio Grinstein
- Program in Cell Biology, The Hospital for Sick Children, Toronto, ON, Canada.,Department of Biochemistry, University of Toronto, Toronto, ON, Canada
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33
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Søe K. Osteoclast Fusion: Physiological Regulation of Multinucleation through Heterogeneity-Potential Implications for Drug Sensitivity. Int J Mol Sci 2020; 21:E7717. [PMID: 33086479 PMCID: PMC7589811 DOI: 10.3390/ijms21207717] [Citation(s) in RCA: 32] [Impact Index Per Article: 6.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/26/2020] [Revised: 10/13/2020] [Accepted: 10/15/2020] [Indexed: 02/06/2023] Open
Abstract
Classically, osteoclast fusion consists of four basic steps: (1) attraction/migration, (2) recognition, (3) cell-cell adhesion, and (4) membrane fusion. In theory, this sounds like a straightforward simple linear process. However, it is not. Osteoclast fusion has to take place in a well-coordinated manner-something that is not simple. In vivo, the complex regulation of osteoclast formation takes place within the bone marrow-in time and space. The present review will focus on considering osteoclast fusion in the context of physiology and pathology. Special attention is given to: (1) regulation of osteoclast fusion in vivo, (2) heterogeneity of osteoclast fusion partners, (3) regulation of multi-nucleation, (4) implications for physiology and pathology, and (5) implications for drug sensitivity and side effects. The review will emphasize that more attention should be given to the human in vivo reality when interpreting the impact of in vitro and animal studies. This should be done in order to improve our understanding of human physiology and pathology, as well as to improve anti-resorptive treatment and reduce side effects.
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Affiliation(s)
- Kent Søe
- Clinical Cell Biology, Department of Pathology, Odense University Hospital, 5000 Odense C, Denmark; ; Tel.: +45-65-41-31-90
- Department of Clinical Research, University of Southern Denmark, 5230 Odense M, Denmark
- Department of Molecular Medicine, University of Southern Denmark, 5230 Odense M, Denmark
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34
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Chloride channel 7 protects from redox status impairment-induced renal tubular epithelial cell apoptosis by activating autophagy. Life Sci 2020; 261:118484. [PMID: 32976885 DOI: 10.1016/j.lfs.2020.118484] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/02/2020] [Revised: 09/08/2020] [Accepted: 09/18/2020] [Indexed: 02/07/2023]
Abstract
AIM Chloride channel 7 (CLC-7), broadly expressed in kidney tissues, affects the lysosome degradation pathway. And redox status impairment contributes to cell apoptosis and activates autophagy flux. This study mainly investigates the role and molecular mechanism of CLC-7 in redox status impairment-induced autophagic flux and apoptosis. MAIN METHODS When NRK52E cells, rat renal tubular epithelial cells, were exposed to H2O2 treatment, apoptosis, autophagy flux, and CLC-7 expression were detected. Further investigation was done to observe the change of apoptosis and autophagy flux in renal cells under overexpression or knocking down of CLC-7. The lysosomes acidity, lysosome enzyme Cathepsin D activity and phosphorylation of Ampk/mTOR were also examined when CLC-7 was overexpressed or knocked down. KEY FINDINGS Redox status impairment induced apoptosis and autophagy flux in NRK52E cells and upregulated CLC-7. Overexpression of CLC-7 increased lysosome acidity and Cathepsin D activity. In cells with CLC-7 overexpression, we observed a significant increase of autophagy flux and decline of apoptosis, as well as an apparent increase of p-Ampk and decrease of p-mTOR. On the contrary, cells with knocking down CLC-7 led to opposite results. SIGNIFICANCES CLC-7 is essential to maintain and enhance acidity and enzyme activity in lysosome. Through activating autophagy flux, it exerts survival against renal tubular epithelial cell apoptosis induced by redox status impairment. Its function to modulate Ampk/mTOR pathway is the possible reason why CLC-7 can trigger autophagy flux.
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Catacuzzeno L, Sforna L, Esposito V, Limatola C, Franciolini F. Ion Channels in Glioma Malignancy. Rev Physiol Biochem Pharmacol 2020; 181:223-267. [DOI: 10.1007/112_2020_44] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
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Zhang M, Li T, Zhu J, Tuo B, Liu X. Physiological and pathophysiological role of ion channels and transporters in the colorectum and colorectal cancer. J Cell Mol Med 2020; 24:9486-9494. [PMID: 32662230 PMCID: PMC7520301 DOI: 10.1111/jcmm.15600] [Citation(s) in RCA: 17] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/30/2020] [Revised: 06/16/2020] [Accepted: 06/18/2020] [Indexed: 12/24/2022] Open
Abstract
The incidence of colorectal cancer has increased annually, and the pathogenesis of this disease requires further investigation. In normal colorectal tissues, ion channels and transporters maintain the water-electrolyte balance and acid/base homeostasis. However, dysfunction of these ion channels and transporters leads to the development and progression of colorectal cancer. Therefore, this review focuses on the progress in understanding the roles of ion channels and transporters in the colorectum and in colorectal cancer, including aquaporins (AQPs), Cl- channels, Cl- / HCO 3 - exchangers, Na+ / HCO 3 - transporters and Na+ /H+ exchangers. The goal of this review is to promote the identification of new targets for the treatment and prognosis of colorectal cancer.
