scholarly journals Alignment of transmembrane regions in the cystic fibrosis transmembrane conductance regulator chloride channel pore

2011 ◽  
Vol 138 (2) ◽  
pp. 165-178 ◽  
Author(s):  
Wuyang Wang ◽  
Yassine El Hiani ◽  
Paul Linsdell
Keyword(s):  
Cystic Fibrosis ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Transmembrane Conductance Regulator ◽  
Patch Clamp Recording ◽  
Transmembrane Conductance ◽  
Three Dimensional Structure ◽  
Cystic Fibrosis Transmembrane ◽  
Pore Lining ◽  
Cytoplasmic Side

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.

Identification of a region of strong discrimination in the pore of CFTR

2001 ◽  
Vol 281 (4) ◽  
pp. L852-L867 ◽  
Author(s):  
Nael A. McCarty ◽  
Zhi-Ren Zhang
Keyword(s):  
Cystic Fibrosis ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Structural Determinants ◽  
Anion Selectivity ◽  
Relative Conductance ◽  
Pore Domain ◽  
Cystic Fibrosis Transmembrane

The variety of methods used to identify the structural determinants of anion selectivity in the cystic fibrosis transmembrane conductance regulator Cl− channel has made it difficult to assemble the data into a coherent framework that describes the three-dimensional structure of the pore. Here, we compare the relative importance of sites previously studied and identify new sites that contribute strongly to anion selectivity. We studied Cl−and substitute anions in oocytes expressing wild-type cystic fibrosis transmembrane conductance regulator or 12-pore-domain mutants to determine relative permeability and relative conductance for 9 monovalent anions and 1 divalent anion. The data indicate that the region of strong discrimination resides between T338 and S341 in transmembrane 6, where mutations affected selectivity between Cl− and both large and small anions. Mutations further toward the extracellular end of the pore only strongly affected selectivity between Cl− and larger anions. Only mutations at S341 affected selectivity between monovalent and divalent anions. The data are consistent with a narrowing of the pore between the extracellular end and a constriction near the middle of the pore.

scholarly journals The Pore Architecture of the Cystic Fibrosis Transmembrane Conductance Regulator Channel Revealed by Co-Mutation in Pore-Forming Transmembrane Regions

2016 ◽  
pp. 505-515
Author(s):  
F. QIAN ◽  
L. LIU ◽  
Z. LIU ◽  
C. LU
Keyword(s):  
Cystic Fibrosis ◽  
Single Channel ◽  
Transmembrane Conductance Regulator ◽  
Patch Clamp Recording ◽  
Transmembrane Conductance ◽  
Channel Pore ◽  
Selective Filter ◽  
Transmembrane Regions ◽  
Cystic Fibrosis Transmembrane ◽  
Insight Into

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.

scholarly journals Functional analysis of the C-terminal boundary of the second nucleotide binding domain of the cystic fibrosis transmembrane conductance regulator and structural implications

2002 ◽  
Vol 366 (2) ◽  
pp. 541-548 ◽  
Author(s):  
Martina GENTZSCH ◽  
Andrei ALEKSANDROV ◽  
Luba ALEKSANDROV ◽  
John R. RIORDAN
Keyword(s):  
Cystic Fibrosis ◽  
Cellular Localization ◽  
Three Dimensional ◽  
Large Family ◽  
Nucleotide Binding ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Abc Proteins ◽  
Binding Domains ◽  
Cystic Fibrosis Transmembrane

The cystic fibrosis transmembrane conductance regulator (CFTR) contains two nucleotide-binding domains (NBDs) or ATP-binding cassettes (ABCs) that characterize a large family of membrane transporters. Although the three-dimensional structures of these domains from several ABC proteins have been determined, this is not the case for CFTR, and hence the domains are defined simply on the basis of sequence alignment. The functional C-terminal boundary of NBD1 of CFTR was located by analysis of chloride channel function [Chan, Csanady, Seto-Young, Nairn and Gadsby (2000) J. Gen. Physiol. 116, 163–180]. However, the boundary between the C-terminal end of NBD2 and sequences further downstream in the whole protein, that are important for its cellular localization and endocytotic turnover, has not been defined. We have now done this by assaying the influence of progressive C-terminal truncations on photolabelling of NBD2 by 8-azido-ATP, which reflects hydrolysis, as well as binding, at that domain, and on NBD2-dependent channel gating itself. The boundary defined in this way is between residues 1420 and 1424, which corresponds to the final β-strand in aligned NBDs whose structures have been determined. Utilization of this information should facilitate the generation of monodisperse NBD2 polypeptides for structural analysis, which until now has not been possible. The established boundary includes within NBD2 a hydrophobic patch of four residues (1413–1416) previously shown to be essential for CFTR maturation and stability [Gentzsch and Riordan (2001) J. Biol. Chem. 276, 1291–1298]. This hydrophobic cluster is conserved in most ABC proteins, and on alignment with ones of known structure constitutes the penultimate β-strand of the domain which is likely to participate in essential structure-stabilizing β-sheet formation.

