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1.  Nonintegral stoichiometry in CFTR gating revealed by a pore-lining mutation 
The Journal of General Physiology  2012;140(4):347-359.
Cystic fibrosis transmembrane conductance regulator (CFTR) is a unique member of the ATP-binding cassette (ABC) protein superfamily. Unlike most other ABC proteins that function as active transporters, CFTR is an ATP-gated chloride channel. The opening of CFTR’s gate is associated with ATP-induced dimerization of its two nucleotide-binding domains (NBD1 and NBD2), whereas gate closure is facilitated by ATP hydrolysis-triggered partial separation of the NBDs. This generally held theme of CFTR gating—a strict coupling between the ATP hydrolysis cycle and the gating cycle—is put to the test by our recent finding of a short-lived, post-hydrolytic state that can bind ATP and reenter the ATP-induced original open state. We accidentally found a mutant CFTR channel that exhibits two distinct open conductance states, the smaller O1 state and the larger O2 state. In the presence of ATP, the transition between the two states follows a preferred O1→O2 order, a telltale sign of a violation of microscopic reversibility, hence demanding an external energy input likely from ATP hydrolysis, as such preferred gating transition was abolished in a hydrolysis-deficient mutant. Interestingly, we also observed a considerable amount of opening events that contain more than one O1→O2 transition, indicating that more than one ATP molecule may be hydrolyzed within an opening burst. We thus conclude a nonintegral stoichiometry between the gating cycle and ATP consumption. Our results lead to a six-state gating model conforming to the classical allosteric mechanism: both NBDs and transmembrane domains hold a certain degree of autonomy, whereas the conformational change in one domain will facilitate the conformational change in the other domain.
doi:10.1085/jgp.201210834
PMCID: PMC3457689  PMID: 22966014
2.  CFTR channel opening by ATP-driven tight dimerization of its nucleotide-binding domains 
Nature  2005;433(7028):876-880.
ABC (ATP-binding cassette) proteins constitute a large family of membrane proteins that actively transport a broad range of substrates. Cystic fibrosis transmembrane conductance regulator (CFTR), the protein dysfunctional in cystic fibrosis, is unique among ABC proteins in that its transmembrane domains comprise an ion channel. Opening and closing of the pore have been linked to ATP binding and hydrolysis at CFTR’s two nucleotide-binding domains, NBD1 and NBD2 (see, for example, refs 1, 2). Isolated NBDs of prokaryotic ABC proteins dimerize upon binding ATP, and hydrolysis of the ATP causes dimer dissociation3–5. Here, using single-channel recording methods on intact CFTR molecules, we directly follow opening and closing of the channel gates, and relate these occurrences to ATP-mediated events in the NBDs. We find that energetic coupling6 between two CFTR residues, expected to lie on opposite sides of its predicted NBD1–NBD2 dimer interface, changes in concert with channel gating status. The two monitored side chains are independent of each other in closed channels but become coupled as the channels open. The results directly link ATP-driven tight dimerization of CFTR’s cytoplasmic nucleotide-binding domains to opening of the ion channel in the transmembrane domains. This establishes a molecular mechanism, involving dynamic restructuring of the NBD dimer interface, that is probably common to all members of the ABC protein superfamily.
doi:10.1038/nature03313
PMCID: PMC2756053  PMID: 15729345
3.  Stable ATP binding mediated by a partial NBD dimer of the CFTR chloride channel 
The Journal of General Physiology  2010;135(5):399-414.
Cystic fibrosis transmembrane conductance regulator (CFTR), a member of the adenosine triphosphate (ATP) binding cassette (ABC) superfamily, is an ATP-gated chloride channel. Like other ABC proteins, CFTR encompasses two nucleotide binding domains (NBDs), NBD1 and NBD2, each accommodating an ATP binding site. It is generally accepted that CFTR’s opening–closing cycles, each completed within 1 s, are driven by rapid ATP binding and hydrolysis events in NBD2. Here, by recording CFTR currents in real time with a ligand exchange protocol, we demonstrated that during many of these gating cycles, NBD1 is constantly occupied by a stably bound ATP or 8-N3-ATP molecule for tens of seconds. We provided evidence that this tightly bound ATP or 8-N3-ATP also interacts with residues in the signature sequence of NBD2, a telltale sign for an event occurring at the NBD1–NBD2 interface. The open state of CFTR has been shown to represent a two-ATP–bound NBD dimer. Our results indicate that upon ATP hydrolysis in NBD2, the channel closes into a “partial NBD dimer” state where the NBD interface remains partially closed, preventing ATP dissociation from NBD1 but allowing the release of hydrolytic products and binding of the next ATP to occur in NBD2. Opening and closing of CFTR can then be coupled to the formation and “partial” separation of the NBD dimer. The tightly bound ATP molecule in NBD1 can occasionally dissociate from the partial dimer state, resulting in a nucleotide-free monomeric state of NBDs. Our data, together with other structural/functional studies of CFTR’s NBDs, suggest that this process is poorly reversible, implying that the channel in the partial dimer state or monomeric state enters the open state through different pathways. We therefore proposed a gating model for CFTR with two distinct cycles. The structural and functional significance of our results to other ABC proteins is discussed.
doi:10.1085/jgp.201010399
PMCID: PMC2860585  PMID: 20421370
4.  The Two ATP Binding Sites of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Play Distinct Roles in Gating Kinetics and Energetics 
The Journal of General Physiology  2006;128(4):413-422.