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Affiliation(s)
- Minglin Zhang
- Department of GastroenterologyAffiliated Hospital of Zunyi Medical UniversityZunyiChina
- Digestive Disease Institute of Guizhou ProvinceZunyiChina
| | - Taolang Li
- Department of Thyroid and Breast SurgeryAffiliated Hospital of Zunyi Medical UniversityZunyiChina
| | - Jiaxing Zhu
- Department of GastroenterologyAffiliated Hospital of Zunyi Medical UniversityZunyiChina
- Digestive Disease Institute of Guizhou ProvinceZunyiChina
| | - Biguang Tuo
- Department of GastroenterologyAffiliated Hospital of Zunyi Medical UniversityZunyiChina
- Digestive Disease Institute of Guizhou ProvinceZunyiChina
| | - Xuemei Liu
- Department of GastroenterologyAffiliated Hospital of Zunyi Medical UniversityZunyiChina
- Digestive Disease Institute of Guizhou ProvinceZunyiChina
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Zhang X, Mao F, Wong NK, Bao Y, Lin Y, Liu K, Li J, Xiang Z, Ma H, Xiao S, Zhang Y, Yu Z. CLIC2α Chloride Channel Orchestrates Immunomodulation of Hemocyte Phagocytosis and Bactericidal Activity in Crassostrea gigas. iScience 2020; 23:101328. [PMID: 32674055 PMCID: PMC7363696 DOI: 10.1016/j.isci.2020.101328] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/14/2020] [Revised: 06/02/2020] [Accepted: 06/26/2020] [Indexed: 02/07/2023] Open
Abstract
Chloride ion plays critical roles in modulating immunological interactions. Herein, we demonstrated that the anion channel CLIC2α mediates Cl− flux to regulate hemocytes functions in the Pacific oyster (Crassostrea gigas). Specifically, during infection by Vibrio parahemolyticus, chloride influx was activated following onset of phagocytosis. Phosphorylation of Akt was stimulated by Cl− ions entering host cells, further contributing to signal transduction regulating internalization of bacteria through the PI3K/Akt signaling pathway. Concomitantly, Cl− entered phagosomes, promoted the acidification and maturation of phagosomes, and contributed to production of HOCl to eradicate engulfed bacteria. Finally, genomic screening reveals CLIC2α as a major Cl− channel gene responsible for regulating Cl− influx in oysters. Knockdown of CLIC2α predictably impeded phagosome acidification and restricted bacterial killing in oysters. In conclusion, our work has established CLIC2α as a prominent regulator of Cl− influx and thus Cl− function in C. gigas in bacterial infection contexts.
Influx of chloride ions is switched on during phagocytosis in oyster hemocytes PI3K/Akt signaling pathway mediates chloride-dependent activation of phagocytosis Cl− promotes phagosomal acidification and HOCl production CLIC2α is the principal chloride channel encoding gene within oyster genome
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Affiliation(s)
- Xiangyu Zhang
- CAS Key Laboratory of Tropical Marine Bio-resources and Ecology, Guangdong Provincial Key Laboratory of Applied Marine Biology, Innovation Academy of South China Sea Ecology and Environmental Engineering (ISEE), South China Sea Institute of Oceanology, Chinese Academy of Science, Guangzhou 510301, P. R. China; Southern Marine Science and Engineering Guangdong Laboratory (Guangzhou), Guangzhou 510301, P. R. China; University of Chinese Academy of Sciences, Beijing 100049, P. R. China
| | - Fan Mao
- CAS Key Laboratory of Tropical Marine Bio-resources and Ecology, Guangdong Provincial Key Laboratory of Applied Marine Biology, Innovation Academy of South China Sea Ecology and Environmental Engineering (ISEE), South China Sea Institute of Oceanology, Chinese Academy of Science, Guangzhou 510301, P. R. China; Southern Marine Science and Engineering Guangdong Laboratory (Guangzhou), Guangzhou 510301, P. R. China
| | - Nai-Kei Wong
- CAS Key Laboratory of Tropical Marine Bio-resources and Ecology, Guangdong Provincial Key Laboratory of Applied Marine Biology, Innovation Academy of South China Sea Ecology and Environmental Engineering (ISEE), South China Sea Institute of Oceanology, Chinese Academy of Science, Guangzhou 510301, P. R. China; National Clinical Research Center for Infectious Diseases, Shenzhen Third People's Hospital, The Second Hospital Affiliated to Southern University of Science and Technology, Shenzhen 518112, P. R. China
| | - Yongbo Bao
- Zhejiang Key Laboratory of Aquatic Germplasm Resources, College of Biological and Environmental Sciences, Zhejiang Wanli University, Ningbo 315100, P. R. China
| | - Yue Lin
- CAS Key Laboratory of Tropical Marine Bio-resources and Ecology, Guangdong Provincial Key Laboratory of Applied Marine Biology, Innovation Academy of South China Sea Ecology and Environmental Engineering (ISEE), South China Sea Institute of Oceanology, Chinese Academy of Science, Guangzhou 510301, P. R. China; Southern Marine Science and Engineering Guangdong Laboratory (Guangzhou), Guangzhou 510301, P. R. China; University of Chinese Academy of Sciences, Beijing 100049, P. R. China
| | - Kunna Liu
- CAS Key Laboratory of Tropical Marine Bio-resources and Ecology, Guangdong Provincial Key Laboratory of Applied Marine Biology, Innovation Academy of South China Sea Ecology and Environmental Engineering (ISEE), South China Sea Institute of Oceanology, Chinese Academy of Science, Guangzhou 510301, P. R. China; Southern Marine Science and Engineering Guangdong Laboratory (Guangzhou), Guangzhou 510301, P. R. China; University of Chinese Academy of Sciences, Beijing 100049, P. R. China
| | - Jun Li
- CAS Key Laboratory of Tropical Marine Bio-resources and Ecology, Guangdong Provincial Key Laboratory of Applied Marine Biology, Innovation Academy of South China Sea Ecology and Environmental Engineering (ISEE), South China Sea Institute of Oceanology, Chinese Academy of Science, Guangzhou 510301, P. R. China; Southern Marine Science and Engineering Guangdong Laboratory (Guangzhou), Guangzhou 510301, P. R. China
| | - Zhiming Xiang
