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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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Kim Y, Song S, Kim M, Sim E. Soft‐wall
ion transfer channel accurately predicts sterically hindered ion channel permeability. B KOREAN CHEM SOC 2022. [DOI: 10.1002/bkcs.12486] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022]
Affiliation(s)
- Youngsam Kim
- Department of Chemistry Yonsei University Seoul South Korea
| | - Suhwan Song
- Department of Chemistry Yonsei University Seoul South Korea
| | - Min‐Cheol Kim
- Department of Chemistry Yonsei University Seoul South Korea
| | - Eunji Sim
- Department of Chemistry Yonsei University Seoul South Korea
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5
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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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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: 164] [Impact Index Per Article: 27.3] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022] Open
Abstract
The cystic fibrosis transmembrane conductance regulator (CFTR) belongs to the ATP binding cassette (ABC) transporter superfamily but functions as an anion channel crucial for salt and water transport across epithelial cells. CFTR dysfunction, because of mutations, causes cystic fibrosis (CF). The anion-selective pore of the CFTR protein is formed by its two transmembrane domains (TMDs) and regulated by its cytosolic domains: two nucleotide binding domains (NBDs) and a regulatory (R) domain. Channel activation requires phosphorylation of the R domain by cAMP-dependent protein kinase (PKA), and pore opening and closing (gating) of phosphorylated channels is driven by ATP binding and hydrolysis at the NBDs. This review summarizes available information on structure and mechanism of the CFTR protein, with a particular focus on atomic-level insight gained from recent cryo-electron microscopic structures and on the molecular mechanisms of channel gating and its regulation. The pharmacological mechanisms of small molecules targeting CFTR's ion channel function, aimed at treating patients suffering from CF and other diseases, are briefly discussed.
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Affiliation(s)
- László Csanády
- Department of Medical Biochemistry, Semmelweis University , Budapest , Hungary ; MTA-SE Ion Channel Research Group, Budapest , Hungary ; Department of Neuroscience, Physiology and Pharmacology, University College London , London , United Kingdom ; and Laboratory of Cardiac/Membrane Physiology, The Rockefeller University , New York, New York
| | - Paola Vergani
- Department of Medical Biochemistry, Semmelweis University , Budapest , Hungary ; MTA-SE Ion Channel Research Group, Budapest , Hungary ; Department of Neuroscience, Physiology and Pharmacology, University College London , London , United Kingdom ; and Laboratory of Cardiac/Membrane Physiology, The Rockefeller University , New York, New York
| | - David C Gadsby
- Department of Medical Biochemistry, Semmelweis University , Budapest , Hungary ; MTA-SE Ion Channel Research Group, Budapest , Hungary ; Department of Neuroscience, Physiology and Pharmacology, University College London , London , United Kingdom ; and Laboratory of Cardiac/Membrane Physiology, The Rockefeller University , New York, New York
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8
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Negoda A, Cowley EA, El Hiani Y, Linsdell P. Conformational change of the extracellular parts of the CFTR protein during channel gating. Cell Mol Life Sci 2018; 75:3027-3038. [PMID: 29441426 PMCID: PMC11105745 DOI: 10.1007/s00018-018-2777-0] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/28/2017] [Revised: 01/24/2018] [Accepted: 02/08/2018] [Indexed: 12/21/2022]
Abstract
Cystic fibrosis can be treated by potentiators, drugs that interact directly with the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel to increase its open probability. These substances likely target key conformational changes occurring during channel opening and closing, however, the molecular bases of these conformational changes, and their susceptibility to manipulation are poorly understood. We have used patch clamp recording to identify changes in the three-dimensional organization of the extracellularly accessible parts of the CFTR protein during channel opening and closing. State-dependent formation of both disulfide bonds and Cd2+ bridges occurred for pairs of cysteine side-chains introduced into the extreme extracellular ends of transmembrane helices (TMs) 1, 6, and 12. Between each of these three TMs, we found that both disulfide bonds and metal bridges formed preferentially or exclusively in the closed state and that these inter-TM cross-links stabilized the closed state. These results indicate that the extracellular ends of these TMs are close together when the channel is closed and that they separate from each other when the channel opens. These findings identify for the first time key conformational changes in the extracellular parts of the CFTR protein that can potentially be manipulated to control channel activity.