Cystic fibrosis transmembrane conductance regulator (CFTR), a member of the ABC (ATP binding cassette) transporter family, is a chloride channel whose activity is controlled by protein kinase–dependent phosphorylation. Opening and closing (gating) of the phosphorylated CFTR is coupled to ATP binding and hydrolysis at CFTR's two nucleotide binding domains (NBD1 and NBD2). Recent studies present evidence that the open channel conformation reflects a head-to-tail dimerization of CFTR's two NBDs as seen in the NBDs of other ABC transporters (Vergani et al., 2005). Whether these two ATP binding sites play an equivalent role in the dynamics of NBD dimerization, and thus in gating CFTR channels, remains unsettled. Based on the crystal structures of NBDs, sequence alignment, and homology modeling, we have identified two critical aromatic amino acids (W401 in NBD1 and Y1219 in NBD2) that coordinate the adenine ring of the bound ATP. Conversion of the W401 residue to glycine (W401G) has little effect on the sensitivity of the opening rate to [ATP], but the same mutation at the Y1219 residue dramatically lowers the apparent affinity for ATP by >50-fold, suggesting distinct roles of these two ATP binding sites in channel opening. The W401G mutation, however, shortens the open time constant. Energetic analysis of our data suggests that the free energy of ATP binding at NBD1, but not at NBD2, contributes significantly to the energetics of the open state. This kinetic and energetic asymmetry of CFTR's two NBDs suggests an asymmetric motion of the NBDs during channel gating. Opening of the channel is initiated by ATP binding at the NBD2 site, whereas separation of the NBD dimer at the NBD1 site constitutes the rate-limiting step in channel closing.
doi:10.1085/jgp.200609622
PMCID: PMC2151577  PMID: 16966475
5.  Identification of a novel post-hydrolytic state in CFTR gating 
The Journal of General Physiology  2012;139(5):359-370.
Adenosine triphosphate (ATP)-binding cassette (ABC) transporters, ubiquitous proteins found in all kingdoms of life, catalyze substrates translocation across biological membranes using the free energy of ATP hydrolysis. Cystic fibrosis transmembrane conductance regulator (CFTR) is a unique member of this superfamily in that it functions as an ATP-gated chloride channel. Despite difference in function, recent studies suggest that the CFTR chloride channel and the exporter members of the ABC protein family may share an evolutionary origin. Although ABC exporters harness the free energy of ATP hydrolysis to fuel a transport cycle, for CFTR, ATP-induced dimerization of its nucleotide-binding domains (NBDs) and subsequent hydrolysis-triggered dimer separation are proposed to be coupled, respectively, to the opening and closing of the gate in its transmembrane domains. In this study, by using nonhydrolyzable ATP analogues, such as pyrophosphate or adenylyl-imidodiphosphate as baits, we captured a short-lived state (state X), which distinguishes itself from the previously identified long-lived C2 closed state by its fast response to these nonhydrolyzable ligands. As state X is caught during the decay phase of channel closing upon washout of the ligand ATP but before the channel sojourns to the C2 closed state, it likely emerges after the bound ATP in the catalysis-competent site has been hydrolyzed and the hydrolytic products have been released. Thus, this newly identified post-hydrolytic state may share a similar conformation of NBDs as the C2 closed state (i.e., a partially separated NBD and a vacated ATP-binding pocket). The significance of this novel state in understanding the structural basis of CFTR gating is discussed.
doi:10.1085/jgp.201210789
PMCID: PMC3343372  PMID: 22508846
6.  The H-loop in the Second Nucleotide-binding Domain of the Cystic Fibrosis Transmembrane Conductance Regulator is Required for Efficient Chloride Channel Closing 
Cellular Physiology and Biochemistry  2010;25(2-3):169-180.
The cystic fibrosis transmembrane conductance regulator (CFTR) is an ATP-binding cassette (ABC) transporter that functions as a cAMP-activated chloride channel. The recent model of CFTR gating predicts that the ATP binding to both nucleotide-binding domains (NBD1 and NBD2) of CFTR is required for the opening of the channel, while the ATP hydrolysis at NBD2 induces subsequent channel closing. In most ABC proteins, efficient hydrolysis of ATP requires the presence of the invariant histidine residue within the H-loop located in the C-terminal part of the NBD. However, the contribution of the corresponding region (H-loop) of NBD2 to the CFTR channel gating has not been examined so far. Here we report that the alanine substitution of the conserved dipeptide HR motif (HR→AA) in the H-loop of NBD2 leads to prolonged open states of CFTR channel, indicating that the H-loop is required for efficient channel closing. On the other hand, the HR→AA substitution lead to the substantial decrease of CFTR-mediated current density (pA/pF) in transfected HEK 293 cells, as recorded in the whole-cell patch-clamp analysis. These results suggest that the H-loop of NBD2, apart from being required for CFTR channel closing, may be involved in regulating CFTR trafficking to the cell surface.
