ABCMdb

ABCC7 p.Glu1371Ser

ClinVar:	c.4111G>T , p.Glu1371* D , Likely pathogenic
CF databases:	c.4111G>T , p.Glu1371* D , CF-causing
Predicted by SNAP2:	A: D (91%), C: D (91%), D: D (80%), F: D (95%), G: D (95%), H: D (95%), I: D (95%), K: D (91%), L: D (95%), M: D (91%), N: D (91%), P: D (95%), Q: D (91%), R: D (95%), S: D (91%), T: D (91%), V: D (91%), W: D (95%), Y: D (95%),
Predicted by PROVEAN:	A: D, C: D, D: N, F: D, G: D, H: D, I: D, K: D, L: D, M: D, N: D, P: D, Q: N, R: D, S: D, T: D, V: D, W: D, Y: D,

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[hide] On the mechanism of MgATP-dependent gating of CFTR... J Gen Physiol. 2003 Jan;121(1):17-36. Vergani P, Nairn AC, Gadsby DC
On the mechanism of MgATP-dependent gating of CFTR Cl- channels.
J Gen Physiol. 2003 Jan;121(1):17-36., [PMID:12508051]

Abstract [show]
CFTR, the product of the gene mutated in cystic fibrosis, is an ATPase that functions as a Cl(-) channel in which bursts of openings separate relatively long interburst closed times (tauib). Channel gating is controlled by phosphorylation and MgATP, but the underlying molecular mechanisms remain controversial. To investigate them, we expressed CFTR channels in Xenopus oocytes and examined, in excised patches, how gating kinetics of phosphorylated channels were affected by changes in [MgATP], by alterations in the chemical structure of the activating nucleotide, and by mutations expected to impair nucleotide hydrolysis and/or diminish nucleotide binding affinity. The rate of opening to a burst (1/tauib) was a saturable function of [MgATP], but apparent affinity was reduced by mutations in either of CFTR's nucleotide binding domains (NBDs): K464A in NBD1, and K1250A or D1370N in NBD2. Burst duration of neither wild-type nor mutant channels was much influenced by [MgATP]. Poorly hydrolyzable nucleotide analogs, MgAMPPNP, MgAMPPCP, and MgATPgammaS, could open CFTR channels, but only to a maximal rate of opening approximately 20-fold lower than attained by MgATP acting on the same channels. NBD2 catalytic site mutations K1250A, D1370N, and E1371S were found to prolong open bursts. Corresponding NBD1 mutations did not affect timing of burst termination in normal, hydrolytic conditions. However, when hydrolysis at NBD2 was impaired, the NBD1 mutation K464A shortened the prolonged open bursts. In light of recent biochemical and structural data, the results suggest that: nucleotide binding to both NBDs precedes channel opening; at saturating nucleotide concentrations the rate of opening to a burst is influenced by the structure of the phosphate chain of the activating nucleotide; normal, rapid exit from bursts occurs after hydrolysis of the nucleotide at NBD2, without requiring a further nucleotide binding step; if hydrolysis at NBD2 is prevented, exit from bursts occurs through a slower pathway, the rate of which is modulated by the structure of the NBD1 catalytic site and its bound nucleotide. Based on these and other results, we propose a mechanism linking hydrolytic and gating cycles via ATP-driven dimerization of CFTR's NBDs.

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7 NBD2 catalytic site mutations K1250A, D1370N, and E1371S were found to prolong open bursts. Login to comment
179 WT (A), D1370N (B), K1250A (C), and E1371S (D) CFTR channels were activated by 5 mM MgATP plus PKA as indicated: burst termination (‫-4.0ف‬pA downward steps) after nucleotide washout was slowed for NBD2 mutants, relative to WT. Login to comment
180 Note persistence of brief (intraburst) closures during K1250A and E1371S bursts, long after nucleotide withdrawal. Login to comment

[hide] A heteromeric complex of the two nucleotide bindin... J Biol Chem. 2004 Oct 1;279(40):41664-9. Epub 2004 Jul 28. Kidd JF, Ramjeesingh M, Stratford F, Huan LJ, Bear CE
A heteromeric complex of the two nucleotide binding domains of cystic fibrosis transmembrane conductance regulator (CFTR) mediates ATPase activity.
J Biol Chem. 2004 Oct 1;279(40):41664-9. Epub 2004 Jul 28., 2004-10-01 [PMID:15284228]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) protein is a member of the ABC superfamily of transporter proteins. Recently, crystal structures of intact, prokaryotic members of this family have been described. These structures suggested that ATP binding and hydrolysis occurs at two sites formed at the interface between their nucleotide binding domains (NBDs). In contrast to the prokaryotic family members, the NBDs of CFTR are asymmetric (both structurally and functionally), and previous to the present studies, it was not clear whether both NBDs are required for ATP hydrolysis. In order to assess the relative roles of the two NBDs of human CFTR, we purified and reconstituted NBD1 and NBD2, separately and together. We found that NBD1 and NBD2 by themselves exhibited relatively low ATPase activity. Co-assembly of NBD1 and NBD2 exhibited a 2-3-fold enhancement in catalytic activity relative to the isolated domains and this increase reflected enhanced ATP turnover (V(max)). These data provide the first direct evidence that heterodimerization of the NBD1 and NBD2 domains of CFTR is required to generate optimal catalytic activity.

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190 Unfortunately, there are no ATPase data available for the NBD2 Walker B mutations D1370N and E1371S, which would be expected to severely impair hydrolysis. Login to comment

[hide] CFTR gating II: Effects of nucleotide binding on t... J Gen Physiol. 2005 Apr;125(4):377-94. Epub 2005 Mar 14. Bompadre SG, Cho JH, Wang X, Zou X, Sohma Y, Li M, Hwang TC
CFTR gating II: Effects of nucleotide binding on the stability of open states.
J Gen Physiol. 2005 Apr;125(4):377-94. Epub 2005 Mar 14., [PMID:15767296]

Abstract [show]
Previously, we demonstrated that ADP inhibits cystic fibrosis transmembrane conductance regulator (CFTR) opening by competing with ATP for a binding site presumably in the COOH-terminal nucleotide binding domain (NBD2). We also found that the open time of the channel is shortened in the presence of ADP. To further study this effect of ADP on the open state, we have used two CFTR mutants (D1370N and E1371S); both have longer open times because of impaired ATP hydrolysis at NBD2. Single-channel kinetic analysis of DeltaR/D1370N-CFTR shows unequivocally that the open time of this mutant channel is decreased by ADP. DeltaR/E1371S-CFTR channels can be locked open by millimolar ATP with a time constant of approximately 100 s, estimated from current relaxation upon nucleotide removal. ADP induces a shorter locked-open state, suggesting that binding of ADP at a second site decreases the locked-open time. To test the functional consequence of the occupancy of this second nucleotide binding site, we changed the [ATP] and performed similar relaxation analysis for E1371S-CFTR channels. Two locked-open time constants can be discerned and the relative distribution of each component is altered by changing [ATP] so that increasing [ATP] shifts the relative distribution to the longer locked-open state. Single-channel kinetic analysis for DeltaR/E1371S-CFTR confirms an [ATP]-dependent shift of the distribution of two locked-open time constants. These results support the idea that occupancy of a second ATP binding site stabilizes the locked-open state. This binding site likely resides in the NH2-terminal nucleotide binding domain (NBD1) because introducing the K464A mutation, which decreases ATP binding affinity at NBD1, into E1371S-CFTR shortens the relaxation time constant. These results suggest that the binding energy of nucleotide at NBD1 contributes to the overall energetics of the open channel conformation.

