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1
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Levring J, Chen J. Structural identification of a selectivity filter in CFTR. Proc Natl Acad Sci U S A 2024; 121:e2316673121. [PMID: 38381791 PMCID: PMC10907310 DOI: 10.1073/pnas.2316673121] [Citation(s) in RCA: 2] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/25/2023] [Accepted: 01/19/2024] [Indexed: 02/23/2024] Open
Abstract
The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel that regulates transepithelial salt and fluid homeostasis. CFTR dysfunction leads to reduced chloride secretion into the mucosal lining of epithelial tissues, thereby causing the inherited disease cystic fibrosis. Although several structures of CFTR are available, our understanding of the ion-conduction pathway is incomplete. In particular, the route that connects the cytosolic vestibule with the extracellular space has not been clearly defined, and the structure of the open pore remains elusive. Furthermore, although many residues have been implicated in altering the selectivity of CFTR, the structure of the "selectivity filter" has yet to be determined. In this study, we identify a chloride-binding site at the extracellular ends of transmembrane helices 1, 6, and 8, where a dehydrated chloride is coordinated by residues G103, R334, F337, T338, and Y914. Alterations to this site, consistent with its function as a selectivity filter, affect ion selectivity, conductance, and open channel block. This selectivity filter is accessible from the cytosol through a large inner vestibule and opens to the extracellular solvent through a narrow portal. The identification of a chloride-binding site at the intra- and extracellular bridging point leads us to propose a complete conductance path that permits dehydrated chloride ions to traverse the lipid bilayer.
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Affiliation(s)
- Jesper Levring
- Laboratory of Membrane Biology and Biophysics, The Rockefeller University, New York, NY10065
| | - Jue Chen
- Laboratory of Membrane Biology and Biophysics, The Rockefeller University, New York, NY10065
- HHMI, The Rockefeller University, New York, NY10065
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2
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Linsdell P. Role of Hydrophobic Amino-Acid Side-Chains in the Narrow Selectivity Filter of the CFTR Chloride Channel Pore in Conductance and Selectivity. J Membr Biol 2023; 256:433-442. [PMID: 37823914 DOI: 10.1007/s00232-023-00294-w] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/26/2023] [Accepted: 09/26/2023] [Indexed: 10/13/2023]
Abstract
Cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel. Structural analysis of CFTR has identified a narrow, hydrophobic region close to the extracellular end of the open channel pore that may function as a selectivity filter. The present study combines comprehensive mutagenesis of hydrophobic amino-acid side-chains within the selectivity filter with functional evaluation of channel Cl- conductance and anion selectivity. Among these hydrophobic amino-acids, one (F337) appears to play a dominant role in determining both conductance and selectivity. Anion selectivity appears to depend on both side-chain size and hydrophobicity at this position. In contrast, conductance is disrupted by all F337 mutations, suggesting that unique interactions between permeating Cl- ions and the native phenylalanine side-chain are important for conductance. Surprisingly, a positively charged lysine side-chain can be substituted for several hydrophobic residues within the selectivity filter (including F337) with only minor changes in pore function, arguing against a crucial role for overall hydrophobicity. These results suggest that localized interactions between permeating anions and amino-acid side-chains within the selectivity filter may be more important in determining pore functional properties than are global features such as overall hydrophobicity.
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Affiliation(s)
- Paul Linsdell
- Department of Physiology & Biophysics, Dalhousie University, PO Box 15000, Halifax, NS, B3H 4R2, Canada.
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3
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Zeng ZW, Linsdell P, Pomès R. Molecular dynamics study of Cl - permeation through cystic fibrosis transmembrane conductance regulator (CFTR). Cell Mol Life Sci 2023; 80:51. [PMID: 36694009 PMCID: PMC9873711 DOI: 10.1007/s00018-022-04621-7] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/31/2021] [Revised: 10/26/2022] [Accepted: 10/31/2022] [Indexed: 01/25/2023]
Abstract
The recent elucidation of atomistic structures of Cl- channel CFTR provides opportunities for understanding the molecular basis of cystic fibrosis. Despite having been activated through phosphorylation and provided with ATP ligands, several near-atomistic cryo-EM structures of CFTR are in a closed state, as inferred from the lack of a continuous passage through a hydrophobic bottleneck region located in the extracellular portion of the pore. Here, we present repeated, microsecond-long molecular dynamics simulations of human CFTR solvated in a lipid bilayer and aqueous NaCl. At equilibrium, Cl- ions enter the channel through a lateral intracellular portal and bind to two distinct cationic sites inside the channel pore but do not traverse the narrow, de-wetted bottleneck. Simulations conducted in the presence of a strong hyperpolarizing electric field led to spontaneous Cl- translocation events through the bottleneck region of the channel, suggesting that the protein relaxed to a functionally open state. Conformational changes of small magnitude involving transmembrane helices 1 and 6 preceded ion permeation through diverging exit routes at the extracellular end of the pore. The pore bottleneck undergoes wetting prior to Cl- translocation, suggesting that it acts as a hydrophobic gate. Although permeating Cl- ions remain mostly hydrated, partial dehydration occurs at the binding sites and in the bottleneck. The observed Cl- pathway is largely consistent with the loci of mutations that alter channel conductance, anion binding, and ion selectivity, supporting the model of the open state of CFTR obtained in the present study.
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Affiliation(s)
- Zhi Wei Zeng
- Molecular Medicine, Hospital for Sick Children, 686 Bay Street, Toronto, ON, M5G 0A4, Canada
- Department of Biochemistry, University of Toronto, Toronto, ON, M5S 1A8, Canada
| | - Paul Linsdell
- Department of Physiology and Biophysics, Dalhousie University, PO Box 15000, Halifax, NS, B3H 1X5, Canada
| | - Régis Pomès
- Molecular Medicine, Hospital for Sick Children, 686 Bay Street, Toronto, ON, M5G 0A4, Canada.
- Department of Biochemistry, University of Toronto, Toronto, ON, M5S 1A8, Canada.
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4
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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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5
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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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6
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Negoda A, Hogan MS, Cowley EA, Linsdell P. Contribution of the eighth transmembrane segment to the function of the CFTR chloride channel pore. Cell Mol Life Sci 2019; 76:2411-2423. [PMID: 30758641 PMCID: PMC11105405 DOI: 10.1007/s00018-019-03043-2] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/13/2018] [Revised: 01/18/2019] [Accepted: 02/06/2019] [Indexed: 12/20/2022]
Abstract
Our molecular understanding of the cystic fibrosis transmembrane conductance regulator (CFTR)-the chloride channel that is mutated in cystic fibrosis-has been greatly enhanced by a number of recent atomic-level structures of the protein in different conformations. One surprising aspect of these structures was the finding that the eighth of CFTR's 12 membrane-spanning segments (TM8) appeared close to the channel pore. Although functional evidence supports a role for other TMs in forming the pore, such a role for TM8 has not previously been reported. Here, we use patch-clamp recording to investigate the functional role of TM8. Using substituted cysteine accessibility mutagenesis, we find that three amino acid side-chains in TM8 (Y913, Y914, and Y917) are exposed to the extracellular, but not the intracellular, solution. Cysteine cross-linking experiments suggest that Y914 and Y917 are in close proximity to L102 (TM1) and F337 (TM6), respectively, suggesting that TM8 contributes to the narrow selectivity filter region of the pore. Different amino acid substitutions suggest that Y914, and to a lesser extent Y917, play important roles in controlling anion flux through the open channel. Furthermore, substitutions that reduce side-chain volume at Y917 severely affect channel gating, resulting in a channel with an extremely unstable open state. Our results suggest that pore-lining TM8 is among the most important TMs controlling the permeation phenotype of the CFTR channel, and also that movement of TM8 may be critically involved in channel gating.
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Affiliation(s)
- Alexander Negoda
- Department of Physiology and Biophysics, Dalhousie University, PO Box 15000, Halifax, NS, B3H 4R2, Canada
| | - Mairin S Hogan
- Department of Physiology and Biophysics, Dalhousie University, PO Box 15000, Halifax, NS, B3H 4R2, Canada
| | - Elizabeth A Cowley
- Department of Physiology and Biophysics, Dalhousie University, PO Box 15000, Halifax, NS, B3H 4R2, Canada
| | - Paul Linsdell
- Department of Physiology and Biophysics, Dalhousie University, PO Box 15000, Halifax, NS, B3H 4R2, Canada.
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7
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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: 171] [Impact Index Per Article: 28.5] [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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8
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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: 74] [Impact Index Per Article: 10.6] [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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9
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Das J, Aleksandrov AA, Cui L, He L, Riordan JR, Dokholyan NV. Transmembrane helical interactions in the CFTR channel pore. PLoS Comput Biol 2017. [PMID: 28640808 PMCID: PMC5501672 DOI: 10.1371/journal.pcbi.1005594] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/23/2022] Open
Abstract
Mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene affect CFTR protein biogenesis or its function as a chloride channel, resulting in dysregulation of epithelial fluid transport in the lung, pancreas and other organs in cystic fibrosis (CF). Development of pharmaceutical strategies to treat CF requires understanding of the mechanisms underlying channel function. However, incomplete 3D structural information on the unique ABC ion channel, CFTR, hinders elucidation of its functional mechanism and correction of cystic fibrosis causing mutants. Several CFTR homology models have been developed using bacterial ABC transporters as templates but these have low sequence similarity to CFTR and are not ion channels. Here, we refine an earlier model in an outward (OWF) and develop an inward (IWF) facing model employing an integrated experimental-molecular dynamics simulation (200 ns) approach. Our IWF structure agrees well with a recently solved cryo-EM structure of a CFTR IWF state. We utilize cysteine cross-linking to verify positions and orientations of residues within trans-membrane helices (TMHs) of the OWF conformation and to reconstruct a physiologically relevant pore structure. Comparison of pore profiles of the two conformations reveal a radius sufficient to permit passage of hydrated Cl- ions in the OWF but not the IWF model. To identify structural determinants that distinguish the two conformations and possible rearrangements of TMHs within them responsible for channel gating, we perform cross-linking by bifunctional reagents of multiple predicted pairs of cysteines in TMH 6 and 12 and 6 and 9. To determine whether the effects of cross-linking on gating observed are the result of switching of the channel from open to close state, we also treat the same residue pairs with monofunctional reagents in separate experiments. Both types of reagents prevent ion currents indicating that pore blockage is primarily responsible.