PMCID: PMC2844707  PMID: 20110677
CFTR; Cystic fibrosis; ABC transporters; Nucleotide-binding domain; H-loop
7.  The H-loop in the Second Nucleotide-binding Domain of the Cystic Fibrosis Transmembrane Conductance Regulator is Required for Efficient Chloride Channel Closing 
The cystic fibrosis transmembrane conductance regulator (CFTR) is an ATP-binding cassette (ABC) transporter that functions as a cAMP-activated chloride channel. The recent model of CFTR gating predicts that the ATP binding to both nucleotide-binding domains (NBD1 and NBD2) of CFTR is required for the opening of the channel, while the ATP hydrolysis at NBD2 induces subsequent channel closing. In most ABC proteins, efficient hydrolysis of ATP requires the presence of the invariant histidine residue within the H-loop located in the C-terminal part of the NBD. However, the contribution of the corresponding region (H-loop) of NBD2 to the CFTR channel gating has not been examined so far. Here we report that the alanine substitution of the conserved dipeptide HR motif (HR→AA) in the H-loop of NBD2 leads to prolonged open states of CFTR channel, indicating that the H-loop is required for efficient channel closing. On the other hand, the HR→AA substitution lead to the substantial decrease of CFTR-mediated current density (pA/pF) in transfected HEK 293 cells, as recorded in the whole-cell patch-clamp analysis. These results suggest that the H-loop of NBD2, apart from being required for CFTR channel closing, may be involved in regulating CFTR trafficking to the cell surface.
doi:10.1159/000276549
PMCID: PMC2844707  PMID: 20110677
CFTR; Cystic fibrosis; ABC transporters; Nucleotide-binding domain; H-loop
8.  Functional Roles of Nonconserved Structural Segments in CFTR's NH2-terminal Nucleotide Binding Domain 
The cystic fibrosis transmembrane conductance regulator (CFTR), encoded by the gene mutated in cystic fibrosis patients, belongs to the family of ATP-binding cassette (ABC) proteins, but, unlike other members, functions as a chloride channel. CFTR is activated by protein kinase A (PKA)-mediated phosphorylation of multiple sites in its regulatory domain, and gated by binding and hydrolysis of ATP at its two nucleotide binding domains (NBD1, NBD2). The recent crystal structure of NBD1 from mouse CFTR (Lewis, H.A., S.G. Buchanan, S.K. Burley, K. Conners, M. Dickey, M. Dorwart, R. Fowler, X. Gao, W.B. Guggino, W.A. Hendrickson, et al. 2004. EMBO J. 23:282–293) identified two regions absent from structures of all other NBDs determined so far, a “regulatory insertion” (residues 404–435) and a “regulatory extension” (residues 639–670), both positioned to impede formation of the putative NBD1–NBD2 dimer anticipated to occur during channel gating; as both segments appeared highly mobile and both contained consensus PKA sites (serine 422, and serines 660 and 670, respectively), it was suggested that their phosphorylation-linked conformational changes might underlie CFTR channel regulation. To test that suggestion, we coexpressed in Xenopus oocytes CFTR residues 1–414 with residues 433–1480, or residues 1–633 with 668–1480, to yield split CFTR channels (called 414+433 and 633+668) that lack most of the insertion, or extension, respectively. In excised patches, regulation of the resulting CFTR channels by PKA and by ATP was largely normal. Both 414+433 channels and 633+668 channels, as well as 633(S422A)+668 channels (lacking both the extension and the sole PKA consensus site in the insertion), were all shut during exposure to MgATP before addition of PKA, but activated like wild type (WT) upon phosphorylation; this indicates that inhibitory regulation of nonphosphorylated WT channels depends upon neither segment. Detailed kinetic analysis of 414+433 channels revealed intact ATP dependence of single-channel gating kinetics, but slightly shortened open bursts and faster closing from the locked-open state (elicited by ATP plus pyrophosphate or ATP plus AMPPNP). In contrast, 633+668 channel function was indistinguishable from WT at both macroscopic and microscopic levels. We conclude that neither nonconserved segment is an essential element of PKA- or nucleotide-dependent regulation.
doi:10.1085/jgp.200409174
PMCID: PMC2217481  PMID: 15596536
ABC transporters; crystal structure; chloride ion-channel gating; phosphorylation; domain boundaries
9.  Involvement of F1296 and N1303 of CFTR in induced-fit conformational change in response to ATP binding at NBD2 
The Journal of General Physiology  2010;136(4):407-423.
The chloride ion channel cystic fibrosis transmembrane conductance regulator (CFTR) displays a typical adenosine trisphosphate (ATP)-binding cassette (ABC) protein architecture comprising two transmembrane domains, two intracellular nucleotide-binding domains (NBDs), and a unique intracellular regulatory domain. Once phosphorylated in the regulatory domain, CFTR channels can open and close when supplied with cytosolic ATP. Despite the general agreement that formation of a head-to-tail NBD dimer drives the opening of the chloride ion pore, little is known about how ATP binding to individual NBDs promotes subsequent formation of this stable dimer. Structural studies on isolated NBDs suggest that ATP binding induces an intra-domain conformational change termed “induced fit,” which is required for subsequent dimerization. We investigated the allosteric interaction between three residues within NBD2 of CFTR, F1296, N1303, and R1358, because statistical coupling analysis suggests coevolution of these positions, and because in crystal structures of ABC domains, interactions between these positions appear to be modulated by ATP binding. We expressed wild-type as well as F1296S, N1303Q, and R1358A mutant CFTR in Xenopus oocytes and studied these channels using macroscopic inside-out patch recordings. Thermodynamic mutant cycles were built on several kinetic parameters that characterize individual steps in the gating cycle, such as apparent affinities for ATP, open probabilities in the absence of ATP, open probabilities in saturating ATP in a mutant background (K1250R), which precludes ATP hydrolysis, as well as the rates of nonhydrolytic closure. Our results suggest state-dependent changes in coupling between two of the three positions (1296 and 1303) and are consistent with a model that assumes a toggle switch–like interaction pattern during the intra-NBD2 induced fit in response to ATP binding. Stabilizing interactions of F1296 and N1303 present before ATP binding are replaced by a single F1296-N1303 contact in ATP-bound states, with similar interaction partner toggling occurring during the much rarer ATP-independent spontaneous openings.
doi:10.1085/jgp.201010434
PMCID: PMC2947058  PMID: 20876359
10.  Characterization of the ATPase and Transport Activities of Purified CFTR 
Biochemistry  2004;43(4):1045-1053.
