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Aljameeli AM, Alsuwayt B, Bharati D, Gohri V, Mohite P, Singh S, Chidrawar V. Chloride channels and mast cell function: pioneering new frontiers in IBD therapy. Mol Cell Biochem 2025:10.1007/s11010-025-05243-w. [PMID: 40038149 DOI: 10.1007/s11010-025-05243-w] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/30/2024] [Accepted: 02/22/2025] [Indexed: 03/06/2025]
Abstract
Emerging evidence indicates that chloride channels (ClCs) significantly affect the pathogenesis of inflammatory bowel disease (IBD) through their regulatory roles in mast cell function and epithelial integrity. IBD, encompassing conditions such as Crohn's disease and ulcerative colitis, involves chronic inflammation of the gastrointestinal tract, where channels influence immune responses, fluid balance, and cellular signalling pathways essential for maintaining mucosal homeostasis. This review examines the specific roles of ClC in mast cells, focussing on the regulation of mast cell activation, degranulation, cytokine release, and immune cell recruitment in inflamed tissues. Key channels, including Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) and ClC-2, are discussed in detail because of their involvement in maintaining intestinal epithelial barrier function, a critical factor disrupted in IBD. For example, CFTR facilitates chloride ion transport across epithelial cells, which is essential for mucosal hydration and maintenance of the intestinal barrier. Reduced CFTR function can compromise this barrier, permitting microbial antigens to penetrate the underlying tissues and triggering excessive immune responses. ClC-2, another chloride channel expressed in mast cells and epithelial cells, supports tight junction integrity, contributes to barrier function, and reduces intestinal permeability. Dysregulation of these channels is linked to altered mast cell activity and excessive release of pro-inflammatory mediators, exacerbating IBD symptoms, such as diarrhoea, abdominal pain, and tissue damage. Here, we review recent pharmacological strategies targeting ClC, including CFTR potentiators and ClC-2 activators, which show the potential to mitigate inflammatory responses. Additionally, experimental approaches for selective modulation of chloride channels in mast cells have been explored. Although targeting ClC offers promising therapeutic avenues, challenges remain in achieving specificity and minimizing side effects. This review highlights the therapeutic potential of Cl channel modulation in mast cells as a novel approach for IBD treatment, aiming to reduce inflammation and restore intestinal homeostasis in affected patients.
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Affiliation(s)
- Ahmed M Aljameeli
- Department of Pharmacy Practice, College of Pharmacy, University of Hafr Al-Batin, Hafr Albatin, Saudi Arabia
| | - Bader Alsuwayt
- Department of Pharmacy Practice, College of Pharmacy, University of Hafr Al-Batin, Hafr Albatin, Saudi Arabia
| | - Deepak Bharati
- AETs St. John Institute of Pharmacy and Research, Palghar, Maharashtra, 401 404, India
| | - Vaishnavi Gohri
- AETs St. John Institute of Pharmacy and Research, Palghar, Maharashtra, 401 404, India
| | - Popat Mohite
- AETs St. John Institute of Pharmacy and Research, Palghar, Maharashtra, 401 404, India.
| | - Sudarshan Singh
- Office of Research Administration, Chiang Mai University, Chiang Mai, 50200, Thailand
- Faculty of Pharmacy, Chiang Mai University, Chiang Mai, 50200, Thailand
| | - Vijay Chidrawar
- School of Pharmacy and Technology Management, SVKM's Narsee Monjee Institute of Management Studies (NMIMS), Deemed-to-University, Green Industrial Park, TSIIC, Polepally, Jadcherla, Hyderabad, Telangana, 509301, India.
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2
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Jiang P, Dai Z, Yang C, Ding L, Li S, Xu X, Cheng C, Wang J, Liu S. CFTR Inhibitors Display Antiviral Activity against Herpes Simplex Virus. Viruses 2024; 16:1308. [PMID: 39205282 PMCID: PMC11360776 DOI: 10.3390/v16081308] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/19/2024] [Revised: 08/10/2024] [Accepted: 08/13/2024] [Indexed: 09/04/2024] Open
Abstract
The cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-dependent Cl- channel, is closely associated with multiple pathogen infections, such as SARS-CoV-2. However, whether the function of the CFTR is involved in herpes simplex virus (HSV) infection has not been reported. To evaluate the association of CFTR activity with HSV infection, the antiviral effect of CFTR inhibitors in epithelial cells and HSV-infected mice was tested in this study. The data showed that treatment with CFTR inhibitors in different concentrations, Glyh-101 (5-20 μM), CFTRi-172 (5-20 μM) and IOWH-032 (5-20 μM), or the gene silence of the CFTR could suppress herpes simplex virus 1 (HSV-1) and herpes simplex virus 2 (HSV-2) replication in human HaCaT keratinocytes cells, and that a CFTR inhibitor, Glyh-101 (10-20 μM), protected mice from HSV-1 and HSV-2 infection. Intracellular Cl- concentration ([Cl-]i) was decreased after HSV infection via the activation of adenylyl cyclase (AC)-cAMP signaling pathways. CFTR inhibitors (20 μM) increased the reduced [Cl-]i caused by HSV infection in host epithelial cells. Additionally, CFTR inhibitors reduced the activity and phosphorylation of SGK1 in infected cells and tissues (from the eye and vagina). Our study found that CFTR inhibitors can effectively suppress HSV-1 and HSV-2 infection, revealing a previously unknown role of CFTR inhibitors in HSV infection and suggesting new perspectives on the mechanisms governing HSV infection in host epithelial cells, as well as leading to potential novel treatments.
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Affiliation(s)
- Ping Jiang
- Guangdong Provincial Key Laboratory of New Drug Screening, Guangzhou Key Laboratory of Drug Research for Emerging Virus Prevention and Treatment, School of Pharmaceutical Sciences, Southern Medical University, Guangzhou 510515, China
- Guangdong Key Laboratory for Research and Development of Natural Drugs, Dongguan Key Laboratory of Traditional Chinese Medicine and New Pharmaceutical Development, School of Pharmacy, Guangdong Medical University, Dongguan 523808, China
| | - Zhong Dai
- Guangdong Key Laboratory for Research and Development of Natural Drugs, Dongguan Key Laboratory of Traditional Chinese Medicine and New Pharmaceutical Development, School of Pharmacy, Guangdong Medical University, Dongguan 523808, China
| | - Chan Yang
- Guangdong Provincial Key Laboratory of New Drug Screening, Guangzhou Key Laboratory of Drug Research for Emerging Virus Prevention and Treatment, School of Pharmaceutical Sciences, Southern Medical University, Guangzhou 510515, China
| | - Liqiong Ding
- School of Pharmaceutical Sciences, Hubei University of Science and Technology, Xianning 437100, China
| | - Songshan Li
- Guangdong Provincial Key Laboratory of New Drug Screening, Guangzhou Key Laboratory of Drug Research for Emerging Virus Prevention and Treatment, School of Pharmaceutical Sciences, Southern Medical University, Guangzhou 510515, China
| | - Xinfeng Xu
- Guangdong Provincial Key Laboratory of New Drug Screening, Guangzhou Key Laboratory of Drug Research for Emerging Virus Prevention and Treatment, School of Pharmaceutical Sciences, Southern Medical University, Guangzhou 510515, China
| | - Chen Cheng
- Guangdong Provincial Key Laboratory of New Drug Screening, Guangzhou Key Laboratory of Drug Research for Emerging Virus Prevention and Treatment, School of Pharmaceutical Sciences, Southern Medical University, Guangzhou 510515, China
| | - Jinshen Wang
- Guangdong Provincial Key Laboratory of New Drug Screening, Guangzhou Key Laboratory of Drug Research for Emerging Virus Prevention and Treatment, School of Pharmaceutical Sciences, Southern Medical University, Guangzhou 510515, China
| | - Shuwen Liu
- Guangdong Provincial Key Laboratory of New Drug Screening, Guangzhou Key Laboratory of Drug Research for Emerging Virus Prevention and Treatment, School of Pharmaceutical Sciences, Southern Medical University, Guangzhou 510515, China
- State Key Laboratory of Organ Failure Research, Guangdong Provincial Institute of Nephrology, Southern Medical University, Guangzhou 510515, China
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Gao X, Yeh HI, Yang Z, Fan C, Jiang F, Howard RJ, Lindahl E, Kappes JC, Hwang TC. Allosteric inhibition of CFTR gating by CFTRinh-172 binding in the pore. Nat Commun 2024; 15:6668. [PMID: 39107303 PMCID: PMC11303713 DOI: 10.1038/s41467-024-50641-1] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/22/2023] [Accepted: 07/09/2024] [Indexed: 08/10/2024] Open
Abstract
Loss-of-function mutations of the CFTR gene cause the life-shortening genetic disease cystic fibrosis (CF), whereas overactivity of CFTR may lead to secretory diarrhea and polycystic kidney disease. While effective drugs targeting the CFTR protein have been developed for the treatment of CF, little progress has been made for diseases caused by hyper-activated CFTR. Here, we solve the cryo-EM structure of CFTR in complex with CFTRinh-172 (Inh-172), a CFTR gating inhibitor with promising potency and efficacy. We find that Inh-172 binds inside the pore of CFTR, interacting with amino acid residues from transmembrane segments (TMs) 1, 6, 8, 9, and 12 through mostly hydrophobic interactions and a salt bridge. Substitution of these residues lowers the apparent affinity of Inh-172. The inhibitor-bound structure reveals re-orientations of the extracellular segment of TMs 1, 8, and 12, supporting an allosteric modulation mechanism involving post-binding conformational changes. This allosteric inhibitory mechanism readily explains our observations that pig CFTR, which preserves all the amino acid residues involved in Inh-172 binding, exhibits a much-reduced sensitivity to Inh-172 and that the apparent affinity of Inh-172 is altered by the CF drug ivacaftor (i.e., VX-770) which enhances CFTR's activity through binding to a site also comprising TM8.
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Affiliation(s)
- Xiaolong Gao
- Dalton Cardiovascular Research Center, University of Missouri-Columbia, Columbia, MO, 65211, USA.
| | - Han-I Yeh
- Dalton Cardiovascular Research Center, University of Missouri-Columbia, Columbia, MO, 65211, USA
- Institute of Pharmacology, National Yang Ming Chiao Tung University, College of Medicine, Taipei, Taiwan
- Membrane Protein Structural Biology Research Center, National Yang Ming Chiao Tung University, Taipei, Taiwan
| | - Zhengrong Yang
- Heersink School of Medicine, University of Alabama School of Medicine, Birmingham, AL, 35233, USA
| | - Chen Fan
- Department of Applied Physics, Science for Life Laboratory, KTH Royal Institute of Technology, Solna, Sweden
- Department of Biochemistry and Biophysics, Science for Life Laboratory, Stockholm University, Solna, Sweden
| | - Fan Jiang
- Heersink School of Medicine, University of Alabama School of Medicine, Birmingham, AL, 35233, USA
| | - Rebecca J Howard
- Department of Applied Physics, Science for Life Laboratory, KTH Royal Institute of Technology, Solna, Sweden
- Department of Biochemistry and Biophysics, Science for Life Laboratory, Stockholm University, Solna, Sweden
| | - Erik Lindahl
- Department of Applied Physics, Science for Life Laboratory, KTH Royal Institute of Technology, Solna, Sweden
- Department of Biochemistry and Biophysics, Science for Life Laboratory, Stockholm University, Solna, Sweden
| | - John C Kappes
- Heersink School of Medicine, University of Alabama School of Medicine, Birmingham, AL, 35233, USA
- Research Service, Birmingham Veterans Affairs Medical Center, Veterans Health Administration, Birmingham, AL, 35233, USA
| | - Tzyh-Chang Hwang
- Dalton Cardiovascular Research Center, University of Missouri-Columbia, Columbia, MO, 65211, USA.
- Institute of Pharmacology, National Yang Ming Chiao Tung University, College of Medicine, Taipei, Taiwan.