- CAS Key Laboratory of Tropical Marine Bio-resources and Ecology, Guangdong Provincial Key Laboratory of Applied Marine Biology, Innovation Academy of South China Sea Ecology and Environmental Engineering (ISEE), South China Sea Institute of Oceanology, Chinese Academy of Science, Guangzhou 510301, P. R. China; Southern Marine Science and Engineering Guangdong Laboratory (Guangzhou), Guangzhou 510301, P. R. China
| | - Haitao Ma
- CAS Key Laboratory of Tropical Marine Bio-resources and Ecology, Guangdong Provincial Key Laboratory of Applied Marine Biology, Innovation Academy of South China Sea Ecology and Environmental Engineering (ISEE), South China Sea Institute of Oceanology, Chinese Academy of Science, Guangzhou 510301, P. R. China; Southern Marine Science and Engineering Guangdong Laboratory (Guangzhou), Guangzhou 510301, P. R. China
| | - Shu Xiao
- CAS Key Laboratory of Tropical Marine Bio-resources and Ecology, Guangdong Provincial Key Laboratory of Applied Marine Biology, Innovation Academy of South China Sea Ecology and Environmental Engineering (ISEE), South China Sea Institute of Oceanology, Chinese Academy of Science, Guangzhou 510301, P. R. China; Southern Marine Science and Engineering Guangdong Laboratory (Guangzhou), Guangzhou 510301, P. R. China
| | - Yang Zhang
- CAS Key Laboratory of Tropical Marine Bio-resources and Ecology, Guangdong Provincial Key Laboratory of Applied Marine Biology, Innovation Academy of South China Sea Ecology and Environmental Engineering (ISEE), South China Sea Institute of Oceanology, Chinese Academy of Science, Guangzhou 510301, P. R. China; Southern Marine Science and Engineering Guangdong Laboratory (Guangzhou), Guangzhou 510301, P. R. China.
| | - Ziniu Yu
- CAS Key Laboratory of Tropical Marine Bio-resources and Ecology, Guangdong Provincial Key Laboratory of Applied Marine Biology, Innovation Academy of South China Sea Ecology and Environmental Engineering (ISEE), South China Sea Institute of Oceanology, Chinese Academy of Science, Guangzhou 510301, P. R. China; Southern Marine Science and Engineering Guangdong Laboratory (Guangzhou), Guangzhou 510301, P. R. China.
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Leisle L, Xu Y, Fortea E, Lee S, Galpin JD, Vien M, Ahern CA, Accardi A, Bernèche S. Divergent Cl - and H + pathways underlie transport coupling and gating in CLC exchangers and channels. eLife 2020; 9:e51224. [PMID: 32343228 PMCID: PMC7274781 DOI: 10.7554/elife.51224] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/20/2019] [Accepted: 04/28/2020] [Indexed: 12/12/2022] Open
Abstract
The CLC family comprises H+-coupled exchangers and Cl- channels, and mutations causing their dysfunction lead to genetic disorders. The CLC exchangers, unlike canonical 'ping-pong' antiporters, simultaneously bind and translocate substrates through partially congruent pathways. How ions of opposite charge bypass each other while moving through a shared pathway remains unknown. Here, we use MD simulations, biochemical and electrophysiological measurements to identify two conserved phenylalanine residues that form an aromatic pathway whose dynamic rearrangements enable H+ movement outside the Cl- pore. These residues are important for H+ transport and voltage-dependent gating in the CLC exchangers. The aromatic pathway residues are evolutionarily conserved in CLC channels where their electrostatic properties and conformational flexibility determine gating. We propose that Cl- and H+ move through physically distinct and evolutionarily conserved routes through the CLC channels and transporters and suggest a unifying mechanism that describes the gating mechanism of both CLC subtypes.
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Affiliation(s)
- Lilia Leisle
- Department of Anesthesiology, Weill Cornell Medical CollegeNew YorkUnited States
| | - Yanyan Xu
- SIB Swiss Institute of Bioinformatics, University of BaselBaselSwitzerland
- Biozentrum, University of BaselBaselSwitzerland
| | - Eva Fortea
- Department of Physiology and Biophysics, Weill Cornell Medical CollegeNew YorkUnited States
| | - Sangyun Lee
- Department of Anesthesiology, Weill Cornell Medical CollegeNew YorkUnited States
| | - Jason D Galpin
- Department of Molecular Physiology and Biophysics, University of Iowa Carver College of MedicineIowa CityUnited States
| | - Malvin Vien
- Department of Anesthesiology, Weill Cornell Medical CollegeNew YorkUnited States
| | - Christopher A Ahern
- Department of Molecular Physiology and Biophysics, University of Iowa Carver College of MedicineIowa CityUnited States
| | - Alessio Accardi
- Department of Anesthesiology, Weill Cornell Medical CollegeNew YorkUnited States
- Department of Physiology and Biophysics, Weill Cornell Medical CollegeNew YorkUnited States
- Department of Biochemistry, Weill Cornell Medical CollegeNew YorkUnited States
| | - Simon Bernèche
- SIB Swiss Institute of Bioinformatics, University of BaselBaselSwitzerland
- Biozentrum, University of BaselBaselSwitzerland
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Chiariello MG, Bolnykh V, Ippoliti E, Meloni S, Olsen JMH, Beck T, Rothlisberger U, Fahlke C, Carloni P. Molecular Basis of CLC Antiporter Inhibition by Fluoride. J Am Chem Soc 2020; 142:7254-7258. [PMID: 32233472 DOI: 10.1021/jacs.9b13588] [Citation(s) in RCA: 22] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/07/2023]
Abstract
CLC channels and transporters conduct or transport various kinds of anions, with the exception of fluoride, which acts as an effective inhibitor. Here, we performed sub-nanosecond DFT-based QM/MM simulations of the E. coli anion/proton exchanger ClC-ec1 and observed that fluoride binds incoming protons within the selectivity filter, with excess protons shared with the gating glutamate E148. Depending on E148 conformation, the competition for the proton can involve either a direct F-/E148 interaction or the modulation of water molecules bridging the two anions. The direct interaction locks E148 in a conformation that does not allow for proton transport, and thus inhibits protein function.