The cystic fibrosis transmembrane conductance regulator (CFTR) functions in vivo as a cAMP-activated chloride channel. A member of the ATP-binding cassette superfamily of membrane transporters, CFTR contains two transmembrane domains (TMDs), two nucleotide-binding domains (NBDs), and a regulatory (R) domain. It is presumed that CFTR couples ATP hydrolysis to channel gating, and as a first step in addressing this issue directly, we have established conditions for purification of biochemical quantities of human CFTR expressed in Sf9 insect cells. Use of an 8-azido-[α32P]-ATP binding and vanadate-trapping assay allowed us to devise conditions to preserve CFTR function during purification of a C-terminal His10-tagged variant after solubilization with lyso-phosphatidylglycerol (1%) and diheptanolylphosphatidylcholine (0.3%) in the presence of excess phospholipid. Study of purified and reconstituted CFTR showed that it binds nucleotide with an efficiency comparable to that of P-glycoprotein, and that it hydrolyzes ATP at rates sufficient to account for presumed in vivo activity (VMax of 58 ± 5 nmol/min/mg protein, KM (MgATP) of 0.15 mM). In further work, we found that neither nucleotide binding nor ATPase activity were altered by phosphorylation (using Protein Kinase A) or dephosphorylation (with Protein Phosphatase 2B); we also observed inhibition (∼40%) of ATP hydrolysis by reduced glutathione, but not by DTT. To evaluate CFTR function as an anion channel, we introduced an in vitro macroscopic assay based on the equilibrium exchange of proteoliposome-entrapped radioactive tracers. This revealed a CFTR-dependent transport of 125I that could be inhibited by known chloride channel blockers; no significant CFTR-dependent transport of [α32P]-ATP was observed. We conclude that heterologous expression of CFTR in Sf9 cells can support manufacture and purification of fully functional CFTR. This should aid in further biochemical characterization of this important molecule.
doi:10.1021/bi035382a
PMCID: PMC2587309  PMID: 14744150
11.  CONTROL OF THE CFTR CHANNEL’S GATES 
Biochemical Society transactions  2005;33(Pt 5):1003-1007.
SYNOPSYS
Unique among ABC (ATP-binding cassette) protein family members, CFTR (cystic fibrosis transmembrane conductance regulator), 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 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, nucleotide photolabeling, and sulfhydryl-specific crosslinking methods 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 nucleotide binding domains 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.
doi:10.1042/BST20051003
PMCID: PMC2728124  PMID: 16246032
CFTR; ATP binding and hydrolysis; single-channel kinetics; ABC protein
12.  The CFTR Ion Channel: Gating, Regulation, and Anion Permeation 
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.
CFTR is the only ATP-binding cassette transporter that is known to be an ion channel. It is also the only ligand-gated channel that consumes its ligand (ATP) during the gating cycle.
doi:10.1101/cshperspect.a009498
PMCID: PMC3530039  PMID: 23284076
13.  ATP and AMP Mutually Influence Their Interaction with the ATP-binding Cassette (ABC) Adenylate Kinase Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) at Separate Binding Sites*  
The Journal of Biological Chemistry  2013;288(38):27692-27701.
Background: Cystic fibrosis transmembrane conductance regulator (CFTR) has adenylate kinase activity (ATP + AMP ⇆ 2 ADP).
Results: ATP enables CFTR photolabeling by 8-N3-AMP, and AMP increases 8-N3-ATP photolabeling at ATP-binding site 2.
Conclusion: AMP interacts with CFTR in an ATP-dependent manner and alters ATP interaction with the adenylate kinase active center ATP-binding site.
Significance: These findings exemplify nucleotide interactions with an ABC adenylate kinase.
Cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel in the ATP-binding cassette (ABC) transporter protein family. In the presence of ATP and physiologically relevant concentrations of AMP, CFTR exhibits adenylate kinase activity (ATP + AMP ⇆ 2 ADP). Previous studies suggested that the interaction of nucleotide triphosphate with CFTR at ATP-binding site 2 is required for this activity. Two other ABC proteins, Rad50 and a structural maintenance of chromosome protein, also have adenylate kinase activity. All three ABC adenylate kinases bind and hydrolyze ATP in the absence of other nucleotides. However, little is known about how an ABC adenylate kinase interacts with ATP and AMP when both are present. Based on data from non-ABC adenylate kinases, we hypothesized that ATP and AMP mutually influence their interaction with CFTR at separate binding sites. We further hypothesized that only one of the two CFTR ATP-binding sites is involved in the adenylate kinase reaction. We found that 8-azidoadenosine 5′-triphosphate (8-N3-ATP) and 8-azidoadenosine 5′-monophosphate (8-N3-AMP) photolabeled separate sites in CFTR. Labeling of the AMP-binding site with 8-N3-AMP required the presence of ATP. Conversely, AMP enhanced photolabeling with 8-N3-ATP at ATP-binding site 2. The adenylate kinase active center probe P1,P5-di(adenosine-5′) pentaphosphate interacted simultaneously with an AMP-binding site and ATP-binding site 2. These results show that ATP and AMP interact with separate binding sites but mutually influence their interaction with the ABC adenylate kinase CFTR. They further indicate that the active center of the adenylate kinase comprises ATP-binding site 2.
doi:10.1074/jbc.M113.479675
PMCID: PMC3779764  PMID: 23921386
ABC Transporter; AMP; ATP; CFTR; Cystic fibrosis; Genetic Diseases
14.  Mutant cycles at CFTR’s non-canonical ATP-binding site support little interface separation during gating 
The Journal of General Physiology  2011;137(6):549-562.