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3 To further study this effect of ADP on the open state, we have used two CFTR mutants (D1370N and E1371S); both have longer open times because of impaired ATP hydrolysis at NBD2. Login to comment
5 ⌬R/E1371S-CFTR channels can be locked open by millimolar ATP with a time constant of ~100 s, estimated from current relaxation upon nucleotide removal. Login to comment
7 To test the functional consequence of the occupancy of this second nucleotide binding site, we changed the [ATP] and performed similar relaxation analysis for E1371S-CFTR channels. Login to comment
9 Single-channel kinetic analysis for ⌬R/E1371S-CFTR confirms an [ATP]-dependent shift of the distribution of two locked-open time constants. Login to comment
11 This binding site likely resides in the NH2-terminal nucleotide binding domain (NBD1) because introducing the K464A mutation, which decreases ATP binding affinity at NBD1, into E1371S-CFTR shortens the relaxation time constant. Login to comment
45 To study in more detail the effect of nucleotide binding on the open time of CFTR, we used both macroscopic current relaxation and single-channel kinetic analysis for CFTR mutants with impaired ATP hydrolysis: specifically, the D1370N and E1371S mutations in NBD2. Login to comment
49 Mutation of this glutamate to serine produces a channel (E1371S) that presents long "locked-open" times (Aleksandrov et al., 2000; Vergani et al., 2003). Login to comment
52 Relaxation analysis of macroscopic ⌬R/E1371S-CFTR currents upon nucleotide removal shows two different relaxation time constants in the presence of ADP and ATP, indicating that ADP induces a different locked-open state. Login to comment
53 Moreover, studies of ⌬R/E1371S-CFTR open time in patches containing a single channel at three different ATP concentrations show a change of the relative frequency of the different open times, suggesting the presence of an ATP-binding site, occupancy of which affects the stability of the open state. Login to comment
54 Similar results were obtained in E1371S mutants constructed in the WT background. Login to comment
56 M A T E R I A L S A N D M E T H O D S Construction of CFTR Mutants CFTR mutations E1371S and D1370N were introduced into the plasmid pBQ4.7 WT-CFTR (Powe et al., 2002) by using the QuikChange kit (Stratagene). Login to comment
57 The 0.9-kb PflMI-XhoI fragments containing the mutations from the pBQ4.7 were used to substitute the corresponding regions in pBudCE4.1 split ⌬R CFTR (Ai et al., 2004) to obtain pBudCE4.1 ⌬R/D1370N and pBudCE4.1 ⌬R/E1371S CFTR. Login to comment
58 Similarly, the 0.9-kb PflMI-EcoRV fragments were used to replace the corresponding ones in pcDNA 3.1 WT-CFTR or pcDNA 3.1 K464A-CFTR (Powe et al., 2002) to generate pcDNA3.1 D1370N, pcDNA 3.1 E1371S, and pcDNA3.1 K464A/ E1371S CFTR constructs. Login to comment
66 For dwell-time analysis of ⌬R/E1371S-CFTR data, we pooled the current records obtained from several patches containing only one channel. Login to comment
103 We therefore constructed another mutant, E1371S, which has an open time on the order of tens or hundreds of seconds because the ATP hydrolysis is abolished at NBD2 (Aleksandrov et al., 2000; Vergani et al., 2003). Login to comment
105 Macroscopic current relaxation for ⌬R/E1371S-CFTR channels in the presence of ATP and ADP. Login to comment
106 (A) A sample trace of current relaxations for ⌬R/E1371S-CFTR channels opened with 1 mM ATP, and subsequently with 1 mM ATP ϩ 2 mM ADP. Login to comment
108 (C) The current decay upon removal of ATP plus ADP is fitted with a double exponential function with time constants of 12.9 Ϯ 0.1 and 105 Ϯ 3 s. ADP Effect on ⌬R/E1371S Mutant We first introduced the E1371S mutation into the ⌬R background in order to study the behavior of this channel in the presence or absence of ADP without having to worry about the possible effect of dephosphorylation on the channel open time. Login to comment
115 Compared with the relaxation time course with ATP alone, the current relaxation of ⌬R/E1371S-CFTR channels opened by ATP plus ADP is faster. Login to comment
124 Macroscopic current relaxation for ⌬R/E1371S-CFTR opened with 10 ␮M ATP. Login to comment
125 ⌬R/ E1371S-CFTR channels were activated with 10 ␮M ATP until the current reached a steady state. Login to comment
130 The dash line represents current relaxation of ⌬R/ E1371S-CFTR upon removal of 1 mM ATP (from Fig. 2 B). Login to comment
133 ATP Concentration Dependence of ⌬R/E1371S Current Relaxation Our experiments with ADP suggest the presence of a nucleotide binding site, occupancy of which by ATP or ADP can affect the stability of the locked-open state. Login to comment
137 Fig. 3 A shows a representative relaxation experiments for ⌬R/E1371S-CFTR channels opened with 10 ␮M ATP. Login to comment
142 Single-channel recording of ⌬R/E1371S-CFTR in the presence of 1 ␮M ATP. Login to comment
151 Single-channel Analysis of ⌬R/ E1371S Mutant Fig. 4 shows a continuous recording of a single ⌬R/ E1371S mutant channel in the presence of 1 mM ATP that lasts for ~50 min. Login to comment
157 Single-channel recording of ⌬R/E1371S-CFTR in the presence of 10 ␮M ATP. Login to comment
172 We also analyzed one long recording (‫08ف‬ min) of ⌬R/E1371S-CFTR at 3 ␮M ATP (Fig. 6 B). Login to comment
183 Single-channel dwell time analysis of ⌬R/ E1371S-CFTR. Login to comment
223 ATP Concentration Dependence of E1371S-CFTR Current Relaxations Before we attempt to specify to which NBD ATP binds to affect the stability of the open state, we need to be sure that the [ATP] dependence of the open state stability is not solely due to deletion of the R domain. Login to comment
224 We therefore characterized the current relaxation for the E1371S mutation in the WT background. Login to comment
227 Unlike ⌬R/E1371S-CFTR, the E1371S mutants in the WT background express very well. Login to comment
233 We would like to point out that the relaxation curve obtained for the ⌬R/E1371S mutant (Fig. 2 B) shows only one exponential decay (␶ ϭ 100 s) at 1 mM. Login to comment
235 Macroscopic current relaxation of E1371S-CFTR currents. Login to comment
236 (A) Sample trace of current relaxations for E1371S-CFTR channels activated with 10 ␮M ATP ϩ PKA, or with 1 mM ATP ϩ PKA. Login to comment
240 semble current for ⌬R/E1371S-CFTR, even with pooling of 47 patches, is still much smaller (‫58ف‬ pA) than the macroscopic currents obtained with the E1371S mutant in the WT background (‫085ف‬ pA). Login to comment
241 Therefore, the minor fast component of the exponential decay is difficult to resolve in the ⌬R/E1371S-CFTR. Login to comment
242 When performing the relaxation experiments in E1371S-CFTR channels, after several minutes of washout, we can still observe a single channel that remains open (Fig. 9). Login to comment
247 Current Relaxations of the K464A/E1371S Mutant According to the most recent model (Scheme 2 in the accompanying paper) for CFTR gating (Vergani et al., 2003), NBD1 has little role in the gating transitions since the off rate is extremely slow, and ATP binding at NDB2 precedes channel opening. Login to comment
249 Kinetic analysis of the last E1371S-CFTR channel that remains open after removal of ATP. Login to comment
250 (A) Sample trace of the current relaxation of E1371S-CFTR channels upon ATP washout. Login to comment
262 The K464A mutation shortens the locked-open time of E1371S-CFTR. Login to comment
263 (A) Sample trace of K464A/E1371S-CFTR channels in the presence of 1 mM ATP ϩ PKA. Login to comment
267 (C) Sample trace of K464A/E1371S-CFTR channels in the presence of 10 ␮M ATP (blue curve). Login to comment
273 Since the K464 mutation has a mild trafficking defect (Cheng et al., 1990; unpublished data), and ⌬R-CFTR already suffers from low expression, we decided to make the K464A/E1371S double mutant construct in the WT background. Login to comment
275 The current decay of the K464A/E1371S-CFTR channel currents is indeed faster than that of E1371S-CFTR, resulting in a shorter relaxation time constant (19.60 Ϯ 0.01 s) upon washout of 1 mM ATP (Fig. 10 B). Login to comment
276 This relaxation time constant is even shorter when the K464A/E1371S-CFTR channel is opened with 10 ␮M ATP (13.95 Ϯ 0.02 s). Login to comment
277 Since the number of K464A/E1371S-CFTR channels is relatively low due to a moderate trafficking defect, it is easier to observe microscopic channel behavior at 10 ␮M ATP (Fig. 10 C). Login to comment
278 As shown previously for ⌬R/E1371S-CFTR (Fig. 5), the current trace reveals that K464A/E1371S-CFTR channels also exhibit numerous brief openings that last for tens to hundreds of milliseconds in the presence of 10 ␮M ATP. Login to comment
281 (A) A representative ⌬R/E1371S-CFTR current trace from an excised inside-out patch exposed to ATP-free solution for several minutes before 1 mM ATP was applied. Login to comment
283 The open times of these spontaneous openings from several patches containing ⌬R/E1371S-CFTR (C) or ⌬R-CFTR (D) were pooled together to construct survivor plots. Login to comment
289 Excised inside-out patches from cells expressing ⌬R/E1371S-CFTR channels were exposed to ATP-free perfusion solution for Ͼ5 min to determine the opening rate of spontaneous opening events. Login to comment
302 Although the E1371S mutation increases the lifetime of ATP-opened channel by Ͼ100-fold, it only minimally affects the open time constant for these spontaneous opening events. Login to comment
303 Despite the similarity between these open time constants for the spontaneous openings and the brief open time constant for the ⌬R/E1371S-CFTR in the presence of 10 ␮M ATP (Fig. 6 D), it is doubtful that the observed events at 10 ␮M ATP solely come from spontaneous openings because they appear more frequently than the spontaneous openings. Login to comment
304 We then quantify the kinetic step to the brief opening events using the ⌬R/ E1371S-CFTR data in the presence of 10 ␮M ATP. Login to comment
313 We used macroscopic current relaxation upon nucleotide removal to quantify the locked-open times for E1371S mutant CFTR. Login to comment
322 In contrast, the macroscopic current relaxation of ⌬R/E1371S-CFTR clearly shows the presence of two components in the relaxation once the channels are opened by ATP plus ADP (Fig. 2), strongly suggesting the presence of two different locked-open states. Login to comment
323 Furthermore, the relaxation of E1371S-CFTR channel currents in the presence of different ATP concentrations reveals the presence of two locked-open states, and an [ATP]-dependent shift in the distribution of each state (confirmed by ⌬R/ E1371S single-channel data). Login to comment
325 Perhaps because of a much smaller current amplitude, we did not resolve two relaxation time constants in the presence of 1 mM ATP for ⌬R/E1371S-CFTR channels. Login to comment
326 If we accept the fact that multiple locked-open states do exist for ⌬R/E1371S mutants at 1 mM ATP as shown by the single-channel analysis (Fig. 6), how can we ascertain that ADP induces another locked-open state rather than simply increases the relative occupancy of the short-lived locked-open state? Login to comment
342 Using similar dwell time analysis, we were able to detect multiple components in open time histograms (Fig. 6) for ⌬R/E1371S mutants. Login to comment
350 This same idea can also explain ADP`s effects on the current relaxation of ⌬R/E1371S mutants (Fig. 2). Login to comment
356 Indeed, the mean lifetime of the spontaneous openings for ⌬R/E1371S is ‫004ف‬ ms. Login to comment
364 Furthermore, mutations that abolish ATP hydrolysis (e.g., K1250A and E1371S) dramatically prolong the open state (Gunderson and Kopito, 1995; Zeltwanger et al., 1999; Powe et al., 2002; Vergani et al., 2003). Login to comment
368 An equivalent observation is also made in the current report for K464A/E1371S mutants. Login to comment
389 Indeed, the hydrolysis-deficient mutant, e.g., E1371S-CFTR, can assume a locked-open state for minutes, whereas WT channels only open for hundreds of milliseconds. Login to comment
394 In the current report, we show that the locked-open time of the E1371S mutant can be significantly shortened by all three maneuvers. Login to comment
397 Furthermore, from the energetic point of view, the absence of ligands at the dimer interface may also explain the short-lived openings observed for ⌬R/ E1371S-CFTR in the absence of ATP. Login to comment
398 Unsettled Issues One of the unsettled issues is the mechanism of short-lived openings of ⌬R/E1371S-CFTR that appear frequently in the presence of micromolar ATP. Login to comment
418 Single-channel kinetic analysis of the ⌬R/E1371S-CFTR mutant also revealed two ATP-dependent closed states (Fig. 6, A and B). Login to comment
429 A difference in closing transitions between WT and hydrolysis-deficient mutant CFTR may also explain the conundrum that ADP exerts a much larger effect on the locked-open time of ⌬R/E1371S channels than on the open time of the ⌬R channels. Login to comment
432 On the other hand, in E1371S-CFTR whose hydrolysis is abolished, thermoen- ergy is used for channel closing. Login to comment

[hide] High affinity ATP/ADP analogues as new tools for s... J Physiol. 2005 Dec 1;569(Pt 2):447-57. Epub 2005 Oct 13. Zhou Z, Wang X, Li M, Sohma Y, Zou X, Hwang TC
High affinity ATP/ADP analogues as new tools for studying CFTR gating.
J Physiol. 2005 Dec 1;569(Pt 2):447-57. Epub 2005 Oct 13., 2005-12-01 [PMID:16223764]

Abstract [show]
Previous studies using non-hydrolysable ATP analogues and hydrolysis-deficient cystic fibrosis transmembrane conductance regulator (CFTR) mutants have indicated that ATP hydrolysis precedes channel closing. Our recent data suggest that ATP binding is also important in modulating the closing rate. This latter hypothesis predicts that ATP analogues with higher binding affinities should stabilize the open state more than ATP. Here we explore the possibility of using N6-modified ATP/ADP analogues as high-affinity ligands for CFTR gating, since these analogues have been shown to be more potent than native ATP/ADP in other ATP-binding proteins. Among the three N6-modified ATP analogues tested, N6-(2-phenylethyl)-ATP (P-ATP) was the most potent, with a K(1/2) of 1.6 +/- 0.4 microm (>50-fold more potent than ATP). The maximal open probability (P(o)) in the presence of P-ATP was approximately 30% higher than that of ATP, indicating that P-ATP also has a higher efficacy than ATP. Single-channel kinetic analysis showed that as [P-ATP] was increased, the opening rate increased, whereas the closing rate decreased. The fact that these two kinetic parameters have different sensitivities to changes of [P-ATP] suggests an involvement of two different ATP-binding sites, a high-affinity site modulating channel closing and a low affinity site controlling channel opening. The effect of P-ATP on the stability of open states was more evident when ATP hydrolysis was abolished, either by mutating the nucleotide-binding domain 2 (NBD2) Walker B glutamate (i.e. E1371) or by using the non-hydrolysable ATP analogue AMP-PNP. Similar strategies to develop nucleotide analogues with a modified adenine ring could be valuable for future studies of CFTR gating.

