ABCMdb

ABCC7 p.Trp401Phe

ClinVar:	c.1202G>A , p.Trp401* D , Pathogenic c.1203G>A , p.Trp401* D , Pathogenic
CF databases:	c.1202G>A or c.1203G>A , p.Trp401* D , CF-causing
Predicted by SNAP2:	A: D (75%), C: D (71%), D: D (85%), E: D (85%), F: N (53%), G: D (85%), H: D (80%), I: D (75%), K: D (85%), L: D (59%), M: D (75%), N: D (85%), P: D (91%), Q: D (80%), R: D (80%), S: D (85%), T: D (85%), V: D (63%), Y: N (57%),
Predicted by PROVEAN:	A: D, C: D, D: D, E: D, F: D, G: D, H: D, I: D, K: D, L: D, M: D, N: D, P: D, Q: D, R: D, S: D, T: D, V: D, Y: D,

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Publications
[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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229 Representative current traces of W401G/⌬F508 (A) and ⌬F508/Y1219G (B)inthepresenceof50␮M P-dATP.C,summaryofthemaximumcurrent-fold increase in activity induced by 10 ␮M and 50 ␮M P-dATP in W401F/⌬F508 and ⌬F508/Y1219G, and 10 ␮M P-dATP in ⌬F508. 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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4 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). Login to comment
34 Here, guided by accumulated structural and functional understanding of NBDs, we first identified W401Y,W401F mutations that strengthened ligand binding in the NBD1 head subdomain. Login to comment
36 The presence of the Asp-551 residue, which disfavors NBD dimer formation, however, raised the question of whether ATP molecule potentiates W401Y,W401F/G551D channels by binding at the NBD interface (i.e. site 1) or interacting only with the monomeric NBD1. Login to comment
37 Our data suggested that the former is likely the case as the effects of ATP on W401F/G551D channels can be further enhanced by the H1348G mutation in the NBD2 tail subdomain. Login to comment
54 For the W401F/H1348G/G551D channel, the steady-state open probability (Po) in the presence of ATP was estimated by stationary noise analysis of macroscopic currents using the equation ␴2 /i ϭ ͑1 - Po͒I (Eq. 1) where ␴2 is the variance, i is the unitary current amplitude, and I is the amplitude of the steady-state currents. Login to comment
62 This can be readily done for WT and W401F/ H1348G/⌬F508 channels (as seen in Fig. 5). Login to comment
100 Interestingly, we found that phenylalanine (W401F) appeared better than tyrosine (W401Y), which was in turn superior to tryptophan, in stabilizing the lock-open state, suggesting that W401Y and W401F mutations might enhance nucleotide-NBD1 interactions. Login to comment
103 We then incorporated W401Y or W401F mutation into G551D channels, intending to help G551D-NBD1 to bind nucleotide more tightly. Login to comment
106 It can be seen that W401F, which yielded a more stable lock-open state in the WT background (Fig. 1D), was also more effective than W401Y in conferring ATP-dependent activation of G551D channels. Login to comment
110 First, the response to ATP seen in W401Y,W401F/G551D channels could be due to an altered ligand-NBD1 interaction as proposed, or alternatively, is a result of a restored ability for ATP to gate CFTR through binding to site 2. Login to comment
112 Moreover, we also found that mutating the Trp-401-equivalent residue in NBD2 (Y1219G), which greatly decreases the NBD2 ATP affinity (19), had little effect on ATP-mediated activation of W401Y,W401F/ G551D channels (supplemental Fig. S4). Login to comment
113 Second, as PPi fails to lock open G551D-containing channels (data not shown), presumably due to the negatively charged Asp-551 side chain in site 2, it is desirable to have another parameter in support of the conjecture that W401Y,W401F mutations do tighten nucleotide binding in G551D-NBD1. Login to comment
114 One possible approach is to fit the current relaxation traces of W401Y,W401F/G551D channels upon removal of the nucleotide (Fig. 2A, inset) as the resulting time constants could potentially reflect the mean nucleotide dwell time in NBD1 of these channels (see also under "Discussion"). Login to comment
118 Does the ATP or PATP molecule that potentiates W401Y,W401F/G551D channels bind in the head subdomain of the monomeric NBD1 or NBD1-NBD2 dimer interface (i.e. site 1)? Login to comment
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
122 These results thus suggest that ATP or PATP resides in site 1 and interacts with both NBD1 head and NBD2 tail subdomains to support gating of W401Y,W401F/G551D channels. Login to comment