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Affiliation(s)
- Maria Gabriella Chiariello
- Institute for Advanced Simulation (IAS-5) and Institute of Neuroscience and Medicine (INM-9), Computational Biomedicine, Forschungszentrum Jülich, 52425 Jülich, Germany.,JARA-HPC, Forschungszentrum Jülich, D-54245 Jülich, Germany
| | - Viacheslav Bolnykh
- Laboratory of Computational Chemistry and Biochemistry, Ecole Polytechnique Fédérale de Lausanne, CH-1015 Lausanne, Switzerland
| | - Emiliano Ippoliti
- Institute for Advanced Simulation (IAS-5) and Institute of Neuroscience and Medicine (INM-9), Computational Biomedicine, Forschungszentrum Jülich, 52425 Jülich, Germany.,JARA-HPC, Forschungszentrum Jülich, D-54245 Jülich, Germany
| | - Simone Meloni
- Dipartimento di Scienze Chimiche e Farmaceutiche, Università degli Studi di Ferrara, Via Luigi Borsari 46, I-44121 Ferrara, Italy
| | - Jógvan Magnus Haugaard Olsen
- Hylleraas Centre for Quantum Molecular Sciences, Department of Chemistry, UiT The Arctic University of Norway, N-9037 Tromsø, Norway
| | - Thomas Beck
- Department of Chemistry, University of Cincinnati, Cincinnati, Ohio 45221, United States
| | - Ursula Rothlisberger
- Laboratory of Computational Chemistry and Biochemistry, Ecole Polytechnique Fédérale de Lausanne, CH-1015 Lausanne, Switzerland
| | - Christoph Fahlke
- Institute of Biological Information Processing (IBI-1), Molekular- und Zellphysiologie, Forschungszentrum Jülich, D-54245 Jülich, Germany
| | - Paolo Carloni
- Institute for Advanced Simulation (IAS-5) and Institute of Neuroscience and Medicine (INM-9), Computational Biomedicine, Forschungszentrum Jülich, 52425 Jülich, Germany.,JARA-HPC, Forschungszentrum Jülich, D-54245 Jülich, Germany.,Department of Physics, RWTH Aachen University, 52056 Aachen, Germany.,Institute of Neuroscience and Medicine (INM-11), Molecular Neuroscience and Neuroimaging, Forschungszentrum Julich, 52425 Julich, Germany
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40
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McKiernan KA, Koster AK, Maduke M, Pande VS. Dynamical model of the CLC-2 ion channel reveals conformational changes associated with selectivity-filter gating. PLoS Comput Biol 2020; 16:e1007530. [PMID: 32226009 PMCID: PMC7145265 DOI: 10.1371/journal.pcbi.1007530] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/19/2018] [Revised: 04/09/2020] [Accepted: 11/05/2019] [Indexed: 12/18/2022] Open
Abstract
This work reports a dynamical Markov state model of CLC-2 "fast" (pore) gating, based on 600 microseconds of molecular dynamics (MD) simulation. In the starting conformation of our CLC-2 model, both outer and inner channel gates are closed. The first conformational change in our dataset involves rotation of the inner-gate backbone along residues S168-G169-I170. This change is strikingly similar to that observed in the cryo-EM structure of the bovine CLC-K channel, though the volume of the intracellular (inner) region of the ion conduction pathway is further expanded in our model. From this state (inner gate open and outer gate closed), two additional states are observed, each involving a unique rotameric flip of the outer-gate residue GLUex. Both additional states involve conformational changes that orient GLUex away from the extracellular (outer) region of the ion conduction pathway. In the first additional state, the rotameric flip of GLUex results in an open, or near-open, channel pore. The equilibrium population of this state is low (∼1%), consistent with the low open probability of CLC-2 observed experimentally in the absence of a membrane potential stimulus (0 mV). In the second additional state, GLUex rotates to occlude the channel pore. This state, which has a low equilibrium population (∼1%), is only accessible when GLUex is protonated. Together, these pathways model the opening of both an inner and outer gate within the CLC-2 selectivity filter, as a function of GLUex protonation. Collectively, our findings are consistent with published experimental analyses of CLC-2 gating and provide a high-resolution structural model to guide future investigations.