- Membrane Protein Structural Biology Research Center, National Yang Ming Chiao Tung University, Taipei, Taiwan.
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Molecular mechanisms of Cystic Fibrosis - how mutations lead to misfunction and guide therapy. Biosci Rep 2022; 42:231430. [PMID: 35707985 PMCID: PMC9251585 DOI: 10.1042/bsr20212006] [Citation(s) in RCA: 19] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/03/2022] [Revised: 06/03/2022] [Accepted: 06/13/2022] [Indexed: 11/17/2022] Open
Abstract
Cystic fibrosis, the most common autosomal recessive disorder in Caucasians, is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encodes a cAMP-activated chloride and bicarbonate channel that regulates ion and water transport in secretory epithelia. Although all mutations lead to the lack or reduction in channel function, the mechanisms through which this occurs are diverse – ranging from lack of full-length mRNA, reduced mRNA levels, impaired folding and trafficking, targeting to degradation, decreased gating or conductance, and reduced protein levels to decreased half-life at the plasma membrane. Here, we review the different molecular mechanisms that cause cystic fibrosis and detail how these differences identify theratypes that can inform the use of directed therapies aiming at correcting the basic defect. In summary, we travel through CFTR life cycle from the gene to function, identifying what can go wrong and what can be targeted in terms of the different types of therapeutic approaches.
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5
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DNA Binding Effects of 2,2'-bipyridine and 1,10-phenanthroline ligands synthesized with benzimidazole copper (II) complexes :Crystal Structure, Molecular Docking, DNA Binding and Anti-Cancer Studies. Polyhedron 2022. [DOI: 10.1016/j.poly.2022.115834] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/23/2022]
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Infield DT, Strickland KM, Gaggar A, McCarty NA. The molecular evolution of function in the CFTR chloride channel. J Gen Physiol 2021; 153:212705. [PMID: 34647973 PMCID: PMC8640958 DOI: 10.1085/jgp.202012625] [Citation(s) in RCA: 13] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/10/2020] [Revised: 08/11/2021] [Accepted: 09/09/2021] [Indexed: 12/13/2022] Open
Abstract
The ATP-binding cassette (ABC) transporter superfamily includes many proteins of clinical relevance, with genes expressed in all domains of life. Although most members use the energy of ATP binding and hydrolysis to accomplish the active import or export of various substrates across membranes, the cystic fibrosis transmembrane conductance regulator (CFTR) is the only known animal ABC transporter that functions primarily as an ion channel. Defects in CFTR, which is closely related to ABCC subfamily members that bear function as bona fide transporters, underlie the lethal genetic disease cystic fibrosis. This article seeks to integrate structural, functional, and genomic data to begin to answer the critical question of how the function of CFTR evolved to exhibit regulated channel activity. We highlight several examples wherein preexisting features in ABCC transporters were functionally leveraged as is, or altered by molecular evolution, to ultimately support channel function. This includes features that may underlie (1) construction of an anionic channel pore from an anionic substrate transport pathway, (2) establishment and tuning of phosphoregulation, and (3) optimization of channel function by specialized ligand–channel interactions. We also discuss how divergence and conservation may help elucidate the pharmacology of important CFTR modulators.
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Affiliation(s)
- Daniel T Infield
- Department of Molecular Physiology and Biophysics, University of Iowa, Iowa City, IA
| | | | - Amit Gaggar
- Department of Medicine, University of Alabama at Birmingham, Birmingham, AL.,Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama at Birmingham, Birmingham, AL.,Program in Protease and Matrix Biology, University of Alabama at Birmingham, Birmingham, AL.,Birmingham Veterans Administration Medical Center, Birmingham, AL
| | - Nael A McCarty
- Department of Pediatrics, Emory University, Atlanta, GA.,Children's Healthcare of Atlanta Center for Cystic Fibrosis and Airways Disease Research, Emory University, Atlanta, GA
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Epithelial barrier function properties of the 16HBE14o- human bronchial epithelial cell culture model. Biosci Rep 2021; 40:226530. [PMID: 32985670 PMCID: PMC7569203 DOI: 10.1042/bsr20201532] [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: 05/20/2020] [Revised: 09/08/2020] [Accepted: 09/25/2020] [Indexed: 01/23/2023] Open
Abstract
The human bronchial epithelial cell line, 16HBE14o- (16HBE), is widely used as a model for respiratory epithelial diseases and barrier function. During differentiation, transepithelial electrical resistance (TER) increased to approximately 800 Ohms × cm2, while 14C-d-mannitol flux rates (Jm) simultaneously decreased. Tight junctions (TJs) were shown by diffusion potential studies to be anion-selective with PC1/PNa = 1.9. Transepithelial leakiness could be induced by the phorbol ester, protein kinase C (PKC) activator, 12-O-tetradecanoylphorbol-13-acetate (TPA), and the proinflammatory cytokine, tumor necrosis factor-α (TNF-α). Basal barrier function could not be improved by the micronutrients, zinc, or quercetin. Of methodological significance, TER was observed to be more variable and to spontaneously, significantly decrease after initial barrier formation, whereas Jm did not significantly fluctuate or increase. Unlike the strong inverse relationship between TER and Jm during differentiation, differentiated cell layers manifested no relationship between TER and Jm. There was also much greater variability for TER values compared with Jm. Investigating the dependence of 16HBE TER on transcellular ion conductance, inhibition of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) chloride channel with GlyH-101 produced a large decrease in short-circuit current (Isc) and a slight increase in TER, but no significant change in Jm. A strong temperature dependence was observed not only for Isc, but also for TER. In summary, research utilizing 16HBE as a model in airway barrier function studies needs to be aware of the complexity of TER as a parameter of barrier function given the influence of CFTR-dependent transcellular conductance on TER.
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Linsdell P. On the relationship between anion binding and chloride conductance in the CFTR anion channel. BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES 2021; 1863:183558. [PMID: 33444622 DOI: 10.1016/j.bbamem.2021.183558] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Subscribe] [Scholar Register] [Received: 10/21/2020] [Revised: 12/21/2020] [Accepted: 12/29/2020] [Indexed: 12/26/2022]
Abstract
Mutations at many sites within the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel pore region result in changes in chloride conductance. Although chloride binding in the pore - as well as interactions between concurrently bound chloride ions - are thought to be important facets of the chloride permeation mechanism, little is known about the relationship between anion binding and chloride conductance. The present work presents a comprehensive investigation of a number of anion binding properties in different pore mutants with differential effects on chloride conductance. When multiple pore mutants are compared, conductance appears best correlated with the ability of anions to bind to the pore when it is already occupied by chloride ions. In contrast, conductance was not correlated with biophysical measures of anion:anion interactions inside the pore. Although these findings suggest anion binding is required for high conductance, mutations that strengthened anion binding had very little effect on conductance, especially at high chloride concentrations, suggesting that the wild-type CFTR pore is already close to saturated with chloride ions. These results are used to support a revised model of chloride permeation in CFTR in which the overall chloride occupancy of multiple loosely-defined chloride binding sites results in high chloride conductance through the pore.
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Affiliation(s)
- Paul Linsdell
- Department of Physiology & Biophysics, Dalhousie University, Halifax, Nova Scotia, Canada.
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de Jonge HR, Ardelean MC, Bijvelds MJC, Vergani P. Strategies for cystic fibrosis transmembrane conductance regulator inhibition: from molecular mechanisms to treatment for secretory diarrhoeas. FEBS Lett 2020; 594:4085-4108. [PMID: 33113586 PMCID: PMC7756540 DOI: 10.1002/1873-3468.13971] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/19/2020] [Revised: 09/22/2020] [Accepted: 10/15/2020] [Indexed: 02/06/2023]
Abstract
Cystic fibrosis transmembrane conductance regulator (CFTR) is an unusual ABC transporter. It acts as an anion‐selective channel that drives osmotic fluid transport across many epithelia. In the gut, CFTR is crucial for maintaining fluid and acid‐base homeostasis, and its activity is tightly controlled by multiple neuro‐endocrine factors. However, microbial toxins can disrupt this intricate control mechanism and trigger protracted activation of CFTR. This results in the massive faecal water loss, metabolic acidosis and dehydration that characterize secretory diarrhoeas, a major cause of malnutrition and death of children under 5 years of age. Compounds that inhibit CFTR could improve emergency treatment of diarrhoeal disease. Drawing on recent structural and functional insight, we discuss how existing CFTR inhibitors function at the molecular and cellular level. We compare their mechanisms of action to those of inhibitors of related ABC transporters, revealing some unexpected features of drug action on CFTR. Although challenges remain, especially relating to the practical effectiveness of currently available CFTR inhibitors, we discuss how recent technological advances might help develop therapies to better address this important global health need.
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Affiliation(s)
- Hugo R. de Jonge
- Department of Gastroenterology & HepatologyErasmus University Medical CenterRotterdamThe Netherlands
| | - Maria C. Ardelean
- Department of Neuroscience, Physiology and PharmacologyUniversity College LondonUK
- Department of Natural SciencesUniversity College LondonUK
| | - Marcel J. C. Bijvelds
- Department of Gastroenterology & HepatologyErasmus University Medical CenterRotterdamThe Netherlands
| | - Paola Vergani
- Department of Neuroscience, Physiology and PharmacologyUniversity College LondonUK
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Strickland KM, Stock G, Cui G, Hwang H, Infield DT, Schmidt-Krey I, McCarty NA, Gumbart JC. ATP-Dependent Signaling in Simulations of a Revised Model of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR). J Phys Chem B 2019; 123:3177-3188. [PMID: 30921517 DOI: 10.1021/acs.jpcb.8b11970] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
Abstract
Cystic fibrosis transmembrane conductance regulator (CFTR) is a member of the ATP-binding cassette (ABC) transporter superfamily that has uniquely evolved to function as a chloride channel. It binds and hydrolyzes ATP at its nucleotide binding domains to form a pore providing a diffusive pathway within its transmembrane domains. CFTR is the only known protein from the ABC superfamily with channel activity, and its dysfunction causes the disease cystic fibrosis. While much is known about the functional aspects of CFTR, significant gaps remain, such as the structure-function relationship underlying signaling of ATP binding. In the present work, we refined an existing homology model using an intermediate-resolution (9 Å) published cryo-electron microscopy map. The newly derived models have been simulated in equilibrium molecular dynamics simulations for a total of 2.5 μs in multiple ATP-occupancy states. Putative conformational movements connecting ATP binding with pore formation are elucidated and quantified. Additionally, new interdomain interactions between E543, K968, and K1292 have been identified and confirmed experimentally; these interactions may be relevant for signaling ATP binding and hydrolysis to the transmembrane domains and induction of pore opening.
