ABCMdb

ABCC7 p.Ser1347Gly

Predicted by SNAP2:	A: D (85%), C: D (91%), D: D (95%), E: D (91%), F: D (95%), G: D (85%), H: D (95%), I: D (95%), K: D (95%), L: D (95%), M: D (91%), N: D (91%), P: D (95%), Q: D (91%), R: D (95%), T: D (91%), V: D (95%), W: D (95%), Y: D (95%),
Predicted by PROVEAN:	A: N, C: D, D: D, E: D, F: D, G: D, H: D, I: D, K: D, L: D, M: D, N: N, P: D, Q: D, R: D, T: N, V: D, W: D, Y: D,

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[hide] State-dependent modulation of CFTR gating by pyrop... J Gen Physiol. 2009 Apr;133(4):405-19. Tsai MF, Shimizu H, Sohma Y, Li M, Hwang TC
State-dependent modulation of CFTR gating by pyrophosphate.
J Gen Physiol. 2009 Apr;133(4):405-19., [PMID:19332621]

Abstract [show]
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 Mg(2+)-dependent manner long after ATP is removed from the cytoplasmic side of excised membrane patches. However, the short-lived open state (tau approximately 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 (tau approximately 30 s) when administrated shortly after the closure of ATP-opened channels. These results indicate the presence of two different closed states (C(1) and C(2)) 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 (C(2)) that can be locked open by MgPPi. The stability of the C(2) state is enhanced when the channel is initially opened by N(6)-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 C(2) state. Taking advantage of the slow opening rate of Y1219G-CFTR, we are able to identify a C(2)-equivalent state (C(2)*), which exists before the channel in the C(1) state is opened by ATP. This closed state responds to MgPPi much more inefficiently than the C(2) 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.

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35 Electrophysiological recordings Before inside-out patch clamp recordings, glass chips containing CHO cells transfected with various CFTR constructs, W401G, Y1219G, S1347G, E1371S, and WT-CFTR, were transferred to a continuously perfused chamber located on the stage of an inverted microscope (Olympus). Login to comment
105 Online supplemental material Fig. S1 shows that S1347G-CFTR has a weaker response to MgPPi than WT. Login to comment
246 Interestingly, we also found that S1347G, a mutation at NBD2 signature sequence, which presumably forms the ATP-binding pocket with NBD1`s Walker A domain upon NBD dimer formation, greatly attenuates the stability of the C2 state, and this reduced stability can also be partly reversed by P-ATP (Fig. S1). Login to comment
440 Supporting this hypothesis, we demonstrated that the C2 state dissipates much faster when residues at either NBD1 (W401G; Fig. 7 B) or the signature sequence of NBD2 were mutated (S1347G; Fig. S1). Login to comment

[hide] Potentiation of disease-associated cystic fibrosis... J Biol Chem. 2010 Jun 25;285(26):19967-75. Epub 2010 Apr 20. Miki H, Zhou Z, Li M, Hwang TC, Bompadre SG
Potentiation of disease-associated cystic fibrosis transmembrane conductance regulator mutants by hydrolyzable ATP analogs.
J Biol Chem. 2010 Jun 25;285(26):19967-75. Epub 2010 Apr 20., 2010-06-25 [PMID:20406820]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel belonging to the ATP-binding cassette transporter superfamily. CFTR is gated by ATP binding and hydrolysis at its two nucleotide-binding domains (NBDs), which dimerize in the presence of ATP to form two ATP-binding pockets (ABP1 and ABP2). Mutations reducing the activity of CFTR result in the genetic disease cystic fibrosis. Two of the most common mutations causing a severe phenotype are G551D and DeltaF508. Previously we found that the ATP analog N(6)-(2-phenylethyl)-ATP (P-ATP) potentiates the activity of G551D by approximately 7-fold. Here we show that 2'-deoxy-ATP (dATP), but not 3'-deoxy-ATP, increases the activity of G551D-CFTR by approximately 8-fold. We custom synthesized N(6)-(2-phenylethyl)-2'-deoxy-ATP (P-dATP), an analog combining the chemical modifications in dATP and P-ATP. This new analog enhances G551D current by 36.2 +/- 5.4-fold suggesting an independent but energetically additive action of these two different chemical modifications. We show that P-dATP binds to ABP1 to potentiate the activity of G551D, and mutations in both sides of ABP1 (W401G and S1347G) decrease its potentiation effect, suggesting that the action of P-dATP takes place at the interface of both NBDs. Interestingly, P-dATP completely rectified the gating abnormality of DeltaF508-CFTR by increasing its activity by 19.5 +/- 3.8-fold through binding to both ABPs. This result highlights the severity of the gating defect associated with DeltaF508, the most prevalent disease-associated mutation. The new analog P-dATP can be not only an invaluable tool to study CFTR gating, but it can also serve as a proof-of-principle that, by combining elements that potentiate the channel activity independently, the increase in chloride transport necessary to reach a therapeutic target is attainable.