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20 By examining macroscopic and microscopic kinetics of a hydrolysis-deficient mutant CFTR (i.e. E1371S), we demonstrate an [ATP]-dependent distribution of the open time constants, indicating that ATP binding can affect the life time of the open state (Bompadre et al. 2005a,b). Login to comment
60 For E1371S-CFTR current relaxation experiments (Fig. 7A), initially channels were activated by PKA plus P-ATP in some patches, while by PKA plus ATP in others. Login to comment
63 Since we did not find time-dependent changes in E1371S-CFTR experiments or in WT-CFTR experiments, data from each set of experiments were pooled for statistical analysis. Login to comment
71 Point mutation E1371S was introduced into the pcDNA3.1 wild-type CFTR by QuikChange XL method (Stratagene, La Jolla, CA, USA). Login to comment
164 Wefurtherdifferentiatedbetweenthesetwopossibilities by using the hydrolysis-deficient mutant CFTR, E1371S (Vergani et al. 2003; Bompadre et al. 2005b). Login to comment
168 Interestingly, macroscopic E1371S-CFTR channel currents can be activated by 50 µm P-ATP and PKA, indicating that P-ATP not only can support ATP-dependent gating, but also can beusedasasubstrateforPKA-dependentphosphorylation of the R domain. Login to comment
171 These results suggest that P-ATP stabilizes the locked open state of E1371S-CFTR due to tight binding. Login to comment
189 Effect of P-ATP on CFTR locked open state A, macroscopic E1371S-CFTR current relaxations upon removal of 50 µM P-ATP or 1 mM ATP. Login to comment
191 B, mean data of the current relaxation experiments of E1371S-CFTR. Login to comment
192 The current relaxation time constants (τrelaxation) for E1371S-CFTR channels are 297.6 ± 34.0 s (n = 5) upon the removal of 50 µM P-ATP, and 118.8 ± 9.4 s (n = 5) upon the removal of 1 mM ATP. Login to comment
224 In addition, K464A mutation decreases the locked open time of hydrolysis-deficient mutants K464A/K1250A and K464A/E1371S (Powe et al. 2002; Vergani et al. 2003; Bompadre et al. 2005b), supporting the idea that the strength of ligand binding at the NBD1 site affects the stability of the open state. Login to comment

[hide] The role of cystic fibrosis transmembrane conducta... J Physiol. 2006 Apr 15;572(Pt 2):347-58. Epub 2006 Feb 16. Cui L, Aleksandrov L, Hou YX, Gentzsch M, Chen JH, Riordan JR, Aleksandrov AA
The role of cystic fibrosis transmembrane conductance regulator phenylalanine 508 side chain in ion channel gating.
J Physiol. 2006 Apr 15;572(Pt 2):347-58. Epub 2006 Feb 16., 2006-04-15 [PMID:16484308]

Abstract [show]
Cystic fibrosis transmembrane conductance regulator (CFTR) is an ion channel employing the ABC transporter structural motif. Deletion of a single residue (Phe508) in the first nucleotide-binding domain (NBD1), which occurs in most patients with cystic fibrosis, impairs both maturation and function of the protein. However, substitution of the Phe508 with small uncharged amino acids, including cysteine, is permissive for maturation. To explore the possible role of the phenylalanine aromatic side chain in channel gating we introduced a cysteine at this position in cysless CFTR, enabling its selective chemical modification by sulfhydryl reagents. Both cysless and wild-type CFTR ion channels have identical mean open times when activated by different nucleotide ligands. Moreover, both channels could be locked in an open state by introducing an ATPase inhibiting mutation (E1371S). However, the introduction of a single cysteine (F508C) prevented the cysless E1371S channel from maintaining the permanently open state, allowing closing to occur. Chemical modification of cysless E1371S/F508C by sulfhydryl reagents was used to probe the role of the side chain in ion channel function. Specifically, benzyl-methanethiosulphonate modification of this variant restored the gating behaviour to that of cysless E1371S containing the wild-type phenylalanine at position 508. This provides the first direct evidence that a specific interaction of the Phe508 aromatic side chain plays a role in determining the residency time in the closed state. Thus, despite the fact that this aromatic side chain is not essential for CFTR folding, it is important in the ion channel function.

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5 Moreover, both channels could be locked in an open state by introducing an ATPase inhibiting mutation (E1371S). Login to comment
6 However, the introduction of a single cysteine (F508C) prevented the cysless E1371S channel from maintaining the permanently open state, allowing closing to occur. Login to comment
7 Chemical modification of cysless E1371S/F508C by sulfhydryl reagents was used to probe the role of the side chain in ion channel function. Login to comment
8 Specifically, benzyl-methanethiosulphonate modification of this variant restored the gating behaviour to that of cysless E1371S containing the wild-type phenylalanine at position 508. Login to comment
141 However, it was possible to circumvent this limitation by employing a variant in which the glutamate residue adjacent to the Walker B aspartate in NBD2 was mutated (E1371S). Login to comment
142 The effect of E1371S substitution on the wild-type CFTR ion channel function is shown in Fig. 6 (upper panel, Po = 0.97 ± 0.02, n = 5) and is in a good agreement with published data (Vergani et al. 2005). Login to comment
144 While cysless E1371S was not completely locked open, its open probability was increased approximately sevenfold relative to the cysless wild-type protein (compare Fig. 4 upper panel, and Fig. 6 middle panel). Login to comment
145 The fact that the essentially non-hydrolytic cysless E1371S channel was able to open and close in a robust mannerisofinterestmechanistically.However,ofpractical importance for the utility of the cysless protein to study the role of the Phe508 residue in gating was the fact that the E1371S substitution also increased the activity of cysless F508C (Fig. 6, lower panel, Po = 0.25 ± 0.03, n = 4). Login to comment
146 Moreover, it was possible to differentiate better between cysless E1371S and cysless E1371S/F508C by using 8BrATP instead of ATP as a ligand (Fig. 7, first panel, Po = 0.97 ± 0.02, n = 4 and second panel (Fig. 7, second panel, Po = 0.71 ± 0.03, n = 4). Login to comment
150 To test this assumption we used the cysless E1371S construct that is locked open with 8BrATP as a reference point (Fig. 7, upper panel). Login to comment
152 The return of an aromatic ring to position 508 by the chemical modification with 50 μm MTSBn restored the locked open state typical of cysless E1371S (Fig. 7,thirdpanel, Po = 0.97 ± 0.02,n = 3).Thisstrongly supports the notion that the aromatic side chain of Phe508 plays a role in channel gating. Login to comment
154 This was found to be the case as the positively charged MTSET not only did not lock the cysless E1371S/F508C channel open but completely ablated gating (Fig. 7, lower trace, Po < 0.01, n = 3). Login to comment
180 Improvement of the cysless CFTR ion channel activity by introducing E1371S in NBD2 The upper panel shows typical single-channel activity of the E1371S mutant at 30◦C. Login to comment
181 The cysless E1371S single-channel record is shown in the middle panel. The influence of the further introduction of F508C in the cysless E1371S background on the ion channel gating is shown in the lower panel. The mean values of Po ± S.E.M. and number of experiments are shown in the text. All records were done at 30◦C and 2 mM ATP. Login to comment
196 Aromatic side chain at position 508 is required to lock the channel open Cysless E1371S ion channel gated by 2 mM 8BrATP is shown in the upper panel. Login to comment
197 Effect of the introduction of F508C in the cysless E1371S background on the ion channel gating is shown in the second panel. The result of the chemical modification of the cysless E1371S/F508C channel by MTSBn at the cis side on the ion channel gating is shown by the arrow in the third panel. The effect of positively charged 2-trimethylammonioethylmethanethiosulphonate (MTSET) on the cysless E1371S/F508C ion channel function is shown by the arrow in the lower panel. The values of Po before and after chemical modification are shown above the traces. Login to comment
201 This could be confirmed using a site II ATPase-inhibited mutant (E1371S) which is locked open in both the wild-type and cysless backgrounds, while the F508C version of cysless E1371S was unable to maintain the locked open state. Login to comment
207 The ability of F508C version of cysless E1371S to maintain the locked open state was fully restored on modificaton of Cys508 with MTSBn. Login to comment

[hide] The two ATP binding sites of cystic fibrosis trans... J Gen Physiol. 2006 Oct;128(4):413-22. Epub 2006 Sep 11. Zhou Z, Wang X, Liu HY, Zou X, Li M, Hwang TC
The two ATP binding sites of cystic fibrosis transmembrane conductance regulator (CFTR) play distinct roles in gating kinetics and energetics.
J Gen Physiol. 2006 Oct;128(4):413-22. Epub 2006 Sep 11., [PMID:16966475]

Abstract [show]
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.