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Affiliation(s)
- Alexander Negoda
- 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
| | - Yassine El Hiani
- 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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9
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Zhou P, Polovitskaya MM, Jentsch TJ. LRRC8 N termini influence pore properties and gating of volume-regulated anion channels (VRACs). J Biol Chem 2018; 293:13440-13451. [PMID: 29925591 PMCID: PMC6120214 DOI: 10.1074/jbc.ra118.002853] [Citation(s) in RCA: 25] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/12/2018] [Revised: 06/19/2018] [Indexed: 12/22/2022] Open
Abstract
Volume-regulated anion channels (VRACs) are crucial for cell volume regulation and have various roles in physiology and pathology. VRACs were recently discovered to be formed by heteromers of leucine-rich repeat–containing 8 (LRRC8) proteins. However, the structural determinants of VRAC permeation and gating remain largely unknown. We show here that the short stretch preceding the first LRRC8 transmembrane domain determines VRAC conductance, ion permeability, and inactivation gating. Substituted-cysteine accessibility studies revealed that several of the first 15 LRRC8 residues are functionally important and exposed to a hydrophilic environment. Substituting glutamate 6 with cysteine decreased the amplitudes of swelling-activated ICl,vol currents, strongly increased iodide-over-chloride permeability, and markedly shifted the voltage dependence of channel inactivation. Importantly, these effects were reversed by 2-sulfonatoethyl methanethiosulfonate, which restores the negative charge at this amino acid position. Cd2+-mediated blocking of ICl,vol in cysteine variants suggested that the LRRC8 N termini come close together in the multimeric channel complex and might form part of the pore. We propose a model in which the N termini of the LRRC8 subunits line the cytoplasmic portion of the VRAC pore, possibly by folding back into the ion permeation pathway.
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Affiliation(s)
- Pingzheng Zhou
- From the Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP) and Max-Delbrück-Centrum für Molekulare Medizin (MDC), D-13125 Berlin, Germany
| | - Maya M Polovitskaya
- From the Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP) and Max-Delbrück-Centrum für Molekulare Medizin (MDC), D-13125 Berlin, Germany.,Graduate Program, Faculty of Biology, Chemistry, and Pharmacy, Freie Universität Berlin, D-14195 Berlin, Germany
| | - Thomas J Jentsch
- From the Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP) and Max-Delbrück-Centrum für Molekulare Medizin (MDC), D-13125 Berlin, Germany, .,Neurocure Cluster of Excellence, Charité Universitätsmedizin, D-10117 Berlin, Germany, and
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10
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Hwang TC, Yeh JT, Zhang J, Yu YC, Yeh HI, Destefano S. Structural mechanisms of CFTR function and dysfunction. J Gen Physiol 2018; 150:539-570. [PMID: 29581173 PMCID: PMC5881446 DOI: 10.1085/jgp.201711946] [Citation(s) in RCA: 85] [Impact Index Per Article: 12.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/16/2017] [Accepted: 03/05/2018] [Indexed: 12/18/2022] Open
Abstract
Hwang et al. integrate new structural insights with prior functional studies to reveal the functional anatomy of CFTR chloride channels. Cystic fibrosis (CF) transmembrane conductance regulator (CFTR) chloride channel plays a critical role in regulating transepithelial movement of water and electrolyte in exocrine tissues. Malfunction of the channel because of mutations of the cftr gene results in CF, the most prevalent lethal genetic disease among Caucasians. Recently, the publication of atomic structures of CFTR in two distinct conformations provides, for the first time, a clear overview of the protein. However, given the highly dynamic nature of the interactions among CFTR’s various domains, better understanding of the functional significance of these structures requires an integration of these new structural insights with previously established biochemical/biophysical studies, which is the goal of this review.