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.
doi:10.1085/jgp.201110608
PMCID: PMC3105517  PMID: 21576373
15.  Demonstration of Phosphoryl Group Transfer Indicates That the ATP-binding Cassette (ABC) Transporter Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Exhibits Adenylate Kinase Activity* 
The Journal of Biological Chemistry  2012;287(43):36105-36110.
Background: Electrophysiological studies indicated that Cl− channel function of cystic fibrosis transmembrane conductance regulator (CFTR) can be coupled to adenylate kinase activity (ATP+AMP ⇆ 2 ADP).
Results: CFTR catalyzes phosphoryl group transfer between a nucleotide triphosphate and a photoactivatable AMP analog.
Conclusion: CFTR exhibits adenylate kinase activity.
Significance: These data demonstrate biochemically that a membrane-bound ABC transporter can function as an adenylate kinase.
Cystic fibrosis transmembrane conductance regulator (CFTR) is a membrane-spanning adenosine 5′-triphosphate (ATP)-binding cassette (ABC) transporter. ABC transporters and other nuclear and cytoplasmic ABC proteins have ATPase activity that is coupled to their biological function. Recent studies with CFTR and two nonmembrane-bound ABC proteins, the DNA repair enzyme Rad50 and a structural maintenance of chromosome (SMC) protein, challenge the model that the function of all ABC proteins depends solely on their associated ATPase activity. Patch clamp studies indicated that in the presence of physiologically relevant concentrations of adenosine 5′-monophosphate (AMP), CFTR Cl− channel function is coupled to adenylate kinase activity (ATP+AMP ⇆ 2 ADP). Work with Rad50 and SMC showed that these enzymes catalyze both ATPase and adenylate kinase reactions. However, despite the supportive electrophysiological results with CFTR, there are no biochemical data demonstrating intrinsic adenylate kinase activity of a membrane-bound ABC transporter. We developed a biochemical assay for adenylate kinase activity, in which the radioactive γ-phosphate of a nucleotide triphosphate could transfer to a photoactivatable AMP analog. UV irradiation could then trap the 32P on the adenylate kinase. With this assay, we discovered phosphoryl group transfer that labeled CFTR, thereby demonstrating its adenylate kinase activity. Our results also suggested that the interaction of nucleotide triphosphate with CFTR at ATP-binding site 2 is required for adenylate kinase activity. These biochemical data complement earlier biophysical studies of CFTR and indicate that the ABC transporter CFTR can function as an adenylate kinase.
doi:10.1074/jbc.M112.408450
PMCID: PMC3476278  PMID: 22948143
ABC Transporter; Bioenergetics; CFTR; Cystic Fibrosis; Genetic Diseases
16.  Structural basis for the channel function of a degraded ABC transporter, CFTR (ABCC7) 
The Journal of General Physiology  2011;138(5):495-507.
Cystic fibrosis transmembrane conductance regulator (CFTR) is a member of the ATP-binding cassette (ABC) transporter superfamily, but little is known about how this ion channel that harbors an uninterrupted ion permeation pathway evolves from a transporter that works by alternately exposing its substrate conduit to the two sides of the membrane. Here, we assessed reactivity of intracellularly applied thiol-specific probes with cysteine residues substituted into the 12th transmembrane segment (TM12) of CFTR. Our experimental data showing high reaction rates of substituted cysteines toward the probes, strong blocker protection of cysteines against reaction, and reaction-induced alterations in channel conductance support the idea that TM12 of CFTR contributes to the lining of the ion permeation pathway. Together with previous work, these findings raise the possibility that pore-lining elements of CFTR involve structural components resembling those that form the substrate translocation pathway of ABC transporters. In addition, comparison of reaction rates in the open and closed states of the CFTR channel leads us to propose that upon channel opening, the wide cytoplasmic vestibule tightens and the pore-lining TM12 rotates along its helical axis. This simple model for gating conformational changes in the inner pore domain of CFTR argues that the gating transition of CFTR and the transport cycle of ABC proteins share analogous conformational changes. Collectively, our data corroborate the popular hypothesis that degradation of the cytoplasmic-side gate turned an ABC transporter into the CFTR channel.
doi:10.1085/jgp.201110705
PMCID: PMC3206304  PMID: 22042986
17.  Conformational changes in the catalytically inactive nucleotide-binding site of CFTR 
A central step in the gating of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is the association of its two cytosolic nucleotide-binding domains (NBDs) into a head-to-tail dimer, with two nucleotides bound at the interface. Channel opening and closing, respectively, are coupled to formation and disruption of this tight NBD dimer. CFTR is an asymmetric adenosine triphosphate (ATP)-binding cassette protein in which the two interfacial-binding sites (composite sites 1 and 2) are functionally different. During gating, the canonical, catalytically active nucleotide-binding site (site 2) cycles between dimerized prehydrolytic (state O1), dimerized post-hydrolytic (state O2), and dissociated (state C) forms in a preferential C→O1→O2→C sequence. In contrast, the catalytically inactive nucleotide-binding site (site 1) is believed to remain associated, ATP-bound, for several gating cycles. Here, we have examined the possibility of conformational changes in site 1 during gating, by studying gating effects of perturbations in site 1.