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18 It is generally agreed that ATP hydrolysis at NBD2 precedes channel closing since mutations (e.g., K1250A and E1371S) that abolish ATP hydrolysis at the NBD2 site drastically prolong the open time (Carson et al., 1995; Gunderson and Kopito, 1995; Zeltwanger et al., 1999; Vergani et al., 2003; Bompadre et al., 2005b). Login to comment
36 The shortening of the open time by the W401G mutation is also seen with the hydrolysis-deficient mutant background (i.e., E1371S), suggesting that the effect of W401G mutation is not through a perturbation of ATP hydrolysis. Login to comment
148 To examine the potential effect of mutation in NBD1 on ATP hydrolysis at NBD2, we introduced the W401G mutation into the E1371S background, a mutant CFTR whose ATP hydrolysis is abolished (Moody et al., 2002; Tombline et al., 2004; Vergani et al., 2005). Login to comment
150 Since the gating cycle of channels in the E1371S background is extremely long, it is technically difficult to do microscopic single-channel kinetic analysis. Login to comment
158 (B) Representative current relaxation traces upon withdrawal of 1 mM ATP plus PKA for E1371S, W401G/E1371S, triple/ E1371S, Y1219G/E1371S, and W401G/Y1219G/E1371S. Login to comment
161 ** indicates P < 0.01 and *** indicates P < 0.001 (compared with E1371S). Login to comment
164 Fig. 3 B shows experiments using current relaxation analysis to estimate the open time constants for E1371S and W401G/E1371S. Login to comment
165 Our results show that the relaxation time constant for W401G/E1371S (59.1 ± 4.6 s, n = 8) is shortened by ‫,%05ف‬ compared with that of E1371S (111.7 ± 12.1 s, n = 15) (Fig. 3 C), suggesting that the shorter open time of W401G is not secondary to an altered ATP hydrolysis rate. Login to comment
166 In contrast, although the Y1219G mutation greatly decreases the apparent affinity of ATP (Fig. 2 C), introducing this mutation into the E1371S background has little effect on the relaxation time constant (107.6 ± 12.4 s, n = 7) (Fig. 3, B and C). Login to comment
167 In addition, W401G/Y1219G/E1371S has a relaxation time constant of 49.0 ± 5.3 s (Fig. 3, B and C), which is similar to that of W401G/E1371S, indicating that W401, but not Y1219, plays a dominant role in modulating the open time. Login to comment
178 We converted all three aromatic amino acids, including W401, F409, and F430 to glycine in the E1371S background and examined current relaxations of the W401G/F409G/F430G/E1371S (or triple/E1371S). Login to comment
179 Compared with the current relaxation of W401G/ E1371S (Fig. 3 B), the triple/E1371S mutation further shortens the time course of current decay. Login to comment
186 Fig. 4 B shows experiments examining current relaxations upon removal of ATP or P-ATP for E1371S, W401G/E1371S, Y1219G/ E1371S, and triple/E1371S. Login to comment
187 As demonstrated previously (Zhou et al., 2005), the relaxation time course upon washout of P-ATP for E1371S is approximately twofold longer than that with ATP. Login to comment
188 P-ATP also increases the relaxation time constant of Y1219G/E1371S by approximately twofold. Login to comment
189 However, this prolongation effect of P-ATP is significantly larger for W401G/E1371S (2.7-fold) and triple/E1371S (greater than fourfold; Fig. 4 C). Login to comment
194 Studies using different mutations that perturb ATP hydrolysis (e.g., K1250A, E1371S) indicate that ATP hydrolysis drives channel closure. Login to comment
217 (B) Representative current relaxation traces of E1371S, W401G/E1371S, triple/ E1371S, and Y1219G/E1371S after withdrawal of 1 mM ATP plus PKA or 50 μM P-ATP plus PKA. Horizontal scale bars represent 200 s. Login to comment
218 (C) The ratio of the relaxation time constant upon withdrawal of 50 μM P-ATP plus PKA to that upon withdrawal of 1 mM ATP plus PKA from the same patch was calculated for E1371S and various mutants in the E1371S background. Login to comment
220 *** indicates P < 0.001 and **** indicates P < 0.0001 (compared with E1371S). Login to comment

[hide] Cystic fibrosis transmembrane conductance regulato... Sheng Li Xue Bao. 2007 Aug 25;59(4):431-42. Bompadre SG, Hwang TC
Cystic fibrosis transmembrane conductance regulator: a chloride channel gated by ATP binding and hydrolysis.
Sheng Li Xue Bao. 2007 Aug 25;59(4):431-42., 2007-08-25 [PMID:17700963]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel that belongs to the ATP-binding cassette (ABC) transporter superfamily. Defective function of CFTR is responsible for cystic fibrosis (CF), the most common lethal autosomal recessive disorder in Caucasian populations. The disease is manifested in defective chloride transport across the epithelial cells in various tissues. To date, more than 1400 different mutations have been identified as CF-associated. CFTR is regulated by phosphorylation in its regulatory (R) domain, and gated by ATP binding and hydrolysis at its two nucleotide-binding domains (NBD1 and NBD2). Recent studies reveal that the NBDs of CFTR may dimerize as observed in other ABC proteins. Upon dimerization of CFTR's two NBDs, in a head-to-tail configuration, the two ATP-binding pockets (ABP1 and ABP2) are formed by the canonical Walker A and B motifs from one NBD and the signature sequence from the partner NBD. Mutations of the amino acids that interact with ATP reveal that the two ABPs play distinct roles in controlling ATP-dependent gating of CFTR. It was proposed that binding of ATP to the ABP2, which is formed by the Walker A and B in NBD2 and the signature sequence in NBD1, is critical for catalyzing channel opening. While binding of ATP to the ABP1 alone may not increase the opening rate, it does contribute to the stabilization of the open channel conformation. Several disease-associated mutations of the CFTR channel are characterized by gating defects. Understanding how CFTR's two NBDs work together to gate the channel could provide considerable mechanistic information for future pharmacological studies, which could pave the way for tailored drug design for therapeutical interventions in CF.

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90 This shortening of the open time by ADP was more evident in the hydrolysis-deficient mutants E1371S and D1370N[38] . Login to comment
194 B: Representative current relaxation traces for E1371S, W401G/E1371S and W401G/ F409G/F430G/E1371S (or triple/E1371S). Login to comment
201 Interestingly, P-ATP also prolongs the open time of wild-type CFTR and a hydrolysis-deficient mutant, E1371S-CFTR, indicating that the effect of P-ATP on the open time is not due to a perturbation of ATP hydrolysis. Login to comment

[hide] CLC-0 and CFTR: chloride channels evolved from tra... Physiol Rev. 2008 Apr;88(2):351-87. Chen TY, Hwang TC
CLC-0 and CFTR: chloride channels evolved from transporters.
Physiol Rev. 2008 Apr;88(2):351-87., [PMID:18391167]

Abstract [show]
CLC-0 and cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channels play important roles in Cl(-) transport across cell membranes. These two proteins belong to, respectively, the CLC and ABC transport protein families whose members encompass both ion channels and transporters. Defective function of members in these two protein families causes various hereditary human diseases. Ion channels and transporters were traditionally viewed as distinct entities in membrane transport physiology, but recent discoveries have blurred the line between these two classes of membrane transport proteins. CLC-0 and CFTR can be considered operationally as ligand-gated channels, though binding of the activating ligands appears to be coupled to an irreversible gating cycle driven by an input of free energy. High-resolution crystallographic structures of bacterial CLC proteins and ABC transporters have led us to a better understanding of the gating properties for CLC and CFTR Cl(-) channels. Furthermore, the joined force between structural and functional studies of these two protein families has offered a unique opportunity to peek into the evolutionary link between ion channels and transporters. A promising byproduct of this exercise is a deeper mechanistic insight into how different transport proteins work at a fundamental level.

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638 In addition, even the hydrolysis-deficient mutant E1371S-CFTR seldom assumes a stable open state in the presence of AMP-PNP alone (Cho and Hwang, unpublished data). Login to comment
684 However, since CFTR mutants whose ATP hydrolysis is abolished (e.g., K1250A, E1371S) (251, 302), once opened by ATP, can remain open for minutes (34, 112, 323, 324, 350; cf. Refs. 42, 251), it is now generally accepted that hydrolysis of ATP at ABP2 closes the channel (97, 358). Login to comment
769 Since the spontaneous openings for the hydrolysis-deficient mutant E1371S are short-lived events (Cho and Hwang, unpublished observations), a stable NBD dimer formation requires the action of ATP presumably at ABP2. Login to comment
810 P-ATP also increases the locked open time of the hydrolysis-deficient mutant E1371S-CFTR, suggesting that this effect of P-ATP is through a tighter binding. Login to comment
820 (360) examined the effect of mutations and a combination of mutations at W401, F409, and F430 residues on the open state (or NBD dimer) stability under the E1371S background. Login to comment
821 The locked open time of E1371S-CFTR was shortened in a graded manner as the number of altered aromatic amino acids was increased. Login to comment
824 (360) were able to show that P-ATP binds to ABP1 to prolong the open time of E1371S-CFTR. Login to comment

[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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10 The idea that ATP hydrolysis precedes channel closing is further supported by the observations that CFTR mutations whose ATPase activity is abrogated (e.g., K1250A and E1371S) (Ramjeesingh et al., 1999) can remain open for minutes (Gunderson and Kopito, 1995; Zeltwanger et al., 1999; Vergani et al., 2003; Bompadre et al., 2005b), and that channel closure is markedly delayed in the presence of nonhydrolyzable ATP analogue AMP-PNP (Hwang et al., 1994), or of inorganic phosphate analogue orthovanadate, which presumably forms a stable complex with the hydrolytic product ADP (Baukrowitz et al., 1994). Login to comment
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
106 The observation that MgPPi induces shorter opening events than ATP in E1371S channels is shown in Fig. S2. Login to comment
375 This result thus suggests that it is MgPPi dissociation from NBD2, but not ATP from NBD1, that is associated with E1371S-CFTR, a mutant whose ATPase activity is abolished (Moody et al., 2002; Tombline et al., 2004; Vergani et al., 2005; Zhou et al., 2006; Stratford et al., 2007). Login to comment
376 We reasoned that if MgPPi elicits longer open bursts due to a slower hydrolysis rate, E1371S mutation should further prolong the burst duration induced by MgPPi. Login to comment
377 However, as can be seen in Fig. S2, although the E1371S mutation dramatically increases the relaxation time constant of the ATP-gated channels (␶ = 126.1 ± 24.2 s; n = 5), the lifetime of MgPPi-induced openings for E1371S channels (1.65 s, ensemble current relaxation from five data) is very close to that of WT-CFTR (~1.5 s in Fig. 1 B). Login to comment
378 This result suggests that both MgPPi-opened WT-CFTR and E1371S-CFTR channels close through a nonhydrolytic pathway. Login to comment
380 Finally, the data also indicate that MgPPi is a poor ligand for CFTR channels because, unlike ATP, it fails to induce a stable open state with E1371S-CFTR. Login to comment

[hide] Strict coupling between CFTR's catalytic cycle and... Proc Natl Acad Sci U S A. 2010 Jan 19;107(3):1241-6. Epub 2009 Dec 4. Csanady L, Vergani P, Gadsby DC
Strict coupling between CFTR's catalytic cycle and gating of its Cl- ion pore revealed by distributions of open channel burst durations.
Proc Natl Acad Sci U S A. 2010 Jan 19;107(3):1241-6. Epub 2009 Dec 4., 2010-01-19 [PMID:19966305]

Abstract [show]
CFTR, the ABC protein defective in cystic fibrosis, functions as an anion channel. Once phosphorylated by protein kinase A, a CFTR channel is opened and closed by events at its two cytosolic nucleotide binding domains (NBDs). Formation of a head-to-tail NBD1/NBD2 heterodimer, by ATP binding in two interfacial composite sites between conserved Walker A and B motifs of one NBD and the ABC-specific signature sequence of the other, has been proposed to trigger channel opening. ATP hydrolysis at the only catalytically competent interfacial site is suggested to then destabilize the NBD dimer and prompt channel closure. But this gating mechanism, and how tightly CFTR channel opening and closing are coupled to its catalytic cycle, remains controversial. Here we determine the distributions of open burst durations of individual CFTR channels, and use maximum likelihood to evaluate fits to equilibrium and nonequilibrium mechanisms and estimate the rate constants that govern channel closure. We examine partially and fully phosphorylated wild-type CFTR channels, and two mutant CFTR channels, each bearing a deleterious mutation in one or other composite ATP binding site. We show that the wild-type CFTR channel gating cycle is essentially irreversible and tightly coupled to the ATPase cycle, and that this coupling is completely destroyed by the NBD2 Walker B mutation D1370N but only partially disrupted by the NBD1 Walker A mutation K464A.