123 The idea that both the head of NBD1 and the tail of NBD2 are involved in ATP-dependent activation of W401Y,W401F/ G551D channels implicates that optimizing the interactions of ATP with the NBD2 tail subdomain may also improve the func- FIGURE 2. Login to comment
124 W401Y and W401F mutations confer ATP-dependent activa- tionofG551Dchannels.A,applicationofATPorPATPsignificantlyincreased currents of W401Y,W401F/G551D channels. Login to comment
125 Insets, current relaxation traces recorded after the removal of ATP (blue box) or PATP (green box) for W401F/ G551D-CFTR. Login to comment
127 C, effects of W401Y,W401F mutations on the relaxation time constants upon removal of ATP or PATP as shown in panel A. Note that for G551D-CFTR (Trp-401), no current decay was seen because the effect of ATP was negligible. Login to comment
133 Moreover, the H1348G mutation further improved the function of W401F/G551D channels so that the application of ATP and PATP increased the basal activity by ϳ25- and ϳ75-fold, respectively (Fig. 3, C and D, and supplemental Fig. S5), further supporting the notion that optimizing ATP binding in site 1 enhances the function of G551D channels. 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
140 C, H1348G enhanced the response of W401F/G551D-CFTR to ATP. Login to comment
141 D, histogram summarizing the ratio of ATP (blue bars)- or PATP (green bars)-induced current over basal current for W401F/G551D channels combined with a mutation in the NBD2 signature motif as marked. Login to comment
142 Dashed lines, the ratio of IATP/IBasal (blue) and IPATP/IBasal (green) for W401F/G551D channels. Login to comment
145 Single-channel kinetics of G551D channels with W401Y,W401F or H1348G mutations. Login to comment
156 W401F and H1348G Mutations Improve the Function of WT and ⌬F508 Channels-To this point, we have demonstrated that optimizing the interactions of ATP with site 1 components, NBD1 head (W401Y and W401F) and NBD2 tail (H1348G), ameliorates the gating defects of G551D channels, which hold a non-functional site 2. Login to comment
160 Indeed, when W401F and H1348G mutations were engineered into WT channels (Fig. 5A), the mean open time of WT-CFTR was more than quadrupled (Fig. 5C) with the already high Po (ϳ0.4) nearly doubled (ϳ0.78, Fig. 5D). Login to comment
162 In either case, W401F/H1348G mutations did not significantly alter the opening rate (Fig. 5E). Login to comment
165 First, ATP-site 1 interactions of the CFTR channel can be strengthened by introducing mutations in both the head domain of NBD1 (i.e. W401Y,W401F) and the tail domain of NBD2 (i.e. H1348G). Login to comment
180 Effects of W401F/H1348G mutations on WT and ⌬F508 channels. Login to comment
181 A, 30-s single-channel recordings of WT and W401F/H1348G channels exposed to 2.75 mM ATP. Login to comment
182 B, current recordings of ⌬F508 and ⌬F508/W401F/ H1348G channels in the presence of 2.75 mM ATP. Login to comment
191 It is this correlation between the chemical nature of mutations and the stability of the lock-open state that grants us the confidence that W401Y,W401F and H1348G mutations, which prolonged the lock-open duration of WT-CFTR, indeed tighten ATP binding in site 1. Login to comment
195 The results shown in Fig. 2C did suggest that W401Y,W401F mutations enhance nucleotide binding in G551D site 1, like in the case of WT site 1. Login to comment
199 Unlike WT and ⌬F508 channels, whose Po in the presence or absence of W401F/H1348G mutations (Fig. 5) can be measured with reasonable accuracy, the G551D-containing channels exhibit a Po too low to be derived from single-channel analysis. Login to comment
204 For instance, that the W401F/H1348G/G551D channel has an IATP/IBasal of ϳ25 indicates that the Po for this mutant is ϳ0.1. Login to comment
205 To verify this value, we performed stationary noise analysis for the W401F/ H1348G/G551D mutant, and the resulting Po of 0.09 Ϯ 0.01 (supplemental Fig. S7) did provide some reassurance. Login to comment
209 We have observed that W401F/H1348G mutations in site 1 prolonged the mean open time of WT-CFTR (Fig. 5C). Login to comment
211 Thus, it appears that a stronger ATP binding in site 1, due to the presence of W401F/H1348G mutations, can allosterically tighten the connection between two NBDs around site 2, thereby slowing down channel closure. Login to comment

