ABCMdb

ABCC7 p.Ala462Phe

Predicted by SNAP2:	C: D (80%), D: D (95%), E: D (95%), F: D (95%), G: D (91%), H: D (95%), I: D (91%), K: D (95%), L: D (95%), M: D (91%), N: D (95%), P: D (95%), Q: D (95%), R: D (95%), S: N (61%), T: D (91%), V: D (91%), W: D (95%), Y: D (95%),
Predicted by PROVEAN:	C: N, D: D, E: N, F: D, G: N, H: D, I: D, K: N, L: D, M: D, N: N, P: D, Q: N, R: D, S: N, T: N, V: D, W: D, Y: D,

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[hide] Normal gating of CFTR requires ATP binding to both... Proc Natl Acad Sci U S A. 2005 Jan 11;102(2):455-60. Epub 2004 Dec 27. Berger AL, Ikuma M, Welsh MJ
Normal gating of CFTR requires ATP binding to both nucleotide-binding domains and hydrolysis at the second nucleotide-binding domain.
Proc Natl Acad Sci U S A. 2005 Jan 11;102(2):455-60. Epub 2004 Dec 27., 2005-01-11 [PMID:15623556]

Abstract [show]
ATP interacts with the two nucleotide-binding domains (NBDs) of CFTR to control gating. However, it is unclear whether gating involves ATP binding alone, or also involves hydrolysis at each NBD. We introduced phenylalanine residues into nonconserved positions of each NBD Walker A motif to sterically prevent ATP binding. These mutations blocked [alpha-(32)P]8-N(3)-ATP labeling of the mutated NBD and reduced channel opening rate without changing burst duration. Introducing cysteine residues at these positions and modifying with N-ethylmaleimide produced the same gating behavior. These results indicate that normal gating requires ATP binding to both NBDs, but ATP interaction with one NBD is sufficient to support some activity. We also studied mutations of the conserved Walker A lysine residues (K464A and K1250A) that prevent hydrolysis. By combining substitutions that block ATP binding with Walker A lysine mutations, we could differentiate the role of ATP binding vs. hydrolysis at each NBD. The K1250A mutation prolonged burst duration; however, blocking ATP binding prevented the long bursts. These data indicate that ATP binding to NBD2 allowed channel opening and that closing was delayed in the absence of hydrolysis. The corresponding NBD1 mutations showed relatively little effect of preventing ATP hydrolysis but a large inhibition of blocking ATP binding. These data suggest that ATP binding to NBD1 is required for normal activity but that hydrolysis has little effect. Our results suggest that both NBDs contribute to channel gating, NBD1 binds ATP but supports little hydrolysis, and ATP binding and hydrolysis at NBD2 are key for normal gating.

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No. Sentence Comment
71 The 6% gels revealed that CFTR-A462F and -K464A produced little band C protein, whereas other variants produced predominantly the band C form. Login to comment
103 The A462F mutation prevented labeling of NBD1; S1248F blocked NBD2 labeling; and the double mutant A462F͞S1248F abolished labeling at both NBDs. Login to comment
154 To assess the gating effects of ATP binding to NBD1, we studied the A462F variant, which blocks NBD1 nucleotide binding. Login to comment
156 A462F reduced Po, as did treating A462C channels with NEM. Login to comment
179 (A) Examples of recordings from CFTR-A462F, and of A462C channels before and after NEM treatment. Login to comment
180 Although the A462F tracing shown has a higher Po than the mean, we chose this example to show the bursts. Login to comment
250 However, that is not what we observed; the A462F mutation completely blocked NBD1 labeling, and S1248F blocked NBD2 labeling. Login to comment
253 Nevertheless, we did find that the A462F mutation in NBD1 diminished labeling of NBD2 and that S1248F slightly reduced NBD1 labeling. Login to comment

[hide] Curcumin opens cystic fibrosis transmembrane condu... J Biol Chem. 2007 Feb 16;282(7):4533-44. Epub 2006 Dec 18. Wang W, Bernard K, Li G, Kirk KL
Curcumin opens cystic fibrosis transmembrane conductance regulator channels by a novel mechanism that requires neither ATP binding nor dimerization of the nucleotide-binding domains.
J Biol Chem. 2007 Feb 16;282(7):4533-44. Epub 2006 Dec 18., 2007-02-16 [PMID:17178710]

Abstract [show]
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.