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78 As a three-parameter fit of scheme 2 to the data in Fig. 1B (and also Fig. 3) did not provide a reliable estimate of this small rate (SI Text), to estimate k-1 we measured the macroscopic closing rates of prephosphorylated K1250A, K1250R, and E1371S mutant channels (e.g., Fig. 2A) upon sudden removal of ATP. Login to comment
79 These rates, obtained as the reciprocals of the time constants of fitted single exponentials (e.g., Fig. 2A, blue line), were 0.044 ± 0.004 s-1 (n = 9) for K1250A (Fig. 2C, blue bar), 0.22 ± 0.01 s-1 (n = 17) for K1250R, and 0.036 ± 0.002 s-1 (n = 16) for E1371S. Login to comment

[hide] The H-loop in the second nucleotide-binding domain... Cell Physiol Biochem. 2010;25(2-3):169-80. Epub 2010 Jan 12. Kloch M, Milewski M, Nurowska E, Dworakowska B, Cutting GR, Dolowy K
The H-loop in the second nucleotide-binding domain of the cystic fibrosis transmembrane conductance regulator is required for efficient chloride channel closing.
Cell Physiol Biochem. 2010;25(2-3):169-80. Epub 2010 Jan 12., [PMID:20110677]

Abstract [show]
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.

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195 Substitution of this conserved glutamate with either serine (E1371S) or glutamine (E1371Q) produces a channel that displays prolonged open times [3, 16, 21, 59]. Login to comment

[hide] Involvement of F1296 and N1303 of CFTR in induced-... J Gen Physiol. 2010 Oct;136(4):407-23. Szollosi A, Vergani P, Csanady L
Involvement of F1296 and N1303 of CFTR in induced-fit conformational change in response to ATP binding at NBD2.
J Gen Physiol. 2010 Oct;136(4):407-23., [PMID:20876359]

Abstract [show]
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.

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242 Although for WT CFTR and for the nonhydrolytic mutant D1370N these two parameters are in rough agreement (Csanády et al., 2010), such comparisons have not yet been done for several other NBD2mutantsdefectiveinATPhydrolysis(e.g.,K1250R, K1250A, E1371S, and E1371Q). Login to comment

[hide] On the mechanism of CFTR inhibition by a thiazolid... J Gen Physiol. 2010 Dec;136(6):659-71. Epub 2010 Nov 15. Kopeikin Z, Sohma Y, Li M, Hwang TC
On the mechanism of CFTR inhibition by a thiazolidinone derivative.
J Gen Physiol. 2010 Dec;136(6):659-71. Epub 2010 Nov 15., [PMID:21078867]

Abstract [show]
The effects of a thiazolidinone derivative, 3-[(3-trifluoromethyl)phenyl]-5-[(4-carboxyphenyl)methylene]-2-thioxo-4-th iazolidinone (or CFTRinh-172), on cystic fibrosis transmembrane conductance regulator (CFTR) gating were studied in excised inside-out membrane patches from Chinese hamster ovary cells transiently expressing wild-type and mutant CFTR. We found that the application of CFTRinh-172 results in an increase of the mean closed time and a decrease of the mean open time of the channel. A hyperbolic relationship between the closing rate and [CFTRinh-172] suggests that CFTRinh-172 does not act as a simple pore blocker. Interestingly, the potency of inhibition increases as the open time of the channel is increased with an IC50 in the low nanomolar range for CFTR channels locked in an open state for tens of seconds. Our studies also provide evidence that CFTRinh-172 can bind to both the open state and the closed state. However, at least one additional step, presumably reflecting inhibitor-induced conformational changes, is required to shut down the conductance after the binding of the inhibitor to the channel. Using the hydrolysis-deficient mutant E1371S as a tool as the closing rate of this mutant is dramatically decreased, we found that CFTRinh-172-dependent inhibition of CFTR channel gating, in two aspects, mimics the inactivation of voltage-dependent cation channels. First, similar to the recovery from inactivation in voltage-gated channels, once CFTR is inhibited by CFTRinh-172, reopening of the channel can be seen upon removal of the inhibitor in the absence of adenosine triphosphate (ATP). Second, ATP induced a biphasic current response on inhibitor-bound closed channels as if the ATP-opened channels "inactivate" despite a continuous presence of ATP. A simplified six-state kinetic scheme can well describe our data, at least qualitatively. Several possible structural mechanisms for the effects of CFTRinh-172 will be discussed.

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23 Using the hydrolysis-deficient mutant E1371S as a tool as the closing rate of this mutant is dramatically decreased, we found that CFTRinh-172-dependent inhibition of CFTR channel gating, in two aspects, mimics the inactivation of voltage-dependent cation channels. Login to comment
139 A similar observation was made with E1371S-CFTR channels locked open with ATP (Fig. 6 B). Login to comment
148 A similar observation was made with E1371S-CFTR, a hydrolysis-deficient mutant with an open time of 100 s (Fig. 5 B). Login to comment
149 Fig. 5 C shows the dose-response relationships of CFTRinh-172 for WT-CFTR locked open with ATP and PPi (green circles), or E1371S-CFTR locked open with ATP (blue squares). Login to comment
171 To further verify this slow recovery, we quantified this recovery phase using a phosphorylation-independent construct characterized previously (i.e., E1371S mutation in a construct without the R domain, or E1371S/R; see Bompadre et al., 2005). Login to comment
172 Again, the time constant of the current recovery for E1371S/R is 6 min (Fig. 6, C and D). Login to comment
173 Because the activity of E1371S/R-CFTR is independent of phosphorylation, in the following sections we will use Figure 6. Slow recovery from inhibition for the locked-open CFTR. Login to comment
175 (D) The diagram shows the average rate of recovery from inhibition by CFTRinh-172 for WT and E1371S CFTR locked in an open state. Login to comment
177 The mean rate constants of recovery are: 0.024 ± 0.003 s1 (n = 17) for ATP-gated WT-CFTR; 0.0042 ± 0.0011 s1 (n = 10) for WT-CFTR locked in an open state with ATP plus PPi; 0.0026 ± 0.0004 s1 (n = 18) for E1371S; and 0.0039 ± 0.0005 s1 (n = 18) for E1371S/R. Figure 5. Inhibition of locked-open CFTR by CFTRinh-172. Login to comment
178 CFTR channels were locked into an open state by using ATP plus PPi for WT-CFTR (A) or ATP for E1371S-CFTR (B). Login to comment
182 (C) Dose-response relationships of CFTRinh-172 for WT-CFTR locked open by PPi (green symbols) and E1371S-CFTR locked open with ATP (blue symbols). Login to comment
196 Again, E1371S-CFTR allows us to address this question. Login to comment
211 Once E1371S-CFTR channels were activated with PKA and ATP, the channels were subsequently inhibited by applying 5 µM CFTRinh-172. Login to comment
216 Some of those brief openings (Fig. 7, blue inset) probably represent ATP-independent events observed normally in the absence of ATP for E1371S-CFTR (Bompadre et al., 2005). Login to comment
218 Based on the idea that the open state represents an NBD dimer (Vergani et al., 2005; Zhou et al., 2006; Tsai et al., 2009), we conclude that CFTRinh-172 does not promote the Figure 7. Reopening of inhibited E1371S-CFTR channels upon removing CFTRinh-172. Login to comment
219 A continuous recording of E1371S-CFTR, a hydrolysis-deficient mutant with a prolonged open time, shows reopening of the inhibited channels in the absence of ATP upon removal of CFTRinh-172. Login to comment
245 However, unlike inhibitor-free E1371S channels that are locked open by ATP, the current from inhibitor-bound channels, first elicited by ATP, dropped by itself within a few seconds, as the inhibitor-bound open channels are now inactivated. Login to comment
246 Again, this observation is some- Figure 8. The binding and inhibition of CFTRinh-172 in the closed state of E1371S/R-CFTR channels. Login to comment
262 E1371S/R CFTR channels were opened with 1 mM ATP and then inhibited with 2 µM CFTRinh-172. Login to comment
284 (A) A continuous current trace of E1371S/R-CFTR channels showing that some of the inhibited channels recover from inhibition without reopening. Login to comment
286 (B) Faster recovery of inhibited E1371S/R-CFTR channels in the absence of ATP. Login to comment
346 In addition to the aforementioned differences between WT and E1371S channels regarding the mechanism of recovery from inactivation, Scheme 3 also predicts differences in inactivation per se. Login to comment
347 For E1371S-CFTR, because the closing rate is much slower than the rate of inactivation (i.e., Oinh→Cinh << Oinh→Iinh), the application of ATP to channels preexposed to CFTRinh-172 (Fig. 8) will generate a biphasic current response as a result of Cinh→Oinh→Iinh. 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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102 We introduced the E1371S mutation, which is known to demolish ATPase activity in ABC proteins including CFTR (Aleksandrov et al. 2000; Vergani et al. 2003; Bompadre et al. 2005), into WT and F508 backgrounds. Login to comment
103 For E1371S channels, the relaxation time constant of the current decay after ATP washout is ~110 s (Fig. 2A and C, Zhou et al. 2006), whereas that of F508/E1371S channels is only 32.45 ± 4.07 s (n = 4) (Fig. 2B and C). Login to comment
104 Although it is unclear why the F508 mutation shows less effect on the stability of NBD dimer under the E1371S background, the shortening of the locked-open time seen in F508/E1371S channels is consistent with the idea that the culprit is a destabilization of the NBD dimer rather than a lower affinity or efficacy of PPi. Login to comment
118 Comparison of the locked-open time of E1371S- and F508/E1371S-CFTR Representative traces of non-hydrolytic E1371S-CFTR (A) and F508/E1371S-CFTR (B) in the presence of 1 mM ATP. Login to comment
119 C, summary of the locked-open times for E1371S-CFTR (n = 15) and F508/E1371S-CFTR (n = 4) (P < 0.01). 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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44 For example, the drastic effect of nonhydrolyzable ATP analogues or mutations (e.g., E1371S or K1250A) that abolish ATP hydrolysis on the open time supports the notion that ATP hydrolysis is coupled to channel Figure 1. An updated model illustrating the relationship between an opening/closing cycle of the gate and ATP consumption in CFTR` s NBDs. Login to comment
80 Consistent with this idea, ATP only induces C→O1→C transitions in E1371S/R352C-CFTR, a hydrolysis-deficient mutant. Login to comment
114 To further test our hypothesis that the dominant O1→O2 transition versus O2→O1 transition is the result of ATP hydrolysis, we engineered the E1371S mutation into R352C-CFTR to abolish ATP hydrolysis (Vergani et al., 2003; Bompadre et al., 2005b) and recorded ATP-dependent opening events. Login to comment
116 This lack of transitions to the O2 state for ATP-induced opening bursts is not caused by the mutation, E1371S, eliminating the O2 state, because in the absence of ATP, the channel opens predominantly into bursts of a larger conductance level corresponding to that of the O2 state (Fig. 3 C). Login to comment
146 (B and C) Representative traces and amplitude histograms for R352C/E1371S-CFTR in the presence (B) or absence (C) of 2.75 mM ATP. Login to comment
257 Macroscopic current traces of E1371S-CFTR (A), R352C/E1371S-CFTR (B), T1246N/E1371S-CFTR (C), and R352C/T1246N/E1371S-CFTR (D). Login to comment
259 The current relaxation was fitted with a single-exponential function resulting in the relaxation time constant for each mutant: 65.6 ± 10.1 s (n = 8) for E1371S-CFTR, 4.9 ± 0.8 s (n = 12) for R352C/ E1371S-CFTR, 7.8 ± 1.6 s (n = 7) for T1246N/E1371S-CFTR, and 2.27 ± 0.27 s (n = 6) for R352C/T1246N/E1371S-CFTR. Login to comment
261 *, P < 0.05 compared with E1371S; #, P < 0.05 between two designated data. Login to comment
293 Here, in Fig. 7, we show that both R352C and T1246N mutations significantly shorten the locked-open time of the hydrolytic-deficient E1371S-CFTR (Fig. 7). Login to comment
43 For example, the drastic effect of nonhydrolyzable ATP analogues or mutations (e.g., E1371S or K1250A) that abolish ATP hydrolysis on the open time supports the notion that ATP hydrolysis is coupled to channel Figure 1.ߓ An updated model illustrating the relationship between an opening/closing cycle of the gate and ATP consumption in CFTR` s NBDs. Login to comment
79 Consistent with this idea, ATP only induces C࢐O1࢐C transitions in E1371S/R352C-CFTR, a hydrolysis-deficient mutant. Login to comment
113 To further test our hypothesis that the dominant O1࢐O2 transition versus O2࢐O1 transition is the result of ATP hydrolysis, we engineered the E1371S mutation into R352C-CFTR to abolish ATP hydrolysis (Vergani et al., 2003; Bompadre et al., 2005b) and recorded ATP-dependent opening events. Login to comment
115 This lack of transitions to the O2 state for ATP-induced opening bursts is not caused by the mutation, E1371S, eliminating the O2 state, because in the absence of ATP, the channel opens predominantly into bursts of a larger conductance level corresponding to that of the O2 state (Fig. 3 C). Login to comment
145 (B and C) Representative traces and amplitude histograms for R352C/E1371S-CFTR in the presence (B) or absence (C) of 2.75 mM ATP. Login to comment
263 *, P < 0.05 compared with E1371S; #, P < 0.05 between two designated data. Login to comment
295 Here, in Fig. 7, we show that both R352C and T1246N mutations significantly shorten the locked-open time of the hydrolytic-deficient E1371S-CFTR (Fig. 7). Login to comment