[hide] The most common cystic fibrosis-associated mutatio... J Physiol. 2011 Jun 1;589(Pt 11):2719-31. Epub 2011 Apr 11. Jih KY, Li M, Hwang TC, Bompadre SG
The most common cystic fibrosis-associated mutation destabilizes the dimeric state of the nucleotide-binding domains of CFTR.
J Physiol. 2011 Jun 1;589(Pt 11):2719-31. Epub 2011 Apr 11., 2011-06-01 [PMID:21486785]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel that belongs to the ATP binding cassette (ABC) superfamily. The deletion of the phenylalanine 508 (DeltaF508-CFTR) is the most common mutation among cystic fibrosis (CF) patients. The mutant channels present a severe trafficking defect, and the few channels that reach the plasma membrane are functionally impaired. Interestingly, an ATP analogue, N6-(2-phenylethyl)-2'-deoxy-ATP (P-dATP), can increase the open probability (Po) to approximately 0.7, implying that the gating defect of DeltaF508 may involve the ligand binding domains, such as interfering with the formation or separation of the dimeric states of the nucleotide-binding domains (NBDs). To test this hypothesis, we employed two approaches developed for gauging the stability of the NBD dimeric states using the patch-clamp technique. We measured the locked-open time induced by pyrophosphate (PPi), which reflects the stability of the full NBD dimer state, and the ligand exchange time for ATP/N6-(2-phenylethyl)-ATP (P-ATP), which measures the stability of the partial NBD dimer state wherein the head of NBD1 and the tail of NBD2 remain associated. We found that both the PPi-induced locked-open time and the ATP/P-ATP ligand exchange time of DeltaF508-CFTR channels are dramatically shortened, suggesting that the DeltaF508 mutation destabilizes the full and partial NBD dimer states. We also tested if mutations that have been shown to improve trafficking of DeltaF508-CFTR, namely the solubilizing mutation F494N/Q637R and DeltaRI (deletion of the regulatory insertion), exert any effects on these newly identified functional defects associated with DeltaF508-CFTR. Our results indicate that although these mutations increase the membrane expression and function of DeltaF508-CFTR, they have limited impact on the stability of both full and partial NBD dimeric states for DeltaF508 channels. The structure-function insights gained from this mechanism may provide clues for future drug design.