scholarly journals CFTR structure, stability, function and regulation

2019 ◽  
Vol 400 (10) ◽  
pp. 1359-1370 ◽  
Author(s):  
Xin Meng ◽  
Jack Clews ◽  
Anca D. Ciuta ◽  
Eleanor R. Martin ◽  
Robert C. Ford
Keyword(s):  
Cystic Fibrosis ◽  
Regulatory Region ◽  
Three Dimensional ◽  
Structure Stability ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Experimental Conditions ◽  
Interacting Protein ◽  
European Origin ◽  
Cystic Fibrosis Transmembrane

Abstract Cystic fibrosis transmembrane conductance regulator (CFTR) is a unique member of the ATP-binding cassette family of proteins because it has evolved into a channel. Mutations in CFTR cause cystic fibrosis, the most common genetic disease in people of European origin. The F508del mutation is found in about 90% of patients and here we present data that suggest its main effect is on CFTR stability rather than on the three-dimensional (3D) folded state. A survey of recent cryo-electron microscopy studies was carried out and this highlighted differences in terms of CFTR conformation despite similarities in experimental conditions. We further studied CFTR structure under various phosphorylation states and with the CFTR-interacting protein NHERF1. The coexistence of outward-facing and inward-facing conformations under a range of experimental conditions was suggested from these data. These results are discussed in terms of structural models for channel gating, and favour the model where the mostly disordered regulatory-region of the protein acts as a channel plug.

Interaction between 2 extracellular loops influences the activity of the cystic fibrosis transmembrane conductance regulator chloride channel

2014 ◽  
Vol 92 (5) ◽  
pp. 390-396 ◽  
Author(s):  
Steven D. Broadbent ◽  
Wuyang Wang ◽  
Paul Linsdell
Keyword(s):  
Cystic Fibrosis ◽  
Chloride Channel ◽  
Transmembrane Conductance Regulator ◽  
Channel Activity ◽  
Patch Clamp Recording ◽  
Transmembrane Conductance ◽  
Channel Function ◽  
Extracellular Loops ◽  
Key Sites ◽  
Cystic Fibrosis Transmembrane

Activity of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is thought to be controlled by cytoplasmic factors. However, recent evidence has shown that overall channel activity is also influenced by extracellular anions that interact directly with the extracellular loops (ECLs) of the CFTR protein. Very little is known about the structure of the ECLs or how substances interacting with these ECLs might affect CFTR function. We used patch-clamp recording to investigate the accessibility of cysteine-reactive reagents to cysteines introduced throughout ECL1 and 2 key sites in ECL4. Furthermore, interactions between ECL1 and ECL4 were investigated by the formation of disulfide crosslinks between cysteines introduced into these 2 regions. Crosslinks could be formed between R899C (in ECL4) and a number of sites in ECL1 in a manner that was dependent on channel activity, suggesting that the relative orientation of these 2 loops changes on activation. Formation of these crosslinks inhibited channel function, suggesting that relative movement of these ECLs is important to normal channel function. Implications of these findings for the effects of mutations in the ECLs that are associated with cystic fibrosis and interactions with extracellular substances that influence channel activity are discussed.

Inhibition of cystic fibrosis transmembrane conductance regulator chloride channel currents by arachidonic acid

2000 ◽  
Vol 78 (6) ◽  
pp. 490-499 ◽  
Author(s):  
Paul Linsdell
Keyword(s):  
Cystic Fibrosis ◽  
Fatty Acids ◽  
Arachidonic Acid ◽  
Fatty Acid ◽  
Chloride Channel ◽  
Transmembrane Conductance Regulator ◽  
Patch Clamp Recording ◽  
Transmembrane Conductance ◽  
High Affinity ◽  
Cystic Fibrosis Transmembrane

Chloride permeation through the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel is inhibited by a number of different classes of organic anions which are able to enter and block the channel pore from its cytoplasmic end. Here I show, using patch clamp recording from CFTR-transfected baby hamster kidney cell lines, that the cis-unsaturated fatty acid arachidonic acid also inhibits CFTR Cl- currents when applied to the cytoplasmic face of excised membrane patches. This inhibition was of a relatively high affinity compared with other known CFTR inhibitors, with an apparent Kd of 6.5 ± 0.9 µM. However, in contrast with known CFTR pore blockers, inhibition by arachidonic acid was only very weakly voltage dependent, and was insensitive to the extracellular Cl- concentration. Arachidonic acid-mediated inhibition of CFTR Cl- currents was not abrogated by inhibitors of lipoxygenases, cyclooxygenases or cytochrome P450, suggesting that arachidonic acid itself, rather than some metabolite, directly affects CFTR. Similar inhibition of CFTR Cl- currents was seen with other fatty acids, with the rank order of potency linoleic [Formula: see text] arachidonic [Formula: see text] oleic > elaidic [Formula: see text] palmitic [Formula: see text] myristic. These results identify fatty acids as novel high affinity modulators of the CFTR Cl- channel.Key words: CFTR, chloride channel, fatty acid, channel block, cystic fibrosis.

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