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Affiliation(s)
- Tzyh-Chang Hwang
- Dalton Cardiovascular Research Center, University of Missouri, Columbia, MO .,Department of Medical Pharmacology and Physiology, University of Missouri, Columbia, MO.,Department of Biological Engineering, University of Missouri, Columbia, MO
| | - Jiunn-Tyng Yeh
- Dalton Cardiovascular Research Center, University of Missouri, Columbia, MO
| | - Jingyao Zhang
- Dalton Cardiovascular Research Center, University of Missouri, Columbia, MO.,Department of Biological Engineering, University of Missouri, Columbia, MO
| | - Ying-Chun Yu
- Dalton Cardiovascular Research Center, University of Missouri, Columbia, MO.,Department of Medical Pharmacology and Physiology, University of Missouri, Columbia, MO
| | - Han-I Yeh
- Dalton Cardiovascular Research Center, University of Missouri, Columbia, MO.,Department of Medical Pharmacology and Physiology, University of Missouri, Columbia, MO
| | - Samantha Destefano
- Dalton Cardiovascular Research Center, University of Missouri, Columbia, MO.,Department of Medical Pharmacology and Physiology, University of Missouri, Columbia, MO
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11
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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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12
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Callebaut I, Hoffmann B, Lehn P, Mornon JP. Molecular modelling and molecular dynamics of CFTR. Cell Mol Life Sci 2017; 74:3-22. [PMID: 27717958 PMCID: PMC11107702 DOI: 10.1007/s00018-016-2385-9] [Citation(s) in RCA: 25] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/27/2016] [Accepted: 09/28/2016] [Indexed: 12/11/2022]
Abstract
The cystic fibrosis transmembrane conductance regulator (CFTR) protein is a member of the ATP-binding cassette (ABC) transporter superfamily that functions as an ATP-gated channel. Considerable progress has been made over the last years in the understanding of the molecular basis of the CFTR functions, as well as dysfunctions causing the common genetic disease cystic fibrosis (CF). This review provides a global overview of the theoretical studies that have been performed so far, especially molecular modelling and molecular dynamics (MD) simulations. A special emphasis is placed on the CFTR-specific evolution of an ABC transporter framework towards a channel function, as well as on the understanding of the effects of disease-causing mutations and their specific modulation. This in silico work should help structure-based drug discovery and design, with a view to develop CFTR-specific pharmacotherapeutic approaches for the treatment of CF in the context of precision medicine.
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Affiliation(s)
- Isabelle Callebaut
- UMR CNRS 7590, Museum National d'Histoire Naturelle, IRD UMR 206, IUC, Case 115, IMPMC, Sorbonne Universités, UPMC Univ Paris 06, 4 Place Jussieu, 75005, Paris Cedex 05, France.
| | - Brice Hoffmann
- UMR CNRS 7590, Museum National d'Histoire Naturelle, IRD UMR 206, IUC, Case 115, IMPMC, Sorbonne Universités, UPMC Univ Paris 06, 4 Place Jussieu, 75005, Paris Cedex 05, France
| | - Pierre Lehn
- INSERM U1078, SFR ScInBioS, Université de Bretagne Occidentale, Brest, France
| | - Jean-Paul Mornon
- UMR CNRS 7590, Museum National d'Histoire Naturelle, IRD UMR 206, IUC, Case 115, IMPMC, Sorbonne Universités, UPMC Univ Paris 06, 4 Place Jussieu, 75005, Paris Cedex 05, France
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13
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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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14
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MLKL forms cation channels. Cell Res 2016; 26:517-28. [PMID: 27033670 PMCID: PMC4856759 DOI: 10.1038/cr.2016.26] [Citation(s) in RCA: 156] [Impact Index Per Article: 17.3] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/05/2015] [Revised: 02/01/2016] [Accepted: 02/01/2016] [Indexed: 12/17/2022] Open
Abstract
The mixed lineage kinase domain-like (MLKL) protein is a key factor in tumor necrosis factor-induced necroptosis. Recent studies on necroptosis execution revealed a commitment role of MLKL in membrane disruption. However, our knowledge of how MLKL functions on membrane remains very limited. Here we demonstrate that MLKL forms cation channels that are permeable preferentially to Mg2+ rather than Ca2+ in the presence of Na+ and K+. Moreover, the N-terminal domain containing six helices (H1-H6) is sufficient to form channels. Using the substituted cysteine accessibility method, we further determine that helix H1, H2, H3, H5 and H6 are transmembrane segments, while H4 is located in the cytoplasm. Finally, MLKL-induced membrane depolarization and cell death exhibit a positive correlation to its channel activity. The Mg2+-preferred permeability and five transmembrane segment topology distinguish MLKL from previously identified Mg2+-permeable channels and thus establish MLKL as a novel class of cation channels.