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105 Tight binding of nucleotides in NBD1 prolongs the channel locked-open time In a previous report (Tsai et al. 2010a), we demonstrated that the locked-open time of WT-CFTR induced by PPi is prolonged by replacing ATP with the high affinity ATP analogue N6 -phenylethyl-ATP (P-ATP), or by introducing 'gain-of-function` mutations to the ATP-binding site 1 (mutations which increase the Po of CFTR, such as W401F and H1348G) as the locked-open state reflects an NBD dimer with ATP-binding site 1 occupied by ATP and ATP-binding site 2 by PPi (Tsai et al. 2009). Login to comment
106 In Fig. 3, we show that the gain-of-function mutations W401F and H1348G (Fig. 3A) and P-ATP (Fig. 3B) also prolong the locked-open time of F508-CFTR channels. Login to comment
107 Compared to F508-CFTR, the double mutant W401F/ F508-CFTR ( F508/DM)prolongedthelocked-opentimeby~2-fold, and the triple mutant W401F/H1348G/ F508-CFTR ( F508/TM) by ~4-fold. Login to comment
127 C, summary of PPi locked-open times for each construct ( F508/DM: W401F/ F508-CFTR, F508/TM: W401F/H1348G/ F508-CFTR). Login to comment

[hide] Nonintegral stoichiometry in CFTR gating revealed ... J Gen Physiol. 2012 Oct;140(4):347-59. Epub 2012 Sep 10. Jih KY, Sohma Y, Hwang TC
Nonintegral stoichiometry in CFTR gating revealed by a pore-lining mutation.
J Gen Physiol. 2012 Oct;140(4):347-59. Epub 2012 Sep 10., [PMID:22966014]

Abstract [show]
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.

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71 Fig. S1 shows the closed- time distribution for R352C-CFTR. Fig. S2 presents dwell-time distributions for O1 and O2 states. Login to comment
72 Fig. S3 demonstrates the effects of the W401F mutation on the single-channel kinetics of R352C-CFTR. Login to comment
130 ATP hydrolysis drives the O1→O2 transition Although our initial observations were made with Cysless/R352C-CFTR, this unique pattern of gating transitions was also seen when we introduced the R352C mutation into the WT background (Fig. 3 A and Table 1). Login to comment
131 One notable difference between R352C- and Cysless/ Ta b l e 1 Summary of opening events by different gating patterns in three CFTR mutants Gating topology O1-O2 O1 O2 O2-O1 (O1-O2) n # Total Cysless/R352C 2.75 mM ATP 100 µM ATP 720 (45%) 663 (56%) 290 (18%) 216 (18%) 175 (11%) 137 (12%) 42 (3%) 32 (3%) 375 (23%) 128 (11%) 1,602 (100%) 1,176 (100%) R352C 2.75 mM ATP 100 µM ATP 834 (55%) 1,246 (59%) 301 (20%) 406 (19%) 173 (11%) 281 (13%) 39 (3%) 45 (2%) 169 (11%) 121 (6%) 1,516 (100%) 2,099 (100%) R352C/W401F 2.75 mM ATP 100 µM ATP 733 (44%) 1,189 (54%) 326 (19%) 367 (17%) 122 (7%) 337 (15%) 28 (2%) 60 (3%) 474 (28%) 232 (11%) 1,683 (100%) 2,185 (100%) Five different gating patterns are illustrated on the top of the table. Login to comment
182 Quantitative analysis of gating events indeed demonstrates a higher percentage of gating events with reentry transitions in W401F/R352C-CFTR (Table 1). Login to comment
186 Prolonged O1 and O2 dwell times were also found in W401F/R352C-CFTR, but to a lesser extent. Login to comment
187 The effect of Cysless and W401F mutations in prolonging the open time of R352C mutant channels is consistent with the observations made for the same mutants in the WT background (Bai et al., 2010; Tsai et al., 2010a). Login to comment
188 D I S C U S S I O N An accidental discovery of the R352C mutation grants us the opportunity to actually "see"-in real time-ATP hydrolysis taking place during CFTR gating as the ordered transition between two distinct levels of open channel conductance (O1 and O2) indicates an input of the free energy from ATP hydrolysis. Login to comment
206 W401F mutation promotes O2→O1 transition In our latest paper (Jih et al., 2012), using nonhydrolyzable ATP analogues (pyrophosphate or adenylyl-imidodi- phosphate) as baits, we captured a post-hydrolytic state (state X), which, like the O2 state in this paper, can reenter the prehydrolytic open state (O1) upon ATP binding to the vacant ABP2. Login to comment
208 Interestingly, a conservative mutation in NBD1 (W401F, both W and F are aromatic amino acids) can enhance the responsiveness of state X to ATP as well as nonhydrolyzable ATP analogues for reasons yet to be elucidated. Login to comment
209 Nevertheless, the striking functional similarities between state X in Jih et al. (2012) and the O2 state in this paper prompt us to test the effect of the W401F mutation on R352C-CFTR. Fig. S3 shows a representative single-channel trace of W401F/R352C-CFTR. Login to comment
210 Compared state C is nearly 1 s for R352C-CFTR. Login to comment
183 Quantitative analysis of gating events indeed demonstrates a higher percentage of gating events with reentry transitions in W401F/R352C-CFTR (Table 1). Login to comment
211 Nevertheless, the striking functional similarities between state X in Jih et al. (2012) and the O2 state in this paper prompt us to test the effect of the W401F mutation on R352C-CFTR. Fig. S3 shows a representative single-channel trace of W401F/R352C-CFTR. Login to comment