[hide] CFTR: An ion channel with a transporter-type energ... J Gen Physiol. 2012 Oct;140(4):343-5. Epub 2012 Sep 10. Tsai MF
CFTR: An ion channel with a transporter-type energy-coupling mechanism.
J Gen Physiol. 2012 Oct;140(4):343-5. Epub 2012 Sep 10., [PMID:22966013]

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50 The authors then took the story one step further by introducing a catalysis-abolishing mutation E1371S into R352C-CFTR; these hydrolysis-deficient channels now open and close reversibly (C→O1→C and C→O2→C), indicating that ATP hydrolysis underlies a unidirectional transition from O1 to O2 (Fig. 1 B). Login to comment
48 The authors then took the story one step further by introducing a catalysis-abolishing mutation E1371S into R352C-CFTR; these hydrolysis-deficient channels now open and close reversibly (C࢐O1࢐C and C࢐O2࢐C), indicating that ATP hydrolysis underlies a unidirectional transition from O1 to O2 (Fig. 1 B). 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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217 These flickering closings have been long thought to be ATP independent, as they can be easily discerned in a complete absence of ATP within an opening burst of hydrolysis-deficient mutants, such as E1371S or K1250A (Carson et al., 1995; Powe et al., 2002; Bompadre et al., 2005b; Vergani et al., 2005). Login to comment

[hide] ATP hydrolysis-dependent asymmetry of the conforma... J Physiol Sci. 2011 Jul;61(4):267-78. doi: 10.1007/s12576-011-0144-0. Epub 2011 Apr 3. Krasilnikov OV, Sabirov RZ, Okada Y
ATP hydrolysis-dependent asymmetry of the conformation of CFTR channel pore.
J Physiol Sci. 2011 Jul;61(4):267-78. doi: 10.1007/s12576-011-0144-0. Epub 2011 Apr 3., [PMID:21461971]

Abstract [show]
Despite substantial efforts, the entire cystic fibrosis transmembrane conductance regulator (CFTR) protein proved to be difficult for structural analysis at high resolution, and little is still known about the actual dimensions of the anion-transporting pathway of CFTR channel. In the present study, we therefore gauged geometrical features of the CFTR Cl(-) channel pore by a nonelectrolyte exclusion technique. Polyethylene glycols with a hydrodynamic radius (R (h)) smaller than 0.95 nm (PEG 300-1,000) added from the intracellular side greatly suppressed the inward unitary anionic conductance, whereas only molecules with R (h) </= 0.62 nm (PEG 200-400) applied extracellularly were able to affect the outward unitary anionic currents. Larger molecules with R (h) = 1.16-1.84 nm (PEG 1,540-3,400) added from either side were completely excluded from the pore and had no significant effect on the single-channel conductance. The cut-off radius of the inner entrance of CFTR channel pore was assessed to be 1.19 +/- 0.02 nm. The outer entrance was narrower with its cut-off radius of 0.70 +/- 0.16 nm and was dilated to 0.93 +/- 0.23 nm when a non-hydrolyzable ATP analog, 5'-adenylylimidodiphosphate (AMP-PNP), was added to the intracellular solution. Thus, it is concluded that the structure of CFTR channel pore is highly asymmetric with a narrower extracellular entrance and that a dilating conformational change of the extracellular entrance is associated with the channel transition to a non-hydrolytic, locked-open state.

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31 This locked-open phenotype is also observed when the ATPase activity of NBD2 is abolished by the mutation of K1250A or E1371S [10, 23, 39]. Login to comment
225 An intriguing question as to whether the mutation at the site essential for ATP hydrolysis in NBD2 (such as K1250A and E1371S) causes a similar conformational change remains for future studies. Login to comment

[hide] A stable ATP binding to the nucleotide binding dom... J Physiol Sci. 2010 Sep;60(5):353-62. doi: 10.1007/s12576-010-0102-2. Epub 2010 Jul 14. Shimizu H, Yu YC, Kono K, Kubota T, Yasui M, Li M, Hwang TC, Sohma Y
A stable ATP binding to the nucleotide binding domain is important for reliable gating cycle in an ABC transporter CFTR.
J Physiol Sci. 2010 Sep;60(5):353-62. doi: 10.1007/s12576-010-0102-2. Epub 2010 Jul 14., [PMID:20628841]

Abstract [show]
Cystic fibrosis transmembrane conductance regulator (CFTR) anion channel, a member of ABC transporter superfamily, gates following ATP-dependent conformational changes of the nucleotide binding domains (NBD). Reflecting the hundreds of milliseconds duration of the channel open state corresponding to the dimerization of two NBDs, macroscopic WT-CFTR currents usually showed a fast, single exponential relaxation upon removal of cytoplasmic ATP. Mutations of tyrosine1219, a residue critical for ATP binding in second NBD (NBD2), induced a significant slow phase in the current relaxation, suggesting that weakening ATP binding affinity at NBD2 increases the probability of the stable open state. The slow phase was effectively diminished by a higher affinity ATP analogue. These data suggest that a stable binding of ATP to NBD2 is required for normal CFTR gating cycle, andthat the instability of ATP binding frequently halts the gating cycle in the open state presumably through a failure of ATP hydrolysis at NBD2.

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16 Contrary to WT-CFTR, CFTR mutants whose ATP hydrolysis at NBD2 is abolished (i.e., E1371S, K1250A), can remain open for minutes [9, 12, 17-20]. Login to comment
106 Since the life time of these long-lasting openings (in tens of seconds) is very similar to that of hydrolysis-deficient CFTR mutants such as K1250A and E1371S [9, 12, 17-20], it seems difficult to explain these events with the conventional theory that ATP hydrolysis closes the channel. Login to comment
118 On the other hand, CFTR channels can open for hundreds of seconds when ATP hydrolysis is abolished by mutations such as E1371S or K1250A [9, 12, 17-20]. Login to comment

[hide] Nonequilibrium gating of CFTR on an equilibrium th... Physiology (Bethesda). 2012 Dec;27(6):351-61. doi: 10.1152/physiol.00026.2012. Jih KY, Hwang TC
Nonequilibrium gating of CFTR on an equilibrium theme.
Physiology (Bethesda). 2012 Dec;27(6):351-61. doi: 10.1152/physiol.00026.2012., [PMID:23223629]

Abstract [show]
Malfunction of cystic fibrosis transmembrane conductance regulator (CFTR), a member of the ABC protein superfamily that functions as an ATP-gated chloride channel, causes the lethal genetic disease, cystic fibrosis. This review focuses on the most recent findings on the gating mechanism of CFTR. Potential clinical relevance and implications to ABC transporter function are also discussed.