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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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Zhang J, Hwang TC. The Fifth Transmembrane Segment of Cystic Fibrosis Transmembrane Conductance Regulator Contributes to Its Anion Permeation Pathway. Biochemistry 2015; 54:3839-50. [PMID: 26024338 DOI: 10.1021/acs.biochem.5b00427] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
Abstract
Previous studies have identified several transmembrane segments (TMs), including TM1, TM3, TM6, TM9, TM11, and TM12, as pore-lining segments in cystic fibrosis transmembrane conductance regulator (CFTR), but the role of TM5 in pore construction remains controversial. In this study, we employed substituted cysteine accessibility methodology (SCAM) to screen the entire TM5 defined by the original topology model and its cytoplasmic extension in a Cysless background. We found six positions (A299, R303, N306, S307, F310, and F311) where engineered cysteines react to intracellular 2-sulfonatoethyl methanethiosulfonate (MTSES⁻). Quantification of the modification rate of engineered cysteines in the presence or absence of ATP suggests that these six residues are accessible in both the open and closed states. Whole-cell experiments with external MTSES⁻ identified only two positive positions (L323 and A326), resulting in a segment containing 11 consecutive amino acids, where substituted cysteines respond to neither internal nor external MTSES⁻, a unique feature not seen previously in CFTR's pore-lining segments. The observation that these positions are inaccessible to channel-permeant thiol-specific reagent [Au(CN)₂]⁻ suggests that this segment of TM5 between F311 and L323 is concealed from the pore by other TMs and/or lipid bilayers. In addition, our data support the idea that the positively charged arginine at position 303 poses a pure electrostatic action in determining the single-channel current amplitude of CFTR and the effect of an open-channel blocker glibencalmide. Collectively, we conclude that the cytoplasmic portion of CFTR's TM5 lines the pore. Our functional data are remarkably consistent with predicted structural arrangements of TM5 in some homology models of CFTR.
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Affiliation(s)
- Jingyao Zhang
- †Department of Biological Engineering, University of Missouri-Columbia, 254 Agricultural Engineering, Columbia, Missouri 65211, United States.,‡Dalton Cardiovascular Research Center, University of Missouri-Columbia, 134 Research Park, Columbia, Missouri 65211, United States
| | - Tzyh-Chang Hwang
- †Department of Biological Engineering, University of Missouri-Columbia, 254 Agricultural Engineering, Columbia, Missouri 65211, United States.,‡Dalton Cardiovascular Research Center, University of Missouri-Columbia, 134 Research Park, Columbia, Missouri 65211, United States.,§Department of Medical Pharmacology and Physiology, University of Missouri-Columbia, Medical Sciences Building, Columbia, Missouri 65212, United States
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17
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Mornon JP, Hoffmann B, Jonic S, Lehn P, Callebaut I. Full-open and closed CFTR channels, with lateral tunnels from the cytoplasm and an alternative position of the F508 region, as revealed by molecular dynamics. Cell Mol Life Sci 2015; 72:1377-403. [PMID: 25287046 PMCID: PMC11113974 DOI: 10.1007/s00018-014-1749-2] [Citation(s) in RCA: 62] [Impact Index Per Article: 6.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/05/2014] [Revised: 09/28/2014] [Accepted: 09/29/2014] [Indexed: 12/17/2022]
Abstract
In absence of experimental 3D structures, several homology models, based on ABC exporter 3D structures, have provided significant insights into the molecular mechanisms underlying the function of the cystic fibrosis transmembrane conductance regulator (CFTR) protein, a chloride channel whose defects are associated with cystic fibrosis (CF). Until now, these models, however, did not furnished much insights into the continuous way that ions could follow from the cytosol to the extracellular milieu in the open form of the channel. Here, we have built a refined model of CFTR, based on the outward-facing Sav1866 experimental 3D structure and integrating the evolutionary and structural information available today. Molecular dynamics simulations revealed significant conformational changes, resulting in a full-open channel, accessible from the cytosol through lateral tunnels displayed in the long intracellular loops (ICLs). At the same time, the region of nucleotide-binding domain 1 in contact with one of the ICLs and carrying amino acid F508, the deletion of which is the most common CF-causing mutation, was found to adopt an alternative but stable position. Then, in a second step, this first stable full-open conformation evolved toward another stable state, in which only a limited displacement of the upper part of the transmembrane helices leads to a closure of the channel, in a conformation very close to that adopted by the Atm1 ABC exporter, in an inward-facing conformation. These models, supported by experimental data, provide significant new insights into the CFTR structure-function relationships and into the possible impact of CF-causing mutations.
