Functional Architecture of the Cytoplasmic Entrance to the Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channel Pore

Hiani, Yassine El; Linsdell, Paul

doi:10.1074/jbc.m115.656181

Cited by 36 publications

(71 citation statements)

References 40 publications

(46 reference statements)

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“…The corresponding residues in zCFTR, K249, and K371, are both located at the cytoplasmic opening ( Figure 5). The same study also showed that positively charged residues along TM10 and TM12, the helices equivalent to TM4 and TM6 in the other half of CFTR, are not involved in comparable favorable electrostatic interactions with chloride ions (El Hiani and Linsdell, 2015). Entirely consistent with these functional studies, we did not observe a lateral portal (like that between TM4 and TM6) between TM10 and TM12 ( Figure S4).…”

Section: Asymmetric Nbd Associationsupporting

confidence: 86%

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Conformational Changes of CFTR upon Phosphorylation and ATP Binding

Zhang

Liu

Chen

2017

Cell

223

375

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The cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel evolved from an ATP-binding cassette transporter. CFTR channel gating is strictly coupled to phosphorylation and ATP hydrolysis. Previously, we reported essentially identical structures of zebrafish and human CFTR in the dephosphorylated, ATP-free form. Here, we present the structure of zebrafish CFTR in the phosphorylated, ATP-bound conformation, determined by cryoelectron microscopy to 3.4 Å resolution. Comparison of the two conformations shows major structural rearrangements leading to channel opening. The phosphorylated regulatory domain is disengaged from its inhibitory position; the nucleotide-binding domains (NBDs) form a "head-to-tail" dimer upon binding ATP; and the cytoplasmic pathway, found closed off in other ATP-binding cassette transporters, is cracked open, consistent with CFTR's unique channel function. Unexpectedly, the extracellular mouth of the ion pore remains closed, indicating that local movements of the transmembrane helices can control ion access to the pore even in the NBD-dimerized conformation.

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Section: Asymmetric Nbd Associationsupporting

confidence: 86%

“…The ion conduction pathway is connected to the cytosol through this opening. Studies of human CFTR have shown that R248 and K370 are important in recruiting chloride ions to the pore (El Hiani and Linsdell, 2015). The corresponding residues in zCFTR, K249, and K371, are both located at the cytoplasmic opening ( Figure 5).…”

Section: Asymmetric Nbd Associationmentioning

confidence: 99%

Conformational Changes of CFTR upon Phosphorylation and ATP Binding

Zhang

Liu

Chen

2017

Cell

223

375

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“…It is worth noting that this region is also found in a model of the open form of the CFTR channel ( Supplementary Figure 1A, RMSD 4.7 Å on 204 Cα, Mornon et al, 2015). This model, supported by experimental data (El Hiani and Linsdell, 2015) and consistent with the cryo-EM 3D structure at the level of individual blocks of TM helices (Hoffmann et al, 2018), gives insight into an alternative conformation of the CFTR channel, with a different configuration of the TM7-TM8 hairpin as well as of the N-terminal extension (lasso in the cryo-EM 3D structure), free for interaction with cytosolic partners (Supplementary Figure 1A).…”

Section: Position Of W361 On the Cftr 3d Structures And Possible Invosupporting

confidence: 77%

Functional and Pharmacological Characterization of the Rare CFTR Mutation W361R

et al. 2020

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Understanding the functional consequence of rare cystic fibrosis (CF) mutations is mandatory for the adoption of precision therapeutic approaches for CF. Here we studied the effect of the very rare CF mutation, W361R, on CFTR processing and function. We applied western blot, patch clamp and pharmacological modulators of CFTR to study the maturation and ion transport properties of pEGFP-WT and mutant CFTR constructs, W361R, F508del and L69H-CFTR, expressed in HEK293 cells. Structural analyses were also performed to study the molecular environment of the W361 residue. Western blot showed that W361R-CFTR was not efficiently processed to a mature band C, similar to F508del CFTR, but unlike F508del CFTR, it did exhibit significant transport activity at the cell surface in response to cAMP agonists. Importantly, W361R-CFTR also responded well to CFTR modulators: its maturation defect was efficiently corrected by VX-809 treatment and its channel activity further potentiated by VX-770. Based on these results, we postulate that W361R is a novel class-2 CF mutation that causes abnormal protein maturation which can be corrected by VX-809, and additionally potentiated by VX-770, two FDA-approved small molecules. At the structural level, W361 is located within a class-2 CF mutation hotspot that includes other mutations that induce variable disease severity. Analysis of the 3D structure of CFTR within a lipid environment indicated that W361, together with other mutations located in this hotspot, is at the edge of a groove which stably accommodates lipid acyl chains. We suggest this lipid environment impacts CFTR folding, maturation and response to CFTR modulators.

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“…Based on atomic homology modeling and molecular dynamics studies, it was proposed that such access may be provided not by a central pathway, but instead by one or more (possibly up to four) lateral entrances between individual transmembrane domain cytoplasmic extensions (TMEs) [27,28]. The first functional evidence for the existence of such cytoplasmic lateral portals to the pore came from substituted cysteine accessibility mutagenesis work that identified porelining, positively charged amino acid side chains within the TMEs and apparently surrounding two potential portals [29]. Subsequent, more detailed biophysical analysis suggested that there is only one functional portal involved in the entry of cytoplasmic Cl − ions into the pore, and indicated that four positively charged residues -K190 (TME3), R248 (TME4), R303 (TME5) and K370 (TME6)play major roles in attracting cytoplasmic Cl − ions to the pore via this portal [30].…”

Section: The Structure Of Cftr Suggests Conformational Transitions Tymentioning

confidence: 99%

“…This portal is facing the viewer in (b), and on the right hand side of the MSDs in (c), indicating cytoplasmic access as indicated by the arrows. (d) A cross-section through the TMEs indicates the location of the portal (arrow), as well as important positively charged amino acid side chains (red; K190 (TME3), R248 (TME4), R303 (TME5) and K370 (TME6) in human CFTR) that have been shown to play functional roles in the electrostatic attraction of cytoplasmic anions to the pore[29,30].…”

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confidence: 99%

Cystic fibrosis transmembrane conductance regulator (CFTR): Making an ion channel out of an active transporter structure

Linsdell

2018

Channels

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Cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR is a member of the ATP-binding cassette (ABC) family of membrane transport proteins, most members of which function as ATP-dependent pumps. CFTR is unique among human ABC proteins in functioning not as a pump, but as an ion channel. Recent structural data has indicated that CFTR shares broadly similar overall architecture and ATP-dependent conformational changes as other ABC proteins. Functional investigations suggest that CFTR has a unique open portal connecting the cytoplasm to the transmembrane channel pore, that allows for a continuous pathway for Cl ions to cross the membrane in one conformation. This lateral portal may be what allows CFTR to function as an ion channel rather than as a pump, suggesting a plausible mechanism by which channel function may have evolved in CFTR.

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Functional Architecture of the Cytoplasmic Entrance to the Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channel Pore

Abstract: Background:The cytoplasmic entrance to the CFTR channel pore is not defined. Results: Functionally important positively charged amino acid side chains attract Cl Ϫ to the cytoplasmic entrance to the pore.

Cited by 36 publications

References 40 publications

Conformational Changes of CFTR upon Phosphorylation and ATP Binding

Conformational Changes of CFTR upon Phosphorylation and ATP Binding

Functional and Pharmacological Characterization of the Rare CFTR Mutation W361R

Cystic fibrosis transmembrane conductance regulator (CFTR): Making an ion channel out of an active transporter structure

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