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8 We show that P-dATP binds to ABP1 to potentiate the activity of G551D, and mutations in both sides of ABP1 (W401G and S1347G) decrease its potentiation effect, suggesting that the action of P-dATP takes place at the interface of both NBDs. Login to comment
147 Mutation of the serine at position 1347 to glycine will likely diminish the interaction of the P-dATP molecule with the signature sequence in NBD2. Login to comment
148 Fig. 4D shows a representative trace for the mutant G551D/S1347G. Login to comment
150 In fact the mutation S1347G in NBD2 decreases the potentiation effect of P-dATP to a similar extent as the W401G mutation in NBD1 (Fig. 4C). Login to comment
163 Representative current traces of G551D/Y1219G (A), W401G/G551D (B), and G551D/S1347G (D) in the presence of 10 ␮M P-dATP. Login to comment
164 C, P-dATP dose-response relationships for G551D (red, F), W401G/G551D (blue, E), G551D/Y1219G (green, Œ), and G551D/S1347G (black, f). Login to comment
169 F, comparison between the -fold increase in the current for G551D (red), W401G/G551D (blue), and G551D/S1347G (black) channels in the presence of saturating concentrations of dATP, P-ATP, and P-dATP. Login to comment

[hide] Stable ATP binding mediated by a partial NBD dimer... J Gen Physiol. 2010 May;135(5):399-414. Tsai MF, Li M, Hwang TC
Stable ATP binding mediated by a partial NBD dimer of the CFTR chloride channel.
J Gen Physiol. 2010 May;135(5):399-414., [PMID:20421370]

Abstract [show]
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-N(3)-ATP molecule for tens of seconds. We provided evidence that this tightly bound ATP or 8-N(3)-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.

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117 Indeed, single S1347G channels, like W401G-CFTR (Fig. 3 A), opened into long bursts without a delay after changing the ligand from ATP to PATP (red trace in Fig. 4 B; channel kinetics summarized in Fig. 4 C), suggesting that the S1347G mutation in NBD2 dramatically shortens the dwell time of ATP in NBD1. Login to comment
123 As in the case for mutations at W401 (Fig. 3 E), more drastic mutations at S1347, e.g., S1347V and S1347G, shorten this time constant to a greater extent than the more conservative time. Login to comment
152 Consistent with this idea, for the ABC protein TAP2 whose corresponding residue of H1348 is Figure 4. Effects of the S1347G mutation on ligand exchange. Login to comment
158 (B) A representative single S1347G-CFTR channel trace from seven similar recordings. Login to comment
160 (D) Macroscopic currents from S1347G channels. Login to comment
189 The current rising phase Figure 7. Changes of WT-CFTR and S1347G-CFTR currents upon 8-N3-ATP/PATP exchange. Login to comment
191 (B) The second-phase current increase seen in A was essentially abolished by the S1347G mutation. Login to comment
204 Because most channels will stay in the open state when exposed to ATP and MgPPi, the subsequent solution change to PATP plus MgPPi will allow us to test whether ligand exchange in NBD1 occurs from the open state. If ATP/PATP exchange does occur in the open state (Fig. 10 B), PATP will further prolong the lock-open time because it is known that the lock-open state with MgPPi bound in NBD2 and PATP in NBD1 is more stable demonstrated biochemically (Aleksandrov et al., 2008), the trapping of 8-N3-ATP in the current study is also dependent on the tail of NBD2 because the S1347G mutation essentially abolished the second phase of current increase elicited by PATP upon switching the ligand from 8-N3-ATP to PATP (Fig. 7 B). Login to comment