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174 Fig. 4 (D and E) shows that the inhibitory effect of bath ATP on the curcumin response was eliminated by introducing a mutation in the Walker A sequence (A462F) in NBD1. Login to comment
176 The lack of effect of ATP on the curcumin activation of the A462F/⌬1198-CFTR construct FIGURE 3. Login to comment
195 D, bath ATP does not inhibit A462F/⌬1198-CFTR channels. Login to comment
196 E, mean data comparing the effects of bath ATP (1.5 mM) on ⌬1198-CFTR (n ϭ 16) and A462F/⌬1198-CFTR (n ϭ 10) activation by 30 ␮M curcumin. Login to comment
198 Note that the absolute currents mediated by A462F/⌬1198-CFTR are lower because the A462F mutation partially disrupts ER processing and cell surface localization (33).3 All of the records are representative of at least three experiments. Login to comment
246 Introducing a mutation in the Walker A motif that was shown previously to disrupt ATP binding to NBD1 (A462F (33)) severely blunted the inhibitory effect of ATP on ⌬1198-CFTR channel activation by curcumin. Login to comment

[hide] The inhibition mechanism of non-phosphorylated Ser... J Biol Chem. 2011 Jan 21;286(3):2171-82. Epub 2010 Nov 8. Wang G
The inhibition mechanism of non-phosphorylated Ser768 in the regulatory domain of cystic fibrosis transmembrane conductance regulator.
J Biol Chem. 2011 Jan 21;286(3):2171-82. Epub 2010 Nov 8., 2011-01-21 [PMID:21059651]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) is a member of the ATP-binding cassette transporters but serves as a chloride channel dysfunctional in cystic fibrosis. The activity of CFTR is tightly controlled not only by ATP-driven dimerization of its nucleotide-binding domains but also by phosphorylation of a unique regulatory (R) domain by protein kinase A (PKA). The R domain has multiple excitatory phosphorylation sites, but Ser(737) and Ser(768) are inhibitory. The underlying mechanism is unclear. Here, sulfhydryl-specific cross-linking strategy was employed to demonstrate that Ser(768) or Ser(737) could interact with outwardly facing hydrophilic residues of cytoplasmic loop 3 regulating channel gating. Furthermore, mutation of these residues to alanines promoted channel opening by curcumin in an ATP-dependent manner even in the absence of PKA. However, mutation of Ser(768) and His(950) with different hydrogen bond donors or acceptors clearly changed ATP- and PKA-dependent channel activity no matter whether curcumin was present or not. More importantly, significant activation of a double mutant H950R/S768R needed only ATP. Finally, in vitro and in vivo single channel recordings suggest that Ser(768) may form a putative hydrogen bond with His(950) of cytoplasmic loop 3 to prevent channel opening by ATP in the non-phosphorylated state and by subsequent cAMP-dependent phosphorylation. These observations support an electron cryomicroscopy-based structural model on which the R domain is closed to cytoplasmic loops regulating channel gating.

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No. Sentence Comment
268 Role of Curcumin in Normal CFTR Gating-A previous study (21) showed that curcumin activates mutant CFTR channels, such as G551D, W1282X, ⌬1198, and A462F. Login to comment

[hide] Ligand-driven vectorial folding of ribosome-bound ... Mol Cell. 2011 Mar 18;41(6):682-92. Khushoo A, Yang Z, Johnson AE, Skach WR
Ligand-driven vectorial folding of ribosome-bound human CFTR NBD1.
Mol Cell. 2011 Mar 18;41(6):682-92., 2011-03-18 [PMID:21419343]