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Affiliation(s)
- Jhuma Das
- Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America
| | - Andrei A. Aleksandrov
- Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America
- Cystic Fibrosis Treatment and Research Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America
| | - Liying Cui
- Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America
- Cystic Fibrosis Treatment and Research Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America
| | - Lihua He
- Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America
- Cystic Fibrosis Treatment and Research Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America
| | - John R. Riordan
- Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America
- Cystic Fibrosis Treatment and Research Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America
- * E-mail: (JRR); (NVD)
| | - Nikolay V. Dokholyan
- Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America
- Cystic Fibrosis Treatment and Research Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America
- * E-mail: (JRR); (NVD)
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Zhang Z, Chen J. Atomic Structure of the Cystic Fibrosis Transmembrane Conductance Regulator. Cell 2017; 167:1586-1597.e9. [PMID: 27912062 DOI: 10.1016/j.cell.2016.11.014] [Citation(s) in RCA: 246] [Impact Index Per Article: 30.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/09/2016] [Revised: 10/28/2016] [Accepted: 11/04/2016] [Indexed: 02/07/2023]
Abstract
The cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel evolved from the ATP-binding cassette (ABC) transporter family. In this study, we determined the structure of zebrafish CFTR in the absence of ATP by electron cryo-microscopy to 3.7 Å resolution. Human and zebrafish CFTR share 55% sequence identity, and 42 of the 46 cystic-fibrosis-causing missense mutational sites are identical. In CFTR, we observe a large anion conduction pathway lined by numerous positively charged residues. A single gate near the extracellular surface closes the channel. The regulatory domain, dephosphorylated, is located in the intracellular opening between the two nucleotide-binding domains (NBDs), preventing NBD dimerization and channel opening. The structure also reveals why many cystic-fibrosis-causing mutations would lead to defects either in folding, ion conduction, or gating and suggests new avenues for therapeutic intervention.
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Affiliation(s)
- Zhe Zhang
- The Rockefeller University and Howard Hughes Medical Institute, 1230 York Avenue, New York, NY 10065, USA
| | - Jue Chen
- The Rockefeller University and Howard Hughes Medical Institute, 1230 York Avenue, New York, NY 10065, USA.
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11
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Negoda A, El Hiani Y, Cowley EA, Linsdell P. Contribution of a leucine residue in the first transmembrane segment to the selectivity filter region in the CFTR chloride channel. BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES 2017; 1859:1049-1058. [DOI: 10.1016/j.bbamem.2017.02.014] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Received: 11/18/2016] [Revised: 02/01/2017] [Accepted: 02/20/2017] [Indexed: 12/15/2022]
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12
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Linsdell P. Architecture and functional properties of the CFTR channel pore. Cell Mol Life Sci 2017; 74:67-83. [PMID: 27699452 PMCID: PMC11107662 DOI: 10.1007/s00018-016-2389-5] [Citation(s) in RCA: 35] [Impact Index Per Article: 4.4] [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: 12/12/2022]
Abstract
The main function of the cystic fibrosis transmembrane conductance regulator (CFTR) is as an ion channel for the movement of small anions across epithelial cell membranes. As an ion channel, CFTR must form a continuous pathway across the cell membrane-referred to as the channel pore-for the rapid electrodiffusional movement of ions. This review summarizes our current understanding of the architecture of the channel pore, as defined by electrophysiological analysis and molecular modeling studies. This includes consideration of the characteristic functional properties of the pore, definition of the overall shape of the entire extent of the pore, and discussion of how the molecular structure of distinct regions of the pore might control different facets of pore function. Comparisons are drawn with closely related proteins that are not ion channels, and also with structurally unrelated proteins with anion channel function. A simple model of pore function is also described.
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Affiliation(s)
- Paul Linsdell
- Department of Physiology and Biophysics, Dalhousie University, PO Box 15000, Halifax, NS, B3H 4R2, Canada.
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13
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Wang H, Cebotaru L, Lee HW, Yang Q, Pollard BS, Pollard HB, Guggino WB. CFTR Controls the Activity of NF-κB by Enhancing the Degradation of TRADD. Cell Physiol Biochem 2016; 40:1063-1078. [PMID: 27960153 DOI: 10.1159/000453162] [Citation(s) in RCA: 27] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 11/11/2016] [Indexed: 01/08/2023] Open
Abstract
BACKGROUND/AIMS Chronic lung infection in cystic fibrosis leads to an inflammatory response that persists because of the chronic presence of bacteria and ultimately leads to a catastrophic failure of lung function. METHODS We use a combination of biochemistry, cell and molecular biology to study the interaction of TRADD, a key adaptor molecule in TNFα signaling, with CFTR in the regulation of NFκB. RESULTS We show that Wt CFTR binds to and colocalizes with TRADD. TRADD is a key signaling intermediate connecting TNFα with activation of NFκB. By contrast, ΔF508 CFTR does not bind to TRADD. NF-κB activation is higher in CFBE expressing ΔF508 CFTR than in cells expressing Wt CFTR. However, this differential effect is abolished when TRADD levels are knocked down. Transfecting Wt CFTR into CFBE cells reduces NF-κB activity. However the reduction is abolished by the CFTR chloride transport inhibitor-172. Consistently, transfecting in the correctly trafficked CFTR conduction mutants G551D or S341A also fail to reduce NFκB activity. Thus CFTR must be functional if it is to regulate NF-κB activity. We also found that TNFα produced a greater increase in NF-κB activity in CFBE cells than in the same cell when Wt CFTR-corrected. Consistently, the effect is also abolished when TRADD is knocked down by shRNA. Thus, Wt CFTR control of TRADD modulates the physiological activation of NF-κB by TNFα. Based on studies with proteosomal and lysosomal inhibitors, the mechanism by which Wt CFTR, but not ΔF508 CFTR, suppresses TRADD is by lysosomal degradation. CONCLUSION We have uncovered a novel mechanism whereby Wt CFTR regulates TNFα signaling by enhancing TRADD degradation. Thus by reducing the levels of TRADD, Wt CFTR suppresses downstream proinflammatory NFκB signaling. By contrast, suppression of NF-κB activation fails in CF cells expressing ΔF508 CFTR.
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Affiliation(s)
- Hua Wang
- Department of Physiology, Medicine, School of Medicine, The Johns Hopkins University, Baltimore, USA
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14
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Qian F, Liu L, Liu Z, Lu C. The pore architecture of the cystic fibrosis transmembrane conductance regulator channel revealed by co-mutation in pore-forming transmembrane regions. Physiol Res 2016; 65:505-15. [PMID: 27070741 DOI: 10.33549/physiolres.933143] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/27/2022] Open
Abstract
The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel contains 12 transmembrane (TM) regions that are presumed to form the channel pore. However, there is no direct evidence clearly illustrating the involvement of these transmembrane regions in the actual CFTR pore structure. To obtain insight into the architecture of the CFTR channel pore, we used patch clamp recording techniques and a strategy of co-mutagenesis of two potential pore-forming transmembrane regions (TM1 and TM6) to investigate the collaboration of these two TM regions. We performed a range of specific functional assays comparing the single channel conductance, anion binding, and anion selectivity properties of the co-mutated CFTR variants, and the results indicated that TM1 and TM6 play vital roles in forming the channel pore and, thus, determine the functional properties of the channel. Furthermore, we provided functional evidence that the amino acid threonine (T338) in TM6 has synergic effects with lysine (K95) in TM1. Therefore, we propose that these two residues have functional collaboration in the CFTR channel pore and may collectively form a selective filter.
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Affiliation(s)
- F Qian
- Laboratory of Neuronal Network and Brain Diseases Modulation, Yangtze University, Jingzhou, Hubei province, China.
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15
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Sorum B, Czégé D, Csanády L. Timing of CFTR pore opening and structure of its transition state. Cell 2015; 163:724-33. [PMID: 26496611 DOI: 10.1016/j.cell.2015.09.052] [Citation(s) in RCA: 49] [Impact Index Per Article: 4.9] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/02/2015] [Revised: 08/25/2015] [Accepted: 09/18/2015] [Indexed: 12/12/2022]
Abstract
In CFTR, the chloride ion channel mutated in cystic fibrosis (CF) patients, pore opening is coupled to ATP-binding-induced dimerization of two cytosolic nucleotide binding domains (NBDs) and closure to dimer disruption following ATP hydrolysis. CFTR opening rate, unusually slow because of its high-energy transition state, is further slowed by CF mutation ΔF508. Here, we exploit equilibrium gating of hydrolysis-deficient CFTR mutant D1370N and apply rate-equilibrium free-energy relationship analysis to estimate relative timing of opening movements in distinct protein regions. We find clear directionality of motion along the longitudinal protein axis and identify an opening transition-state structure with the NBD dimer formed but the pore still closed. Thus, strain at the NBD/pore-domain interface, the ΔF508 mutation locus, underlies the energetic barrier for opening. Our findings suggest a therapeutic opportunity to stabilize this transition-state structure pharmacologically in ΔF508-CFTR to correct its opening defect, an essential step toward restoring CFTR function.