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Affiliation(s)
- Kerry M Strickland
- School of Chemistry and Biochemistry , Georgia Institute of Technology , Atlanta , Georgia 30332 , United States
| | - Gorman Stock
- School of Chemistry and Biochemistry , Georgia Institute of Technology , Atlanta , Georgia 30332 , United States
| | - Guiying Cui
- Division of Pulmonology, Allergy and Immunology, Cystic Fibrosis, and Sleep, Department of Pediatrics, Center for Cystic Fibrosis and Airways Disease Research, Emory+Children's Pediatric Research Center , Emory University School of Medicine and Children's Healthcare of Atlanta , Atlanta , Georgia 30322 , United States
| | - Hyea Hwang
- School of Materials Science and Engineering , Georgia Institute of Technology , Atlanta , Georgia 30332 , United States
| | - Daniel T Infield
- Division of Pulmonology, Allergy and Immunology, Cystic Fibrosis, and Sleep, Department of Pediatrics, Center for Cystic Fibrosis and Airways Disease Research, Emory+Children's Pediatric Research Center , Emory University School of Medicine and Children's Healthcare of Atlanta , Atlanta , Georgia 30322 , United States
| | - Ingeborg Schmidt-Krey
- School of Chemistry and Biochemistry , Georgia Institute of Technology , Atlanta , Georgia 30332 , United States.,School of Biological Sciences , Georgia Institute of Technology , Atlanta , Georgia 30332 , United States.,Parker H. Petit Institute for Bioengineering and Bioscience , Georgia Institute of Technology , Atlanta , Georgia 30332 , United States
| | - Nael A McCarty
- Division of Pulmonology, Allergy and Immunology, Cystic Fibrosis, and Sleep, Department of Pediatrics, Center for Cystic Fibrosis and Airways Disease Research, Emory+Children's Pediatric Research Center , Emory University School of Medicine and Children's Healthcare of Atlanta , Atlanta , Georgia 30322 , United States.,Parker H. Petit Institute for Bioengineering and Bioscience , Georgia Institute of Technology , Atlanta , Georgia 30332 , United States
| | - James C Gumbart
- School of Chemistry and Biochemistry , Georgia Institute of Technology , Atlanta , Georgia 30332 , United States.,School of Biological Sciences , Georgia Institute of Technology , Atlanta , Georgia 30332 , United States.,Parker H. Petit Institute for Bioengineering and Bioscience , Georgia Institute of Technology , Atlanta , Georgia 30332 , United States.,School of Physics , Georgia Institute of Technology , Atlanta , Georgia 30332 , United States
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11
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Csanády L, Vergani P, Gadsby DC. STRUCTURE, GATING, AND REGULATION OF THE CFTR ANION CHANNEL. Physiol Rev 2019; 99:707-738. [PMID: 30516439 DOI: 10.1152/physrev.00007.2018] [Citation(s) in RCA: 164] [Impact Index Per Article: 27.3] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022] Open
Abstract
The cystic fibrosis transmembrane conductance regulator (CFTR) belongs to the ATP binding cassette (ABC) transporter superfamily but functions as an anion channel crucial for salt and water transport across epithelial cells. CFTR dysfunction, because of mutations, causes cystic fibrosis (CF). The anion-selective pore of the CFTR protein is formed by its two transmembrane domains (TMDs) and regulated by its cytosolic domains: two nucleotide binding domains (NBDs) and a regulatory (R) domain. Channel activation requires phosphorylation of the R domain by cAMP-dependent protein kinase (PKA), and pore opening and closing (gating) of phosphorylated channels is driven by ATP binding and hydrolysis at the NBDs. This review summarizes available information on structure and mechanism of the CFTR protein, with a particular focus on atomic-level insight gained from recent cryo-electron microscopic structures and on the molecular mechanisms of channel gating and its regulation. The pharmacological mechanisms of small molecules targeting CFTR's ion channel function, aimed at treating patients suffering from CF and other diseases, are briefly discussed.
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Affiliation(s)
- László Csanády
- Department of Medical Biochemistry, Semmelweis University , Budapest , Hungary ; MTA-SE Ion Channel Research Group, Budapest , Hungary ; Department of Neuroscience, Physiology and Pharmacology, University College London , London , United Kingdom ; and Laboratory of Cardiac/Membrane Physiology, The Rockefeller University , New York, New York
| | - Paola Vergani
- Department of Medical Biochemistry, Semmelweis University , Budapest , Hungary ; MTA-SE Ion Channel Research Group, Budapest , Hungary ; Department of Neuroscience, Physiology and Pharmacology, University College London , London , United Kingdom ; and Laboratory of Cardiac/Membrane Physiology, The Rockefeller University , New York, New York
| | - David C Gadsby
- Department of Medical Biochemistry, Semmelweis University , Budapest , Hungary ; MTA-SE Ion Channel Research Group, Budapest , Hungary ; Department of Neuroscience, Physiology and Pharmacology, University College London , London , United Kingdom ; and Laboratory of Cardiac/Membrane Physiology, The Rockefeller University , New York, New York
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12
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Functional characterization reveals that zebrafish CFTR prefers to occupy closed channel conformations. PLoS One 2018; 13:e0209862. [PMID: 30596737 PMCID: PMC6312236 DOI: 10.1371/journal.pone.0209862] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/24/2018] [Accepted: 12/12/2018] [Indexed: 12/19/2022] Open
Abstract
Cystic fibrosis transmembrane conductance regulator (CFTR), the culprit behind the genetic disease cystic fibrosis (CF), is a phosphorylation-activated, but ATP-gated anion channel. Studies of human CFTR over the past two decades have provided an in-depth understanding of how CFTR works as an ion channel despite its structural resemblance to ABC transporters. Recently-solved cryo-EM structures of unphosphorylated human and zebrafish CFTR (hCFTR and zCFTR), as well as phosphorylated ATP-bound zebrafish and human CFTR offer an unprecedented opportunity to understand CFTR's function at a molecular level. Interestingly, despite millions of years of phylogenetic distance between human and zebrafish, the structures of zCFTR and hCFTR exhibit remarkable similarities. In the current study, we characterized biophysical and pharmacological properties of zCFTR with the patch-clamp technique, and showed surprisingly very different functional properties between these two orthologs. First, while hCFTR has a single-channel conductance of 8.4 pS with a linear I-V curve, zCFTR shows an inwardly-rectified I-V relationship with a single-channel conductance of ~3.5 pS. Second, single-channel gating behaviors of phosphorylated zCFTR are very different from those of hCFTR, featuring a very low open probability Po (0.03 ± 0.02, vs. ~0.50 for hCFTR) with exceedingly long closed events and brief openings. In addition, unlike hCFTR where each open burst is clearly defined with rare short-lived flickery closures, the open bursts of zCFTR are not easily resolved. Third, although abolishing ATP hydrolysis by replacing the catalytic glutamate with glutamine (i.e., E1372Q) drastically prolongs the open bursts defined by the macroscopic relaxation analysis in zCFTR, the Po within a "locked-open" burst of E1372Q-zCFTR is only ~ 0.35 (vs. Po > 0.94 in E1371Q-hCFTR). Collectively, our data not only provide a reasonable explanation for the unexpected closed-state structure of phosphorylated E1372Q-zCFTR with a canonical ATP-bound dimer of the nucleotide binding domains (NBDs), but also implicate significant structural and functional differences between these two evolutionarily distant orthologs.
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13
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Hoffmann B, Elbahnsi A, Lehn P, Décout JL, Pietrucci F, Mornon JP, Callebaut I. Combining theoretical and experimental data to decipher CFTR 3D structures and functions. Cell Mol Life Sci 2018; 75:3829-3855. [PMID: 29779042 PMCID: PMC11105360 DOI: 10.1007/s00018-018-2835-7] [Citation(s) in RCA: 21] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/31/2017] [Revised: 05/04/2018] [Accepted: 05/07/2018] [Indexed: 12/15/2022]
Abstract
Cryo-electron microscopy (cryo-EM) has recently provided invaluable experimental data about the full-length cystic fibrosis transmembrane conductance regulator (CFTR) 3D structure. However, this experimental information deals with inactive states of the channel, either in an apo, quiescent conformation, in which nucleotide-binding domains (NBDs) are widely separated or in an ATP-bound, yet closed conformation. Here, we show that 3D structure models of the open and closed forms of the channel, now further supported by metadynamics simulations and by comparison with the cryo-EM data, could be used to gain some insights into critical features of the conformational transition toward active CFTR forms. These critical elements lie within membrane-spanning domains but also within NBD1 and the N-terminal extension, in which conformational plasticity is predicted to occur to help the interaction with filamin, one of the CFTR cellular partners.
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Affiliation(s)
- Brice Hoffmann
- Sorbonne Université, Muséum National d'Histoire Naturelle, UMR CNRS 7590, IRD, Institut de Minéralogie, de Physique des Matériaux et de Cosmochimie, IMPMC, 75005, Paris, France
- Iktos, Paris, France
| | - Ahmad Elbahnsi
- Sorbonne Université, Muséum National d'Histoire Naturelle, UMR CNRS 7590, IRD, Institut de Minéralogie, de Physique des Matériaux et de Cosmochimie, IMPMC, 75005, Paris, France
| | - Pierre Lehn
- INSERM U1078, SFR ScInBioS, Université de Bretagne Occidentale, Brest, France
| | | | - Fabio Pietrucci
- Sorbonne Université, Muséum National d'Histoire Naturelle, UMR CNRS 7590, IRD, Institut de Minéralogie, de Physique des Matériaux et de Cosmochimie, IMPMC, 75005, Paris, France
| | - Jean-Paul Mornon
- Sorbonne Université, Muséum National d'Histoire Naturelle, UMR CNRS 7590, IRD, Institut de Minéralogie, de Physique des Matériaux et de Cosmochimie, IMPMC, 75005, Paris, France.
| | - Isabelle Callebaut
- Sorbonne Université, Muséum National d'Histoire Naturelle, UMR CNRS 7590, IRD, Institut de Minéralogie, de Physique des Matériaux et de Cosmochimie, IMPMC, 75005, Paris, France
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14
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Han ST, Rab A, Pellicore MJ, Davis EF, McCague AF, Evans TA, Joynt AT, Lu Z, Cai Z, Raraigh KS, Hong JS, Sheppard DN, Sorscher EJ, Cutting GR. Residual function of cystic fibrosis mutants predicts response to small molecule CFTR modulators. JCI Insight 2018; 3:121159. [PMID: 30046002 DOI: 10.1172/jci.insight.121159] [Citation(s) in RCA: 70] [Impact Index Per Article: 10.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/22/2018] [Accepted: 06/12/2018] [Indexed: 12/24/2022] Open
Abstract
Treatment of individuals with cystic fibrosis (CF) has been transformed by small molecule therapies that target select pathogenic variants in the CF transmembrane conductance regulator (CFTR). To expand treatment eligibility, we stably expressed 43 rare missense CFTR variants associated with moderate CF from a single site in the genome of human CF bronchial epithelial (CFBE41o-) cells. The magnitude of drug response was highly correlated with residual CFTR function for the potentiator ivacaftor, the corrector lumacaftor, and ivacaftor-lumacaftor combination therapy. Response of a second set of 16 variants expressed stably in Fischer rat thyroid (FRT) cells showed nearly identical correlations. Subsets of variants were identified that demonstrated statistically significantly higher responses to specific treatments. Furthermore, nearly all variants studied in CFBE cells (40 of 43) and FRT cells (13 of 16) demonstrated greater response to ivacaftor-lumacaftor combination therapy than either modulator alone. Together, these variants represent 87% of individuals in the CFTR2 database with at least 1 missense variant. Thus, our results indicate that most individuals with CF carrying missense variants are (a) likely to respond modestly to currently available modulator therapy, while a small fraction will have pronounced responses, and (b) likely to derive the greatest benefit from combination therapy.
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Affiliation(s)
- Sangwoo T Han
- McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA
| | - Andras Rab
- Department of Pediatrics, Emory University School of Medicine, Atlanta, Georgia, USA
| | - Matthew J Pellicore
- McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA
| | - Emily F Davis
- McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA
| | - Allison F McCague
- McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA
| | - Taylor A Evans
- McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA
| | - Anya T Joynt
- McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA
| | - Zhongzhou Lu
- McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA
| | - Zhiwei Cai
- School of Physiology, Pharmacology and Neuroscience, University of Bristol, Bristol, United Kingdom
| | - Karen S Raraigh
- McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA
| | - Jeong S Hong
- Department of Pediatrics, Emory University School of Medicine, Atlanta, Georgia, USA
| | - David N Sheppard
- School of Physiology, Pharmacology and Neuroscience, University of Bristol, Bristol, United Kingdom
| | - Eric J Sorscher
- Department of Pediatrics, Emory University School of Medicine, Atlanta, Georgia, USA
| | - Garry R Cutting
- McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA
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15
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Hwang TC, Yeh JT, Zhang J, Yu YC, Yeh HI, Destefano S. Structural mechanisms of CFTR function and dysfunction. J Gen Physiol 2018; 150:539-570. [PMID: 29581173 PMCID: PMC5881446 DOI: 10.1085/jgp.201711946] [Citation(s) in RCA: 85] [Impact Index Per Article: 12.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/16/2017] [Accepted: 03/05/2018] [Indexed: 12/18/2022] Open
Abstract
Hwang et al. integrate new structural insights with prior functional studies to reveal the functional anatomy of CFTR chloride channels. Cystic fibrosis (CF) transmembrane conductance regulator (CFTR) chloride channel plays a critical role in regulating transepithelial movement of water and electrolyte in exocrine tissues. Malfunction of the channel because of mutations of the cftr gene results in CF, the most prevalent lethal genetic disease among Caucasians. Recently, the publication of atomic structures of CFTR in two distinct conformations provides, for the first time, a clear overview of the protein. However, given the highly dynamic nature of the interactions among CFTR’s various domains, better understanding of the functional significance of these structures requires an integration of these new structural insights with previously established biochemical/biophysical studies, which is the goal of this review.