Abstract [show]
The mechanism by which protein folding is coupled to biosynthesis is a critical, but poorly understood, aspect of protein conformational diseases. Here we use fluorescence resonance energy transfer (FRET) to characterize tertiary structural transitions of nascent polypeptides and show that the first nucleotide-binding domain (NBD1) of human CFTR, whose folding is defective in cystic fibrosis, folds via a cotranslational multistep pathway as it is synthesized on the ribosome. Folding begins abruptly as NBD1 residues 389-500 emerge from the ribosome exit tunnel, initiating compaction of a small, N-terminal alpha/beta-subdomain. Real-time kinetics of synchronized nascent chains revealed that subdomain folding is rapid, occurs coincident with synthesis, and is facilitated by direct ATP binding to the nascent polypeptide. These findings localize the major CF defect late in the NBD1 folding pathway and establish a paradigm wherein a cellular ligand promotes vectorial domain folding by facilitating an energetically favored local peptide conformation.

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114 In addition, mutations that inhibit ATP binding to NBD1 in full-length CFTR (W401A, A462F, and K464A [Berger et al., 2005]) also inhibited binding to truncated NBD1 (Figures 5E and 5F). Login to comment
205 The CFP-NBD1 ATP-binding mutant contained W401A, A462F, and K464A mutations in the ATP-binding site as predicted by the crystal structure (2BBO). Login to comment

[hide] Demonstration of Phosphoryl Group Transfer Indicat... J Biol Chem. 2012 Oct 19;287(43):36105-10. doi: 10.1074/jbc.M112.408450. Epub 2012 Sep 4. Randak CO, Ver Heul AR, Welsh MJ
Demonstration of Phosphoryl Group Transfer Indicates That the ATP-binding Cassette (ABC) Transporter Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Exhibits Adenylate Kinase Activity.
J Biol Chem. 2012 Oct 19;287(43):36105-10. doi: 10.1074/jbc.M112.408450. Epub 2012 Sep 4., [PMID:22948143]

Abstract [show]
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 &lrarr2; 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 gamma-phosphate of a nucleotide triphosphate could transfer to a photoactivatable AMP analog. UV irradiation could then trap the (32)P 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.

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No. Sentence Comment
165 We could not assess the effect of the homologous mutation in ATP-binding site 1 (A462F mutation) on adenylate kinase activity because that mutation affected intracellular CFTR processing to an extent that we were unable to detect the mutant CFTR protein in our membrane preparations by Western blot. Login to comment
164 We could not assess the effect of the homologous mutation in ATP-binding site 1 (A462F mutation) on adenylate kinase activity because that mutation affected intracellular CFTR processing to an extent that we were unable to detect the mutant CFTR protein in our membrane preparations by Western blot. Login to comment

[hide] ATP and AMP mutually influence their interaction w... J Biol Chem. 2013 Sep 20;288(38):27692-701. doi: 10.1074/jbc.M113.479675. Epub 2013 Aug 6. Randak CO, Dong Q, Ver Heul AR, Elcock AH, Welsh MJ
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.
J Biol Chem. 2013 Sep 20;288(38):27692-701. doi: 10.1074/jbc.M113.479675. Epub 2013 Aug 6., [PMID:23921386]

Abstract [show]
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 &lrarr2; 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 P(1),P(5)-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.

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No. Sentence Comment
94 Recordings from patches containing very few channels (A462F CFTR) with up to five simultaneous channel openings were low pass-filtered at 500 Hz for analysis. Login to comment
238 Berger et al. (15) found that substituting alanine at position 462 in NBD1 with phenylalanine (A462F mutation; Fig. 9B, left) abolished nucleotide interaction with ATP-binding site 1. Login to comment
241 The A462F, but not the S1248F mutation interfered with processing and trafficking to the cell membrane (supplemental Fig. S1), and hence, the number of channels in excised membrane patches was small; therefore, we quantified channel activity as NPo. Login to comment
242 We found that Ap5A reduced the NPo of A462F CFTR (Fig. 9B, middle and right). Login to comment
261 B, left, model of A462F CFTR. Login to comment
262 Middle, current recording from one excised inside-out membrane patch containing at least two A462F CFTR channels perfused on cytosolic surface with ATP and Ap5A as indicated. Login to comment
268 Right, NPo of A462F CFTR with 0.3 mM ATP and PKA present in the bath solution before and after adding 1 mM Ap5A. Login to comment