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Affiliation(s)
- Ben Sorum
- Department of Medical Biochemistry, Semmelweis University, Tűzoltó u. 37-47, Budapest 1094, Hungary; MTA-SE Ion Channel Research Group, Semmelweis University, Tűzoltó u. 37-47, Budapest 1094, Hungary
| | - Dávid Czégé
- MTA-SE Ion Channel Research Group, Semmelweis University, Tűzoltó u. 37-47, Budapest 1094, Hungary
| | - László Csanády
- Department of Medical Biochemistry, Semmelweis University, Tűzoltó u. 37-47, Budapest 1094, Hungary; MTA-SE Ion Channel Research Group, Semmelweis University, Tűzoltó u. 37-47, Budapest 1094, Hungary.
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16
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Cui G, McCarty NA. Murine and human CFTR exhibit different sensitivities to CFTR potentiators. Am J Physiol Lung Cell Mol Physiol 2015. [PMID: 26209275 DOI: 10.1152/ajplung.00181.2015] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/09/2023] Open
Abstract
Development of therapeutic molecules with clinical efficacy as modulators of defective CFTR includes efforts to identify potentiators that can overcome or repair the gating defect in mutant CFTR channels. This has taken a great leap forward with the identification of the potentiator VX-770, now available to patients as "Kalydeco." Other small molecules with different chemical structure also are capable of potentiating the activity of either wild-type or mutant CFTR, suggesting that there are features of the protein that may be targeted to achieve stimulation of channel activity by structurally diverse compounds. However, neither the mechanisms by which these compounds potentiate mutant CFTR nor the site(s) where these compounds bind have been identified. This knowledge gap partly reflects the lack of appropriate experimental models to provide clues toward the identification of binding sites. Here, we have compared the channel behavior and response to novel and known potentiators of human CFTR (hCFTR) and murine (mCFTR) expressed in Xenopus oocytes. Both hCFTR and mCFTR were blocked by GlyH-101 from the extracellular side, but mCFTR activity was increased with GlyH-101 applied directly to the cytoplasmic side. Similarly, glibenclamide only exhibited a blocking effect on hCFTR but both blocked and potentiated mCFTR in excised membrane patches and in intact oocytes. The clinically used CFTR potentiator VX-770 transiently increased hCFTR by ∼13% but potentiated mCFTR significantly more strongly. Our results suggest that mCFTR pharmacological sensitivities differ from hCFTR, which will provide a useful tool for identifying the binding sites and mechanism for these potentiators.
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Affiliation(s)
- Guiying Cui
- Division of Pulmonology, Allergy/Immunology, Cystic Fibrosis, and Sleep, Department of Pediatrics, Emory + Children's Center for Cystic Fibrosis and Airways Disease Research, Emory University School of Medicine and Children's Healthcare of Atlanta, Atlanta, Georgia
| | - Nael A McCarty
- Division of Pulmonology, Allergy/Immunology, Cystic Fibrosis, and Sleep, Department of Pediatrics, Emory + Children's Center for Cystic Fibrosis and Airways Disease Research, Emory University School of Medicine and Children's Healthcare of Atlanta, Atlanta, Georgia
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17
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Linsdell P. Interactions between permeant and blocking anions inside the CFTR chloride channel pore. BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES 2015; 1848:1573-90. [DOI: 10.1016/j.bbamem.2015.04.004] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Received: 01/13/2015] [Revised: 04/08/2015] [Accepted: 04/09/2015] [Indexed: 12/19/2022]
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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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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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Abstract
Cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), a member of the ATP-binding cassette (ABC) family of membrane transport proteins. CFTR is unique among ABC proteins in that it functions not as an active transporter but as an ATP-gated Cl(-) channel. As an ion channel, the function of the CFTR transmembrane channel pore that mediates Cl(-) movement has been studied in great detail. On the other hand, only low resolution structural data is available on the transmembrane parts of the protein. The structure of the channel pore has, however, been modeled on the known structure of active transporter ABC proteins. Currently, significant barriers exist to building a unified view of CFTR pore structure and function. Reconciling functional data on the channel with indirect structural data based on other proteins with very different transport functions and substrates has proven problematic. This review summarizes current structural and functional models of the CFTR Cl(-) channel pore, including a comprehensive review of previous electrophysiological investigations of channel structure and function. In addition, functional data on the three-dimensional arrangement of pore-lining helices, as well as contemporary hypotheses concerning conformational changes in the pore that occur during channel opening and closing, are discussed. Important similarities and differences between different models of the pore highlight current gaps in our knowledge of CFTR structure and function. In order to fill these gaps, structural and functional models of the membrane-spanning pore need to become better integrated.
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Affiliation(s)
- Paul Linsdell
- Department of Physiology & Biophysics, Dalhousie University , Halifax, Nova Scotia , Canada
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21
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Hwang TC, Kirk KL. The CFTR ion channel: gating, regulation, and anion permeation. Cold Spring Harb Perspect Med 2013; 3:a009498. [PMID: 23284076 DOI: 10.1101/cshperspect.a009498] [Citation(s) in RCA: 98] [Impact Index Per Article: 8.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/23/2022]
Abstract
Cystic fibrosis transmembrane conductance regulator (CFTR) is an ATP-gated anion channel with two remarkable distinctions. First, it is the only ATP-binding cassette (ABC) transporter that is known to be an ion channel--almost all others function as transport ATPases. Second, CFTR is the only ligand-gated channel that consumes its ligand (ATP) during the gating cycle--a consequence of its enzymatic activity as an ABC transporter. We discuss these special properties of CFTR in the context of its evolutionary history as an ABC transporter. Other topics include the mechanisms by which CFTR gating is regulated by phosphorylation of its unique regulatory domain and our current view of the CFTR permeation pathway (or pore). Understanding these basic operating principles of the CFTR channel is central to defining the mechanisms of action of prospective cystic fibrosis drugs and to the development of new, rational treatment strategies.
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Affiliation(s)
- Tzyh-Chang Hwang
- Dalton Cardiovascular Research Center, University of Missouri-Columbia, Columbia, MO 65211, USA
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22
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Dalton J, Kalid O, Schushan M, Ben-Tal N, Villà-Freixa J. New model of cystic fibrosis transmembrane conductance regulator proposes active channel-like conformation. J Chem Inf Model 2012; 52:1842-53. [PMID: 22747419 DOI: 10.1021/ci2005884] [Citation(s) in RCA: 68] [Impact Index Per Article: 5.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/23/2022]
Abstract
The cystic fibrosis transmembrane conductance regulator (CFTR) is an unusual ABC transporter, functioning as a chloride channel critical for fluid homeostasis in multiple organs. Disruption of CFTR function is associated with cystic fibrosis making it an attractive therapeutic target. In addition, CFTR blockers are being developed as potential antidiarrheals. CFTR drug discovery is hampered by the lack of high resolution structural data, and considerable efforts have been invested in modeling the channel structure. Although previously published CFTR models that have been made publicly available mostly agree with experimental data relating to the overall structure, they present the channel in an outward-facing conformation that does not agree with expected properties of a "channel-like" structure. Here, we make available a model of CFTR in such a "channel-like" conformation, derived by a unique modeling approach combining restrained homology modeling and ROSETTA refinement. In contrast to others, the present model is in agreement with expected channel properties such as pore shape, dimensions, solvent accessibility, and experimentally derived distances. We have used the model to explore the interaction of open channel blockers within the pore, revealing a common binding mode and ionic interaction with K95, in agreement with experimental data. The binding-site was further validated using a virtual screening enrichment experiment, suggesting the model might be suitable for drug discovery. In addition, we subjected the model to a molecular dynamics simulation, revealing previously unaddressed salt-bridge interactions that may be important for structure stability and pore-lining residues that may take part in Cl(-) conductance.
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Affiliation(s)
- James Dalton
- Computational Biochemistry and Biophysics Laboratory, Research Unit on Biomedical Informatics, IMIM Hospital del Mar and Universitat Pompeu Fabra, C/Doctor Aiguader, 88, 08003 Barcelona, Catalunya, Spain
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23
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Norimatsu Y, Ivetac A, Alexander C, Kirkham J, O’Donnell N, Dawson DC, Sansom MS. Cystic fibrosis transmembrane conductance regulator: a molecular model defines the architecture of the anion conduction path and locates a "bottleneck" in the pore. Biochemistry 2012; 51:2199-212. [PMID: 22352759 PMCID: PMC3316148 DOI: 10.1021/bi201888a] [Citation(s) in RCA: 50] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/23/2022]
Abstract
We developed molecular models for the cystic fibrosis transmembrane conductance regulator chloride channel based on the prokaryotic ABC transporter, Sav1866. Here we analyze predicted pore geometry and side-chain orientations for TM3, TM6, TM9, and TM12, with particular attention being paid to the location of the rate-limiting barrier for anion conduction. Side-chain orientations assayed by cysteine scanning were found to be from 77 to 90% in accord with model predictions. The predicted geometry of the anion conduction path was defined by a space-filling model of the pore and confirmed by visualizing the distribution of water molecules from a molecular dynamics simulation. The pore shape is that of an asymmetric hourglass, comprising a shallow outward-facing vestibule that tapers rapidly toward a narrow "bottleneck" linking the outer vestibule to a large inner cavity extending toward the cytoplasmic extent of the lipid bilayer. The junction between the outer vestibule and the bottleneck features an outward-facing rim marked by T338 in TM6 and I1131 in TM12, consistent with the observation that cysteines at both of these locations reacted with both channel-permeant and channel-impermeant, thiol-directed reagents. Conversely, cysteines substituted for S341 in TM6 or T1134 in TM12, predicted by the model to lie below the rim of the bottleneck, were found to react exclusively with channel-permeant reagents applied from the extracellular side. The predicted dimensions of the bottleneck are consistent with the demonstrated permeation of Cl(-), pseudohalide anions, water, and urea.
