Control of the CFTR channel's gates

2005 ◽  
Vol 33 (5) ◽  
pp. 1003-1007 ◽  
Author(s):  
P. Vergani ◽  
C. Basso ◽  
M. Mense ◽  
A.C. Nairn ◽  
D.C. Gadsby
Keyword(s):  
Cystic Fibrosis ◽  
Ion Channel ◽  
Single Channel ◽  
Atp Binding ◽  
Transmembrane Conductance Regulator ◽  
Binding Domains ◽  
Abc Protein ◽  
Channel Opening ◽  
Atp Binding And Hydrolysis ◽  
Opening And Closing

Unique among ABC (ATP-binding cassette) protein family members, CFTR (cystic fibrosis transmembrane conductance regulator), also termed ABCC7, encoded by the gene mutated in cystic fibrosis patients, functions as an ion channel. Opening and closing of its anion-selective pore are linked to ATP binding and hydrolysis at CFTR's two NBDs (nucleotide-binding domains), NBD1 and NBD2. Isolated NBDs of prokaryotic ABC proteins form homodimers upon binding ATP, but separate after hydrolysis of the ATP. By combining mutagenesis with single-channel recording and nucleotide photolabelling on intact CFTR molecules, we relate opening and closing of the channel gates to ATP-mediated events in the NBDs. In particular, we demonstrate that two CFTR residues, predicted to lie on opposite sides of its anticipated NBD1–NBD2 heterodimer interface, are energetically coupled when the channels open but are independent of each other in closed channels. This directly links ATP-driven tight dimerization of CFTR's cytoplasmic NBDs to opening of the ion channel in the transmembrane domains. Evolutionary conservation of the energetically coupled residues in a manner that preserves their ability to form a hydrogen bond argues that this molecular mechanism, involving dynamic restructuring of the NBD dimer interface, is shared by all members of the ABC protein superfamily.

scholarly journals Cysteine accessibility probes timing and extent of NBD separation along the dimer interface in gating CFTR channels

2015 ◽  
Vol 145 (4) ◽  
pp. 261-283 ◽  
Author(s):  
Luiz A. Poletto Chaves ◽  
David C. Gadsby
Keyword(s):  
Atp Hydrolysis ◽  
Atp Binding ◽  
Transmembrane Conductance Regulator ◽  
Dimer Interface ◽  
Binding Domains ◽  
Channel Opening ◽  
Order Of Magnitude ◽  
Signature Sequence ◽  
Dependent Modification ◽  
Opening And Closing

Cystic fibrosis transmembrane conductance regulator (CFTR) channel opening and closing are driven by cycles of adenosine triphosphate (ATP) binding–induced formation and hydrolysis-triggered disruption of a heterodimer of its cytoplasmic nucleotide-binding domains (NBDs). Although both composite sites enclosed within the heterodimer interface contain ATP in an open CFTR channel, ATP hydrolysis in the sole catalytically competent site causes channel closure. Opening of the NBD interface at that site then allows ADP–ATP exchange. But how frequently, and how far, the NBD surfaces separate at the other, inactive composite site remains unclear. We assessed separation at each composite site by monitoring access of nucleotide-sized hydrophilic, thiol-specific methanothiosulfonate (MTS) reagents to interfacial target cysteines introduced into either LSGGQ-like ATP-binding cassette signature sequence (replacing equivalent conserved serines: S549 and S1347). Covalent MTS-dependent modification of either cysteine while channels were kept closed by the absence of ATP impaired subsequent opening upon ATP readdition. Modification while channels were opening and closing in the presence of ATP caused macroscopic CFTR current to decline at the same speed as when the unmodified channels shut upon sudden ATP withdrawal. These results suggest that the target cysteines can be modified only in closed channels; that after modification the attached MTS adduct interferes with ATP-mediated opening; and that modification in the presence of ATP occurs rapidly once channels close, before they can reopen. This interpretation was corroborated by the finding that, for either cysteine target, the addition of the hydrolysis-impairing mutation K1250R (catalytic site Walker A Lys) similarly slowed, by an order of magnitude, channel closing on ATP removal and the speed of modification by MTS reagent in ATP. We conclude that, in every CFTR channel gating cycle, the NBD dimer interface separates simultaneously at both composite sites sufficiently to allow MTS reagents to access both signature-sequence serines. Relatively rapid modification of S1347C channels by larger reagents—MTS-glucose, MTS-biotin, and MTS-rhodamine—demonstrates that, at the noncatalytic composite site, this separation must exceed 8 Å.