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Affiliation(s)
- Tzyh-Chang Hwang
- Dalton Cardiovascular Research Center, University of Missouri, Columbia, MO .,Department of Medical Pharmacology and Physiology, University of Missouri, Columbia, MO.,Department of Biological Engineering, University of Missouri, Columbia, MO
| | - Jiunn-Tyng Yeh
- Dalton Cardiovascular Research Center, University of Missouri, Columbia, MO
| | - Jingyao Zhang
- Dalton Cardiovascular Research Center, University of Missouri, Columbia, MO.,Department of Biological Engineering, University of Missouri, Columbia, MO
| | - Ying-Chun Yu
- Dalton Cardiovascular Research Center, University of Missouri, Columbia, MO.,Department of Medical Pharmacology and Physiology, University of Missouri, Columbia, MO
| | - Han-I Yeh
- Dalton Cardiovascular Research Center, University of Missouri, Columbia, MO.,Department of Medical Pharmacology and Physiology, University of Missouri, Columbia, MO
| | - Samantha Destefano
- Dalton Cardiovascular Research Center, University of Missouri, Columbia, MO.,Department of Medical Pharmacology and Physiology, University of Missouri, Columbia, MO
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16
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Callebaut I, Hoffmann B, Mornon JP. The implications of CFTR structural studies for cystic fibrosis drug development. Curr Opin Pharmacol 2017; 34:112-118. [PMID: 29096277 DOI: 10.1016/j.coph.2017.09.006] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/28/2017] [Revised: 09/12/2017] [Accepted: 09/14/2017] [Indexed: 02/08/2023]
Abstract
Development of Cystic Fibrosis Transmembrane conductance Regulator (CFTR) modulators, targeting the root cause of cystic fibrosis (CF), represents a challenge in the era of personalized medicine, as CFTR mutations lead to a variety of phenotypes, which likely require different, specific treatments. CF drug development is also complicated by the need to preserve the right balance between stability and flexibility, required for optimal function of the CFTR protein. In this review, we highlight how structural data can be exploited in this context to understand the molecular mechanisms of disease-associated mutations, to characterize the mechanisms of action of known modulators and to rationalize the search for novel, specific compounds.
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Affiliation(s)
- Isabelle Callebaut
- CNRS UMR7590, Sorbonne Universités, Université Pierre et Marie Curie - Paris 6 - MNHN - IRD - IUC, Paris, France.
| | - Brice Hoffmann
- CNRS UMR7590, Sorbonne Universités, Université Pierre et Marie Curie - Paris 6 - MNHN - IRD - IUC, Paris, France
| | - Jean-Paul Mornon
- CNRS UMR7590, Sorbonne Universités, Université Pierre et Marie Curie - Paris 6 - MNHN - IRD - IUC, Paris, France
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17
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Callebaut I, Hoffmann B, Lehn P, Mornon JP. Molecular modelling and molecular dynamics of CFTR. Cell Mol Life Sci 2017; 74:3-22. [PMID: 27717958 PMCID: PMC11107702 DOI: 10.1007/s00018-016-2385-9] [Citation(s) in RCA: 25] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/27/2016] [Accepted: 09/28/2016] [Indexed: 12/11/2022]
Abstract
The cystic fibrosis transmembrane conductance regulator (CFTR) protein is a member of the ATP-binding cassette (ABC) transporter superfamily that functions as an ATP-gated channel. Considerable progress has been made over the last years in the understanding of the molecular basis of the CFTR functions, as well as dysfunctions causing the common genetic disease cystic fibrosis (CF). This review provides a global overview of the theoretical studies that have been performed so far, especially molecular modelling and molecular dynamics (MD) simulations. A special emphasis is placed on the CFTR-specific evolution of an ABC transporter framework towards a channel function, as well as on the understanding of the effects of disease-causing mutations and their specific modulation. This in silico work should help structure-based drug discovery and design, with a view to develop CFTR-specific pharmacotherapeutic approaches for the treatment of CF in the context of precision medicine.
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Affiliation(s)
- Isabelle Callebaut
- UMR CNRS 7590, Museum National d'Histoire Naturelle, IRD UMR 206, IUC, Case 115, IMPMC, Sorbonne Universités, UPMC Univ Paris 06, 4 Place Jussieu, 75005, Paris Cedex 05, France.
| | - Brice Hoffmann
- UMR CNRS 7590, Museum National d'Histoire Naturelle, IRD UMR 206, IUC, Case 115, IMPMC, Sorbonne Universités, UPMC Univ Paris 06, 4 Place Jussieu, 75005, Paris Cedex 05, France
| | - Pierre Lehn
- INSERM U1078, SFR ScInBioS, Université de Bretagne Occidentale, Brest, France
| | - Jean-Paul Mornon
- UMR CNRS 7590, Museum National d'Histoire Naturelle, IRD UMR 206, IUC, Case 115, IMPMC, Sorbonne Universités, UPMC Univ Paris 06, 4 Place Jussieu, 75005, Paris Cedex 05, France
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18
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Abstract
CFTR protein is an ion channel regulated by cAMP-dependent phosphorylation and expressed in many types of epithelial cells. CFTR-mediated chloride and bicarbonate secretion play an important role in the respiratory and gastrointestinal systems. Pharmacological modulators of CFTR represent promising drugs for a variety of diseases. In particular, correctors and potentiators may restore the activity of CFTR in cystic fibrosis patients. Potentiators are also potentially useful to improve mucociliary clearance in patients with chronic obstructive pulmonary disease. On the other hand, CFTR inhibitors may be useful to block fluid and electrolyte loss in secretory diarrhea and slow down the progression of polycystic kidney disease.
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Affiliation(s)
- Olga Zegarra-Moran
- U.O.C. Genetica Medica, Istituto Giannina Gaslini, Via Gerolamo Gaslini 5, 16147, Genoa, Italy
| | - Luis J V Galietta
- U.O.C. Genetica Medica, Istituto Giannina Gaslini, Via Gerolamo Gaslini 5, 16147, Genoa, Italy.
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19
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Cil O, Phuan PW, Gillespie AM, Lee S, Tradtrantip L, Yin J, Tse M, Zachos NC, Lin R, Donowitz M, Verkman AS. Benzopyrimido-pyrrolo-oxazine-dione CFTR inhibitor (R)-BPO-27 for antisecretory therapy of diarrheas caused by bacterial enterotoxins. FASEB J 2016; 31:751-760. [PMID: 27871064 DOI: 10.1096/fj.201600891r] [Citation(s) in RCA: 40] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/07/2016] [Accepted: 10/24/2016] [Indexed: 12/15/2022]
Abstract
Secretory diarrheas caused by bacterial enterotoxins, including cholera and traveler's diarrhea, remain a major global health problem. Inappropriate activation of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel occurs in these diarrheas. We previously reported that the benzopyrimido-pyrrolo-oxazinedione (R)-BPO-27 inhibits CFTR chloride conductance with low-nanomolar potency. Here, we demonstrate using experimental mouse models and human enterocyte cultures the potential utility of (R)-BPO-27 for treatment of secretory diarrheas caused by cholera and Escherichia coli enterotoxins. (R)-BPO-27 fully blocked CFTR chloride conductance in epithelial cell cultures and intestine after cAMP agonists, cholera toxin, or heat-stable enterotoxin of E. coli (STa toxin), with IC50 down to ∼5 nM. (R)-BPO-27 prevented cholera toxin and STa toxin-induced fluid accumulation in small intestinal loops, with IC50 down to 0.1 mg/kg. (R)-BPO-27 did not impair intestinal fluid absorption or inhibit other major intestinal transporters. Pharmacokinetics in mice showed >90% oral bioavailability with sustained therapeutic serum levels for >4 h without the significant toxicity seen with 7-d administration at 5 mg/kg/d. As evidence to support efficacy in human diarrheas, (R)-BPO-27 blocked fluid secretion in primary cultures of enteroids from human small intestine and anion current in enteroid monolayers. These studies support the potential utility of (R)-BPO-27 for therapy of CFTR-mediated secretory diarrheas.-Cil, O., Phuan, P.-W., Gillespie, A. M., Lee, S., Tradtrantip, L., Yin, J., Tse, M., Zachos, N. C., Lin, R., Donowitz, M., Verkman, A. S. Benzopyrimido-pyrrolo-oxazine-dione CFTR inhibitor (R)-BPO-27 for antisecretory therapy of diarrheas caused by bacterial enterotoxins.
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Affiliation(s)
- Onur Cil
- Department of Medicine, University of California San Francisco, San Francisco, California, USA.,Department of Physiology, University of California San Francisco, San Francisco, California, USA
| | - Puay-Wah Phuan
- Department of Medicine, University of California San Francisco, San Francisco, California, USA.,Department of Physiology, University of California San Francisco, San Francisco, California, USA
| | - Anne Marie Gillespie
- Department of Medicine, University of California San Francisco, San Francisco, California, USA.,Department of Physiology, University of California San Francisco, San Francisco, California, USA
| | - Sujin Lee
- Department of Medicine, University of California San Francisco, San Francisco, California, USA.,Department of Physiology, University of California San Francisco, San Francisco, California, USA
| | - Lukmanee Tradtrantip
- Department of Medicine, University of California San Francisco, San Francisco, California, USA.,Department of Physiology, University of California San Francisco, San Francisco, California, USA
| | - Jianyi Yin
- Department of Physiology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA; and.,Gastroenterology Division, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA
| | - Ming Tse
- Department of Physiology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA; and.,Gastroenterology Division, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA
| | - Nicholas C Zachos
- Department of Physiology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA; and.,Gastroenterology Division, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA
| | - Ruxian Lin
- Department of Physiology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA; and.,Gastroenterology Division, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA
| | - Mark Donowitz
- Department of Physiology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA; and.,Gastroenterology Division, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA
| | - Alan S Verkman
- Department of Medicine, University of California San Francisco, San Francisco, California, USA; .,Department of Physiology, University of California San Francisco, San Francisco, California, USA
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20
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Infield DT, Cui G, Kuang C, McCarty NA. Positioning of extracellular loop 1 affects pore gating of the cystic fibrosis transmembrane conductance regulator. Am J Physiol Lung Cell Mol Physiol 2015; 310:L403-14. [PMID: 26684250 DOI: 10.1152/ajplung.00259.2015] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/28/2015] [Accepted: 12/16/2015] [Indexed: 02/06/2023] Open
Abstract
The cystic fibrosis (CF) transmembrane conductance regulator (CFTR) is a chloride ion channel, the dysfunction of which directly leads to the life-shortening disease CF. Extracellular loop 1 (ECL1) of CFTR contains several residues involved in stabilizing the open state of the channel; some, including D110, are sites of disease-associated gating mutations. Structures from related proteins suggest that the position of CFTR's extracellular loops may change considerably during gating. To better understand the roles of ECL1 in CFTR function, we utilized functional cysteine cross-linking to determine the effects of modulation of D110C-CFTR and of a double mutant of D110C with K892C in extracellular loop 4 (ECL4). The reducing agent DTT elicited a large potentiation of the macroscopic conductance of D110C/K892C-CFTR, likely due to breakage of a spontaneous disulfide bond between C110 and C892. DTT-reduced D110C/K892C-CFTR was rapidly inhibited by binding cadmium ions with high affinity, suggesting that these residues frequently come in close proximity in actively gating channels. Effects of DTT and cadmium on modulation of pore gating were demonstrated at the single-channel level. Finally, disulfided D110C/K892C-CFTR channels were found to be less sensitive than wild-type or DTT-treated D110C/K892C-CFTR channels to stimulation by IBMX, suggesting an impact of this conformational restriction on channel activation by phosphorylation. The results are best explained in the context of a model of CFTR gating wherein stable channel opening requires correct positioning of functional elements structurally influenced by ECL1.