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Affiliation(s)
- Yohei Norimatsu
- Department of Physiology and Pharmacology, Oregon Health & Science University, Portland, Oregon 97239
| | - Anthony Ivetac
- Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, U.K
| | - Christopher Alexander
- Department of Physiology and Pharmacology, Oregon Health & Science University, Portland, Oregon 97239
| | - John Kirkham
- Department of Physiology and Pharmacology, Oregon Health & Science University, Portland, Oregon 97239
| | - Nicolette O’Donnell
- Department of Physiology and Pharmacology, Oregon Health & Science University, Portland, Oregon 97239
| | - David C. Dawson
- Department of Physiology and Pharmacology, Oregon Health & Science University, Portland, Oregon 97239
| | - Mark S.P. Sansom
- Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, U.K
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Bai Y, Li M, Hwang TC. Structural basis for the channel function of a degraded ABC transporter, CFTR (ABCC7). ACTA ACUST UNITED AC 2012; 138:495-507. [PMID: 22042986 PMCID: PMC3206304 DOI: 10.1085/jgp.201110705] [Citation(s) in RCA: 55] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/01/2023]
Abstract
Cystic fibrosis transmembrane conductance regulator (CFTR) is a member of the ATP-binding cassette (ABC) transporter superfamily, but little is known about how this ion channel that harbors an uninterrupted ion permeation pathway evolves from a transporter that works by alternately exposing its substrate conduit to the two sides of the membrane. Here, we assessed reactivity of intracellularly applied thiol-specific probes with cysteine residues substituted into the 12th transmembrane segment (TM12) of CFTR. Our experimental data showing high reaction rates of substituted cysteines toward the probes, strong blocker protection of cysteines against reaction, and reaction-induced alterations in channel conductance support the idea that TM12 of CFTR contributes to the lining of the ion permeation pathway. Together with previous work, these findings raise the possibility that pore-lining elements of CFTR involve structural components resembling those that form the substrate translocation pathway of ABC transporters. In addition, comparison of reaction rates in the open and closed states of the CFTR channel leads us to propose that upon channel opening, the wide cytoplasmic vestibule tightens and the pore-lining TM12 rotates along its helical axis. This simple model for gating conformational changes in the inner pore domain of CFTR argues that the gating transition of CFTR and the transport cycle of ABC proteins share analogous conformational changes. Collectively, our data corroborate the popular hypothesis that degradation of the cytoplasmic-side gate turned an ABC transporter into the CFTR channel.
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Affiliation(s)
- Yonghong Bai
- Dalton Cardiovascular Research Center, University of Missouri-Columbia, Columbia, MO 65211, USA
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Cui G, Song B, Turki HW, McCarty NA. Differential contribution of TM6 and TM12 to the pore of CFTR identified by three sulfonylurea-based blockers. Pflugers Arch 2011; 463:405-18. [PMID: 22160394 DOI: 10.1007/s00424-011-1035-1] [Citation(s) in RCA: 32] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/20/2011] [Revised: 09/14/2011] [Accepted: 09/30/2011] [Indexed: 02/06/2023]
Abstract
Previous studies suggested that four transmembrane domains 5, 6, 11, 12 make the greatest contribution to forming the pore of the CFTR chloride channel. We used excised, inside-out patches from oocytes expressing CFTR with alanine-scanning mutagenesis in amino acids in TM6 and TM12 to probe CFTR pore structure with four blockers: glibenclamide (Glyb), glipizide (Glip), tolbutamide (Tolb), and Meglitinide. Glyb and Glip blocked wildtype (WT)-CFTR in a voltage-, time-, and concentration-dependent manner. At V (M) = -120 mV with symmetrical 150 mM Cl(-) solution, fractional block of WT-CFTR by 50 μM Glyb and 200 μM Glip was 0.64 ± 0.03 (n = 7) and 0.48 ± 0.02 (n = 7), respectively. The major effects on block by Glyb and Glip were found with mutations at F337, S341, I344, M348, and V350 of TM6. Under similar conditions, fractional block of WT-CFTR by 300 μM Tolb was 0.40 ± 0.04. Unlike Glyb, Glip, and Meglitinide, block by Tolb lacked time-dependence (n = 7). We then tested the effects of alanine mutations in TM12 on block by Glyb and Glip; the major effects were found at N1138, T1142, V1147, N1148, S1149, S1150, I1151, and D1152. From these experiments, we infer that amino acids F337, S341, I344, M348, and V350 of TM6 face the pore when the channel is in the open state, while the amino acids of TM12 make less important contributions to pore function. These data also suggest that the region between F337 and S341 forms the narrow part of the CFTR pore.
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Affiliation(s)
- Guiying Cui
- Division of Pulmonology, Allergy/Immunology, Cystic Fibrosis, and Sleep, Department of Pediatrics, Center for Cystic Fibrosis Research, Children's Healthcare of Atlanta, Emory University School of Medicine, 2015 Uppergate Drive, Atlanta, GA 30322, USA
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26
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Stahl M, Stahl K, Brubacher MB, Forrest JN. Divergent CFTR orthologs respond differently to the channel inhibitors CFTRinh-172, glibenclamide, and GlyH-101. Am J Physiol Cell Physiol 2011; 302:C67-76. [PMID: 21940661 DOI: 10.1152/ajpcell.00225.2011] [Citation(s) in RCA: 27] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
Comparison of diverse orthologs is a powerful tool to study the structure and function of channel proteins. We investigated the response of human, killifish, pig, and shark cystic fibrosis transmembrane conductance regulator (CFTR) to specific inhibitors of the channel: CFTR(inh)-172, glibenclamide, and GlyH-101. In three systems, including organ perfusion of the shark rectal gland, primary cultures of shark rectal gland tubules, and expression studies of each ortholog in cRNA microinjected Xenopus laevis oocytes, we observed fundamental differences in the sensitivity to inhibition by these channel blockers. In organ perfusion studies, shark CFTR was insensitive to inhibition by CFTR(inh)-172. This insensitivity was also seen in short-circuit current experiments with cultured rectal gland tubular epithelial cells (maximum inhibition 4 ± 1.3%). In oocyte expression studies, shark CFTR was again insensitive to CFTR(inh)-172 (maximum inhibition 10.3 ± 2.5% at 25 μM), pig CFTR was insensitive to glibenclamide (maximum inhibition 18.4 ± 4.4% at 250 μM), and all orthologs were sensitive to GlyH-101. The amino acid residues considered responsible by previous site-directed mutagenesis for binding of the three inhibitors are conserved in the four CFTR isoforms studied. These experiments demonstrate a profound difference in the sensitivity of different orthologs of CFTR proteins to inhibition by CFTR blockers that cannot be explained by mutagenesis of single amino acids. We believe that the potency of the inhibitors CFTR(inh)-172, glibenclamide, and GlyH-101 on the CFTR chloride channel protein is likely dictated by the local environment and the three-dimensional structure of additional residues that form the vestibules, the chloride pore, and regulatory regions of the channel.
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Affiliation(s)
- Maximilian Stahl
- Nephrology Division, Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut 06510-3222, USA
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Functional arrangement of the 12th transmembrane region in the CFTR chloride channel pore based on functional investigation of a cysteine-less CFTR variant. Pflugers Arch 2011; 462:559-71. [PMID: 21796338 DOI: 10.1007/s00424-011-0998-2] [Citation(s) in RCA: 38] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/26/2011] [Revised: 06/27/2011] [Accepted: 07/15/2011] [Indexed: 12/25/2022]
Abstract
The membrane-spanning part of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel comprises 12 transmembrane (TM) α-helices, arranged into two pseudo-symmetrical groups of six. While TM6 in the N-terminal TMs is known to line the pore and to make an important contribution to channel properties, much less is known about its C-terminal counterpart, TM12. We have used patch clamp recording to investigate the accessibility of cytoplasmically applied cysteine-reactive reagents to cysteines introduced along the length of TM12 in a cysteine-less variant of CFTR. We find that methanethiosulfonate (MTS) reagents irreversibly modify cysteines substituted for TM12 residues N1138, M1140, S1141, T1142, Q1144, W1145, V1147, N1148, and S1149 when applied to the cytoplasmic side of open channels. Cysteines sensitive to internal MTS reagents were not modified by extracellular [2-(trimethylammonium)ethyl] MTS, consistent with MTS reagent impermeability. Both S1141C and T1142C could be modified by intracellular [2-sulfonatoethyl] MTS prior to channel activation; however, N1138C and M1140C, located deeper into the pore from its cytoplasmic end, were modified only after channel activation. Comparison of these results with previous work on CFTR-TM6 allows us to develop a model of the relative positions, functional contributions, and alignment of these two important TMs lining the CFTR pore. We also propose a mechanism by which these seemingly structurally symmetrical TMs make asymmetric contributions to the functional properties of the channel pore.