scholarly journals Functional and pharmacological importance of the composite ATP binding site 1 in CFTR chloride channels

2010 ◽  
Author(s):  
◽  
Ming-Feng Tsai
Keyword(s):  
Cystic Fibrosis ◽  
Chloride Channels ◽  
Molecular Model ◽  
Chloride Ion ◽  
Atp Binding ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Atp Binding Site ◽  
Binding Domains ◽  
Opening And Closing

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride ion channel whose defects cause the deadly genetic disease cystic fibrosis (CF). Like other ATP binding cassette (ABC) proteins, CFTR encompasses two cytoplasmic nucleotide binding domains (NBDs). Upon ATP binding, the two NBDs can coalesce into a head-to-tail dimer with ATP buried at two interfacial composite sites (sites 1 and 2). Although evidence suggests that gating of CFTR is mainly controlled by site 2, the role of site 1 remains less understood. I have used pyrophosphate as a probe or adopted a ligand exchange protocol to investigate ATP binding status in site 1 in real time. With these novel approaches, I have identified a “partial” NBD dimer state mediated by an ATP molecule tightly bound in site 1. A molecular model of CFTR gating was then established with opening and closing of CFTR coupled to the formation and partial separation of the NBD dimer. Moreover, I discovered several mutations that enhance ATP binding in site 1 and demonstrated that the activity of CF-associated mutant channels, Î"F508- and G551D-CFTR, can be significantly improved by these mutations, thus providing evidence that site 1 is a potential target for developing pharmaceutical reagents to treat patients with CF.

scholarly journals Curcumin Opens Cystic Fibrosis Transmembrane Conductance Regulator Channels by a Novel Mechanism That Requires neither ATP Binding nor Dimerization of the Nucleotide-binding Domains

2006 ◽  
Vol 282 (7) ◽  
pp. 4533-4544 ◽  
Author(s):  
Wei Wang ◽  
Karen Bernard ◽  
Ge Li ◽  
Kevin L. Kirk
Keyword(s):  
Cystic Fibrosis ◽  
Nucleotide Binding ◽  
Salt Transport ◽  
Atp Binding ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Binding Domains ◽  
Channel Opening ◽  
Cystic Fibrosis Transmembrane ◽  
R Domain

Cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels are essential mediators of salt transport across epithelia. Channel opening normally requires ATP binding to both nucleotide-binding domains (NBDs), probable dimerization of the two NBDs, and phosphorylation of the R domain. How phosphorylation controls channel gating is unknown. Loss-of-function mutations in the CFTR gene cause cystic fibrosis; thus, there is considerable interest in compounds that improve mutant CFTR function. Here we investigated the mechanism by which CFTR is activated by curcumin, a natural compound found in turmeric. Curcumin opened CFTR channels by a novel mechanism that required neither ATP nor the second nucleotide-binding domain (NBD2). Consequently, this compound potently activated CF mutant channels that are defective for the normal ATP-dependent mode of gating (e.g. G551D and W1282X), including channels that lack NBD2. The stimulation of NBD2 deletion mutants by curcumin was strongly inhibited by ATP binding to NBD1, which implicates NBD1 as a plausible activation site. Curcumin activation became irreversible during prolonged exposure to this compound following which persistently activated channels gated dynamically in the absence of any agonist. Although CFTR activation by curcumin required neither ATP binding nor heterodimerization of the two NBDs, it was strongly dependent on prior channel phosphorylation by protein kinase A. Curcumin is a useful functional probe of CFTR gating that opens mutant channels by circumventing the normal requirements for ATP binding and NBD heterodimerization. The phosphorylation dependence of curcumin activation indicates that the R domain can modulate channel opening without affecting ATP binding to the NBDs or their heterodimerization.