[hide] Identification of a novel post-hydrolytic state in... J Gen Physiol. 2012 May;139(5):359-70. doi: 10.1085/jgp.201210789. Epub 2012 Apr 16. Jih KY, Sohma Y, Li M, Hwang TC
Identification of a novel post-hydrolytic state in CFTR gating.
J Gen Physiol. 2012 May;139(5):359-70. doi: 10.1085/jgp.201210789. Epub 2012 Apr 16., [PMID:22508846]

Abstract [show]
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.

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89 Fig. S3 shows the open dwell-time histograms of WT- and W401F-CFTR. Login to comment
118 On the other hand, mutating the residue Trp401 (W401), which forms ring-ring stacking interactions with ATP in ABP1 (Lewis et al., 2004; Zhou et al., 2006), to phenylalanine significantly increased the propensity of state X in response to PPi (i.e., a higher percentage of channels locked open from state X; WT, 49 ± 2% and n = 13; W401F, 59 ± 2% and n = 13; P < 0.05). Login to comment
120 Therefore, opposite to P-ATP, the W401F mutation enhances the responsiveness of state X to PPi but may destabilize the C2 state. Login to comment
135 It is noteworthy that the success rate of this line of experiments is lower for WT channels (see supplemental Discussion for more details), but similar observations were made for WT-CFTR (Fig. S1), indicating that such open X-locked-open transitions are not just idiosyncratic for W401F mutant channels. Login to comment
147 However, the conserved W401F mutation (see below), which significantly prolongs the open time (Tsai et al., 2010a), makes such experiments more feasible. Login to comment
148 In the presence of ATP, the W401F-CFTR channel exhibits similar behavior as WT-CFTR: the channel opens into bursts that last for hundreds of milliseconds to seconds, and each opening burst is separated by a long interburst that also closes in the range of hundreds of milliseconds (Fig. 6). Login to comment
157 (B) A similar protocol was conducted with W401F-CFTR channels. Login to comment
163 For reasons unclear at this moment, the W401F mutation could somehow improve the responsiveness of state X to PPi (Fig. 5) and therefore presumably to ATP as well. Login to comment
164 We therefore quantified the open burst time of W401F-CFTR at different [ATP]. Login to comment
166 Furthermore, the notion that a prolonged open burst time seen with the W401F mutation is caused by reentry of the channel from state X predicts a disparity between the mean open time estimated from microscopic analysis and the relaxation time constant obtained from macroscopic current decay upon ATP removal, which prohibits reentry to occur. Login to comment
167 Fig. 7 (B and C) shows that when fitting the current relaxation of W401F mac roscopic current after washout of 10 mM ATP, the time constant is almost identical to the mean open time of W401F channels in the presence of micromolar ATP. Login to comment
168 Figure 6. Single-channel ligand exchange for W401F-CFTR. Login to comment
169 (A and C) A single W401F-CFTR was activated by ATP, and the ligand was switched to a 1-s PPi (A) or AMP-PNP (C) pulse in the open state. Login to comment
188 As shown in Fig. 5, the W401F mutation and the high-affinity ATP analogue P-ATP, both acting on ABP1, affect state X and the C2 state very differently. Login to comment
193 Figure 7. The mean open time of W401F-CFTR is [ATP] dependent. Login to comment
194 (A) Microscopic current traces of W401F-CFTR in the presence of 10 mM (top), 1 mM (middle), or 100 µM (bottom) ATP. Login to comment
195 (B) Mean open time of W401F-CFTR at different [ATP] (closed circles). Login to comment
197 (C)Macroscopic current of W401F-CFTR was activated by 10 mM ATP to a steady state. Login to comment
205 Furthermore, the prolonged open burst time in the presence of higher [ATP] (>1 mM; P < 0.05 when comparing 1 and 10 mM) for W401F-CFTR indicates that the lifetime of state X as a closed state must be indistinguishable from that of the flickers so that closed events corresponding to state X are excluded by the analysis. Login to comment
223 By comparing WT-CFTR and W401F-CFTR, the increasing reentry events (X→X→O1) results in differences only in the mean open time and the tail of the fitted curve (Fig. S3), which is supplanted with an increased number of long opening bursts (>1,000 ms). Login to comment