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Functional differences in pore properties between wild-type and cysteine-less forms of the CFTR chloride channel. J Membr Biol 2011; 243:15-23. [PMID: 21796426 DOI: 10.1007/s00232-011-9388-0] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/03/2011] [Accepted: 07/13/2011] [Indexed: 12/23/2022]
Abstract
Studies of the structure and function of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel have been advanced by the development of functional channel variants in which all 18 endogenous cysteine residues have been mutated ("cys-less" CFTR). However, cys-less CFTR has a slightly higher single-channel conductance than wild-type CFTR, raising questions as to the suitability of cys-less as a model of the wild-type CFTR pore. We used site-directed mutagenesis and patch-clamp recording to investigate the origin of this conductance difference and to determine the extent of functional differences between wild-type and cys-less CFTR channel permeation properties. Our results suggest that the conductance difference is the result of a single substitution, of C343: the point mutant C343S has a conductance similar to cys-less, whereas the reverse mutation, S343C in a cys-less background, restores wild-type conductance levels. Other cysteine substitutions (C128S, C225S, C376S, C866S) were without effect. Substitution of other residues for C343 suggested that conductance is dependent on amino acid side chain volume at this position. A range of other functional pore properties, including interactions with channel blockers (Au[CN] (2) (-) , 5-nitro-2-[3-phenylpropylamino]benzoic acid, suramin) and anion permeability, were not significantly different between wild-type and cys-less CFTR. Our results suggest that functional differences between these two CFTR constructs are of limited scale and scope and result from a small change in side chain volume at position 343. These results therefore support the use of cys-less as a model of the CFTR pore region.
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Bai Y, Li M, Hwang TC. Dual roles of the sixth transmembrane segment of the CFTR chloride channel in gating and permeation. ACTA ACUST UNITED AC 2010; 136:293-309. [PMID: 20805575 PMCID: PMC2931150 DOI: 10.1085/jgp.201010480] [Citation(s) in RCA: 71] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
Cystic fibrosis transmembrane conductance regulator (CFTR) is the only member of the adenosine triphosphate-binding cassette (ABC) transporter superfamily that functions as a chloride channel. Previous work has suggested that the external side of the sixth transmembrane segment (TM6) plays an important role in governing chloride permeation, but the function of the internal side remains relatively obscure. Here, on a cysless background, we performed cysteine-scanning mutagenesis and modification to screen the entire TM6 with intracellularly applied thiol-specific methanethiosulfonate reagents. Single-channel amplitude was reduced in seven cysteine-substituted mutants, suggesting a role of these residues in maintaining the pore structure for normal ion permeation. The reactivity pattern of differently charged reagents suggests that the cytoplasmic part of TM6 assumes a secondary structure of an alpha helix, and that reactive sites (341, 344, 345, 348, 352, and 353) reside in two neighboring faces of the helix. Although, as expected, modification by negatively charged reagents inhibits anion permeation, interestingly, modification by positively charged reagents of cysteine thiolates on one face (344, 348, and 352) of the helix affects gating. For I344C and M348C, the open time was prolonged and the closed time was shortened after modification, suggesting that depositions of positive charges at these positions stabilize the open state but destabilize the closed state. For R352C, which exhibited reduced single-channel amplitude, modifications by two positively charged reagents with different chemical properties completely restored the single-channel amplitude but had distinct effects on both the open time and the closed time. These results corroborate the idea that a helix rotation of TM6, which has been proposed to be part of the molecular motions during transport cycles in other ABC transporters, is associated with gating of the CFTR pore.
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Affiliation(s)
- Yonghong Bai
- Dalton Cardiovascular Research Center, Department of Biological Engineering, University of Missouri-Columbia, Columbia, MO 65211, USA
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Li MS, Holstead RG, Wang W, Linsdell P. Regulation of CFTR chloride channel macroscopic conductance by extracellular bicarbonate. Am J Physiol Cell Physiol 2010; 300:C65-74. [PMID: 20926782 DOI: 10.1152/ajpcell.00290.2010] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/10/2023]
Abstract
The CFTR contributes to Cl⁻ and HCO₃⁻ transport across epithelial cell apical membranes. The extracellular face of CFTR is exposed to varying concentrations of Cl⁻ and HCO₃⁻ in epithelial tissues, and there is evidence that CFTR is sensitive to changes in extracellular anion concentrations. Here we present functional evidence that extracellular Cl⁻ and HCO₃⁻ regulate anion conduction in open CFTR channels. Using cell-attached and inside-out patch-clamp recordings from constitutively active mutant E1371Q-CFTR channels, we show that voltage-dependent inhibition of CFTR currents in intact cells is significantly stronger when the extracellular solution contains HCO₃⁻ than when it contains Cl⁻. This difference appears to reflect differences in the ability of extracellular HCO₃⁻ and Cl⁻ to interact with and repel intracellular blocking anions from the pore. Strong block by endogenous cytosolic anions leading to reduced CFTR channel currents in intact cells occurs at physiologically relevant HCO₃⁻ concentrations and membrane potentials and can result in up to ∼50% inhibition of current amplitude. We propose that channel block by cytosolic anions is a previously unrecognized, physiologically relevant mechanism of channel regulation that confers on CFTR channels sensitivity to different anions in the extracellular fluid. We further suggest that this anion sensitivity represents a feedback mechanism by which CFTR-dependent anion secretion could be regulated by the composition of the secretions themselves. Implications for the mechanism and regulation of CFTR-dependent secretion in epithelial tissues are discussed.
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Affiliation(s)
- Man-Song Li
- Department of Physiology and Biophysics, Dalhousie University, Halifax, Nova Scotia, Canada
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31
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Identification of RL-TGR, a coreceptor involved in aversive chemical signaling. Proc Natl Acad Sci U S A 2010; 107:12339-44. [PMID: 20566865 DOI: 10.1073/pnas.1000343107] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022] Open
Abstract
Chemical signaling plays an important role in predator-prey interactions and feeding dynamics. Like other organisms that are sessile or slow moving, some marine sponges contain aversive compounds that defend these organisms from predation. We sought to identify and characterize a fish chemoreceptor that detects one of these compounds. Using expression cloning in Xenopus oocytes coexpressing the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel, the beta-2 adrenergic receptor (beta(2)AR), and fractions of a zebrafish cDNA library, we isolated a cDNA clone encoding receptor activity-modifying protein (RAMP)-like triterpene glycoside receptor (RL-TGR), a novel coreceptor involved in signaling in response to triterpene glycosides. This coreceptor appears to be structurally and functionally related to RAMPs, a family of coreceptors that physically associate with and modify the activity of G protein-coupled receptors (GPCRs). In membranes from formoside-responsive oocytes, RL-TGR was immunoprecipitated in an apparent complex with beta(2)AR. In HEK293 cells, coexpression of beta(2)AR induced the trafficking of RL-TGR from the cytoplasm to the plasma membrane. These results suggest that RL-TGR in the predatory fish physically associates with the beta(2)AR or another, more physiologically relevant GPCR and modifies its pharmacology to respond to triterpene glycosides found in sponges that serve as a potential food source for the fish. RL-TGR forms a coreceptor that responds to a chemical defense compound in the marine environment, and its discovery might lead the way to the identification of other receptors that mediate chemical defense signaling.
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Haack KKV, Tougas MR, Jones KT, El-Dahr SS, Radhakrishna H, McCarty NA. A Novel Bioassay for Detecting GPCR Heterodimerization. ACTA ACUST UNITED AC 2010; 15:251-60. [DOI: 10.1177/1087057109360254] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
Many G-protein-coupled receptors (GPCRs) have been shown to form heteromeric complexes primarily by biochemical methods, including competitive radioligand binding assays or measurements of changes in second-messenger concentration in lysed cells. These results are often cell line specific, and the expression of other cell surface proteins makes it difficult to detect potential functional consequences of GPCR interaction. Here, 2-electrode voltage clamping in Xenopus oocytes was used as a bioassay to explore heterodimerization of bradykinin type 2 receptor (Bk2R) and beta 2 adrenergic receptor (β2AR), using chloride channels as outputs for receptor activation. The data show for the first time that these 2 receptors heterodimerize with functional consequences. Stimulation with bradykinin induced activation of Gαq- and transactivation of Gαs-coupled pathways in oocytes expressing Bk2R and β2AR. To corroborate these data, potential receptor interaction was examined in PC12 cells, a cell line that endogenously expresses both receptors, and confirmed that stimulation with bradykinin transactivates β2AR. In both oocytes and PC12 cells, transactivation was ablated by Bk2R or β2AR inverse agonists, suggesting that transactivation occurred directly through both receptors. This is the first evidence of Bk2R/β2AR physical interaction, forming a functional heterodimer. The oocyte system may prove highly useful for exploration of GPCR heterodimerization and the functional consequences thereof.