scholarly journals Mutant cycles at CFTR’s non-canonical ATP-binding site support little interface separation during gating

2011 ◽  
Vol 137 (6) ◽  
pp. 549-562 ◽  
Author(s):  
Andras Szollosi ◽  
Daniella R. Muallem ◽  
László Csanády ◽  
Paola Vergani
Keyword(s):  
Atp Binding ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Atp Binding Site ◽  
Binding Domains ◽  
Gating Model ◽  
Channel Opening ◽  
Independent Technique ◽  
Atp Binding And Hydrolysis ◽  
Thermodynamic Coupling

Cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel belonging to the adenosine triphosphate (ATP)-binding cassette (ABC) superfamily. ABC proteins share a common molecular mechanism that couples ATP binding and hydrolysis at two nucleotide-binding domains (NBDs) to diverse functions. This involves formation of NBD dimers, with ATP bound at two composite interfacial sites. In CFTR, intramolecular NBD dimerization is coupled to channel opening. Channel closing is triggered by hydrolysis of the ATP molecule bound at composite site 2. Site 1, which is non-canonical, binds nucleotide tightly but is not hydrolytic. Recently, based on kinetic arguments, it was suggested that this site remains closed for several gating cycles. To investigate movements at site 1 by an independent technique, we studied changes in thermodynamic coupling between pairs of residues on opposite sides of this site. The chosen targets are likely to interact based on both phylogenetic analysis and closeness on structural models. First, we mutated T460 in NBD1 and L1353 in NBD2 (the corresponding site-2 residues become energetically coupled as channels open). Mutation T460S accelerated closure in hydrolytic conditions and in the nonhydrolytic K1250R background; mutation L1353M did not affect these rates. Analysis of the double mutant showed additive effects of mutations, suggesting that energetic coupling between the two residues remains unchanged during the gating cycle. We next investigated pairs 460–1348 and 460–1375. Although both mutations H1348A and H1375A produced dramatic changes in hydrolytic and nonhydrolytic channel closing rates, in the corresponding double mutants these changes proved mostly additive with those caused by mutation T460S, suggesting little change in energetic coupling between either positions 460–1348 or positions 460–1375 during gating. These results provide independent support for a gating model in which ATP-bound composite site 1 remains closed throughout the gating cycle.

scholarly journals Catalyst-like modulation of transition states for CFTR channel opening and closing: New stimulation strategy exploits nonequilibrium gating

2014 ◽  
Vol 143 (2) ◽  
pp. 269-287 ◽  
Author(s):  
László Csanády ◽  
Beáta Töröcsik
Keyword(s):  
Cystic Fibrosis ◽  
Ion Channels ◽  
Single Channel ◽  
Transition States ◽  
Chloride Ion ◽  
Open Probability ◽  
Gating Mechanism ◽  
Channel Opening ◽  
The Stability ◽  
Opening And Closing

Cystic fibrosis transmembrane conductance regulator (CFTR) is the chloride ion channel mutated in cystic fibrosis (CF) patients. It is an ATP-binding cassette protein, and its resulting cyclic nonequilibrium gating mechanism sets it apart from most other ion channels. The most common CF mutation (ΔF508) impairs folding of CFTR but also channel gating, reducing open probability (Po). This gating defect must be addressed to effectively treat CF. Combining single-channel and macroscopic current measurements in inside-out patches, we show here that the two effects of 5-nitro-2-(3-phenylpropylamino)benzoate (NPPB) on CFTR, pore block and gating stimulation, are independent, suggesting action at distinct sites. Furthermore, detailed kinetic analysis revealed that NPPB potently increases Po, also of ΔF508 CFTR, by affecting the stability of gating transition states. This finding is unexpected, because for most ion channels, which gate at equilibrium, altering transition-state stabilities has no effect on Po; rather, agonists usually stimulate by stabilizing open states. Our results highlight how for CFTR, because of its unique cyclic mechanism, gating transition states determine Po and offer strategic targets for potentiator compounds to achieve maximal efficacy.