[hide] Optimization of the degenerated interfacial ATP bi... J Biol Chem. 2010 Nov 26;285(48):37663-71. Epub 2010 Sep 22. Tsai MF, Jih KY, Shimizu H, Li M, Hwang TC
Optimization of the degenerated interfacial ATP binding site improves the function of disease-related mutant cystic fibrosis transmembrane conductance regulator (CFTR) channels.
J Biol Chem. 2010 Nov 26;285(48):37663-71. Epub 2010 Sep 22., 2010-11-26 [PMID:20861014]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel, an ATP binding cassette (ABC) protein whose defects cause the deadly genetic disease cystic fibrosis (CF), encompasses two nucleotide binding domains (NBD1 and NBD2). Recent studies indicate that in the presence of ATP, the two NBDs coalesce into a dimer, trapping an ATP molecule in each of the two interfacial composite ATP binding sites (site 1 and site 2). Experimental evidence also suggests that CFTR gating is mainly controlled by ATP binding and hydrolysis in site 2, whereas site 1, which harbors several non-canonical substitutions in ATP-interacting motifs, is considered degenerated. The CF-associated mutation G551D, by introducing a bulky and negatively charged side chain into site 2, completely abolishes ATP-induced openings of CFTR. Here, we report a strategy to optimize site 1 for ATP binding by converting two amino acid residues to ABC consensus (i.e. H1348G) or more commonly seen residues in other ABC proteins (i.e. W401Y,W401F). Introducing either one or both of these mutations into G551D-CFTR confers ATP responsiveness for this disease-associated mutant channel. We further showed that the same maneuver also improved the function of WT-CFTR and the most common CF-associated DeltaF508 channels, both of which rely on site 2 for gating control. Thus, our results demonstrated that the degenerated site 1 can be rebuilt to complement or support site 2 for CFTR function. Possible approaches for developing CFTR potentiators targeting site 1 will be discussed.

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120 Interestingly, we found that non-conservative mutations L1346Q and S1347G (Fig. 3A) and G1349I greatly reduced the nucleotide-dependent activation of W401F/G551D channels. Login to comment
121 For example, ATP and PATP, which increased the basal activity of W401F/G551D-CFTR by ϳ12-fold (Fig. 3D, blue dashed line) and ϳ30-fold (green dashed line), respectively, led to only ϳ2.5-and ϳ6-fold current increases when the S1347G mutation was present (Fig. 3D). Login to comment
139 A, the S1347G mutation diminished the response of W401F/G551D channels to ATP or PATP. B, incorporating the H1348G mutation into G551D-CFTR conferred responsiveness to ATP. Login to comment
189 These mutations include W401G,W401I (Fig. 1, B-D), which eliminate a ring-ring stacking interaction, S1347G (supplemental Fig. S6), which may break a hydrogen bond between ATP and the NBD2 signature motif, and G1349I (supplemental Fig. S6), whose side chain likely protrudes into site 1 and causes a steric clash with ATP (22-24). Login to comment

[hide] Cysteine accessibility probes timing and extent of... J Gen Physiol. 2015 Apr;145(4):261-83. doi: 10.1085/jgp.201411347. Chaves LA, Gadsby DC
Cysteine accessibility probes timing and extent of NBD separation along the dimer interface in gating CFTR channels.
J Gen Physiol. 2015 Apr;145(4):261-83. doi: 10.1085/jgp.201411347., [PMID:25825169]

Abstract [show]
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 A.

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328 Even in CFTR channels, the nonconservative mutation S1347G in the NBD2 signature sequence diminished open burst duration, and open probability, by only about one third (Tsai et al., 2010). Login to comment