[hide] Vx-770 potentiates CFTR function by promoting deco... Proc Natl Acad Sci U S A. 2013 Mar 12;110(11):4404-9. doi: 10.1073/pnas.1215982110. Epub 2013 Feb 25. Jih KY, Hwang TC
Vx-770 potentiates CFTR function by promoting decoupling between the gating cycle and ATP hydrolysis cycle.
Proc Natl Acad Sci U S A. 2013 Mar 12;110(11):4404-9. doi: 10.1073/pnas.1215982110. Epub 2013 Feb 25., [PMID:23440202]

Abstract [show]
Vx-770 (Ivacaftor), a Food and Drug Administration (FDA)-approved drug for clinical application to patients with cystic fibrosis (CF), shifts the paradigm from conventional symptomatic treatments to therapeutics directly tackling the root of the disease: functional defects of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel caused by pathogenic mutations. The underlying mechanism for the action of Vx-770 remains elusive partly because this compound not only increases the activity of wild-type (WT) channels whose gating is primarily controlled by ATP binding/hydrolysis, but also improves the function of G551D-CFTR, a disease-associated mutation that abolishes CFTR's responsiveness to ATP. Here we provide a unified theory to account for this dual effect of Vx-770. We found that Vx-770 enhances spontaneous, ATP-independent activity of WT-CFTR to a similar magnitude as its effects on G551D channels, a result essentially explaining Vx-770's effect on G551D-CFTR. Furthermore, Vx-770 increases the open time of WT-CFTR in an [ATP]-dependent manner. This distinct kinetic effect is accountable with a newly proposed CFTR gating model depicting an [ATP]-dependent "reentry" mechanism that allows CFTR shuffling among different open states by undergoing multiple rounds of ATP hydrolysis. We further examined the effect of Vx-770 on R352C-CFTR, a unique mutant that allows direct observation of hydrolysis-triggered gating events. Our data corroborate that Vx-770 increases the open time of WT-CFTR by stabilizing a posthydrolytic open state and thereby fosters decoupling between the gating cycle and ATP hydrolysis cycle. The current study also suggests that this unique mechanism of drug action can be further exploited to develop strategies that enhance the function of CFTR.