scholarly journals Modulation of CFTR gating by permeant ions

2014 ◽  
Vol 145 (1) ◽  
pp. 47-60 ◽  
Author(s):  
Han-I Yeh ◽  
Jiunn-Tyng Yeh ◽  
Tzyh-Chang Hwang
Keyword(s):  
Atp Hydrolysis ◽  
Single Channel ◽  
Common Mechanism ◽  
Transmembrane Conductance Regulator ◽  
Open Probability ◽  
Binding Domains ◽  
Gating Model ◽  
Sites Of Action ◽  
Opening And Closing ◽  
Closing Rate

Cystic fibrosis transmembrane conductance regulator (CFTR) is unique among ion channels in that after its phosphorylation by protein kinase A (PKA), its ATP-dependent gating violates microscopic reversibility caused by the intimate involvement of ATP hydrolysis in controlling channel closure. Recent studies suggest a gating model featuring an energetic coupling between opening and closing of the gate in CFTR’s transmembrane domains and association and dissociation of its two nucleotide-binding domains (NBDs). We found that permeant ions such as nitrate can increase the open probability (Po) of wild-type (WT) CFTR by increasing the opening rate and decreasing the closing rate. Nearly identical effects were seen with a construct in which activity does not require phosphorylation of the regulatory domain, indicating that nitrate primarily affects ATP-dependent gating steps rather than PKA-dependent phosphorylation. Surprisingly, the effects of nitrate on CFTR gating are remarkably similar to those of VX-770 (N-(2,4-Di-tert-butyl-5-hydroxyphenyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide), a potent CFTR potentiator used in clinics. These include effects on single-channel kinetics of WT CFTR, deceleration of the nonhydrolytic closing rate, and potentiation of the Po of the disease-associated mutant G551D. In addition, both VX-770 and nitrate increased the activity of a CFTR construct lacking NBD2 (ΔNBD2), indicating that these gating effects are independent of NBD dimerization. Nonetheless, whereas VX-770 is equally effective when applied from either side of the membrane, nitrate potentiates gating mainly from the cytoplasmic side, implicating a common mechanism for gating modulation mediated through two separate sites of action.

scholarly journals ATP hydrolysis-driven gating in cystic fibrosis transmembrane conductance regulator

2008 ◽  
Vol 364 (1514) ◽  
pp. 247-255 ◽  
Author(s):  
Daniella Muallem ◽  
Paola Vergani
Keyword(s):  
Cystic Fibrosis ◽  
Conformational Changes ◽  
Atp Hydrolysis ◽  
Atp Binding ◽  
Functional Difference ◽  
Transmembrane Conductance Regulator ◽  
Cellular Functions ◽  
Transmembrane Conductance ◽  
Binding Domains ◽  
Cystic Fibrosis Transmembrane

Proteins belonging to the ATP-binding cassette superfamily couple ATP binding and hydrolysis at conserved nucleotide-binding domains (NBDs) to diverse cellular functions. Most superfamily members are transporters, while cystic fibrosis transmembrane conductance regulator (CFTR), alone, is an ion channel. Despite this functional difference, recent results have suggested that CFTR shares a common molecular mechanism with other members. ATP binds to partial binding sites on the surface of the two NBDs, which then associate to form a NBD dimer, with complete composite catalytic sites now buried at the interface. ATP hydrolysis and γ-phosphate dissociation, with the loss of molecular contacts linking the two sides of the composite site, trigger dimer dissociation. The conformational signals generated by NBD dimer formation and dissociation are transmitted to the transmembrane domains where, in transporters, they drive the cycle of conformational changes that translocate the substrate across the membrane; in CFTR, they result in opening and closing (gating) of the ion-permeation pathway.