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Affiliation(s)
- Jean-Paul Mornon
- IMPMC, Sorbonne Universités, UPMC Univ Paris 06, UMR CNRS 7590, Museum National d’Histoire Naturelle, IRD UMR 206, IUC, Case 115, 4 Place Jussieu, 75005 Paris Cedex 05, France
| | - Brice Hoffmann
- IMPMC, Sorbonne Universités, UPMC Univ Paris 06, UMR CNRS 7590, Museum National d’Histoire Naturelle, IRD UMR 206, IUC, Case 115, 4 Place Jussieu, 75005 Paris Cedex 05, France
| | - Slavica Jonic
- IMPMC, Sorbonne Universités, UPMC Univ Paris 06, UMR CNRS 7590, Museum National d’Histoire Naturelle, IRD UMR 206, IUC, Case 115, 4 Place Jussieu, 75005 Paris Cedex 05, France
| | - Pierre Lehn
- INSERM U1078, SFR ScInBioS, Université de Bretagne Occidentale, Brest, France
| | - Isabelle Callebaut
- IMPMC, Sorbonne Universités, UPMC Univ Paris 06, UMR CNRS 7590, Museum National d’Histoire Naturelle, IRD UMR 206, IUC, Case 115, 4 Place Jussieu, 75005 Paris Cedex 05, France
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18
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Qian F, Li T, Yang F, Liu L. Stoichiometry and novel gating mechanism within the cystic fibrosis transmembrane conductance regulator channel. Exp Physiol 2014; 99:1611-23. [DOI: 10.1113/expphysiol.2014.081034] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/27/2023]
Affiliation(s)
- Feng Qian
- Department of Medical Function; School of Medicine; Yangtze University; Jingzhou Hubei Province 434023 China
| | - Tao Li
- Department of Biology; College of Chemistry and Life Sciences; Zhejiang Normal University; Jinhua Zhejiang Province 321004 China
| | - Fei Yang
- Department of Medical Function; School of Medicine; Yangtze University; Jingzhou Hubei Province 434023 China
| | - Lian Liu
- Department of Medical Function; School of Medicine; Yangtze University; Jingzhou Hubei Province 434023 China
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19
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El Hiani Y, Linsdell P. Conformational changes opening and closing the CFTR chloride channel: insights from cysteine scanning mutagenesis. Biochem Cell Biol 2014; 92:481-8. [PMID: 25367045 DOI: 10.1139/bcb-2014-0038] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/01/2023] Open
Abstract
Cystic fibrosis, the most common lethal genetic disease affecting young people in North America, is caused by failure of the chloride ion channel known as CFTR (cystic fibrosis transmembrane conductance regulator). CFTR belongs to the large family of ATP-binding cassette (ABC) membrane transporters. In CFTR, ATP-driven events at the nucleotide-binding domains (NBDs) open and close a gate that controls chloride permeation. However, the conformational changes concomitant with opening and closing of the CFTR gate are unknown. Diverse techniques including substituted cysteine accessibility method, disulfide cross-linking, and patch-clamp recording have been used to explore CFTR channel structure. Here, we consider the architecture of both the open and the closed CFTR channel. We review how CFTR channel structure changes between the closed and the open channel conformations and portray the relative function of both cytoplasmic and vestigial gates during the gating cycle. Understanding how the CFTR channel gates chloride permeation is central for understanding how CFTR defects lead to CF. Such knowledge opens the door for novel ways to maximize CFTR channel activity in a CF setting.