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Affiliation(s)
- Karla K. V. Haack
- School of Biology, Georgia Institute of Technology, Atlanta, Georgia
- Department of Pediatrics, Emory University, and Children’s Healthcare of Atlanta, Atlanta, Georgia
| | | | - Kymry T. Jones
- School of Biology, Georgia Institute of Technology, Atlanta, Georgia
| | - Samir S. El-Dahr
- Department of Pediatrics, Tulane University, New Orleans, Louisiana
| | | | - Nael A. McCarty
- School of Biology, Georgia Institute of Technology, Atlanta, Georgia
- Department of Pediatrics, Emory University, and Children’s Healthcare of Atlanta, Atlanta, Georgia
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Zhou JJ, Li MS, Qi J, Linsdell P. Regulation of conductance by the number of fixed positive charges in the intracellular vestibule of the CFTR chloride channel pore. ACTA ACUST UNITED AC 2010; 135:229-45. [PMID: 20142516 PMCID: PMC2828907 DOI: 10.1085/jgp.200910327] [Citation(s) in RCA: 62] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/15/2023]
Abstract
Rapid chloride permeation through the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel is dependent on the presence of fixed positive charges in the permeation pathway. Here, we use site-directed mutagenesis and patch clamp recording to show that the functional role played by one such positive charge (K95) in the inner vestibule of the pore can be "transplanted" to a residue in a different transmembrane (TM) region (S1141). Thus, the mutant channel K95S/S1141K showed Cl(-) conductance and open-channel blocker interactions similar to those of wild-type CFTR, thereby "rescuing" the effects of the charge-neutralizing K95S mutation. Furthermore, the function of K95C/S1141C, but not K95C or S1141C, was inhibited by the oxidizing agent copper(II)-o-phenanthroline, and this inhibition was reversed by the reducing agent dithiothreitol, suggesting disulfide bond formation between these two introduced cysteine side chains. These results suggest that the amino acid side chains of K95 (in TM1) and S1141 (in TM12) are functionally interchangeable and located closely together in the inner vestibule of the pore. This allowed us to investigate the functional effects of increasing the number of fixed positive charges in this vestibule from one (in wild type) to two (in the S1141K mutant). The S1141K mutant had similar Cl(-) conductance as wild type, but increased susceptibility to channel block by cytoplasmic anions including adenosine triphosphate, pyrophosphate, 5-nitro-2-(3-phenylpropylamino)benzoic acid, and Pt(NO(2))(4)(2-) in inside-out membrane patches. Furthermore, in cell-attached patch recordings, apparent voltage-dependent channel block by cytosolic anions was strengthened by the S1141K mutation. Thus, the Cl(-) channel function of CFTR is maximal with a single fixed positive charge in this part of the inner vestibule of the pore, and increasing the number of such charges to two causes a net decrease in overall Cl(-) transport through a combination of failure to increase Cl(-) conductance and increased susceptibility to channel block by cytosolic substances.
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Affiliation(s)
- Jing-Jun Zhou
- Department of Physiology and Biophysics, Dalhousie University, Halifax, Nova Scotia B3H 1X5, Canada
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Alexander C, Ivetac A, Liu X, Norimatsu Y, Serrano JR, Landstrom A, Sansom M, Dawson DC. Cystic fibrosis transmembrane conductance regulator: using differential reactivity toward channel-permeant and channel-impermeant thiol-reactive probes to test a molecular model for the pore. Biochemistry 2009; 48:10078-88. [PMID: 19754156 PMCID: PMC2765204 DOI: 10.1021/bi901314c] [Citation(s) in RCA: 64] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/19/2023]
Abstract
The sixth transmembrane segment (TM6) of the CFTR chloride channel has been intensively investigated. The effects of amino acid substitutions and chemical modification of engineered cysteines (cysteine scanning) on channel properties strongly suggest that TM6 is a key component of the anion-conducting pore, but previous cysteine-scanning studies of TM6 have produced conflicting results. Our aim was to resolve these conflicts by combining a screening strategy based on multiple, thiol-directed probes with molecular modeling of the pore. CFTR constructs were screened for reactivity toward both channel-permeant and channel-impermeant thiol-directed reagents, and patterns of reactivity in TM6 were mapped onto two new, molecular models of the CFTR pore: one based on homology modeling using Sav1866 as the template and a second derived from the first by molecular dynamics simulation. Comparison of the pattern of cysteine reactivity with model predictions suggests that nonreactive sites are those where the TM6 side chains are occluded by other TMs. Reactive sites, in contrast, are generally situated such that the respective amino acid side chains either project into the predicted pore or lie within a predicted extracellular loop. Sites where engineered cysteines react with both channel-permeant and channel-impermeant probes occupy the outermost extent of TM6 or the predicted TM5-6 loop. Sites where cysteine reactivity is limited to channel-permeant probes occupy more cytoplasmic locations. The results provide an initial validation of two, new molecular models for CFTR and suggest that molecular dynamics simulation will be a useful tool for unraveling the structural basis of anion conduction by CFTR.
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Affiliation(s)
- Christopher Alexander
- Department of Physiology and Pharmacology, Oregon Health and Science University, Portland, Oregon 97239, USA
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Mornon JP, Lehn P, Callebaut I. Molecular models of the open and closed states of the whole human CFTR protein. Cell Mol Life Sci 2009; 66:3469-86. [PMID: 19707853 PMCID: PMC11115851 DOI: 10.1007/s00018-009-0133-0] [Citation(s) in RCA: 127] [Impact Index Per Article: 7.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/25/2009] [Revised: 07/17/2009] [Accepted: 08/12/2009] [Indexed: 12/15/2022]
Abstract
Cystic fibrosis transmembrane conductance regulator (CFTR), involved in cystic fibrosis (CF), is a chloride channel belonging to the ATP-binding cassette (ABC) superfamily. Using the experimental structure of Sav1866 as template, we previously modeled the human CFTR structure, including membrane-spanning domains (MSD) and nucleotide-binding domains (NBD), in an outward-facing conformation (open channel state). Here, we constructed a model of the CFTR inward-facing conformation (closed channel) on the basis of the recent corrected structures of MsbA and compared the structural features of those two states of the channel. Interestingly, the MSD:NBD coupling interfaces including F508 (DeltaF508 being the most common CF mutation) are mainly left unchanged. This prediction, completed by the modeling of the regulatory R domain, is supported by experimental data and provides a molecular basis for a better understanding of the functioning of CFTR, especially of the structural features that make CFTR the unique channel among the ABC transporters.
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Affiliation(s)
- Jean-Paul Mornon
- IMPMC, UMR7590, CNRS, Universités Pierre et Marie Curie-Paris 6 et Denis Diderot-Paris 7, 140 rue de Lourmel, Paris, France
| | - Pierre Lehn
- INSERM U613, IFR148 ScInBioS, Université de Bretagne Occidentale, Brest, France
| | - Isabelle Callebaut
- IMPMC, UMR7590, CNRS, Universités Pierre et Marie Curie-Paris 6 et Denis Diderot-Paris 7, 140 rue de Lourmel, Paris, France
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Zhou JJ, Linsdell P. Evidence that extracellular anions interact with a site outside the CFTR chloride channel pore to modify channel properties. Can J Physiol Pharmacol 2009; 87:387-95. [PMID: 19448737 DOI: 10.1139/y09-023] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/19/2022]
Abstract
Extracellular anions enter into the pore of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel, interacting with binding sites on the pore walls and with other anions inside the pore. There is increasing evidence that extracellular anions may also interact with sites away from the channel pore to influence channel properties. We have used site-directed mutagenesis and patch-clamp recording to identify residues that influence interactions with external anions. Anion interactions were assessed by the ability of extracellular Pt(NO2)42- ions to weaken the pore-blocking effect of intracellular Pt(NO2)42- ions, a long-range ion-ion interaction that does not appear to reflect ion interactions inside the pore. We found that mutations that remove positive charges in the 4th extracellular loop of CFTR (K892Q and R899Q) significantly alter the interaction between extracellular and intracellular Pt(NO2)42- ions. These mutations do not affect unitary Cl- conductance or block of single-channel currents by extracellular Pt(NO2)42- ions, however, suggesting that the mutated residues are not in the channel pore region. These results suggest that extracellular anions can regulate CFTR pore properties by binding to a site outside the pore region, probably by a long-range conformational change. Our findings also point to a novel function of the long 4th extracellular loop of the CFTR protein in sensing and (or) responding to anions in the extracellular solution.
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Affiliation(s)
- Jing-Jun Zhou
- Department of Physiology and Biophysics, Dalhousie University, 5850 College Street, Halifax, NS B3H 1X5, Canada
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Fatehi M, Linsdell P. Novel residues lining the CFTR chloride channel pore identified by functional modification of introduced cysteines. J Membr Biol 2009; 228:151-64. [PMID: 19381710 DOI: 10.1007/s00232-009-9167-3] [Citation(s) in RCA: 34] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/02/2009] [Accepted: 03/24/2009] [Indexed: 12/18/2022]
Abstract
Substituted cysteine accessibility mutagenesis (SCAM) has been used widely to identify pore-lining amino acid side chains in ion channel proteins. However, functional effects on permeation and gating can be difficult to separate, leading to uncertainty concerning the location of reactive cysteine side chains. We have combined SCAM with investigation of the charge-dependent effects of methanethiosulfonate (MTS) reagents on the functional permeation properties of cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channels. We find that cysteines substituted for seven out of 21 continuous amino acids in the eleventh and twelfth transmembrane (TM) regions can be modified by external application of positively charged [2-(trimethylammonium)ethyl] MTS bromide (MTSET) and negatively charged sodium [2-sulfonatoethyl] MTS (MTSES). Modification of these cysteines leads to changes in the open channel current-voltage relationship at both the macroscopic and single-channel current levels that reflect specific, charge-dependent effects on the rate of Cl(-) permeation through the channel from the external solution. This approach therefore identifies amino acid side chains that lie within the permeation pathway. Cysteine mutagenesis of pore-lining residues also affects intrapore anion binding and anion selectivity, giving more information regarding the roles of these residues. Our results demonstrate a straightforward method of screening for pore-lining amino acids in ion channels. We suggest that TM11 contributes to the CFTR pore and that the extracellular loop between TMs 11 and 12 lies close to the outer mouth of the pore.