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Affiliation(s)
- Daniel T Infield
- Division of Pulmonology, Allergy and Immunology, Cystic Fibrosis, and Sleep, Department of Pediatrics, Center for Cystic Fibrosis and Airways Disease Research, Emory+Children's Pediatric Research Center, Emory University School of Medicine and Children's Healthcare of Atlanta, Atlanta, Georgia; and Graduate Division of Biological and Biomedical Sciences, Emory University, Atlanta, Georgia
| | - Guiying Cui
- Division of Pulmonology, Allergy and Immunology, Cystic Fibrosis, and Sleep, Department of Pediatrics, Center for Cystic Fibrosis and Airways Disease Research, Emory+Children's Pediatric Research Center, Emory University School of Medicine and Children's Healthcare of Atlanta, Atlanta, Georgia; and
| | - Christopher Kuang
- Division of Pulmonology, Allergy and Immunology, Cystic Fibrosis, and Sleep, Department of Pediatrics, Center for Cystic Fibrosis and Airways Disease Research, Emory+Children's Pediatric Research Center, Emory University School of Medicine and Children's Healthcare of Atlanta, Atlanta, Georgia; and
| | - Nael A McCarty
- Division of Pulmonology, Allergy and Immunology, Cystic Fibrosis, and Sleep, Department of Pediatrics, Center for Cystic Fibrosis and Airways Disease Research, Emory+Children's Pediatric Research Center, Emory University School of Medicine and Children's Healthcare of Atlanta, Atlanta, Georgia; and
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21
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Wei S, Roessler BC, Icyuz M, Chauvet S, Tao B, Hartman JL, Kirk KL. Long-range coupling between the extracellular gates and the intracellular ATP binding domains of multidrug resistance protein pumps and cystic fibrosis transmembrane conductance regulator channels. FASEB J 2015; 30:1247-62. [PMID: 26606940 DOI: 10.1096/fj.15-278382] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/31/2015] [Accepted: 11/16/2015] [Indexed: 12/22/2022]
Abstract
The ABCC transporter subfamily includes pumps, the long and short multidrug resistance proteins (MRPs), and an ATP-gated anion channel, the cystic fibrosis transmembrane conductance regulator (CFTR). We show that despite their thermodynamic differences, these ABCC transporter subtypes use broadly similar mechanisms to couple their extracellular gates to the ATP occupancies of their cytosolic nucleotide binding domains. A conserved extracellular phenylalanine at this gate was a prime location for producing gain of function (GOF) mutants of a long MRP in yeast (Ycf1p cadmium transporter), a short yeast MRP (Yor1p oligomycin exporter), and human CFTR channels. Extracellular gate mutations rescued ATP binding mutants of the yeast MRPs and CFTR by increasing ATP sensitivity. Control ATPase-defective MRP mutants could not be rescued by this mechanism. A CFTR double mutant with an extracellular gate mutation plus a cytosolic GOF mutation was highly active (single-channel open probability >0.3) in the absence of ATP and protein kinase A, each normally required for CFTR activity. We conclude that all 3 ABCC transporter subtypes use similar mechanisms to couple their extracellular gates to ATP occupancy, and highly active CFTR channels that bypass defects in ATP binding or phosphorylation can be produced.
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Affiliation(s)
- Shipeng Wei
- *Department of Cell, Developmental, and Integrative Biology, Department of Genetics, and Department of Neurobiology, Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama at Birmingham, Birmingham, Alabama, USA
| | - Bryan C Roessler
- *Department of Cell, Developmental, and Integrative Biology, Department of Genetics, and Department of Neurobiology, Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama at Birmingham, Birmingham, Alabama, USA
| | - Mert Icyuz
- *Department of Cell, Developmental, and Integrative Biology, Department of Genetics, and Department of Neurobiology, Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama at Birmingham, Birmingham, Alabama, USA
| | - Sylvain Chauvet
- *Department of Cell, Developmental, and Integrative Biology, Department of Genetics, and Department of Neurobiology, Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama at Birmingham, Birmingham, Alabama, USA
| | - Binli Tao
- *Department of Cell, Developmental, and Integrative Biology, Department of Genetics, and Department of Neurobiology, Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama at Birmingham, Birmingham, Alabama, USA
| | - John L Hartman
- *Department of Cell, Developmental, and Integrative Biology, Department of Genetics, and Department of Neurobiology, Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama at Birmingham, Birmingham, Alabama, USA
| | - Kevin L Kirk
- *Department of Cell, Developmental, and Integrative Biology, Department of Genetics, and Department of Neurobiology, Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama at Birmingham, Birmingham, Alabama, USA
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22
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Kim Y, Anderson MO, Park J, Lee MG, Namkung W, Verkman AS. Benzopyrimido-pyrrolo-oxazine-dione (R)-BPO-27 Inhibits CFTR Chloride Channel Gating by Competition with ATP. Mol Pharmacol 2015; 88:689-96. [PMID: 26174774 PMCID: PMC4576686 DOI: 10.1124/mol.115.098368] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/10/2015] [Accepted: 07/13/2015] [Indexed: 12/16/2022] Open
Abstract
We previously reported that benzopyrimido-pyrrolo-oxazinedione BPO-27 [6-(5-bromofuran-2-yl)-7,9-dimethyl-8,10-dioxo-11-phenyl-7,8,9,10-tetrahydro-6H-benzo[b]pyrimido [4',5':3,4]pyrrolo [1,2-d][1,4]oxazine-2-carboxylic acid] inhibits the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel with low nanomolar potency and reduces cystogenesis in a model of polycystic kidney disease. We used computational chemistry and patch-clamp to show that enantiomerically pure (R)-BPO-27 inhibits CFTR by competition with ATP, whereas (S)-BPO-27 is inactive. Docking computations using a homology model of CFTR structure suggested that (R)-BPO-27 binds near the canonical ATP binding site, and these findings were supported by molecular dynamics simulations showing a lower binding energy for the (R) versus (S) stereoisomers. Three additional lower-potency BPO-27 analogs were modeled in a similar fashion, with the binding energies predicted in the correct order. Whole-cell patch-clamp studies showed linear CFTR currents with a voltage-independent (R)-BPO-27 block mechanism. Single-channel recordings in inside-out patches showed reduced CFTR channel open probability and increased channel closed time by (R)-BPO-27 without altered unitary channel conductance. At a concentration of (R)-BPO-27 that inhibited CFTR chloride current by ∼50%, the EC50 for ATP activation of CFTR increased from 0.27 to 1.77 mM but was not changed by CFTRinh-172 [4-[[4-oxo-2-thioxo-3-[3-trifluoromethyl)phenyl]-5-thiazolidinylidene]methyl]benzoic acid], a thiazolidinone CFTR inhibitor that acts at a site distinct from the ATP binding site. Our results suggest that (R)-BPO-27 inhibition of CFTR involves competition with ATP.
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Affiliation(s)
- Yonjung Kim
- Department of Pharmacology, Brain Korea 21 PLUS Project for Medical Sciences, Yonsei University College of Medicine, Seoul, Korea (Y.K., M.G.L.); Department of Chemistry and Biochemistry, San Francisco State University, San Francisco, California (M.O.A.); College of Pharmacy, Yonsei Institute of Pharmaceutical Sciences, Yonsei University, Incheon, Korea (J.P., W.N.); and Departments of Medicine and Physiology, University of California, San Francisco, California (A.S.V.)
| | - Marc O Anderson
- Department of Pharmacology, Brain Korea 21 PLUS Project for Medical Sciences, Yonsei University College of Medicine, Seoul, Korea (Y.K., M.G.L.); Department of Chemistry and Biochemistry, San Francisco State University, San Francisco, California (M.O.A.); College of Pharmacy, Yonsei Institute of Pharmaceutical Sciences, Yonsei University, Incheon, Korea (J.P., W.N.); and Departments of Medicine and Physiology, University of California, San Francisco, California (A.S.V.)
| | - Jinhong Park
- Department of Pharmacology, Brain Korea 21 PLUS Project for Medical Sciences, Yonsei University College of Medicine, Seoul, Korea (Y.K., M.G.L.); Department of Chemistry and Biochemistry, San Francisco State University, San Francisco, California (M.O.A.); College of Pharmacy, Yonsei Institute of Pharmaceutical Sciences, Yonsei University, Incheon, Korea (J.P., W.N.); and Departments of Medicine and Physiology, University of California, San Francisco, California (A.S.V.)
| | - Min Goo Lee
- Department of Pharmacology, Brain Korea 21 PLUS Project for Medical Sciences, Yonsei University College of Medicine, Seoul, Korea (Y.K., M.G.L.); Department of Chemistry and Biochemistry, San Francisco State University, San Francisco, California (M.O.A.); College of Pharmacy, Yonsei Institute of Pharmaceutical Sciences, Yonsei University, Incheon, Korea (J.P., W.N.); and Departments of Medicine and Physiology, University of California, San Francisco, California (A.S.V.)
| | - Wan Namkung
- Department of Pharmacology, Brain Korea 21 PLUS Project for Medical Sciences, Yonsei University College of Medicine, Seoul, Korea (Y.K., M.G.L.); Department of Chemistry and Biochemistry, San Francisco State University, San Francisco, California (M.O.A.); College of Pharmacy, Yonsei Institute of Pharmaceutical Sciences, Yonsei University, Incheon, Korea (J.P., W.N.); and Departments of Medicine and Physiology, University of California, San Francisco, California (A.S.V.)
| | - A S Verkman
- Department of Pharmacology, Brain Korea 21 PLUS Project for Medical Sciences, Yonsei University College of Medicine, Seoul, Korea (Y.K., M.G.L.); Department of Chemistry and Biochemistry, San Francisco State University, San Francisco, California (M.O.A.); College of Pharmacy, Yonsei Institute of Pharmaceutical Sciences, Yonsei University, Incheon, Korea (J.P., W.N.); and Departments of Medicine and Physiology, University of California, San Francisco, California (A.S.V.)
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23
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Sandal M, Behrens M, Brockhoff A, Musiani F, Giorgetti A, Carloni P, Meyerhof W. Evidence for a Transient Additional Ligand Binding Site in the TAS2R46 Bitter Taste Receptor. J Chem Theory Comput 2015; 11:4439-49. [PMID: 26575934 DOI: 10.1021/acs.jctc.5b00472] [Citation(s) in RCA: 55] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
Abstract
Most human G protein coupled receptors (GPCRs) are activated by small molecules binding to their 7-transmembrane (7-TM) helix bundle. They belong to basally diverging branches: the 25 bitter taste 2 receptors and most members of the very large rhodopsin-like/class A GPCRs subfamily. Some members of the class A branch have been suggested to feature not only an orthosteric agonist-binding site but also a more extracellular or "vestibular" site, involved in the binding process. Here we use a hybrid molecular mechanics/coarse-grained (MM/CG) molecular dynamics approach on a widely studied bitter taste receptor (TAS2R46) receptor in complex with its agonist strychnine. Three ∼1 μs molecular simulation trajectories find two sites hosting the agonist, which together elucidate experimental data measured previously and in this work. This mechanism shares similarities with the one suggested for the evolutionarily distant class A GPCRs. It might be instrumental for the remarkably broad but specific spectrum of agonists of these chemosensory receptors.