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Affiliation(s)
- Yassine El Hiani
- Department of Physiology & Biophysics, Dalhousie University, Halifax, NS B3H 4R2, Canada
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20
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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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21
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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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22
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Wang W, El Hiani Y, Rubaiy HN, Linsdell P. Relative contribution of different transmembrane segments to the CFTR chloride channel pore. Pflugers Arch 2013; 466:477-90. [PMID: 23955087 DOI: 10.1007/s00424-013-1317-x] [Citation(s) in RCA: 30] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/07/2013] [Revised: 06/18/2013] [Accepted: 06/18/2013] [Indexed: 12/16/2022]
Abstract
The membrane-spanning part of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel comprises 12 transmembrane (TM) α-helices, arranged in 2 symmetrical groups of 6. However, those TMs that line the channel pore are not completely defined. We used patch clamp recording to compare the accessibility of cysteine-reactive reagents to cysteines introduced into different TMs. Several residues in TM11 were accessible to extracellular and/or intracellular cysteine reactive reagents; however, no reactive cysteines were identified in TMs 5 or 11. Two accessible residues in TM11 (T1115C and S1118C) were found to be more readily modified from the extracellular solution in closed channels, but more readily modified from the intracellular solution in open channels, as previously reported for T338C in TM6. However, the effects of mutagenesis at S1118 (TM11) on a range of pore functional properties were relatively minor compared to the large effects of mutagenesis at T338 (TM6). Our results suggest that the CFTR pore is lined by TM11 but not by TM5 or TM7. Comparison with previous works therefore suggests that the pore is lined by TMs 1, 6, 11, and 12, suggesting that the structure of the open channel pore is asymmetric in terms of the contributions of different TMs. Although TMs 6 and 11 appear to undergo similar conformational changes during channel opening and closing, the influence of these two TMs on the functional properties of the narrowest region of the pore is clearly unequal.
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Affiliation(s)
- Wuyang Wang
- Department of Physiology and Biophysics, Dalhousie University, PO Box 15000 Halifax, Nova Scotia, B3H 4R2, Canada
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23
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Denny RA, Gavrin LK, Saiah E. Recent developments in targeting protein misfolding diseases. Bioorg Med Chem Lett 2013; 23:1935-44. [PMID: 23454013 DOI: 10.1016/j.bmcl.2013.01.089] [Citation(s) in RCA: 31] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/16/2012] [Revised: 01/18/2013] [Accepted: 01/21/2013] [Indexed: 10/27/2022]
Abstract
Protein misfolding is an emerging field that crosses multiple therapeutic areas and causes many serious diseases. As the biological pathways of protein misfolding become more clearly elucidated, small molecule approaches in this arena are gaining increased attention. This manuscript will survey current small molecules from the literature that are known to modulate misfolding, stabilization or proteostasis. Specifically, the following targets and approaches will be discussed: CFTR, glucocerebrosidase, modulation of toxic oligomers, serum amyloid P (SAP) sections and HSF1 activators.
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Affiliation(s)
- Rajiah Aldrin Denny
- BioTherapeutics Chemistry, Pfizer Worldwide Medicinal Chemistry, 200 CambridgePark Drive, Cambridge, MA 02140, USA
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24
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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: 97] [Impact Index Per Article: 8.1] [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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25
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Furukawa-Hagiya T, Furuta T, Chiba S, Sohma Y, Sakurai M. The power stroke driven by ATP binding in CFTR as studied by molecular dynamics simulations. J Phys Chem B 2012; 117:83-93. [PMID: 23214920 DOI: 10.1021/jp308315w] [Citation(s) in RCA: 30] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/07/2023]
Abstract
Cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel belonging to the ATP binding cassette (ABC) protein superfamily. Currently, it remains unclear how ATP binding causes the opening of the channel gate at the molecular level. To clarify this mechanism, we first constructed an atomic model of the inward-facing CFTR using the X-ray structures of other ABC proteins. Molecular dynamics (MD) simulations were then performed to explore the structure and dynamics of the inward-facing CFTR in a membrane environment. In the MgATP-bound state, two nucleotide-binding domains (NBDs) formed a head-to-tail type of dimer, in which the ATP molecules were sandwiched between the Walker A and signature motifs. Alternatively, one of the final MD structures in the apo state was similar to that of a "closed-apo" conformation found in the X-ray analysis of ATP-free MsbA. Principal component analysis for the MD trajectory indicated that NBD dimerization causes significant structural and dynamical changes in the transmembrane domains (TMDs), which is likely indicative of the formation of a chloride ion access path. This study suggests that the free energy gain from ATP binding acts as a driving force not only for NBD dimerization but also for NBD-TMD concerted motions.