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Affiliation(s)
- Keri A. McKiernan
- Department of Chemistry, Stanford University, Stanford, California, United States of America
| | - Anna K. Koster
- Department of Chemistry, Stanford University, Stanford, California, United States of America
- Department of Molecular & Cellular Physiology, Stanford University, Stanford, California, United States of America
| | - Merritt Maduke
- Department of Molecular & Cellular Physiology, Stanford University, Stanford, California, United States of America
| | - Vijay S. Pande
- Department of Bioengineering, Stanford University, Stanford, California, United States of America
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Lee JH, Wolfe DM, Darji S, McBrayer MK, Colacurcio DJ, Kumar A, Stavrides P, Mohan PS, Nixon RA. β2-adrenergic Agonists Rescue Lysosome Acidification and Function in PSEN1 Deficiency by Reversing Defective ER-to-lysosome Delivery of ClC-7. J Mol Biol 2020; 432:2633-2650. [PMID: 32105735 DOI: 10.1016/j.jmb.2020.02.021] [Citation(s) in RCA: 19] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/16/2019] [Revised: 02/11/2020] [Accepted: 02/12/2020] [Indexed: 02/06/2023]
Abstract
Lysosomal dysfunction is considered pathogenic in Alzheimer disease (AD). Loss of presenilin-1 (PSEN1) function causing AD impedes acidification via defective vacuolar ATPase (vATPase) V0a1 subunit delivery to lysosomes. We report that isoproterenol (ISO) and related β2-adrenergic agonists reacidify lysosomes in PSEN1 Knock out (KO) cells and fibroblasts from PSEN1 familial AD patients, which restores lysosomal proteolysis, calcium homeostasis, and normal autophagy flux. We identify a novel rescue mechanism involving Portein Kinase A (PKA)-mediated facilitation of chloride channel-7 (ClC-7) delivery to lysosomes which reverses markedly lowered chloride (Cl-) content in PSEN1 KO lysosomes. Notably, PSEN1 loss of function impedes Endoplasmic Reticulum (ER)-to-lysosome delivery of ClC-7. Transcriptomics of PSEN1-deficient cells reveals strongly downregulated ER-to-lysosome transport pathways and reversibility by ISO, thus accounting for lysosomal Cl- deficits that compound pH elevation due to deficient vATPase and its rescue by β2-adrenergic agonists. Our findings uncover a broadened PSEN1 role in lysosomal ion homeostasis and novel pH modulation of lysosomes through β2-adrenergic regulation of ClC-7, which can potentially be modulated therapeutically.
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Affiliation(s)
- Ju-Hyun Lee
- Center for Dementia Research, Nathan S. Kline Institute, Orangeburg, NY, 10962, USA; Department of Psychiatry, Langone Medical Center, New York, NY, 10016, USA.
| | - Devin M Wolfe
- Center for Dementia Research, Nathan S. Kline Institute, Orangeburg, NY, 10962, USA
| | - Sandipkumar Darji
- Center for Dementia Research, Nathan S. Kline Institute, Orangeburg, NY, 10962, USA
| | - Mary Kate McBrayer
- Center for Dementia Research, Nathan S. Kline Institute, Orangeburg, NY, 10962, USA
| | - Daniel J Colacurcio
- Center for Dementia Research, Nathan S. Kline Institute, Orangeburg, NY, 10962, USA
| | - Asok Kumar
- Center for Dementia Research, Nathan S. Kline Institute, Orangeburg, NY, 10962, USA
| | - Philip Stavrides
- Center for Dementia Research, Nathan S. Kline Institute, Orangeburg, NY, 10962, USA
| | - Panaiyur S Mohan
- Center for Dementia Research, Nathan S. Kline Institute, Orangeburg, NY, 10962, USA; Department of Psychiatry, Langone Medical Center, New York, NY, 10016, USA
| | - Ralph A Nixon
- Center for Dementia Research, Nathan S. Kline Institute, Orangeburg, NY, 10962, USA; Department of Psychiatry, Langone Medical Center, New York, NY, 10016, USA; Department of Cell Biology, Langone Medical Center, New York, NY, 10016, USA; Neuroscience Institute, New York University, New York, NY 10016, USA.
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Morales F, Pusch M. An Up-to-Date Overview of the Complexity of Genotype-Phenotype Relationships in Myotonic Channelopathies. Front Neurol 2020; 10:1404. [PMID: 32010054 PMCID: PMC6978732 DOI: 10.3389/fneur.2019.01404] [Citation(s) in RCA: 30] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/24/2019] [Accepted: 12/23/2019] [Indexed: 12/11/2022] Open
Abstract
Myotonic disorders are inherited neuromuscular diseases divided into dystrophic myotonias and non-dystrophic myotonias (NDM). The latter is a group of dominant or recessive diseases caused by mutations in genes encoding ion channels that participate in the generation and control of the skeletal muscle action potential. Their altered function causes hyperexcitability of the muscle membrane, thereby triggering myotonia, the main sign in NDM. Mutations in the genes encoding voltage-gated Cl− and Na+ channels (respectively, CLCN1 and SCN4A) produce a wide spectrum of phenotypes, which differ in age of onset, affected muscles, severity of myotonia, degree of hypertrophy, and muscle weakness, disease progression, among others. More than 200 CLCN1 and 65 SCN4A mutations have been identified and described, but just about half of them have been functionally characterized, an approach that is likely extremely helpful to contribute to improving the so-far rather poor clinical correlations present in NDM. The observed poor correlations may be due to: (1) the wide spectrum of symptoms and overlapping phenotypes present in both groups (Cl− and Na+ myotonic channelopathies) and (2) both genes present high genotypic variability. On the one hand, several mutations cause a unique and reproducible phenotype in most patients. On the other hand, some mutations can have different inheritance pattern and clinical phenotypes in different families. Conversely, different mutations can be translated into very similar phenotypes. For these reasons, the genotype-phenotype relationships in myotonic channelopathies are considered complex. Although the molecular bases for the clinical variability present in myotonic channelopathies remain obscure, several hypotheses have been put forward to explain the variability, which include: (a) differential allelic expression; (b) trans-acting genetic modifiers; (c) epigenetic, hormonal, or environmental factors; and (d) dominance with low penetrance. Improvements in clinical tests, the recognition of the different phenotypes that result from particular mutations and the understanding of how a mutation affects the structure and function of the ion channel, together with genetic screening, is expected to improve clinical correlation in NDMs.