Previous work showed that channel closure is slowed, both under hydrolytic and nonhydrolytic conditions, by occupancy of site 1 by N6-(2-phenylethyl)-ATP (P-ATP) as well as by the site-1 mutation H1348A (NBD2 signature sequence). Here, we found that P-ATP prolongs wild-type (WT) CFTR burst durations by selectively slowing (>2×) transition O1→O2 and decreases the nonhydrolytic closing rate (transition O1→C) of CFTR mutants K1250A (∼4×) and E1371S (∼3×). Mutation H1348A also slowed (∼3×) the O1→O2 transition in the WT background and decreased the nonhydrolytic closing rate of both K1250A (∼3×) and E1371S (∼3×) background mutants. Neither P-ATP nor the H1348A mutation affected the 1:1 stoichiometry between ATP occlusion and channel burst events characteristic to WT CFTR gating in ATP. The marked effect that different structural perturbations at site 1 have on both steps O1→C and O1→O2 suggests that the overall conformational changes that CFTR undergoes upon opening and coincident with hydrolysis at the active site 2 include significant structural rearrangement at site 1.
doi:10.1085/jgp.201210954
PMCID: PMC3691448  PMID: 23752332
18.  The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) 
The Journal of Biological Chemistry  2011;286(49):42647-42654.
Background: Cystic fibrosis is a disease where mutations in the cftr gene lead to loss of a chloride channel.
Results: The CFTR transmembrane domains show an outward facing configuration.
Conclusion: The map shows regions that probably represent the channel's gate and its regulatory region.
Significance: Residues associated with changes in channel function and disease are adjacent to the gate.
Cystic fibrosis affects about 1 in 2500 live births and involves loss of transmembrane chloride flux due to a lack of a membrane protein channel termed the cystic fibrosis transmembrane conductance regulator (CFTR). We have studied CFTR structure by electron crystallography. The data were compared with existing structures of other ATP-binding cassette transporters. The protein was crystallized in the outward facing state and resembled the well characterized Sav1866 transporter. We identified regions in the CFTR map, not accounted for by Sav1866, which were potential locations for the regulatory region as well as the channel gate. In this analysis, we were aided by the fact that the unit cell was composed of two molecules not related by crystallographic symmetry. We also identified regions in the fitted Sav1866 model that were missing from the map, hence regions that were either disordered in CFTR or differently organized compared with Sav1866. Apart from the N and C termini, this indicated that in CFTR, the cytoplasmic end of transmembrane helix 5/11 and its associated loop could be partly disordered (or alternatively located).
doi:10.1074/jbc.M111.292268
PMCID: PMC3234965  PMID: 21931164
CFTR; Cystic Fibrosis; Electron Microscopy (EM); Ion Channels; Protein Structure
19.  Catalyst-like modulation of transition states for CFTR channel opening and closing: New stimulation strategy exploits nonequilibrium gating 
The Journal of General Physiology  2014;143(2):269-287.
Two gating transition states determine open probability of CFTR (the chloride channel mutated in cystic fibrosis), defining strategic targets for therapeutic intervention.
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.
doi:10.1085/jgp.201311089
PMCID: PMC4001772  PMID: 24420771
20.  State-dependent modulation of CFTR gating by pyrophosphate 
The Journal of General Physiology  2009;133(4):405-419.
Cystic fibrosis transmembrane conductance regulator (CFTR) is an adenosine triphosphate (ATP)-gated chloride channel. ATP-induced dimerization of CFTR's two nucleotide-binding domains (NBDs) has been shown to reflect the channel open state, whereas hydrolysis of ATP is associated with channel closure. Pyrophosphate (PPi), like nonhydrolytic ATP analogues, is known to lock open the CFTR channel for tens of seconds when applied with ATP. Here, we demonstrate that PPi by itself opens the CFTR channel in a Mg2+-dependent manner long after ATP is removed from the cytoplasmic side of excised membrane patches. However, the short-lived open state (τ ∼1.5 s) induced by MgPPi suggests that MgPPi alone does not support a stable NBD dimer configuration. Surprisingly, MgPPi elicits long-lasting opening events (τ ∼30 s) when administrated shortly after the closure of ATP-opened channels. These results indicate the presence of two different closed states (C1 and C2) upon channel closure and a state-dependent effect of MgPPi on CFTR gating. The relative amount of channels entering MgPPi-induced long-open bursts during the ATP washout phase decreases over time, indicating a time-dependent dissipation of the closed state (C2) that can be locked open by MgPPi. The stability of the C2 state is enhanced when the channel is initially opened by N6-phenylethyl-ATP, a high affinity ATP analogue, but attenuated by W401G mutation, which likely weakens ATP binding to NBD1, suggesting that an ATP molecule remains bound to the NBD1 site in the C2 state. Taking advantage of the slow opening rate of Y1219G-CFTR, we are able to identify a C2-equivalent state (C2*), which exists before the channel in the C1 state is opened by ATP. This closed state responds to MgPPi much more inefficiently than the C2 state. Finally, we show that MgAMP-PNP exerts its effects on CFTR gating via a similar mechanism as MgPPi. The structural and functional significance of our findings is discussed.
doi:10.1085/jgp.200810186
PMCID: PMC2699106  PMID: 19332621
21.  G551D and G1349D, Two CF-associated Mutations in the Signature Sequences of CFTR, Exhibit Distinct Gating Defects 
The Journal of General Physiology  2007;129(4):285-298.