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Affiliation(s)
- Tomoka Furukawa-Hagiya
- Center for Biological Resources and Informatics, Tokyo Institute of Technology, 4259-B-62, Nagatsuta-cho, Midori-ku, Yokohama, 226-8501, Japan
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26
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Wang W, Linsdell P. Relative movements of transmembrane regions at the outer mouth of the cystic fibrosis transmembrane conductance regulator channel pore during channel gating. J Biol Chem 2012; 287:32136-46. [PMID: 22843683 DOI: 10.1074/jbc.m112.385096] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/19/2022] Open
Abstract
Multiple transmembrane (TM) segments line the pore of the cystic fibrosis transmembrane conductance regulator Cl(-) channel; however, the relative alignment of these TMs and their relative movements during channel gating are unknown. To gain three-dimensional structural information on the outer pore, we have used patch clamp recording to study the proximity of pairs of cysteine side chains introduced into TMs 6 and 11, using both disulfide cross-linking and Cd(2+) coordination. Following channel activation, disulfide bonds could apparently be formed between three cysteine pairs (of 15 studied): R334C/T1122C, R334C/G1127C, and T338C/S1118C. To examine the state dependence of cross-linking, we combined these cysteine mutations with a nucleotide-binding domain mutation (E1371Q) that stabilizes the channel open state. Investigation of the effects of the E1371Q mutation on disulfide bond formation and Cd(2+) coordination suggests that although R334C/T1122C and T338C/S1118C are closer together in the channel open state, R334C/G1127C are close together and can form disulfide bonds only when the channel is closed. These results provide important new information on the three-dimensional structure of the outer mouth of the cystic fibrosis transmembrane conductance regulator channel pore: TMs 6 and 11 are close enough together to form disulfide bonds in both open and closed channels. Moreover, the altered relative locations of residues in open and in closed channels that we infer allow us to propose that channel opening and closing may be associated with a relative translational movement of TMs 6 and 11, with TM6 moving "down" (toward the cytoplasm) during channel opening.
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Affiliation(s)
- Wuyang Wang
- Department of Physiology and Biophysics, Dalhousie University, Halifax, Nova Scotia B3H 4R2, Canada
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27
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El Hiani Y, Linsdell P. Role of the juxtamembrane region of cytoplasmic loop 3 in the gating and conductance of the cystic fibrosis transmembrane conductance regulator chloride channel. Biochemistry 2012; 51:3971-81. [PMID: 22545782 PMCID: PMC3381012 DOI: 10.1021/bi300065z] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
Abstract
Opening and closing of the cystic fibrosis transmembrane conductance regulator chloride channel are controlled by interactions of ATP with its cytoplasmic nucleotide binding domains (NBDs). The NBDs are connected to the transmembrane pore via four cytoplasmic loops. These loops have been suggested to play roles both in channel gating and in forming a cytoplasmic extension of the channel pore. To investigate the structure and function of one of these cytoplasmic loops, we have used patch clamp recording to investigate the accessibility of cytoplasmically applied cysteine-reactive reagents to cysteines introduced into loop 3. We find that methanethiosulfonate (MTS) reagents modify cysteines introduced at 14 of 16 sites studied in the juxtamembrane region of loop 3, in all cases leading to inhibition of channel function. In most cases, both the functional effects of modification and the rate of modification were similar for negatively and positively charged MTS reagents. Single-channel recordings indicated that, at all sites, inhibition was the result of an MTS reagent-induced decrease in channel open probability; in no case was the Cl(-) conductance of open channels altered by modification. These results indicate that loop 3 is readily accessible to the cytoplasm and support the involvement of this region in the control of channel gating. However, our results do not support the hypothesis that this region is close enough to the Cl(-) permeation pathway to exert any influence on permeating Cl(-) ions. We propose that either the cytoplasmic pore is very wide or cytoplasmic Cl(-) ions use other routes to access the transmembrane pore.