Fluoride stimulates cystic fibrosis transmembrane conductance regulator Cl− channel activity

1998 ◽  
Vol 274 (3) ◽  
pp. L305-L312 ◽  
Author(s):  
Herbert A. Berger ◽  
Sue M. Travis ◽  
Michael J. Welsh
Keyword(s):  
Cystic Fibrosis ◽  
Single Channel ◽  
Single Channels ◽  
Dependent Manner ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Binding Domains ◽  
Cytosolic Domains ◽  
Cystic Fibrosis Transmembrane ◽  
Dose Dependent

While studying the regulation of the cystic fibrosis transmembrane conductance regulator (CFTR), we found that addition of F− to the cytosolic surface of excised, inside-out membrane patches reversibly increased Cl− current in a dose-dependent manner. Stimulation required prior phosphorylation and the presence of ATP. F− increased current even in the presence of deferoxamine, which chelates Al3+, suggesting that stimulation was not due to A[Formula: see text]. F− also stimulated current in a CFTR variant that lacked a large part of the R domain, suggesting that the effect was not mediated via this domain. Studies of single channels showed that F−increased the open-state probability by slowing channel closure from bursts of activity; the mean closed time between bursts and single-channel conductance was not altered. These results suggested that F− influenced regulation by the cytosolic domains, most likely the nucleotide-binding domains (NBDs). Consistent with this, we found that mutation of a conserved Walker lysine in NBD2 changed the relative stimulatory effect of F− compared with wild-type CFTR, whereas mutation of the Walker lysine in NBD1 had no effect. Based on these and previous data, we speculate that F− interacts with CFTR, possibly via NBD2, and slows the rate of channel closure.

scholarly journals Positioning of extracellular loop 1 affects pore gating of the cystic fibrosis transmembrane conductance regulator

2016 ◽  
Vol 310 (5) ◽  
pp. L403-L414 ◽  
Author(s):  
Daniel T. Infield ◽  
Guiying Cui ◽  
Christopher Kuang ◽  
Nael A. McCarty
Keyword(s):  
Cystic Fibrosis ◽  
Single Channel ◽  
Chloride Ion ◽  
Transmembrane Conductance Regulator ◽  
Extracellular Loop ◽  
Transmembrane Conductance ◽  
Conformational Restriction ◽  
Channel Opening ◽  
Cf Transmembrane Conductance Regulator ◽  
Related Proteins

The cystic fibrosis (CF) transmembrane conductance regulator (CFTR) is a chloride ion channel, the dysfunction of which directly leads to the life-shortening disease CF. Extracellular loop 1 (ECL1) of CFTR contains several residues involved in stabilizing the open state of the channel; some, including D110, are sites of disease-associated gating mutations. Structures from related proteins suggest that the position of CFTR's extracellular loops may change considerably during gating. To better understand the roles of ECL1 in CFTR function, we utilized functional cysteine cross-linking to determine the effects of modulation of D110C-CFTR and of a double mutant of D110C with K892C in extracellular loop 4 (ECL4). The reducing agent DTT elicited a large potentiation of the macroscopic conductance of D110C/K892C-CFTR, likely due to breakage of a spontaneous disulfide bond between C110 and C892. DTT-reduced D110C/K892C-CFTR was rapidly inhibited by binding cadmium ions with high affinity, suggesting that these residues frequently come in close proximity in actively gating channels. Effects of DTT and cadmium on modulation of pore gating were demonstrated at the single-channel level. Finally, disulfided D110C/K892C-CFTR channels were found to be less sensitive than wild-type or DTT-treated D110C/K892C-CFTR channels to stimulation by IBMX, suggesting an impact of this conformational restriction on channel activation by phosphorylation. The results are best explained in the context of a model of CFTR gating wherein stable channel opening requires correct positioning of functional elements structurally influenced by ECL1.

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