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Affiliation(s)
- Mohammad Fatehi
- Department of Physiology and Biophysics, Dalhousie University, 5850 College Street, Halifax, NS B3H 1X5, Canada
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Evolutionary and functional divergence between the cystic fibrosis transmembrane conductance regulator and related ATP-binding cassette transporters. Proc Natl Acad Sci U S A 2008; 105:18865-70. [PMID: 19020075 DOI: 10.1073/pnas.0806306105] [Citation(s) in RCA: 52] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/29/2023] Open
Abstract
The cystic fibrosis transmembrane conductance regulator (CFTR) is a member of the ATP-binding cassette (ABC) transporter superfamily, an ancient family of proteins found in all phyla. In nearly all cases, ABC proteins are transporters that couple the hydrolysis of ATP to the transmembrane movement of substrate via an alternating access mechanism. In contrast, CFTR is best known for its activity as an ATP-dependent chloride channel. We asked why CFTR, which shares the domain architecture of ABC proteins that function as transporters, exhibits functional divergence. We compared CFTR protein sequences to those of other ABC transporters, which identified the ABCC4 proteins as the closest mammalian paralogs, and used statistical analysis of the CFTR-ABCC4 multiple sequence alignment to identify the specific domains and residues most likely to be involved in the evolutionary transition from transporter to channel activity. Among the residues identified as being involved in CFTR functional divergence, by virtue of being both CFTR-specific and conserved among all CFTR orthologs, was R352 in the sixth transmembrane helix (TM6). Patch-clamp experiments show that R352 interacts with D993 in TM9 to stabilize the open-channel state; D993 is absolutely conserved between CFTRs and ABCC4s. These data suggest that CFTR channel activity evolved, at least in part, by converting the conformational changes associated with binding and hydrolysis of ATP, as are found in true ABC Transporters, into an open permeation pathway by means of intraprotein interactions that stabilize the open state. This analysis sets the stage for understanding the evolutionary and functional relationships that make CFTR a unique ABC transporter protein.
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Tang L, Fatehi M, Linsdell P. Mechanism of direct bicarbonate transport by the CFTR anion channel. J Cyst Fibros 2008; 8:115-21. [PMID: 19019741 DOI: 10.1016/j.jcf.2008.10.004] [Citation(s) in RCA: 79] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/09/2008] [Revised: 10/20/2008] [Accepted: 10/20/2008] [Indexed: 01/11/2023]
Abstract
BACKGROUND CFTR contributes to HCO(3)(-) transport in epithelial cells both directly (by HCO(3)(-) permeation through the channel) and indirectly (by regulating Cl(-)/HCO(3)(-) exchange proteins). While loss of HCO(3)(-) transport is highly relevant to cystic fibrosis, the relative importance of direct and indirect HCO(3)(-) transport it is currently unknown. METHODS Patch clamp recordings from membrane patches excised from cells heterologously expressing wild type and mutant forms of human CFTR were used to isolate directly CFTR-mediated HCO(3)(-) transport and characterize its functional properties. RESULTS The permeability of HCO(3)(-) was approximately 25% that of Cl(-) and was invariable under all ionic conditions studied. CFTR-mediated HCO(3)(-) currents were inhibited by open channel blockers DNDS, glibenclamide and suramin, and these inhibitions were affected by mutations within the channel pore. Cystic fibrosis mutations previously associated with disrupted cellular HCO(3)(-) transport did not affect direct HCO(3)(-) permeability. CONCLUSIONS Cl(-) and HCO(3)(-) share a common transport pathway in CFTR, and selectivity between Cl(-) and HCO(3)(-) is independent of ionic conditions. The mechanism of transport is therefore effectively identical for both ions. We suggest that mutations in CFTR that cause cystic fibrosis by selectively disrupting HCO(3)(-) transport do not impair direct CFTR-mediated HCO(3)(-) transport, but may predominantly alter CFTR regulation of other HCO(3)(-) transport pathways.
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Affiliation(s)
- Lin Tang
- Department of Physiology & Biophysics, Dalhousie University, Nova Scotia, Canada
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40
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Zhou JJ, Fatehi M, Linsdell P. Identification of positive charges situated at the outer mouth of the CFTR chloride channel pore. Pflugers Arch 2008; 457:351-60. [PMID: 18449561 DOI: 10.1007/s00424-008-0521-6] [Citation(s) in RCA: 38] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/29/2008] [Revised: 04/14/2008] [Accepted: 04/17/2008] [Indexed: 11/30/2022]
Abstract
We have used site-directed mutagenesis and functional analysis to identify positively charged amino acid residues in the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel that interact with extracellular anions. Mutation of two positively charged arginine residues in the first extracellular loop (ECL) of CFTR, R104, and R117, as well as lysine residue K335 in the sixth transmembrane region, leads to inward rectification of the current-voltage relationship and decreased single channel conductance. These effects are dependent on the charge of the substituted side chain and on the Cl(-) concentration, suggesting that these positive charges normally act to concentrate extracellular Cl(-) ions near the outer mouth of the pore. Side chain charge-dependent effects are mimicked by manipulating charge in situ by mutating these amino acids to cysteine followed by covalent modification with charged cysteine-reactive reagents, confirming the location of these side chains within the pore outer vestibule. State-independent modification of R104C and R117C suggests that these residues are located at the outermost part of the pore. We suggest that ECL1 contributes to the CFTR pore external vestibule and that positively charged amino acid side chains in this region act to attract Cl(-) ions into the pore. In contrast, we find no evidence that fixed positive charges in other ECLs contribute to the permeation properties of the pore.
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Affiliation(s)
- Jing-Jun Zhou
- Department of Physiology & Biophysics, Dalhousie University, Halifax, NS, B3H 1X5, Canada
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Cohen SAP, Hatt H, Kubanek J, McCarty NA. Reconstitution of a chemical defense signaling pathway in a heterologous system. ACTA ACUST UNITED AC 2008; 211:599-605. [PMID: 18245637 DOI: 10.1242/jeb.009225] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
Abstract
Chemical signaling plays an important role in ecological interactions, such as communication and predator-prey dynamics. Since sessile species cannot physically escape predators, many contain compounds that deter predation; however, it is largely unknown how predators physiologically detect deterrent chemicals. Few studies have investigated ecologically relevant aversive taste responses in any predator. Our objective was to determine if a signaling pathway for detecting marine sponge-derived deterrent compounds could be reconstituted in a heterologous expression system to ultimately facilitate investigation of the molecular mechanism of such an aversive behavioral response. Zebrafish (Danio rerio) rejected artificial diets laced with sponge chemical defense compounds that were previously shown to deter a generalist marine predator, Thalassoma bifasciatum, suggesting that zebrafish can recognize deterrent compounds relevant to coral reef systems. Transcripts made from a zebrafish cDNA library were expressed in a heterologous system, Xenopus laevis oocytes, and tested for chemoreceptor activation via electrophysiology, using the cystic fibrosis transmembrane conductance regulator (CFTR) as a reporter. Oocytes expressing gene sequences from the library and CFTR exhibited a CFTR-like electrophysiological response to formoside and ectyoplasides A and B, sponge defense compounds. Therefore, the chemical defense-activated signaling pathway can be reconstituted in Xenopus oocytes. Kinetics of the responses suggested that the responses to formoside and ectyoplasides A and B were receptor-mediated and capable of using the G(alphas) signaling pathway in this system. This bioassay has the potential to lead to the identification of genes that encode receptors capable of interacting with deterrent chemicals, which would enable understanding of predator detection of chemical defenses.
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Cui G, Zhang ZR, O'Brien ARW, Song B, McCarty NA. Mutations at arginine 352 alter the pore architecture of CFTR. J Membr Biol 2008; 222:91-106. [PMID: 18421494 PMCID: PMC2474774 DOI: 10.1007/s00232-008-9105-9] [Citation(s) in RCA: 46] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/29/2007] [Accepted: 03/21/2008] [Indexed: 01/11/2023]
Abstract
Arginine 352 (R352) in the sixth transmembrane domain of the cystic fibrosis transmembrane conductance regulator (CFTR) previously was reported to form an anion/cation selectivity filter and to provide positive charge in the intracellular vestibule. However, mutations at this site have nonspecific effects, such as inducing susceptibility of endogenous cysteines to chemical modification. We hypothesized that R352 stabilizes channel structure and that charge-destroying mutations at this site disrupt pore architecture, with multiple consequences. We tested the effects of mutations at R352 on conductance, anion selectivity and block by the sulfonylurea drug glipizide, using recordings of wild-type and mutant channels. Charge-altering mutations at R352 destabilized the open state and altered both selectivity and block. In contrast, R352K-CFTR was similar to wild-type. Full conductance state amplitude was similar to that of wild-type CFTR in all mutants except R352E, suggesting that R352 does not itself form an anion coordination site. In an attempt to identify an acidic residue that may interact with R352, we found that permeation properties were similarly affected by charge-reversing mutations at D993. Wild-type-like properties were rescued in R352E/D993R-CFTR, suggesting that R352 and D993 in the wild-type channel may interact to stabilize pore architecture. Finally, R352A-CFTR was sensitive to modification by externally applied MTSEA+, while wild-type and R352E/D993R-CFTR were not. These data suggest that R352 plays an important structural role in CFTR, perhaps reflecting its involvement in forming a salt bridge with residue D993.
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Affiliation(s)
- Guiying Cui
- School of Biology, Georgia Institute of Technology, Atlanta, GA 30332-0230, USA.
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Fatehi M, Linsdell P. State-dependent Access of Anions to the Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channel Pore. J Biol Chem 2008; 283:6102-9. [DOI: 10.1074/jbc.m707736200] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/23/2023] Open
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44
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Ravna AW, Sylte I, Sager G. A molecular model of a putative substrate releasing conformation of multidrug resistance protein 5 (MRP5). Eur J Med Chem 2008; 43:2557-67. [PMID: 18313803 DOI: 10.1016/j.ejmech.2008.01.015] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/01/2007] [Revised: 10/08/2007] [Accepted: 01/07/2008] [Indexed: 01/31/2023]
Abstract
The ATP-binding cassette (ABC) transporter multidrug resistance protein 5 (MRP5) contributes to the cellular export of organic anions, including guanosine 3'-5' cyclic monophosphate (cGMP). The structural knowledge of this protein is limited, and in lack of an MRP5 X-ray structure, a model of MRP5 was constructed based on the homology with the bacterial ABC transporter Sav1866 from Staphylococcus aureus, which has been crystallised in an outward-facing, substrate releasing conformation. Two putative binding sites were identified, and docking of cGMP indicated that TMHs 1-3, 6, 11 and 12 were in contact with the ligands in binding site 1, while TMHs 1, 3, 5-8 were in contact with the ligands in binding site 2. The proposed MRP5 model may be used for further experimental studies of the molecular structure and function of this member of the ABC-transporter superfamily.