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157 Summary of the effects of Vx-770 on the gating patterns exhibited in R352C-CFTR and W401F/R352C-CFTR Total Experimental condition Without Vx-770* R352C (%) 834 (55) 301 (20) 173 (11) 39 (3) 169 (11) 1,516 (100) R352C/W401F (%) 733 (44) 326 (19) 122 (7) 28 (2) 474 (28) 1,683 (100) With 200 nM Vx-770 R352C (%) 578 (57) 126 (12) 73 (7) 20 (2) 217 (21) 1,014 (100) R352C/W401F (%) 411 (37) 162 (15) 79 (7) 31 (3) 425 (38) 1,108 (100) Five different categories of the gating pattern are illustrated. Login to comment
161 *Data for R352C-CFTR and R352C/W401F-CFTR in the absence of Vx-770 were taken from ref. 24. gate in the absence of ATP, C2 ࢒ O2) primarily affected by Vx-770 are supposed to take place in CFTR`s TMDs (24). Login to comment
178 Lately, we discovered that W401F, a conserved mutation in NBD1, increases the reentry frequency without significantly altering the O2 ࢐ C2 transition (24). Login to comment
179 Although how the W401F mutation modifies the function of NBDs is unclear, this finding does raise the possibility that manipulating NBD function can complement the action of Vx-770. Login to comment
180 As shown in Fig. S3 and Table 1, the W401F mutation almost doubles the reentry frequency of R352C-CFTR and also significantly enhances the effect of Vx-770 (Fig. 5, Table 1 and Fig. S3). Login to comment
208 W401F mutation and Vx-770 work additively on the reentry pathway. Login to comment
209 Representative single-channel traces for R352C/W401F-CFTR treated with 2.75 mM ATP in the absence (A) or presence (B) of 200 nM Vx-770. Login to comment

[hide] Conformational changes in the catalytically inacti... J Gen Physiol. 2013 Jul;142(1):61-73. doi: 10.1085/jgp.201210954. Epub 2013 Jun 10. Csanady L, Mihalyi C, Szollosi A, Torocsik B, Vergani P
Conformational changes in the catalytically inactive nucleotide-binding site of CFTR.
J Gen Physiol. 2013 Jul;142(1):61-73. doi: 10.1085/jgp.201210954. Epub 2013 Jun 10., [PMID:23752332]

Abstract [show]
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 N(6)-(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 (>2x) transition O1-->O2 and decreases the nonhydrolytic closing rate (transition O1-->C) of CFTR mutants K1250A ( approximately 4x) and E1371S ( approximately 3x). Mutation H1348A also slowed ( approximately 3x) the O1-->O2 transition in the WT background and decreased the nonhydrolytic closing rate of both K1250A ( approximately 3x) and E1371S ( approximately 3x) 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.

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49 In contrast, [ATP]-dependent prolongation of open burst durations for the W401F CFTR mutant (Jih et al., 2012b), or for WT CFTR gating in the presence of the potentiator compound Vx-770 (Jih and Hwang, 2013), has led to the suggestion that post-hydrolytic ADP/ATP exchange in site 2 might occur without intervening pore closure, thereby increasing the coupling ratio by allowing more than one ATP to be hydrolyzed in site 2 within a single gating cycle (Jih et al., 2012a). Login to comment
111 Channels opened by Because for another site-1 mutant (W401F) mean burst durations were shown to increase at high millimolar ATP concentrations (Jih et al., 2012b), we tested whether that was the case for H1348A CFTR. Login to comment
160 Because the W401F mutation also resides in site 1, we asked whether a reentry mechanism might explain the longer burst durations in P-ATP or in the presence of the H1348A mutation. Login to comment
164 Recent studies suggested a novel mechanism for a prolongation of steady-state CFTR burst durations for NBD1 mutant W401F (Jih et al., 2012a,b), by proposing the presence of a short time window at the end of each burst during which the hydrolysis products ADP and phosphate at site 2 can be replaced by a new ATP molecule without intervening channel gate closure. Login to comment
194 Because one such mutation (W401F) resides in site 1, we have evaluated the possibility that the H1348A mutation, or P-ATP bound in site 1, might also act by such a mechanism. Login to comment