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Affiliation(s)
- Massimo Sandal
- Computational Biophysics, German Research School for Simulation Sciences , 52425 Jülich, Germany
| | - Maik Behrens
- Department of Molecular Genetics, German Institute of Human Nutrition Potsdam-Rehbruecke (DIfE) , Arthur-Scheunert-Allee 114-116, 14558 Nuthetal, Germany
| | - Anne Brockhoff
- Department of Molecular Genetics, German Institute of Human Nutrition Potsdam-Rehbruecke (DIfE) , Arthur-Scheunert-Allee 114-116, 14558 Nuthetal, Germany
| | - Francesco Musiani
- Scuola Internazionale Superiore di Studi Avanzati (SISSA/ISAS) , Via Bonomea 265, 34151 Trieste, Italy.,Laboratory of Bioinorganic Chemistry, Department of Pharmacy and Biotechnology (FaBIT), University of Bologna , Viale Giuseppe Fanin 40, I-40127, Bologna, Italy
| | - Alejandro Giorgetti
- Computational Biophysics, German Research School for Simulation Sciences , 52425 Jülich, Germany.,Department of Biotechnology, University of Verona , Ca' Vignal 1, Strada Le Grazie 15, 37134 Verona, Italy
| | - Paolo Carloni
- Computational Biophysics, German Research School for Simulation Sciences , 52425 Jülich, Germany
| | - Wolfgang Meyerhof
- Department of Molecular Genetics, German Institute of Human Nutrition Potsdam-Rehbruecke (DIfE) , Arthur-Scheunert-Allee 114-116, 14558 Nuthetal, Germany
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24
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Zhang J, Hwang TC. The Fifth Transmembrane Segment of Cystic Fibrosis Transmembrane Conductance Regulator Contributes to Its Anion Permeation Pathway. Biochemistry 2015; 54:3839-50. [PMID: 26024338 DOI: 10.1021/acs.biochem.5b00427] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
Abstract
Previous studies have identified several transmembrane segments (TMs), including TM1, TM3, TM6, TM9, TM11, and TM12, as pore-lining segments in cystic fibrosis transmembrane conductance regulator (CFTR), but the role of TM5 in pore construction remains controversial. In this study, we employed substituted cysteine accessibility methodology (SCAM) to screen the entire TM5 defined by the original topology model and its cytoplasmic extension in a Cysless background. We found six positions (A299, R303, N306, S307, F310, and F311) where engineered cysteines react to intracellular 2-sulfonatoethyl methanethiosulfonate (MTSES⁻). Quantification of the modification rate of engineered cysteines in the presence or absence of ATP suggests that these six residues are accessible in both the open and closed states. Whole-cell experiments with external MTSES⁻ identified only two positive positions (L323 and A326), resulting in a segment containing 11 consecutive amino acids, where substituted cysteines respond to neither internal nor external MTSES⁻, a unique feature not seen previously in CFTR's pore-lining segments. The observation that these positions are inaccessible to channel-permeant thiol-specific reagent [Au(CN)₂]⁻ suggests that this segment of TM5 between F311 and L323 is concealed from the pore by other TMs and/or lipid bilayers. In addition, our data support the idea that the positively charged arginine at position 303 poses a pure electrostatic action in determining the single-channel current amplitude of CFTR and the effect of an open-channel blocker glibencalmide. Collectively, we conclude that the cytoplasmic portion of CFTR's TM5 lines the pore. Our functional data are remarkably consistent with predicted structural arrangements of TM5 in some homology models of CFTR.
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Affiliation(s)
- Jingyao Zhang
- †Department of Biological Engineering, University of Missouri-Columbia, 254 Agricultural Engineering, Columbia, Missouri 65211, United States.,‡Dalton Cardiovascular Research Center, University of Missouri-Columbia, 134 Research Park, Columbia, Missouri 65211, United States
| | - Tzyh-Chang Hwang
- †Department of Biological Engineering, University of Missouri-Columbia, 254 Agricultural Engineering, Columbia, Missouri 65211, United States.,‡Dalton Cardiovascular Research Center, University of Missouri-Columbia, 134 Research Park, Columbia, Missouri 65211, United States.,§Department of Medical Pharmacology and Physiology, University of Missouri-Columbia, Medical Sciences Building, Columbia, Missouri 65212, United States
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25
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Cai Z, Palmai-Pallag T, Khuituan P, Mutolo MJ, Boinot C, Liu B, Scott-Ward TS, Callebaut I, Harris A, Sheppard DN. Impact of the F508del mutation on ovine CFTR, a Cl- channel with enhanced conductance and ATP-dependent gating. J Physiol 2015; 593:2427-46. [PMID: 25763566 DOI: 10.1113/jp270227] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/22/2015] [Accepted: 03/02/2015] [Indexed: 12/20/2022] Open
Abstract
KEY POINTS Malfunction of the cystic fibrosis transmembrane conductance regulator (CFTR), a gated pathway for chloride movement, causes the common life-shortening genetic disease cystic fibrosis (CF). Towards the development of a sheep model of CF, we have investigated the function of sheep CFTR. We found that sheep CFTR was noticeably more active than human CFTR, while the most common CF mutation, F508del, had reduced impact on sheep CFTR function. Our results demonstrate that subtle changes in protein structure have marked effects on CFTR function and the consequences of the CF mutation F508del. ABSTRACT Cross-species comparative studies are a powerful approach to understanding the epithelial Cl(-) channel cystic fibrosis transmembrane conductance regulator (CFTR), which is defective in the genetic disease cystic fibrosis (CF). Here, we investigate the single-channel behaviour of ovine CFTR and the impact of the most common CF mutation, F508del-CFTR, using excised inside-out membrane patches from transiently transfected CHO cells. Like human CFTR, ovine CFTR formed a weakly inwardly rectifying Cl(-) channel regulated by PKA-dependent phosphorylation, inhibited by the open-channel blocker glibenclamide. However, for three reasons, ovine CFTR was noticeably more active than human CFTR. First, single-channel conductance was increased. Second, open probability was augmented because the frequency and duration of channel openings were increased. Third, with enhanced affinity and efficacy, ATP more strongly stimulated ovine CFTR channel gating. Consistent with these data, the CFTR modulator phloxine B failed to potentiate ovine CFTR Cl(-) currents. Similar to its impact on human CFTR, the F508del mutation caused a temperature-sensitive folding defect, which disrupted ovine CFTR protein processing and reduced membrane stability. However, the F508del mutation had reduced impact on ovine CFTR channel gating in contrast to its marked effects on human CFTR. We conclude that ovine CFTR forms a regulated Cl(-) channel with enhanced conductance and ATP-dependent channel gating. This phylogenetic analysis of CFTR structure and function demonstrates that subtle changes in structure have pronounced effects on channel function and the consequences of the CF mutation F508del.
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Affiliation(s)
- Zhiwei Cai
- School of Physiology and Pharmacology, University of Bristol, Medical Sciences Building, University Walk, Bristol, BS8 1TD, UK
| | - Timea Palmai-Pallag
- Human Molecular Genetics Program, Lurie Children's Research Center and Department of Pediatrics, Northwestern University Feinberg School of Medicine, Chicago, IL, 60614, USA.,Harris Laboratory, formerly at the Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK
| | - Pissared Khuituan
- School of Physiology and Pharmacology, University of Bristol, Medical Sciences Building, University Walk, Bristol, BS8 1TD, UK.,Center of Calcium and Bone Research, Department of Physiology, Faculty of Science, Mahidol University, Bangkok, 10400, Thailand
| | - Michael J Mutolo
- Human Molecular Genetics Program, Lurie Children's Research Center and Department of Pediatrics, Northwestern University Feinberg School of Medicine, Chicago, IL, 60614, USA
| | - Clément Boinot
- Institut de Physiologie et Biologie Cellulaires, Université de Poitiers, CNRS FRE 3511, 86022, Poitiers, France
| | - Beihui Liu
- School of Physiology and Pharmacology, University of Bristol, Medical Sciences Building, University Walk, Bristol, BS8 1TD, UK
| | - Toby S Scott-Ward
- School of Physiology and Pharmacology, University of Bristol, Medical Sciences Building, University Walk, Bristol, BS8 1TD, UK
| | - Isabelle Callebaut
- IMPMC, Sorbonne Universités - UPMC Univ Paris 06, UMR CNRS 7590, Museum National d'Histoire Naturelle, IRD UMR 206, IUC, 75005, Paris, France
| | - Ann Harris
- Human Molecular Genetics Program, Lurie Children's Research Center and Department of Pediatrics, Northwestern University Feinberg School of Medicine, Chicago, IL, 60614, USA
| | - David N Sheppard
- School of Physiology and Pharmacology, University of Bristol, Medical Sciences Building, University Walk, Bristol, BS8 1TD, UK
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26
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Wang G. Interplay between Inhibitory Ferric and Stimulatory Curcumin Regulates Phosphorylation-Dependent Human Cystic Fibrosis Transmembrane Conductance Regulator and ΔF508 Activity. Biochemistry 2015; 54:1558-66. [DOI: 10.1021/bi501318h] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
Affiliation(s)
- Guangyu Wang
- Department of Physiology
and Biophysics and Gregory Fleming James Cystic Fibrosis Research
Center, University of Alabama at Birmingham School of Medicine, Birmingham, Alabama 35294, United States
- Department of Physiology
and Membrane Biology, University of California School of Medicine, Davis, California 95616, United States
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27
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Abstract
Experimental and computational studies have painted a picture of the chloride permeation pathway in cystic fibrosis transmembrane conductance regulator (CFTR) as a short narrow tunnel flanked by wider inner and outer vestibules. Although these studies also identified a number of transmembrane segments (TMs) as pore-lining, the exact location of CFTR's gate(s) remains unknown. Here, using a channel-permeant probe, [Au(CN)2](-), we provide evidence that CFTR bears a gate that coincides with the predicted narrow section of the pore defined as residues 338-341 in TM6. Specifically, cysteines introduced cytoplasmic to the narrow region (i.e., positions 344 in TM6 and 1148 in TM12) can be modified by intracellular [Au(CN)2](-) in both open and closed states, corroborating the conclusion that the internal vestibule does not harbor a gate. However, cysteines engineered to positions external to the presumed narrow region (e.g., 334, 335, and 337 in TM6) are all nonreactive toward cytoplasmic [Au(CN)2](-) in the absence of ATP, whereas they can be better accessed by extracellular [Au(CN)2](-) when the open probability is markedly reduced by introducing a second mutation, G1349D. As [Au(CN)2](-) and chloride ions share the same permeation pathway, these results imply a gate is situated between amino acid residues 337 and 344 along TM6, encompassing the very segment that may also serve as the selectivity filter for CFTR. The unique position of a gate in the middle of the ion translocation pathway diverges from those seen in ATP-binding cassette (ABC) transporters and thus distinguishes CFTR from other members of the ABC transporter family.
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28
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Ju M, Scott-Ward TS, Liu J, Khuituan P, Li H, Cai Z, Husbands SM, Sheppard DN. Loop diuretics are open-channel blockers of the cystic fibrosis transmembrane conductance regulator with distinct kinetics. Br J Pharmacol 2014; 171:265-78. [PMID: 24117047 DOI: 10.1111/bph.12458] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/11/2013] [Revised: 09/21/2013] [Accepted: 09/26/2013] [Indexed: 12/26/2022] Open
Abstract
BACKGROUND AND PURPOSE Loop diuretics are widely used to inhibit the Na(+), K(+), 2Cl(-) co-transporter, but they also inhibit the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel. Here, we investigated the mechanism of CFTR inhibition by loop diuretics and explored the effects of chemical structure on channel blockade. EXPERIMENTAL APPROACH Using the patch-clamp technique, we tested the effects of bumetanide, furosemide, piretanide and xipamide on recombinant wild-type human CFTR. KEY RESULTS When added to the intracellular solution, loop diuretics inhibited CFTR Cl(-) currents with potency approaching that of glibenclamide, a widely used CFTR blocker with some structural similarity to loop diuretics. To begin to study the kinetics of channel blockade, we examined the time dependence of macroscopic current inhibition following a hyperpolarizing voltage step. Like glibenclamide, piretanide blockade of CFTR was time and voltage dependent. By contrast, furosemide blockade was voltage dependent, but time independent. Consistent with these data, furosemide blocked individual CFTR Cl(-) channels with 'very fast' speed and drug-induced blocking events overlapped brief channel closures, whereas piretanide inhibited individual channels with 'intermediate' speed and drug-induced blocking events were distinct from channel closures. CONCLUSIONS AND IMPLICATIONS Structure-activity analysis of the loop diuretics suggests that the phenoxy group present in bumetanide and piretanide, but absent in furosemide and xipamide, might account for the different kinetics of channel block by locking loop diuretics within the intracellular vestibule of the CFTR pore. We conclude that loop diuretics are open-channel blockers of CFTR with distinct kinetics, affected by molecular dimensions and lipophilicity.