Mutations in the gene encoding cystic fibrosis transmembrane conductance regulator (CFTR) result in cystic fibrosis (CF). CFTR is a chloride channel that is regulated by phosphorylation and gated by ATP binding and hydrolysis at its nucleotide binding domains (NBDs). G551D-CFTR, the third most common CF-associated mutation, has been characterized as having a lower open probability (Po) than wild-type (WT) channels. Patients carrying the G551D mutation present a severe clinical phenotype. On the other hand, G1349D, also a mutant with gating dysfunction, is associated with a milder clinical phenotype. Residues G551 and G1349 are located at equivalent positions in the highly conserved signature sequence of each NBD. The physiological importance of these residues lies in the fact that the signature sequence of one NBD and the Walker A and B motifs from the other NBD form the ATP-binding pocket (ABP1 and ABP2, named after the location of the Walker A motif) once the two NBDs dimerize. Our studies show distinct gating characteristics for these mutants. The G551D mutation completely eliminates the ability of ATP to increase the channel activity, and the observed activity is ∼100-fold smaller than WT-CFTR. G551D-CFTR does not respond to ADP, AMP-PNP, or changes in [Mg2+]. The low activity of G551D-CFTR likely represents the rare ATP-independent gating events seen with WT channels long after the removal of ATP. G1349D-CFTR maintains ATP dependence, albeit with a Po ∼10-fold lower than WT. Interestingly, compared to WT results, the ATP dose–response relationship of G1349D-CFTR is less steep and shows a higher apparent affinity for ATP. G1349D data could be well described by a gating model that predicts that binding of ATP at ABP1 hinders channel opening. Thus, our data provide a quantitative explanation at the single-channel level for different phenotypes presented by patients carrying these two mutations. In addition, these results support the idea that CFTR's two ABPs play distinct functional roles in gating.
doi:10.1085/jgp.200609667
PMCID: PMC2151620  PMID: 17353351
22.  Reversible Silencing of CFTR Chloride Channels by Glutathionylation 
The Journal of General Physiology  2005;125(2):127-141.
The cystic fibrosis transmembrane conductance regulator (CFTR) is a phosphorylation- and ATP-dependent chloride channel that modulates salt and water transport across lung and gut epithelia. The relationship between CFTR and oxidized forms of glutathione is of potential interest because reactive glutathione species are produced in inflamed epithelia where they may be modulators or substrates of CFTR. Here we show that CFTR channel activity in excised membrane patches is markedly inhibited by several oxidized forms of glutathione (i.e., GSSG, GSNO, and glutathione treated with diamide, a strong thiol oxidizer). Three lines of evidence indicate that the likely mechanism for this inhibitory effect is glutathionylation of a CFTR cysteine (i.e., formation of a mixed disulfide with glutathione): (a) channels could be protected from inhibition by pretreating the patch with NEM (a thiol alkylating agent) or by lowering the bath pH; (b) inhibited channels could be rescued by reducing agents (e.g., DTT) or by purified glutaredoxins (Grxs; thiol disulfide oxidoreductases) including a mutant Grx that specifically reduces mixed disulfides between glutathione and cysteines within proteins; and (c) reversible glutathionylation of CFTR polypeptides in microsomes could be detected biochemically under the same conditions. At the single channel level, the primary effect of reactive glutathione species was to markedly inhibit the opening rates of individual CFTR channels. CFTR channel inhibition was not obviously dependent on phosphorylation state but was markedly slowed when channels were first “locked open” by a poorly hydrolyzable ATP analogue (AMP-PNP). Consistent with the latter finding, we show that the major site of inhibition is cys-1344, a poorly conserved cysteine that lies proximal to the signature sequence in the second nucleotide binding domain (NBD2) of human CFTR. This region is predicted to participate in ATP-dependent channel opening and to be occluded in the nucleotide-bound state of the channel based on structural comparisons to related ATP binding cassette transporters. Our results demonstrate that human CFTR channels are reversibly inhibited by reactive glutathione species, and support an important role of the region proximal to the NBD2 signature sequence in ATP-dependent channel opening.
doi:10.1085/jgp.200409115
PMCID: PMC2217496  PMID: 15657297
cystic fibrosis transmembrane conductance regulator; ABC transporter; glutathione; glutaredoxin; redox
23.  Molecular modeling of the heterodimer of human CFTR’s nucleotide-binding domains using a protein–protein docking approach 
We have presented a new protein–protein docking approach to model heterodimeric structures based on the conformations of the monomeric units. The conventional modeling method relies on superimposing two monomeric structures onto the crystal structure of a homologous protein dimer. The resulting structure may exhibit severe backbone clashes at the dimeric interface depending on the backbone dissimilarity between the target and template proteins. Our method overcomes the backbone clashing problem and requires no a priori knowledge of the dimeric structure of a homologous protein. Here we used human Cystic Fibrosis Transmembrane conductance Regulator (CFTR), a chloride channel whose dysfunction causes cystic fibrosis, for illustration. The two intracellular nucleotide-binding domains (NBDs) of CFTR control the opening and closing of the channel. Yet, the structure of the CFTR’s NBD1–NBD2 complex has not been experimentally determined. Thus, correct modeling of this heterodimeric structure is valuable for understanding CFTR functions and would have potential applications for drug design for cystic fibrosis treatment. Based on the crystal structure of human CFTR’s NBD1, we constructed a model of the NBD1–NBD2 complex. The constructed model is consistent with the dimeric mode observed in the crystal structures of other ABC transporters. To verify our structural model, an ATP substrate was docked into the nucleotide-binding site. The predicted binding mode shows consistency with related crystallographic findings and CFTR functional studies. Finally, genistein, an agent that enhances CFTR activity, though the mechanism for such enhancement is unclear, was docked to the model. Our predictions agreed with genistein’s bell-shaped dose-response relationship. Potential mutagenesis experiments were proposed for understanding the potentiation mechanism of genistein and for providing insightful information for drug design targeting at CFTR. The method used in this study can be applied to modeling studies of other dimeric protein structures.