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Affiliation(s)
- Fernando Morales
- Instituto de Investigaciones en Salud, Universidad de Costa, San José, Costa Rica
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A highly-selective chloride microelectrode based on a mercuracarborand anion carrier. Sci Rep 2019; 9:18860. [PMID: 31827130 PMCID: PMC6906508 DOI: 10.1038/s41598-019-54885-6] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/06/2019] [Accepted: 11/20/2019] [Indexed: 11/08/2022] Open
Abstract
The chloride gradient plays an important role in regulating cell volume, membrane potential, pH, secretion, and the reversal potential of inhibitory glycine and GABAA receptors. Measurement of intracellular chloride activity, \documentclass[12pt]{minimal}
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\begin{document}$${{\boldsymbol{a}}}_{{\boldsymbol{Cl}}}^{{\boldsymbol{i}}}$$\end{document}aCli, using liquid membrane ion-selective microelectrodes (ISM), however, has been limited by the physiochemical properties of Cl− ionophores which have caused poor stability, drift, sluggish response times, and interference from other biologically relevant anions. Most importantly, intracellular \documentclass[12pt]{minimal}
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\begin{document}$${\bf{HC}}{{\bf{O}}}_{{\bf{3}}}^{-}$$\end{document}HCO3− may be up to 4 times more abundant than Cl− (e.g. skeletal muscle) which places severe constraints on the required selectivity of a Cl− – sensing ISM. Previously, a sensitive and highly-selective Cl− sensor was developed in a polymeric membrane electrode using a trinuclear Hg(II) complex containing carborane-based ligands, [9]-mercuracarborand-3, or MC3 for short. Here, we have adapted the use of the MC3 anion carrier in a liquid membrane ion-selective microelectrode and show the MC3-ISM has a linear Nernstian response over a wide range of aCl (0.1 mM to 100 mM), is highly selective for Cl− over other biological anions or inhibitors of Cl− transport, and has a 10% to 90% settling time of 3 sec. Importantly, over the physiological range of aCl (1 mM to 100 mM) the potentiometric response of the MC3-ISM is insensitive to \documentclass[12pt]{minimal}
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\begin{document}$${\bf{HC}}{{\bf{O}}}_{{\bf{3}}}^{-}$$\end{document}HCO3− or changes in pH. Finally, we demonstrate the biological application of an MC3-ISM by measuring intracellular aCl, and the response to an external Cl-free challenge, for an isolated skeletal muscle fiber.
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Araújo CET, Oliveira CMC, Barbosa JD, Oliveira-Filho JP, Resende LAL, Badial PR, Araujo-Junior JP, McCue ME, Borges AS. A large intragenic deletion in the CLCN1 gene causes Hereditary Myotonia in pigs. Sci Rep 2019; 9:15632. [PMID: 31666547 PMCID: PMC6821760 DOI: 10.1038/s41598-019-51286-7] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/18/2019] [Accepted: 09/11/2019] [Indexed: 12/14/2022] Open
Abstract
Mutations in the CLCN1 gene are the primary cause of non-dystrophic Hereditary Myotonia in several animal species. However, there are no reports of Hereditary Myotonia in pigs to date. Therefore, the objective of the present study was to characterize the clinical and molecular findings of Hereditary Myotonia in an inbred pedigree. The clinical, electromyographic, histopathological, and molecular findings were evaluated. Clinically affected pigs presented non-dystrophic recessive Hereditary Myotonia. Nucleotide sequence analysis of the entire coding region of the CLCN1 gene revealed the absence of the exons 15 and 16 in myotonic animals. Analysis of the genomic region flanking the deletion unveiled a large intragenic deletion of 4,165 nucleotides. Interestingly, non-related, non-myotonic pigs expressed transcriptional levels of an alternate transcript (i.e., X2) that was identical to the deleted X1 transcript of myotonic pigs. All myotonic pigs and their progenitors were homozygous recessive and heterozygous, respectively, for the 4,165-nucleotide deletion. This is the first study reporting Hereditary Myotonia in pigs and characterizing its clinical and molecular findings. Moreover, to the best of our knowledge, Hereditary Myotonia has never been associated with a genomic deletion in the CLCN1 gene in any other species.
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Affiliation(s)
- C E T Araújo
- São Paulo State University (UNESP), School of Veterinary Medicine and Animal Science, Botucatu, São Paulo, Brazil
| | - C M C Oliveira
- Instituto de Medicina Veterinária, Universidade Federal do Pará, Campus Castanhal, PA, Brazil
| | - J D Barbosa
- Instituto de Medicina Veterinária, Universidade Federal do Pará, Campus Castanhal, PA, Brazil
| | - J P Oliveira-Filho
- São Paulo State University (UNESP), School of Veterinary Medicine and Animal Science, Botucatu, São Paulo, Brazil
| | - L A L Resende
- São Paulo State University (UNESP), Medical School, Botucatu, Brazil
| | - P R Badial
- Department of Pathobiology and Population Medicine, College of Veterinary Medicine, Mississippi State University, Starkville, MS, USA
| | - J P Araujo-Junior
- São Paulo State University (UNESP), Institute of Bioscience, Botucatu, Brazil
| | - M E McCue
- College of Veterinary Medicine, University of Minnesota, St Paul, Minnesota, 55108, USA
| | - A S Borges
- São Paulo State University (UNESP), School of Veterinary Medicine and Animal Science, Botucatu, São Paulo, Brazil.
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45
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Astaburuaga R, Quintanar Haro OD, Stauber T, Relógio A. A Mathematical Model of Lysosomal Ion Homeostasis Points to Differential Effects of Cl - Transport in Ca 2+ Dynamics. Cells 2019; 8:E1263. [PMID: 31623161 PMCID: PMC6848924 DOI: 10.3390/cells8101263] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/01/2019] [Revised: 10/11/2019] [Accepted: 10/13/2019] [Indexed: 12/20/2022] Open
Abstract
The establishment and maintenance of ion gradients between the interior of lysosomes and the cytosol are crucial for numerous cellular and organismal functions. Numerous ion transport proteins ensure the required variation in luminal concentrations of the different ions along the endocytic pathway to fit the needs of the organelles. Failures in keeping proper ion homeostasis have pathological consequences. Accordingly, several human diseases are caused by the dysfunction of ion transporters. These include osteopetrosis, caused by the dysfunction of Cl-/H+ exchange by the lysosomal transporter ClC-7. To better understand how chloride transport affects lysosomal ion homeostasis and how its disruption impinges on lysosomal function, we developed a mathematical model of lysosomal ion homeostasis including Ca2+ dynamics. The model recapitulates known biophysical properties of ClC-7 and enables the investigation of its differential activation kinetics on lysosomal ion homeostasis. We show that normal functioning of ClC-7 supports the acidification process, is associated with increased luminal concentrations of sodium, potassium, and chloride, and leads to a higher Ca2+ uptake and release. Our model highlights the role of ClC-7 in lysosomal acidification and shows the existence of differential Ca2+ dynamics upon perturbations of Cl-/H+ exchange and its activation kinetics, with possible pathological consequences.