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Affiliation(s)
- Min Ju
- School of Physiology and Pharmacology, University of Bristol, Bristol, UK
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29
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LaRusch J, Jung J, General IJ, Lewis MD, Park HW, Brand RE, Gelrud A, Anderson MA, Banks PA, Conwell D, Lawrence C, Romagnuolo J, Baillie J, Alkaade S, Cote G, Gardner TB, Amann ST, Slivka A, Sandhu B, Aloe A, Kienholz ML, Yadav D, Barmada MM, Bahar I, Lee MG, Whitcomb DC. Mechanisms of CFTR functional variants that impair regulated bicarbonate permeation and increase risk for pancreatitis but not for cystic fibrosis. PLoS Genet 2014; 10:e1004376. [PMID: 25033378 PMCID: PMC4102440 DOI: 10.1371/journal.pgen.1004376] [Citation(s) in RCA: 130] [Impact Index Per Article: 11.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/26/2013] [Accepted: 03/10/2014] [Indexed: 02/07/2023] Open
Abstract
CFTR is a dynamically regulated anion channel. Intracellular WNK1-SPAK activation causes CFTR to change permeability and conductance characteristics from a chloride-preferring to bicarbonate-preferring channel through unknown mechanisms. Two severe CFTR mutations (CFTRsev) cause complete loss of CFTR function and result in cystic fibrosis (CF), a severe genetic disorder affecting sweat glands, nasal sinuses, lungs, pancreas, liver, intestines, and male reproductive system. We hypothesize that those CFTR mutations that disrupt the WNK1-SPAK activation mechanisms cause a selective, bicarbonate defect in channel function (CFTRBD) affecting organs that utilize CFTR for bicarbonate secretion (e.g. the pancreas, nasal sinus, vas deferens) but do not cause typical CF. To understand the structural and functional requirements of the CFTR bicarbonate-preferring channel, we (a) screened 984 well-phenotyped pancreatitis cases for candidate CFTRBD mutations from among 81 previously described CFTR variants; (b) conducted electrophysiology studies on clones of variants found in pancreatitis but not CF; (c) computationally constructed a new, complete structural model of CFTR for molecular dynamics simulation of wild-type and mutant variants; and (d) tested the newly defined CFTRBD variants for disease in non-pancreas organs utilizing CFTR for bicarbonate secretion. Nine variants (CFTR R74Q, R75Q, R117H, R170H, L967S, L997F, D1152H, S1235R, and D1270N) not associated with typical CF were associated with pancreatitis (OR 1.5, p = 0.002). Clones expressed in HEK 293T cells had normal chloride but not bicarbonate permeability and conductance with WNK1-SPAK activation. Molecular dynamics simulations suggest physical restriction of the CFTR channel and altered dynamic channel regulation. Comparing pancreatitis patients and controls, CFTRBD increased risk for rhinosinusitis (OR 2.3, p<0.005) and male infertility (OR 395, p<<0.0001). WNK1-SPAK pathway-activated increases in CFTR bicarbonate permeability are altered by CFTRBD variants through multiple mechanisms. CFTRBD variants are associated with clinically significant disorders of the pancreas, sinuses, and male reproductive system.
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Affiliation(s)
- Jessica LaRusch
- Department of Medicine, Division of Gastroenterology, Hepatology and Nutrition, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America
| | - Jinsei Jung
- Department of Pharmacology and Brain Korea 21 Plus Project for Medical Science, Yonsei University College of Medicine, Seoul, Korea
| | - Ignacio J. General
- Department of Computational & Systems Biology, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America
| | - Michele D. Lewis
- Division of Gastroenterology and Hepatology, Mayo Clinic, Jacksonville, Florida, United States of America
| | - Hyun Woo Park
- Department of Pharmacology and Brain Korea 21 Plus Project for Medical Science, Yonsei University College of Medicine, Seoul, Korea
| | - Randall E. Brand
- Department of Medicine, Division of Gastroenterology, Hepatology and Nutrition, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America
| | - Andres Gelrud
- Department of Medicine, Division of Gastroenterology, Hepatology and Nutrition, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America
| | - Michelle A. Anderson
- Department of Medicine, University of Michigan, Ann Arbor, Michigan, United States of America
| | - Peter A. Banks
- Division of Gastroenterology, Brigham and Women's Hospital, Boston, Massachusetts, United States of America
| | - Darwin Conwell
- Division of Gastroenterology, Brigham and Women's Hospital, Boston, Massachusetts, United States of America
| | - Christopher Lawrence
- Digestive Disease Center, Medical University of South Carolina, Charleston, South Carolina, United States of America
| | - Joseph Romagnuolo
- Digestive Disease Center, Medical University of South Carolina, Charleston, South Carolina, United States of America
| | - John Baillie
- Department of Medicine, Duke University Medical Center, Durham, North Carolina, United States of America
| | - Samer Alkaade
- Department of Internal Medicine, St. Louis University School of Medicine, St Louis, Missouri, United States of America
| | - Gregory Cote
- Department of Medicine, Indiana University School of Medicine, Indianapolis, Indiana, United States of America
| | - Timothy B. Gardner
- Dartmouth-Hitchcock Medical Center, Hanover, New Hampshire, United States of America
| | - Stephen T. Amann
- North Mississippi Medical Center, Tupelo, Mississippi, United States of America
| | - Adam Slivka
- Department of Medicine, Division of Gastroenterology, Hepatology and Nutrition, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America
| | - Bimaljit Sandhu
- Division of Gastroenterology, Hepatology and Nutrition, Virginia Commonwealth University Medical Center, Richmond, Virginia, United States of America
| | - Amy Aloe
- Department of Medicine, Division of Gastroenterology, Hepatology and Nutrition, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America
| | - Michelle L. Kienholz
- Department of Medicine, Division of Gastroenterology, Hepatology and Nutrition, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America
| | - Dhiraj Yadav
- Department of Medicine, Division of Gastroenterology, Hepatology and Nutrition, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America
| | - M. Michael Barmada
- Department of Human Genetics, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America
| | - Ivet Bahar
- Department of Computational & Systems Biology, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America
| | - Min Goo Lee
- Department of Pharmacology and Brain Korea 21 Plus Project for Medical Science, Yonsei University College of Medicine, Seoul, Korea
| | - David C. Whitcomb
- Department of Medicine, Division of Gastroenterology, Hepatology and Nutrition, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America
- Department of Human Genetics, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America
- Department of Cell Biology and Molecular Physiology, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America
- * E-mail:
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30
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Cui G, Rahman KS, Infield DT, Kuang C, Prince CZ, McCarty NA. Three charged amino acids in extracellular loop 1 are involved in maintaining the outer pore architecture of CFTR. ACTA ACUST UNITED AC 2014; 144:159-79. [PMID: 25024266 PMCID: PMC4113900 DOI: 10.1085/jgp.201311122] [Citation(s) in RCA: 27] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/26/2022]
Abstract
Disease-associated mutation of charged amino acids in extracellular loop 1 of CFTR may reduce chloride flow by damaging the outer pore architecture. The cystic fibrosis (CF) transmembrane conductance regulator (CFTR) bears six extracellular loops (ECL1–6); ECL1 is the site of several mutations associated with CF. Mutation R117H has been reported to reduce current amplitude, whereas D110H, E116K, and R117C/L/P may impair channel stability. We hypothesized that these amino acids might not be directly involved in ion conduction and permeation but may contribute to stabilizing the outer vestibule architecture in CFTR. We used cRNA injected oocytes combined with electrophysiological techniques to test this hypothesis. Mutants bearing cysteine at these sites were not functionally modified by extracellular MTS reagents and were blocked by GlyH-101 similarly to WT-CFTR. These results suggest that these three residues do not contribute directly to permeation in CFTR. In contrast, mutants D110R-, E116R-, and R117A-CFTR exhibited instability of the open state and significantly shortened burst duration compared with WT-CFTR and failed to be locked into the open state by AMP-PNP (adenosine 5′-(β,γ-imido) triphosphate); charge-retaining mutants showed mainly the full open state with comparably longer open burst duration. These interactions suggest that these ECL1 residues might be involved in maintaining the outer pore architecture of CFTR. A CFTR homology model suggested that E116 interacts with R104 in both the closed and open states, D110 interacts with K892 in the fully closed state, and R117 interacts with E1126 in the open state. These interactions were confirmed experimentally. The results suggest that D110, E116, and R117 may contribute to stabilizing the architecture of the outer pore of CFTR by interactions with other charged residues.
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Affiliation(s)
- Guiying Cui
- Division of Pulmonary, Allergy and Immunology, Cystic Fibrosis, and Sleep, Department of Pediatrics, Center for Cystic Fibrosis and Airways Disease Research, Emory+Children's Pediatric Research Center, Emory University School of Medicine and Children's Healthcare of Atlanta, Atlanta, GA 30322
| | - Kazi S Rahman
- Parker H. Petit Institute for Bioengineering and Bioscience and School of Biology, Georgia Institute of Technology, Atlanta, GA 30332 Parker H. Petit Institute for Bioengineering and Bioscience and School of Biology, Georgia Institute of Technology, Atlanta, GA 30332
| | - Daniel T Infield
- Division of Pulmonary, Allergy and Immunology, Cystic Fibrosis, and Sleep, Department of Pediatrics, Center for Cystic Fibrosis and Airways Disease Research, Emory+Children's Pediatric Research Center, Emory University School of Medicine and Children's Healthcare of Atlanta, Atlanta, GA 30322
| | - Christopher Kuang
- Division of Pulmonary, Allergy and Immunology, Cystic Fibrosis, and Sleep, Department of Pediatrics, Center for Cystic Fibrosis and Airways Disease Research, Emory+Children's Pediatric Research Center, Emory University School of Medicine and Children's Healthcare of Atlanta, Atlanta, GA 30322
| | - Chengyu Z Prince
- Division of Pulmonary, Allergy and Immunology, Cystic Fibrosis, and Sleep, Department of Pediatrics, Center for Cystic Fibrosis and Airways Disease Research, Emory+Children's Pediatric Research Center, Emory University School of Medicine and Children's Healthcare of Atlanta, Atlanta, GA 30322
| | - Nael A McCarty
- Division of Pulmonary, Allergy and Immunology, Cystic Fibrosis, and Sleep, Department of Pediatrics, Center for Cystic Fibrosis and Airways Disease Research, Emory+Children's Pediatric Research Center, Emory University School of Medicine and Children's Healthcare of Atlanta, Atlanta, GA 30322 Parker H. Petit Institute for Bioengineering and Bioscience and School of Biology, Georgia Institute of Technology, Atlanta, GA 30332
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31
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Linsdell P. Cystic fibrosis transmembrane conductance regulator chloride channel blockers: Pharmacological, biophysical and physiological relevance. World J Biol Chem 2014; 5:26-39. [PMID: 24600512 PMCID: PMC3942540 DOI: 10.4331/wjbc.v5.i1.26] [Citation(s) in RCA: 25] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/02/2013] [Revised: 11/15/2013] [Accepted: 12/11/2013] [Indexed: 02/05/2023] Open
Abstract
Dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel causes cystic fibrosis, while inappropriate activity of this channel occurs in secretory diarrhea and polycystic kidney disease. Drugs that interact directly with CFTR are therefore of interest in the treatment of a number of disease states. This review focuses on one class of small molecules that interacts directly with CFTR, namely inhibitors that act by directly blocking chloride movement through the open channel pore. In theory such compounds could be of use in the treatment of diarrhea and polycystic kidney disease, however in practice all known substances acting by this mechanism to inhibit CFTR function lack either the potency or specificity for in vivo use. Nevertheless, this theoretical pharmacological usefulness set the scene for the development of more potent, specific CFTR inhibitors. Biophysically, open channel blockers have proven most useful as experimental probes of the structure and function of the CFTR chloride channel pore. Most importantly, the use of these blockers has been fundamental in developing a functional model of the pore that includes a wide inner vestibule that uses positively charged amino acid side chains to attract both permeant and blocking anions from the cell cytoplasm. CFTR channels are also subject to this kind of blocking action by endogenous anions present in the cell cytoplasm, and recently this blocking effect has been suggested to play a role in the physiological control of CFTR channel function, in particular as a novel mechanism linking CFTR function dynamically to the composition of epithelial cell secretions. It has also been suggested that future drugs could target this same pathway as a way of pharmacologically increasing CFTR activity in cystic fibrosis. Studying open channel blockers and their mechanisms of action has resulted in significant advances in our understanding of CFTR as a pharmacological target in disease states, of CFTR channel structure and function, and of how CFTR activity is controlled by its local environment.