doi:10.1016/j.jmgm.2008.12.005
PMCID: PMC2758335  PMID: 19167254
Molecular modeling; Molecular docking; CFTR
24.  Mutations at the Signature Sequence of CFTR Create a Cd2+-gated Chloride Channel 
The canonical sequence LSGGQ, also known as the signature sequence, defines the adenosine triphosphate (ATP)-binding cassette transporter superfamily. Crystallographic studies reveal that the signature sequence, together with the Walker A and Walker B motifs, forms the ATP-binding pocket upon dimerization of the two nucleotide-binding domains (NBDs) in a head-to-tail configuration. The importance of the signature sequence is attested by the fact that a glycine to aspartate mutation (i.e., G551D) in cystic fibrosis transmembrane conductance regulator (CFTR) results in a severe phenotype of cystic fibrosis. We previously showed that the G551D mutation completely eliminates ATP-dependent gating of the CFTR chloride channel. Here, we report that micromolar [Cd2+] can dramatically increase the activity of G551D-CFTR in the absence of ATP. This effect of Cd2+ is not seen in wild-type channels or in G551A. Pretreatment of G551D-CFTR with the cysteine modification reagent 2-aminoethyl methane thiosulfonate hydrobromide protects the channel from Cd2+ activation, suggesting an involvement of endogenous cysteine residue(s) in mediating this effect of Cd2+. The mutants G551C, L548C, and S549C, all in the signature sequence of CFTR's NBD1, show robust response to Cd2+. On the other hand, negligible effects of Cd2+ were seen with T547C, Q552C, and R553C, indicating that a specific region of the signature sequence is involved in transmitting the signal of Cd2+ binding to the gate. Collectively, these results suggest that the effect of Cd2+ is mediated by a metal bridge formation between yet to be identified cysteine residue(s) and the engineered aspartate or cysteine in the signature sequence. We propose that the signature sequence serves as a switch that transduces the signal of ligand binding to the channel gate.
doi:10.1085/jgp.200810049
PMCID: PMC2606936  PMID: 19114635
25.  Severed Molecules Functionally Define the Boundaries of the Cystic Fibrosis Transmembrane Conductance Regulator's Nh2-Terminal Nucleotide Binding Domain 
The Journal of General Physiology  2000;116(2):163-180.
The cystic fibrosis transmembrane conductance regulator is a Cl− channel that belongs to the family of ATP-binding cassette proteins. The CFTR polypeptide comprises two transmembrane domains, two nucleotide binding domains (NBD1 and NBD2), and a regulatory (R) domain. Gating of the channel is controlled by kinase-mediated phosphorylation of the R domain and by ATP binding, and, likely, hydrolysis at the NBDs. Exon 13 of the CFTR gene encodes amino acids (aa's) 590–830, which were originally ascribed to the R domain. In this study, CFTR channels were severed near likely NH2- or COOH-terminal boundaries of NBD1. CFTR channel activity, assayed using two-microelectrode voltage clamp and excised patch recordings, provided a sensitive measure of successful assembly of each pair of channel segments as the sever point was systematically shifted along the primary sequence. Substantial channel activity was taken as an indication that NBD1 was functionally intact. This approach revealed that the COOH terminus of NBD1 extends beyond aa 590 and lies between aa's 622 and 634, while the NH2 terminus of NBD1 lies between aa's 432 and 449. To facilitate biochemical studies of the expressed proteins, a Flag epitope was added to the NH2 termini of full length CFTR, and of CFTR segments truncated before the normal COOH terminus (aa 1480). The functionally identified NBD1 boundaries are supported by Western blotting, coimmunoprecipitation, and deglycosylation studies, which showed that an NH2-terminal segment representing aa's 3–622 (Flag3-622) or 3–633 (Flag3-633) could physically associate with a COOH-terminal fragment representing aa's 634–1480 (634-1480); however, the latter fragment was glycosylated to the mature form only in the presence of Flag3-633. Similarly, 433-1480 could physically associate with Flag3-432 and was glycosylated to the mature form; however, 449-1480 protein seemed unstable and could hardly be detected even when expressed with Flag3-432. In excised-patch recordings, all functional severed CFTR channels displayed the hallmark characteristics of CFTR, including the requirement of phosphorylation and exposure to MgATP for gating, ability to be locked open by pyrophosphate or AMP-PNP, small single channel conductances, and high apparent affinity of channel opening by MgATP. Our definitions of the boundaries of the NBD1 domain in CFTR are supported by comparison with the solved NBD structures of HisP and RbsA.
PMCID: PMC2229491  PMID: 10919864
adenosine triphosphate-binding cassette transporter; domain structure; chloride channel; gating kinetics; coimmunoprecipitation

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