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87 Nonetheless, for hydrolysis-deficient mutants such as E1371S- and K1250A-CFTR, ATP is still capable of activating these channels and the open probability (Po) of these hydrolysis-deficient mutants is even higher than that of WT-CFTR, suggesting that, under an equilibrium condition when ATP hydrolysis is absent, CFTR can still function fairly well. Login to comment
161 The idea that the C &#a1; O1 &#a1; O2 &#a1; C preferred transition is driven by ATP hydrolysis is supported not only by the time asymmetry but also by the observation that the O1 &#a1; O2 transition seen in an opening burst is abolished when the hydrolysis-deficient mutation (E1371S) is engineered into the R352C background. Login to comment
170 Thus, in the absence of ATP hydrolysis, e.g., in E1371S-CFTR, the channel works simply because ATP-induced dimerization of NBDs promotes gate opening and the Po is exceedingly high because the channel is trapped in the stable O1 state. 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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19 Here, we found that P-ATP prolongs wild-type (WT) CFTR burst durations by selectively slowing (>2&#d7;) transition O1࢐O2 and decreases the nonhydrolytic closing rate (transition O1࢐C) of CFTR mutants K1250A (&#e07a;4&#d7;) and E1371S (&#e07a;3&#d7;). Login to comment
20 Mutation H1348A also slowed (&#e07a;3&#d7;) the O1࢐O2 transition in the WT background and decreased the nonhydrolytic closing rate of both K1250A (&#e07a;3&#d7;) and E1371S (&#e07a;3&#d7;) background mutants. Login to comment
35 M A T E R I A L S A N D M E T H O D S Molecular biology Human WT CFTR and CFTR segment 433-1480 in the pGEMHE plasmid (Chan et al., 2000) served as templates for mutants H1348A, K1250A, E1371S, K1250A/H1348A, E1371S/H1348A, E1371S/K464A, and 433-1480(K1250A), which were created using the QuikChange kit (Agilent Technologies). Login to comment
57 For the nonhydrolytic mutants (K1250A, E1371S, and double mutants), the decay time courses after nucleotide removal often required a double-exponential function-of the form I(t) = I0(A1exp(&#e032;t/&#e074;1) + (1 &#e032; A1)exp(&#e032;t/&#e074;2))-with two slow time constants for a satisfactory fit (e.g., Fig. 3, B and E), suggesting the presence of two populations of open-channel bursts. Login to comment
66 Fig. S2 shows the effect of the K464A mutation on nonhydrolytic closing rate measured in the E1371S background. Login to comment
107 Thus, the nonhydrolytic closing rate (Fig. 3, C and F, bars; calculated from the fitted relaxation time constants as described in Materials and methods) was similarly affected by both site-1 perturbations, and this was true regardless of whether the K1250A (Fig. 3 C) or the E1371S (Fig. 3 F) mutant was chosen as the nonhydrolytic model; i.e., both site-1 perturbations decreased this rate by two- to threefold. Login to comment
115 We therefore compared the effects of our site-1 perturbations on the closing rates of two nonhydrolytic mutants, NBD2 Walker A mutant K1250A (Fig. 3, A-C) and NBD2 Walker B mutant E1371S (Fig. 3, D-F). Login to comment
117 (A and D) Macroscopic currents of prephosphorylated K1250A (A) and E1371S (D) CFTR channels elicited by exposure (bars) to either 10 mM ATP alternating with 50 &#b5;M P-ATP (A) or 2 mM ATP alternating with 10 &#b5;M P-ATP (D); the fivefold higher nucleotide concentrations for the K1250A constructs were used to compensate for the large decrease in apparent ATP affinity caused by this mutation (Vergani et al., 2003). Login to comment
119 (B and E) Macroscopic currents of prephosphorylated K1250A/ H1348A (B) and E1371S/ H1348A (E) CFTR channels elicited by transient exposure (bars) to either 10 mM (B) or 2 mM (E) ATP. Login to comment
122 (C and F) Nonhydrolytic closing rates of channels opened by ATP (blue bars) or P-ATP (red bars), or of channels bearing the H1348A mutation opened by ATP (green bars), measured in the K1250A (C) or E1371S (F) background. Login to comment
219 Interestingly, the K464A mutation, which perturbs site 1 by removing the conserved Walker A lysine, was also shown to affect the energetics of both of the C1࢒O1 and O1࢐O2 gating steps (Csan&#e1;dy et al., 2010), although in a different way: in this mutant, rate k1 decreased approximately fourfold, whereas the rate of nonhydrolytic closure, in a K1250A mutant background, increased by &#e07a;10-fold (this is also replicated in the E1371S background; Fig. S2). Login to comment

[hide] Catalyst-like modulation of transition states for ... J Gen Physiol. 2014 Feb;143(2):269-87. doi: 10.1085/jgp.201311089. Epub 2014 Jan 13. Csanady L, Torocsik B
Catalyst-like modulation of transition states for CFTR channel opening and closing: new stimulation strategy exploits nonequilibrium gating.
J Gen Physiol. 2014 Feb;143(2):269-87. doi: 10.1085/jgp.201311089. Epub 2014 Jan 13., [PMID:24420771]

Abstract [show]
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 (DeltaF508) 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 DeltaF508 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.

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69 (E, G, I, and K) Slowly decaying macroscopic "locked-open" currents of E1371S CFTR in the absence of bath ATP; brief applications of 210 &#b5;M NPPB or 20 mM MOPS&#e032; at various voltages (E and G) or of various blocker concentrations at &#e032;120 mV (I and K). Login to comment
84 A convenient macroscopic pore block assay is afforded by the nonhydrolytic E1371S CFTR mutant, which lacks the catalytic glutamate in site 2. Login to comment
86 Because they are active in resting cells (compare to Zhou et al., 2010), excision of E1371S multichannel patches into an ATP-free solution typically uncovers large, slowly decaying currents devoid of gating fluctuations (Fig. 1, E, G, I, and K). Login to comment
207 (C) Segments of unitary current at +60 mV from a single locked-open E1371S CFTR channel in symmetrical &#e07a;140-mM Cl&#e032; at three different cytosolic pH values, with and without cytosolically applied 210 &#b5;M NPPB, displayed at two bandwidths (fc, filter corner frequency). Login to comment
222 In fact, because of its dual action on both i and Po, fractional reduction of macroscopic current by NPPB is not a good measure of pore block, unless observed on "locked-open" channels, which are not gating (Po of &#e07a;1), such as surviving E1371S (Fig. 1, E and I) or K1250A (Fig. 4 D, inset) channels after the removal of ATP. Login to comment
335 Finally, rate O1࢐C1 represents the slow rate of nonhydrolytic closure, modeled by the closing rates of nonhydrolytic mutants K1250A (Fig. 4 A) or E1371S (Fig. 1 K), or of WT channels that have been locked open by ATP plus pyrophosphate (Fig. S2); the time constant of this slow process is &#e07a;30 s. Login to comment

[hide] Combined effects of VX-770 and VX-809 on several f... J Cyst Fibros. 2014 Sep;13(5):508-14. doi: 10.1016/j.jcf.2014.04.003. Epub 2014 May 3. Kopeikin Z, Yuksek Z, Yang HY, Bompadre SG
Combined effects of VX-770 and VX-809 on several functional abnormalities of F508del-CFTR channels.
J Cyst Fibros. 2014 Sep;13(5):508-14. doi: 10.1016/j.jcf.2014.04.003. Epub 2014 May 3., [PMID:24796242]

Abstract [show]
BACKGROUND: The most common cystic fibrosis-associated mutation, the deletion of phenylalanine 508 (F508del), results in channels with poor membrane expression and impaired function. VX-770, a clinically approved drug for treatment of CF patients carrying the G551D mutation, and VX-809, a corrector shown in vitro to increase membrane expression of mutant channels, are currently undergoing clinical trials, but functional data at the molecular level is still lacking. METHODS: The effect of VX-770 and VX-809 on the multiple functional defects of F508del-CFTR was assessed via excised inside-out patch-clamp experiments. RESULTS: VX-770 completely restores the low opening-rate of F508del-CFTR, with smaller open-time increase, in temperature-corrected and VX-809-treated channels. The shorter locked-open time of hydrolysis-deficient F508del-CFTR is also prolonged by VX-770. VX-809 does not improve channel function by itself as previously reported. CONCLUSIONS: The results from these studies can be interpreted as an equilibrium shift toward the open-channel conformation of F508del-CFTR channels.

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63 We introduced the E1371S mutation, which abolishes ATPase activity [24], in WT and F508del-CFTR channels and expressed them in CHO cells. Login to comment
64 The duration of the locked-open time of the channels is calculated from the time- constants of F508del/E1371S and E1371S-CFTR current relaxations upon ATP withdrawal. Login to comment
66 The relaxation time constant increases about 60% for both F508del/E1371S and E1371S-CFTR, suggesting that the effect may not be specific to the mutant channel. Login to comment
68 When used in conjunction with 200 nM VX-770, the locked open time of F508del/E1371S channels can be further increased to 108 &#b1; 12 s (n = 14), a value Fig. 1. Effect of 200 nM VX-770 on temperature-corrected F508del-CFTR channels expressed in CHO cells. Login to comment
75 Here and below the dashed line represent the base line. similar to that of E1371S-CFTR channels in the absence of VX-770, indicating that VX-770 alone is not sufficient to fully restore the stability of the open-channel conformation of F508del channels when hydrolysis is abolished. Login to comment
124 Representative traces for F508del/E1371S (A.) Login to comment
125 and E1371S (B.) Login to comment
128 C F508del/E1371S-CFTR current relaxation after 10 bc;M P-ATP + 200 nM VX-770 withdrawal and its exponential fit (green line) shown in comparison with representative fit for the relaxation after ATP + VX-770 (red line). Login to comment
129 D. summary of the effect of 200 nM VX-770 on the open time of F508del/E1371S and E1371S CFTR (n = 9-15). Login to comment

[hide] A cocktail drug therapy for patients with cystic f... J Cyst Fibros. 2014 Sep;13(5):489-90. doi: 10.1016/j.jcf.2014.07.002. Epub 2014 Jul 24. Chen JH
A cocktail drug therapy for patients with cystic fibrosis?
J Cyst Fibros. 2014 Sep;13(5):489-90. doi: 10.1016/j.jcf.2014.07.002. Epub 2014 Jul 24., [PMID:25088968]

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14 Second, the mutation E1371S, which greatly stimulates channel activity by preventing ATP hydrolysis-mediated channel closure, had modest effects on the action of VX-770 [6]. Login to comment

[hide] An electrostatic interaction at the tetrahelix bun... J Biol Chem. 2014 Oct 31;289(44):30364-78. doi: 10.1074/jbc.M114.595710. Epub 2014 Sep 4. Wang W, Roessler BC, Kirk KL
An electrostatic interaction at the tetrahelix bundle promotes phosphorylation-dependent cystic fibrosis transmembrane conductance regulator (CFTR) channel opening.
J Biol Chem. 2014 Oct 31;289(44):30364-78. doi: 10.1074/jbc.M114.595710. Epub 2014 Sep 4., [PMID:25190805]

Abstract [show]
The CFTR channel is an essential mediator of electrolyte transport across epithelial tissues. CFTR opening is promoted by ATP binding and dimerization of its two nucleotide binding domains (NBDs). Phosphorylation of its R domain (e.g. by PKA) is also required for channel activity. The CFTR structure is unsolved but homology models of the CFTR closed and open states have been produced based on the crystal structures of evolutionarily related ABC transporters. These models predict the formation of a tetrahelix bundle of intracellular loops (ICLs) during channel opening. Here we provide evidence that residues E267 in ICL2 and K1060 in ICL4 electrostatically interact at the interface of this predicted bundle to promote CFTR opening. Mutations or a thiol modifier that introduced like charges at these two positions substantially inhibited ATP-dependent channel opening. ATP-dependent activity was rescued by introducing a second site gain of function (GOF) mutation that was previously shown to promote ATP-dependent and ATP-independent opening (K978C). Conversely, the ATP-independent activity of the K978C GOF mutant was inhibited by charge- reversal mutations at positions 267 or 1060 either in the presence or absence of NBD2. The latter result indicates that this electrostatic interaction also promotes unliganded channel opening in the absence of ATP binding and NBD dimerization. Charge-reversal mutations at either position markedly reduced the PKA sensitivity of channel activation implying strong allosteric coupling between bundle formation and R domain phosphorylation. These findings support important roles of the tetrahelix bundle and the E267-K1060 electrostatic interaction in phosphorylation-dependent CFTR gating.