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Affiliation(s)
- Rosario Astaburuaga
- Institute for Theoretical Biology (ITB), Charité-Universitätsmedizin Berlin, Corporate Member of the Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, 10115 Berlin, Germany.
- Medical Department of Hematology, Oncology and Tumor Immunology, Molekulares Krebsforschungzentrum (MKFZ), Charité-Universitätsmedizin Berlin, Corporate Member of the Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, 13353 Berlin, Germany.
| | - Orlando Daniel Quintanar Haro
- Institute for Theoretical Biology (ITB), Charité-Universitätsmedizin Berlin, Corporate Member of the Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, 10115 Berlin, Germany.
- Freie Universität Berlin, Institute of Chemistry and Biochemistry, 14195 Berlin, Germany.
| | - Tobias Stauber
- Freie Universität Berlin, Institute of Chemistry and Biochemistry, 14195 Berlin, Germany.
- Department of Human Medicine, Medical School Hamburg, 20457 Hamburg, Germany.
| | - Angela Relógio
- Institute for Theoretical Biology (ITB), Charité-Universitätsmedizin Berlin, Corporate Member of the Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, 10115 Berlin, Germany.
- Medical Department of Hematology, Oncology and Tumor Immunology, Molekulares Krebsforschungzentrum (MKFZ), Charité-Universitätsmedizin Berlin, Corporate Member of the Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, 13353 Berlin, Germany.
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46
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Mutation of external glutamate residue reveals a new intermediate transport state and anion binding site in a CLC Cl -/H + antiporter. Proc Natl Acad Sci U S A 2019; 116:17345-17354. [PMID: 31409705 DOI: 10.1073/pnas.1901822116] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022] Open
Abstract
The CLC family of proteins are involved in a variety of physiological processes to control cellular chloride concentration. Two distinct classes of CLC proteins, Cl- channels and Cl-/H+ antiporters, have been functionally and structurally investigated over the last several decades. Previous studies have suggested that the conformational heterogeneity of the critical glutamate residue, Gluex, could explain the transport cycle of CLC-type Cl-/H+ antiporters. However, the presence of multiple conformations (Up, Middle, and Down) of the Gluex has been suggested from combined structural snapshots of 2 different CLC antiporters: CLC-ec1 from Escherichia coli and cmCLC from a thermophilic red alga, Cyanidioschyzon merolae Thus, we aimed to investigate further the heterogeneity of Gluex-conformations in CLC-ec1, the most deeply studied CLC antiporter, at both functional and structural levels. Here, we show that the crystal structures of the Gluex mutant E148D and wild-type CLC-ec1 with varying anion concentrations suggest a structural intermediate, the "Midlow" conformation. We also found that an extra anion can be located above the external Cl--binding site in the E148D mutant when the anion concentration is high. Moreover, we observed that a carboxylate in solution can occupy either the external or central Cl--binding site in the ungated E148A mutant using an anomalously detectable short carboxylic acid, bromoacetate. These results lend credibility to the idea that the Gluex can take at least 3 distinct conformational states during the transport cycle of a single CLC antiporter.
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Rao MC. Physiology of Electrolyte Transport in the Gut: Implications for Disease. Compr Physiol 2019; 9:947-1023. [PMID: 31187895 DOI: 10.1002/cphy.c180011] [Citation(s) in RCA: 28] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
We now have an increased understanding of the genetics, cell biology, and physiology of electrolyte transport processes in the mammalian intestine, due to the availability of sophisticated methodologies ranging from genome wide association studies to CRISPR-CAS technology, stem cell-derived organoids, 3D microscopy, electron cryomicroscopy, single cell RNA sequencing, transgenic methodologies, and tools to manipulate cellular processes at a molecular level. This knowledge has simultaneously underscored the complexity of biological systems and the interdependence of multiple regulatory systems. In addition to the plethora of mammalian neurohumoral factors and their cross talk, advances in pyrosequencing and metagenomic analyses have highlighted the relevance of the microbiome to intestinal regulation. This article provides an overview of our current understanding of electrolyte transport processes in the small and large intestine, their regulation in health and how dysregulation at multiple levels can result in disease. Intestinal electrolyte transport is a balance of ion secretory and ion absorptive processes, all exquisitely dependent on the basolateral Na+ /K+ ATPase; when this balance goes awry, it can result in diarrhea or in constipation. The key transporters involved in secretion are the apical membrane Cl- channels and the basolateral Na+ -K+ -2Cl- cotransporter, NKCC1 and K+ channels. Absorption chiefly involves apical membrane Na+ /H+ exchangers and Cl- /HCO3 - exchangers in the small intestine and proximal colon and Na+ channels in the distal colon. Key examples of our current understanding of infectious, inflammatory, and genetic diarrheal diseases and of constipation are provided. © 2019 American Physiological Society. Compr Physiol 9:947-1023, 2019.