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Affiliation(s)
- Aina Westrheim Ravna
- Department of Pharmacology, Institute of Medical Biology, Faculty of Medicine, University of Tromsø, MH-Building, Breivika, N-9037 Tromsø, Norway
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45
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Imaging CFTR in its native environment. Pflugers Arch 2007; 456:163-77. [DOI: 10.1007/s00424-007-0399-8] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/07/2007] [Accepted: 11/09/2007] [Indexed: 12/18/2022]
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46
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St Aubin CN, Zhou JJ, Linsdell P. Identification of a second blocker binding site at the cytoplasmic mouth of the cystic fibrosis transmembrane conductance regulator chloride channel pore. Mol Pharmacol 2007; 71:1360-8. [PMID: 17293558 DOI: 10.1124/mol.106.031732] [Citation(s) in RCA: 35] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022] Open
Abstract
Chloride transport by the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel is inhibited by a broad range of substances that bind within a wide inner vestibule in the pore and physically occlude Cl(-) permeation. Binding of many of these so-called open-channel blockers involves electrostatic interactions with a positively charged lysine residue (Lys95) located in the pore. Here, we use site-directed mutagenesis to identify a second blocker binding site located at the cytoplasmic mouth of the pore. Mutagenesis of a positively charged arginine at the cytoplasmic mouth of the pore, Arg303, leads to significant weakening of the blocking effects of suramin, a large negatively charged organic molecule. Apparent suramin affinity is correlated with the side chain charge at this position, consistent with an electrostatic interaction. In contrast, block by suramin is unaffected by mutagenesis of Lys95, suggesting that it does not approach close to this important pore-forming lysine residue. We propose that the CFTR pore inner vestibule contains two distinct blocker binding sites. Relatively small organic anions enter deeply into the pore to interact with Lys95, causing an open-channel block that is sensitive to both the membrane potential and the extracellular Cl(-) concentration. Larger anionic molecules can become lodged in the cytoplasmic mouth of the pore where they interact with Arg303, causing a distinct type of open-channel block that is insensitive to membrane potential or extracellular Cl(-) ions. The pore may narrow significantly between the locations of these two blocker binding sites.
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Affiliation(s)
- Chantal N St Aubin
- Department of Physiology and Biophysics, Dalhousie University, Halifax, Nova Scotia, Canada
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47
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Ge N, Linsdell P. Interactions between impermeant blocking ions in the cystic fibrosis transmembrane conductance regulator chloride channel pore: evidence for anion-induced conformational changes. J Membr Biol 2006; 210:31-42. [PMID: 16794779 DOI: 10.1007/s00232-005-7028-2] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/22/2005] [Accepted: 02/03/2006] [Indexed: 02/08/2023]
Abstract
It is well known that extracellular Cl(-) ions can weaken the inhibitory effects of intracellular open channel blockers in the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel pore. This effect is frequently attributed to repulsive ion-ion interactions inside the pore. However, since Cl(-) ions are permeant in CFTR, it is also possible that extracellular Cl(-) ions are directly competing with intracellular blocking ions for a common binding site; thus, this does not provide direct evidence for multiple, independent anion binding sites in the pore. To test for the possible through-space nature of ion-ion interactions inside the CFTR pore, we investigated the interaction between impermeant anions applied to either end of the pore. We found that inclusion of low concentrations of impermeant Pt(NO(2))(4) (2-) ions in the extracellular solution weaken the blocking effects of three different intracellular blockers [Pt(NO(2))(4) (2-), glibenclamide and 5-nitro-2-(3-phenylpropylamino)benzoic acid] without affecting their apparent voltage dependence. However, the effects of extracellular Pt(NO(2))(4) (2-) ions are too strong to be accounted for by simple competitive models of ion binding inside the pore. In addition, extracellular Fe(CN)(6) (3-) ions, which do not appear to enter the pore, also weaken the blocking effects of intracellular Pt(NO(2))(4) (2-) ions. In contrast to previous models that invoked interactions between anions bound concurrently inside the pore, we propose that Pt(NO(2))(4) (2-) and Fe(CN)(6) (3-) binding to an extracellularly accessible site outside of the channel permeation pathway alters the structure of an intracellular anion binding site, leading to weakened binding of intracellular blocking ions.
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Affiliation(s)
- Ning Ge
- Department of Physiology and Biophysics, Dalhousie University, Halifax, Nova Scotia B3H 1X5, Canada
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48
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Frelet A, Klein M. Insight in eukaryotic ABC transporter function by mutation analysis. FEBS Lett 2006; 580:1064-84. [PMID: 16442101 DOI: 10.1016/j.febslet.2006.01.024] [Citation(s) in RCA: 63] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/15/2005] [Revised: 01/10/2006] [Accepted: 01/10/2006] [Indexed: 11/21/2022]
Abstract
With regard to structure-function relations of ATP-binding cassette (ABC) transporters several intriguing questions are in the spotlight of active research: Why do functional ABC transporters possess two ATP binding and hydrolysis domains together with two ABC signatures and to what extent are the individual nucleotide-binding domains independent or interacting? Where is the substrate-binding site and how is ATP hydrolysis functionally coupled to the transport process itself? Although much progress has been made in the elucidation of the three-dimensional structures of ABC transporters in the last years by several crystallographic studies including novel models for the nucleotide hydrolysis and translocation catalysis, site-directed mutagenesis as well as the identification of natural mutations is still a major tool to evaluate effects of individual amino acids on the overall function of ABC transporters. Apart from alterations in characteristic sequence such as Walker A, Walker B and the ABC signature other parts of ABC proteins were subject to detailed mutagenesis studies including the substrate-binding site or the regulatory domain of CFTR. In this review, we will give a detailed overview of the mutation analysis reported for selected ABC transporters of the ABCB and ABCC subfamilies, namely HsCFTR/ABCC7, HsSUR/ABCC8,9, HsMRP1/ABCC1, HsMRP2/ABCC2, ScYCF1 and P-glycoprotein (Pgp)/MDR1/ABCB1 and their effects on the function of each protein.
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Affiliation(s)
- Annie Frelet
- Zurich Basel Plant Science Center, University of Zurich, Plant Biology, Zollikerstrasse 107, CH-8008 Zurich, Switzerland
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49
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Abstract
Cl(-) channels are widely found anion pores that are regulated by a variety of signals and that play various roles. On the basis of molecular biologic findings, ligand-gated Cl(-) channels in synapses, cystic fibrosis transmembrane conductors (CFTRs) and ClC channel types have been established, followed by bestrophin and possibly by tweety, which encode Ca(2+)-activated Cl(-) channels. The ClC family has been shown to possess a variety of functions, including stabilization of membrane potential, excitation, cell-volume regulation, fluid transport, protein degradation in endosomal vesicles and possibly cell growth. The molecular structure of Cl(-) channel types varies from 1 to 12 transmembrane segments. By means of computer-based prediction, functional Cl(-) channels have been synthesized artificially, revealing that many possible ion pores are hidden in channel, transporter or unidentified hydrophobic membrane proteins. Thus, novel Cl(-)-conducting pores may be occasionally discovered, and evidence from molecular biologic studies will clarify their physiologic and pathophysiologic roles.
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Affiliation(s)
- M Suzuki
- Department of Pharmacology, Division of Molecular Pharmacology, Jichi Medical School, Tochigi 329-0498, Japan.
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50
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Fuller MD, Zhang ZR, Cui G, McCarty NA. The block of CFTR by scorpion venom is state-dependent. Biophys J 2005; 89:3960-75. [PMID: 16183882 PMCID: PMC1366962 DOI: 10.1529/biophysj.105.060731] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/04/2005] [Accepted: 09/13/2005] [Indexed: 02/01/2023] Open
Abstract
Cystic fibrosis transmembrane conductance regulator (CFTR) adenosine triphosphate-dependent chloride channels are expressed in epithelial cells and are associated with a number of genetic disorders, including cystic fibrosis. Venom of the scorpion Leirus quinquestriatus hebraeus reversibly inhibits CFTR when applied to its cytoplasmic surface. To examine the state-dependence of inhibition we recorded wild-type and mutant CFTR channel currents using inside-out membrane patches from Xenopus oocytes. Application of either venom or diphenylamine-2-carboxylate to channels that were either activated (open) or resting (closed) indicate primarily closed state-dependent inhibition of CFTR by venom, whereas diphenylamine-2-carboxylate showed no state-dependence of block. Efficacy of venom-mediated macroscopic current inhibition was inversely related to channel activity. Analysis of single-channel and macropatch data indicated that venom could either inhibit channel opening, if it binds during an interburst closed state or in the absence of cytosolic adenosine triphosphate, or introduce new intraburst closed states, if it binds during an open event. The on-rate of venom binding for intraburst block could be modulated by changing CFTR activity with vanadate or adenylyl-imidodiphosphate, or by introducing the Walker A mutation K1250A. These findings represent the first description of state-dependent inhibition of CFTR and suggest that the active toxin could be used as a tool to study the conformational changes that occur during CFTR gating.
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Affiliation(s)
- Matthew D Fuller
- Program in Molecular and Systems Pharmacology, Emory University, Atlanta, Georgia 30332-0230, USA
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