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Affiliation(s)
- Yassine El Hiani
- Department of Physiology and Biophysics, Dalhousie University , Halifax, Nova Scotia B3H 4R2, Canada
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28
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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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29
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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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Wang W, El Hiani Y, Linsdell P. Alignment of transmembrane regions in the cystic fibrosis transmembrane conductance regulator chloride channel pore. ACTA ACUST UNITED AC 2011; 138:165-78. [PMID: 21746847 PMCID: PMC3149817 DOI: 10.1085/jgp.201110605] [Citation(s) in RCA: 53] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/23/2022]
Abstract
Different transmembrane (TM) α helices are known to line the pore of the cystic fibrosis TM conductance regulator (CFTR) Cl− channel. However, the relative alignment of these TMs in the three-dimensional structure of the pore is not known. We have used patch-clamp recording to investigate the accessibility of cytoplasmically applied cysteine-reactive reagents to cysteines introduced along the length of the pore-lining first TM (TM1) of a cysteine-less variant of CFTR. We find that methanethiosulfonate (MTS) reagents irreversibly modify cysteines substituted for TM1 residues K95, Q98, P99, and L102 when applied to the cytoplasmic side of open channels. Residues closer to the intracellular end of TM1 (Y84–T94) were not apparently modified by MTS reagents, suggesting that this part of TM1 does not line the pore. None of the internal MTS reagent-reactive cysteines was modified by extracellular [2-(trimethylammonium)ethyl] MTS. Only K95C, closest to the putative intracellular end of TM1, was apparently modified by intracellular [2-sulfonatoethyl] MTS before channel activation. Comparison of these results with recent work on CFTR-TM6 suggests a relative alignment of these two important TMs along the axis of the pore. This alignment was tested experimentally by formation of disulfide bridges between pairs of cysteines introduced into these two TMs. Currents carried by the double mutants K95C/I344C and Q98C/I344C, but not by the corresponding single-site mutants, were inhibited by the oxidizing agent copper(II)-o-phenanthroline. This inhibition was irreversible on washing but could be reversed by the reducing agent dithiothreitol, suggesting disulfide bond formation between the introduced cysteine side chains. These results allow us to develop a model of the relative positions, functional contributions, and alignment of two important TMs lining the CFTR pore. Such functional information is necessary to understand and interpret the three-dimensional structure of the pore.
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Affiliation(s)
- Wuyang Wang
- Department of Physiology and Biophysics, Dalhousie University, Halifax, Nova Scotia B3H 1X5, Canada
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El Hiani Y, Linsdell P. Changes in accessibility of cytoplasmic substances to the pore associated with activation of the cystic fibrosis transmembrane conductance regulator chloride channel. J Biol Chem 2010; 285:32126-40. [PMID: 20675380 DOI: 10.1074/jbc.m110.113332] [Citation(s) in RCA: 52] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022] Open
Abstract
Opening of the cystic fibrosis transmembrane conductance regulator Cl(-) channel is dependent both on phosphorylation and on ATP binding and hydrolysis. However, the mechanisms by which these cytoplasmic regulatory factors open the Cl(-) channel pore are not known. We have used patch clamp recording to investigate the accessibility of cytoplasmically applied cysteine-reactive reagents to cysteines introduced along the length of the pore-lining sixth transmembrane region (TM6) of a cysteine-less variant of cystic fibrosis transmembrane conductance regulator. We find that methanethiosulfonate (MTS) reagents modify irreversibly cysteines substituted for TM6 residues Phe-337, Thr-338, Ser-341, Ile-344, Val-345, Met-348, Ala-349, Arg-352, and Gln-353 when applied to the cytoplasmic side of open channels. However, the apparent rate of modification by internal [2-sulfonatoethyl] methanethiosulfonate (MTSES), a negatively charged MTS reagent, is dependent on the activation state of the channels. In particular, cysteines introduced far along the axis of TM6 from the inside (T338C, S341C, I344C) showed no evidence of significant modification even after prolonged pretreatment of non-activated channels with internal MTSES. In contrast, cysteines introduced closer to the inside of TM6 (V345C, M348C) were readily modified in both activated and non-activated channels. Access of a permeant anion, Au(CN)(2)(-), to T338C was similarly dependent upon channel activation state. The pattern of MTS modification we observe allows us to designate different pore-lining amino acid side chains to distinct functional regions of the channel pore. One logical interpretation of these findings is that cytoplasmic access to residues at the narrowest region of the pore changes concomitant with activation.
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Affiliation(s)
- Yassine El Hiani
- Department of Physiology and Biophysics, Dalhousie University, Halifax, Nova Scotia B3H 1X5, Canada
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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: 60] [Impact Index Per Article: 4.0] [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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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: 117] [Impact Index Per Article: 7.3] [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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