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Affiliation(s)
- Mrinalini C Rao
- Department of Physiology and Biophysics, University of Illinois at Chicago, Chicago, Illinois, USA
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48
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Weston MR, Mindell JA. Characterizing chloride-dependent acidification in brain clathrin-coated vesicles 1. Biochem Cell Biol 2019; 97:315-324. [PMID: 30383978 PMCID: PMC8404411 DOI: 10.1139/bcb-2018-0142] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022] Open
Abstract
Endocytic organelles maintain their acidic pH using the V-type ATPase proton pump. However, proton accumulation across the membrane generates a voltage and requires the movement of an additional ion, known as a counterion, to dissipate charge buildup. The role of counterion movement in endosomes is not clear, but a subpopulation of early endosomes, clathrin-coated vesicles (CCVs), has previously been shown to use external chloride (Cl-) to allow V-ATPase-dependent acidification. We aimed to determine the identity and function of this presumed Cl- transporting protein. Our sample of highly enriched bovine brain CCVs exhibited V-type ATPase-facilitated acidification in the presence of external Cl-, independent of the monovalent cations present. While unsuccessful at identifying the mechanism of anion transport, we used glutamate-facilitated acidification, density gradients, and mass spectrometry to show that most brain CCVs are synaptic vesicles, complementing results from earlier studies that argued similarity only on the basis on protein content. The source of Cl--dependent acidification in brain CCVs may be vGLUT1, a synaptic vesicle glutamate transporter with known Cl- permeability, although CCVs in other tissues are likely to utilize different proteins to facilitate acidification.
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Affiliation(s)
- Mary R. Weston
- Membrane Transport Biophysics Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland, United States of America 20892
| | - Joseph A. Mindell
- Membrane Transport Biophysics Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland, United States of America 20892
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49
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Wang K, Preisler SS, Zhang L, Cui Y, Missel JW, Grønberg C, Gotfryd K, Lindahl E, Andersson M, Calloe K, Egea PF, Klaerke DA, Pusch M, Pedersen PA, Zhou ZH, Gourdon P. Structure of the human ClC-1 chloride channel. PLoS Biol 2019; 17:e3000218. [PMID: 31022181 PMCID: PMC6483157 DOI: 10.1371/journal.pbio.3000218] [Citation(s) in RCA: 55] [Impact Index Per Article: 9.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/24/2018] [Accepted: 03/22/2019] [Indexed: 11/18/2022] Open
Abstract
ClC-1 protein channels facilitate rapid passage of chloride ions across cellular membranes, thereby orchestrating skeletal muscle excitability. Malfunction of ClC-1 is associated with myotonia congenita, a disease impairing muscle relaxation. Here, we present the cryo-electron microscopy (cryo-EM) structure of human ClC-1, uncovering an architecture reminiscent of that of bovine ClC-K and CLC transporters. The chloride conducting pathway exhibits distinct features, including a central glutamate residue ("fast gate") known to confer voltage-dependence (a mechanistic feature not present in ClC-K), linked to a somewhat rearranged central tyrosine and a narrower aperture of the pore toward the extracellular vestibule. These characteristics agree with the lower chloride flux of ClC-1 compared with ClC-K and enable us to propose a model for chloride passage in voltage-dependent CLC channels. Comparison of structures derived from protein studied in different experimental conditions supports the notion that pH and adenine nucleotides regulate ClC-1 through interactions between the so-called cystathionine-β-synthase (CBS) domains and the intracellular vestibule ("slow gating"). The structure also provides a framework for analysis of mutations causing myotonia congenita and reveals a striking correlation between mutated residues and the phenotypic effect on voltage gating, opening avenues for rational design of therapies against ClC-1-related diseases.
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Affiliation(s)
- Kaituo Wang
- Department of Biomedical Sciences, University of Copenhagen, Copenhagen, Denmark
- Department of Microbiology, Immunology & Molecular Genetics, University of California at Los Angeles, Los Angeles, California
- California NanoSystems Institute, University of California at Los Angeles, Los Angeles, California
| | | | - Liying Zhang
- Department of Biomedical Sciences, University of Copenhagen, Copenhagen, Denmark
| | - Yanxiang Cui
- Department of Microbiology, Immunology & Molecular Genetics, University of California at Los Angeles, Los Angeles, California
- California NanoSystems Institute, University of California at Los Angeles, Los Angeles, California
| | - Julie Winkel Missel
- Department of Biomedical Sciences, University of Copenhagen, Copenhagen, Denmark
| | - Christina Grønberg
- Department of Biomedical Sciences, University of Copenhagen, Copenhagen, Denmark
| | - Kamil Gotfryd
- Department of Biomedical Sciences, University of Copenhagen, Copenhagen, Denmark
| | - Erik Lindahl
- Department of Biochemistry & Biophysics, Stockholm University, Stockholm, Sweden
| | | | - Kirstine Calloe
- Department of Veterinary and Animal Sciences, University of Copenhagen, Frederiksberg, Denmark
| | - Pascal F. Egea
- Department of Biological Chemistry, University of California at Los Angeles, Los Angeles, California
| | - Dan Arne Klaerke
- Department of Veterinary and Animal Sciences, University of Copenhagen, Frederiksberg, Denmark
| | - Michael Pusch
- Institute of Biophysics, Consiglio Nazionale delle Ricerche, Genova, Italy
| | | | - Z. Hong Zhou
- Department of Microbiology, Immunology & Molecular Genetics, University of California at Los Angeles, Los Angeles, California
- California NanoSystems Institute, University of California at Los Angeles, Los Angeles, California
| | - Pontus Gourdon
- Department of Biomedical Sciences, University of Copenhagen, Copenhagen, Denmark
- Department of Experimental Medical Science, Lund University, Lund, Sweden
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50
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Teulon J, Planelles G, Sepúlveda FV, Andrini O, Lourdel S, Paulais M. Renal Chloride Channels in Relation to Sodium Chloride Transport. Compr Physiol 2018; 9:301-342. [DOI: 10.1002/cphy.c180024] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
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