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32
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Thiagarajah JR, Ko EA, Tradtrantip L, Donowitz M, Verkman A. Discovery and development of antisecretory drugs for treating diarrheal diseases. Clin Gastroenterol Hepatol 2014; 12:204-9. [PMID: 24316107 PMCID: PMC3935719 DOI: 10.1016/j.cgh.2013.12.001] [Citation(s) in RCA: 38] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
Diarrheal diseases constitute a significant global health burden and are a major cause of childhood mortality and morbidity. Treatment of diarrheal disease has centered on the replacement of fluid and electrolyte losses using oral rehydration solutions. Although oral rehydration solutions have been highly successful, significant mortality and morbidity due to diarrheal disease remains. Secretory diarrheas, such as those caused by bacterial and viral enterotoxins, result from activation of cyclic nucleotide and/or Ca(2+) signaling pathways in intestinal epithelial cells, enterocytes, which increase the permeability of Cl(-) channels at the lumen-facing membrane. Additionally, there is often a parallel reduction in intestinal Na(+) absorption. Inhibition of enterocyte Cl(-) channels, including the cystic fibrosis transmembrane conductance regulator and Ca(2+)-activated Cl(-) channels, represents an attractive strategy for antisecretory drug therapy. High-throughput screening of synthetic small-molecule collections has identified several classes of Cl(-) channel inhibitors that show efficacy in animal models of diarrhea but remain to be tested clinically. In addition, several natural product extracts with Cl(-) channel inhibition activity have shown efficacy in diarrhea models. However, a number of challenges remain to translate the promising bench science into clinically useful therapeutics, including efficiently targeting orally administered drugs to enterocytes during diarrhea, funding development costs, and carrying out informative clinical trials. Nonetheless, Cl(-) channel inhibitors may prove to be effective adjunctive therapy in a broad spectrum of clinical diarrheas, including acute infectious and drug-related diarrheas, short bowel syndrome, and congenital enteropathies.
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Affiliation(s)
- Jay R. Thiagarajah
- Departments of Medicine and Physiology, University of California, San Francisco, CA, USA, 94143-0521,Department of Gastroenterology, Hepatology and Nutrition, Children's Hospital Boston, Harvard Medical School, Boston, MA 02115
| | - Eun-A Ko
- Departments of Medicine and Physiology, University of California, San Francisco, CA, USA, 94143-0521
| | - Lukmanee Tradtrantip
- Departments of Medicine and Physiology, University of California, San Francisco, CA, USA, 94143-0521
| | - Mark Donowitz
- Departments of Physiology and Medicine, Gastroenterology Division, Johns Hopkins University School of Medicine, Baltimore, MD 21205
| | - A.S. Verkman
- Departments of Medicine and Physiology, University of California, San Francisco, CA, USA, 94143-0521
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Csanády L, Töröcsik B. Catalyst-like modulation of transition states for CFTR channel opening and closing: new stimulation strategy exploits nonequilibrium gating. ACTA ACUST UNITED AC 2014; 143:269-87. [PMID: 24420771 PMCID: PMC4001772 DOI: 10.1085/jgp.201311089] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/05/2023]
Abstract
Two gating transition states determine open probability of CFTR (the chloride channel mutated in cystic fibrosis), defining strategic targets for therapeutic intervention. Cystic fibrosis transmembrane conductance regulator (CFTR) is the chloride ion channel mutated in cystic fibrosis (CF) patients. It is an ATP-binding cassette protein, and its resulting cyclic nonequilibrium gating mechanism sets it apart from most other ion channels. The most common CF mutation (ΔF508) impairs folding of CFTR but also channel gating, reducing open probability (Po). This gating defect must be addressed to effectively treat CF. Combining single-channel and macroscopic current measurements in inside-out patches, we show here that the two effects of 5-nitro-2-(3-phenylpropylamino)benzoate (NPPB) on CFTR, pore block and gating stimulation, are independent, suggesting action at distinct sites. Furthermore, detailed kinetic analysis revealed that NPPB potently increases Po, also of ΔF508 CFTR, by affecting the stability of gating transition states. This finding is unexpected, because for most ion channels, which gate at equilibrium, altering transition-state stabilities has no effect on Po; rather, agonists usually stimulate by stabilizing open states. Our results highlight how for CFTR, because of its unique cyclic mechanism, gating transition states determine Po and offer strategic targets for potentiator compounds to achieve maximal efficacy.
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Affiliation(s)
- László Csanády
- Department of Medical Biochemistry and 2 MTA-SE Ion Channel Research Group, Semmelweis University, Budapest H-1094, Hungary
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Verkman AS, Synder D, Tradtrantip L, Thiagarajah JR, Anderson MO. CFTR inhibitors. Curr Pharm Des 2013; 19:3529-41. [PMID: 23331030 DOI: 10.2174/13816128113199990321] [Citation(s) in RCA: 24] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/31/2012] [Accepted: 01/16/2013] [Indexed: 12/16/2022]
Abstract
The cystic fibrosis transmembrane conductance regulator (CFTR) protein is a cAMP-regulated Cl- channel whose major function is to facilitate epithelial fluid secretion. Loss-of-function mutations in CFTR cause the genetic disease cystic fibrosis. CFTR is required for transepithelial fluid transport in certain secretory diarrheas, such as cholera, and for cyst expansion in autosomal dominant polycystic kidney disease. High-throughput screening has yielded CFTR inhibitors of the thiazolidinone, glycine hydrazide and quinoxalinedione chemical classes. The glycine hydrazides target the extracellular CFTR pore, whereas the thiazolidinones and quinoxalinediones act at the cytoplasmic surface. These inhibitors have been widely used in cystic fibrosis research to study CFTR function at the cell and organ levels. The most potent CFTR inhibitor has IC50 of approximately 4 nM. Studies in animal models support the development of CFTR inhibitors for antisecretory therapy of enterotoxin-mediated diarrheas and polycystic kidney disease.
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Affiliation(s)
- Alan S Verkman
- University of California-San Francisco, CA 94143-0521, U.S.A.
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Rahman KS, Cui G, Harvey SC, McCarty NA. Modeling the conformational changes underlying channel opening in CFTR. PLoS One 2013; 8:e74574. [PMID: 24086355 PMCID: PMC3785483 DOI: 10.1371/journal.pone.0074574] [Citation(s) in RCA: 42] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/31/2013] [Accepted: 08/05/2013] [Indexed: 12/22/2022] Open
Abstract
Mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator protein (CFTR) cause cystic fibrosis (CF), the most common life-shortening genetic disease among Caucasians. Although general features of the structure of CFTR have been predicted from homology models, the conformational changes that result in channel opening and closing have yet to be resolved. We created new closed- and open-state homology models of CFTR, and performed targeted molecular dynamics simulations of the conformational transitions in a channel opening event. The simulations predict a conformational wave that starts at the nucleotide binding domains and ends with the formation of an open conduction pathway. Changes in side-chain interactions are observed in all major domains of the protein, and experimental confirmation was obtained for a novel intra-protein salt bridge that breaks near the end of the transition. The models and simulation add to our understanding of the mechanism of ATP-dependent gating in this disease-relevant ion channel.
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Affiliation(s)
- Kazi S. Rahman
- Petit Institute of Bioengineering and Bioscience and School of Biology, Georgia Institute of Technology, Atlanta, Georgia, United States of America
| | - Guiying Cui
- Department of Pediatrics and Center for Cystic Fibrosis Research, Emory University and Children's Healthcare of Atlanta, Inc., Atlanta, Georgia, United States of America
| | - Stephen C. Harvey
- Petit Institute of Bioengineering and Bioscience and School of Biology, Georgia Institute of Technology, Atlanta, Georgia, United States of America
| | - Nael A. McCarty
- Department of Pediatrics and Center for Cystic Fibrosis Research, Emory University and Children's Healthcare of Atlanta, Inc., Atlanta, Georgia, United States of America
- * E-mail:
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Chloride channel-targeted therapy for secretory diarrheas. Curr Opin Pharmacol 2013; 13:888-94. [PMID: 23992767 DOI: 10.1016/j.coph.2013.08.005] [Citation(s) in RCA: 38] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/25/2013] [Revised: 07/30/2013] [Accepted: 08/01/2013] [Indexed: 12/18/2022]
Abstract
Secretory diarrheas caused by bacterial and viral enterotoxins remain a significant cause of morbidity and mortality. Enterocyte Cl(-) channels represent an attractive class of targets for diarrhea therapy, as they are the final, rate-limiting step in enterotoxin-induced fluid secretion in the intestine. Activation of cyclic nucleotide and/or Ca(2+) signaling pathways in secretory diarrheas increases the conductance of Cl(-) channels at the enterocyte luminal membrane, which include the cystic fibrosis transmembrane conductance regulator (CFTR) and Ca(2+)-activated Cl(-) channels (CaCCs). High-throughput screens have yielded several chemical classes of small molecule CFTR and CaCC inhibitors that show efficacy in animal models of diarrheas. Natural-product diarrhea remedies with Cl(-) channel inhibition activity have also been identified, with one product recently receiving FDA approval for HIV-associated diarrhea.
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Billet A, Mornon JP, Jollivet M, Lehn P, Callebaut I, Becq F. CFTR: effect of ICL2 and ICL4 amino acids in close spatial proximity on the current properties of the channel. J Cyst Fibros 2013; 12:737-45. [PMID: 23478129 DOI: 10.1016/j.jcf.2013.02.002] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/14/2012] [Revised: 01/18/2013] [Accepted: 02/05/2013] [Indexed: 12/15/2022]
Abstract
BACKGROUND CFTR is the only ABC transporter functioning as a chloride (Cl(-)) channel. We studied molecular determinants, which might distinguish CFTR from standard ABC transporters, and focused on the interface formed by the intracellular loops from the membrane spanning domains. METHODS Residues from ICL2 and ICL4 in close proximity were targeted, and their involvement in the functioning of CFTR was studied by whole cell patch clamp recording. RESULTS We identified 2 pairs of amino acids, at the extremity of the bundle formed by the four intracellular loops, whose mutation i) decreases the Cl(-) current of CFTR (couple E267-K1060) or ii) increases it with a change of the electrophysiological signature (couple S263-V1056). CONCLUSIONS These results highlight the critical role of these ICL residues in the assembly of the different domains and/or in the Cl(-) permeation pathway of CFTR.
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Affiliation(s)
- Arnaud Billet
- Institut de Physiologie et Biologie Cellulaires, Université de Poitiers, CNRS, Poitiers, France
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