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179 Combining a Catalytic Mutation with the E267R Mutation Rescues ATP-dependent Activity but Also Provides a Clue that R Domain Conformational Changes May Be Coupled to Bundle Formation-We next combined one of the charge-reversal mutations (E267R) with an NBD2 mutation (E1371S) that inhibits ATP hydrolysis at site 2 (Fig. 8). Login to comment
180 The E1371S mutation stabilizes the NBD dimer in the presence of ATP and greatly prolongs the open channel bursts (39, 40). Login to comment
181 Our interest was to determine if the E267R mutation inhibited the activity of the E1371S catalytic mutant and/or destabilized the NBD dimer in the absence of ATP hydrolysis. Login to comment
182 Instead, however, the E267R/ E1371S-CFTR double mutant behaved similarly to the E1371S-CFTR single mutant with respect to: (i) high control currents in the presence of normally saturating ATP and PKA; (ii) negligible responses to potentiators presumably because the currents were already maximal under control conditions; and (iii) very slow deactivation upon ATP removal (঄deactivaton b0e; 5 min), indicative of a very tight NBD dimer (Fig. 8A; compare with FIGURE 3. Login to comment
194 CFTR Gating Mechanism 30370 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 289ߦNUMBER 44ߦOCTOBER 31, 2014 at SEMMELWEIS UNIV OF MEDICINE on December 4, E1371S-CFTR data in Refs. 39, 40). Login to comment
196 One interesting difference between the single and double E1371S mutants emerged when we assayed these constructs in the absence of bath PKA (Fig. 8, B-D). Login to comment
198 Indeed, we observed macroscopic currents for E1371S-CFTR channels in the absence of bath PKA that averaged more than 10% of the maximal currents measured after potentiator addition (Fig. 8, C and D). Login to comment
200 Combining the E267R charge-reversal mutation with the E1371S mutation virtually abolished these PKA-independent currents (Fig. 8, B and D). Login to comment
273 The lack of effect on the stability of the activated state is consistent with the virtually complete rescue of the ATP-dependent activity of the E267R bundle mutant that we observed when we combined this mutation with the E1371S catalytic mutation. Login to comment
275 The E267R/E1371S double mutant exhibited high steady-state control currents following activation by ATP and PKA, small relative activation by potentiators and very slow deactivation upon ATP washout similar to that reported for the E1371S single mutant. Login to comment
303 First, in PKA titration experiments the single charge-reversal mutants required much more FIGURE 8. Interplay between the E267R mutation and a site 2 catalytic mutation, E1371S. Login to comment
304 A, macroscopic record showing that the E267R/E1371S double mutant exhibited large control currents, very small stimulation by potentiators and very slow deactivation upon ATP removal. Login to comment
306 These characteristics are similar to those previously reported for the single E1371S catalytic mutant (39, 40). Login to comment
307 B and C, macroscopic records showing that the E267R substitution reduced the baseline current prior to addition of PKA (units/ml) that can be detected for the E1371S-CFTR construct. Login to comment

[hide] Structure-activity analysis of a CFTR channel pote... J Gen Physiol. 2014 Oct;144(4):321-36. doi: 10.1085/jgp.201411246. Csanady L, Torocsik B
Structure-activity analysis of a CFTR channel potentiator: Distinct molecular parts underlie dual gating effects.
J Gen Physiol. 2014 Oct;144(4):321-36. doi: 10.1085/jgp.201411246., [PMID:25267914]

Abstract [show]
The cystic fibrosis (CF) transmembrane conductance regulator (CFTR) is a member of the ATP-binding cassette transporter superfamily that functions as an epithelial chloride channel. Gating of the CFTR ion conduction pore involves a conserved irreversible cyclic mechanism driven by ATP binding and hydrolysis at two cytosolic nucleotide-binding domains (NBDs): formation of an intramolecular NBD dimer that occludes two ATP molecules opens the pore, whereas dimer disruption after ATP hydrolysis closes it. CFTR dysfunction resulting from inherited mutations causes CF. The most common CF mutation, deletion of phenylalanine 508 (DeltaF508), impairs both protein folding and processing and channel gating. Development of DeltaF508 CFTR correctors (to increase cell surface expression) and potentiators (to enhance open probability, Po) is therefore a key focus of CF research. The practical utility of 5-nitro-2-(3-phenylpropylamino)benzoate (NPPB), one of the most efficacious potentiators of DeltaF508 CFTR identified to date, is limited by its pore-blocking side effect. NPPB-mediated stimulation of Po is unique in that it involves modulation of gating transition state stability. Although stabilization by NPPB of the transition state for pore opening enhances both the rate of channel opening and the very slow rate of nonhydrolytic closure, because of CFTR's cyclic gating mechanism, the net effect is Po stimulation. In addition, slowing of ATP hydrolysis by NPPB delays pore closure, further enhancing Po. Here we show that NPPB stimulates gating at a site outside the pore and that these individual actions of NPPB on CFTR are fully attributable to one or the other of its two complementary molecular parts, 3-nitrobenzoate (3NB) and 3-phenylpropylamine (3PP), both of which stimulate Po: the pore-blocking 3NB selectively stabilizes the transition state for opening, whereas the nonblocking 3PP selectively slows the ATP hydrolysis step. Understanding structure-activity relationships of NPPB might prove useful for designing potent, clinically relevant CFTR potentiators.

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49 pore-block measurements on E1371S (see Fig. 2 A) or K1250A CFTR (see Figs. 3 and 4), surviving currents of channels opened in resting oocytes as the result of endogenous phosphorylation were also used (Csan&#e1;dy and T&#f6;r&#f6;csik, 2014). Login to comment
83 A convenient macroscopic assay for measuring fractional effects on average ion flux rates through bursting channels is provided by nonhydrolytic mutant CFTR channels such as E1371S (Vergani et al., 2003) or K1250A. Login to comment
86 As expected, application of MOPS&#e032; at a concentration of 80 mM, approximately ninefold its own KI (Fig. 2 A, green bars), reversibly reduced macroscopic current through locked-open E1371S channels by almost 90% (Fig. 2 A). Login to comment
87 In the presence of 80 mM MOPS&#e032; , addition of NPPB (Fig. 2 A, brown bars) further suppressed locked-open E1371S currents in a dose-dependent manner; however, further fractional reduction by 210 &#b5;M NPPB (&#e07a;10-fold its own KI) was only &#e07a;50% (Fig. 2 A, yellow box magnified in inset). Login to comment
110 (A) Macroscopic locked-open E1371S CFTR current at &#e032;120 mV after removal of ATP from the bath; exposures to various NPPB concentrations (brown bars) in the continued presence of 80 mM MOPS&#e032; (green bar; magnified in yellow inset). Login to comment

[hide] The cystic fibrosis transmembrane conductance regu... Pflugers Arch. 2015 Aug;467(8):1783-94. doi: 10.1007/s00424-014-1618-8. Epub 2014 Oct 4. Broadbent SD, Ramjeesingh M, Bear CE, Argent BE, Linsdell P, Gray MA
The cystic fibrosis transmembrane conductance regulator is an extracellular chloride sensor.
Pflugers Arch. 2015 Aug;467(8):1783-94. doi: 10.1007/s00424-014-1618-8. Epub 2014 Oct 4., [PMID:25277268]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl(-) channel that governs the quantity and composition of epithelial secretions. CFTR function is normally tightly controlled as dysregulation can lead to life-threatening diseases such as secretory diarrhoea and cystic fibrosis. CFTR activity is regulated by phosphorylation of its cytosolic regulatory (R) domain, and ATP binding and hydrolysis at two nucleotide-binding domains (NBDs). Here, we report that CFTR activity is also controlled by extracellular Cl(-) concentration ([Cl(-)]o). Patch clamp current recordings show that a rise in [Cl(-)]o stimulates CFTR channel activity, an effect conferred by a single arginine residue, R899, in extracellular loop 4 of the protein. Using NBD mutants and ATP dose response studies in WT channels, we determined that [Cl(-)]o sensing was linked to changes in ATP binding energy at NBD1, which likely impacts NBD dimer stability. Biochemical measurements showed that increasing [Cl(-)]o decreased the intrinsic ATPase activity of CFTR mainly through a reduction in maximal ATP turnover. Our studies indicate that sensing [Cl(-)]o is a novel mechanism for regulating CFTR activity and suggest that the luminal ionic environment is an important physiological arbiter of CFTR function, which has significant implications for salt and fluid homeostasis in epithelial tissues.

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112 To explore the role of phosphorylation further, we studied the effect of deleting the R domain from CFTR (residues 634-836) [12, 7], which removes all the major PKA/PKC Table 1 Summary of the FSK stimulation of whole cell currents and Erev shifts observed with the CFTR constructs used in this study CFTR Construct n FSK Stimulation (%&#b1;SEM) Erev shift (mV&#b1;SEM) WT (50 bc;M ATP) 5 180&#b1;96 15.0&#b1;3.6 WT (100 bc;M ATP) 6 12,000&#b1;6,000 15.2&#b1;3.0 WT (300 bc;M ATP) 8 1,200&#b1;600 17.0&#b1;3.0 WT (1 mM ATP) 24 13,000&#b1;6,000 23.7&#b1;1.8 WT (1.3 mM ATP) 9 1,400&#b1;900 16.7&#b1;2.6 WT (2 mM ATP) 24 6,100&#b1;5,300 16.7&#b1;1.6 WT (5 mM ATP) 7 1,600&#b1;1,000 20.1&#b1;4.4 WT (50 bc;M ATP + 50 bc;M P-ATP) 7 224&#b1;130 15.3&#b1;1.0 WT + Genistein 4 7,600&#b1;5,200 26.1&#b1;5.4 WT + AMP-PNP 5 2,800&#b1;2,500 21.8&#b1;5.5 WT (3 mM MgCl2) 7 28,000&#b1;17,000 18.3&#b1;3.1 R104Q 5 4,600&#b1;1,600 28.6&#b1;4.7 K114C 5 12,000&#b1;6,700 29.2&#b1;3.0 R117Q 4 33,000&#b1;20,000 30.1&#b1;3.4 K329A 5 13,000&#b1;10,000 33.7&#b1;2.1 R334Q 9 13,000&#b1;6,700 27.3&#b1;2.9 K335A 5 3,200&#b1;1,500 20.8&#b1;7.1 W401G 7 2,600&#b1;1,800 18.5&#b1;4.8 Delta-R (No Stim) 5 - 25.1&#b1;2.7 Delta-R (No FSK, Genistein) 5 140&#b1;13 22.7&#b1;3.0 Delta-R (FSK, No Genistein) 4 89&#b1;14 15.6&#b1;6.0 Delta-R (FSK + Genistein) 6 639&#b1;432 25.1&#b1;4.9 Delta-R-E1371S (No FSK) 9 - 21.4&#b1;4.8 Delta-R-E1371S (FSK) 4 2,600&#b1;1,400 15.3&#b1;4.7 K892Q 7 16,000&#b1;9,500 36.8&#b1;4.8 R899E 4 1,200&#b1;400 25.0&#b1;2.7 R899K 4 1,600&#b1;900 26.6&#b1;2.9 R899Q 7 5,400&#b1;2,800 30.0&#b1;1.3 R899Q + AMP-PNP 4 72,000&#b1;50,000 15.2&#b1;2.8 R899Q-E1371Q (No FSK) 4 - 18.4&#b1;5.9 R899Q-E1371Q (FSK) 6 107&#b1;48 15.6&#b1;3.0 R1128Q 6 14,000&#b1;6,100 41.1&#b1;4.2 Y1219G 6 3,200&#b1;2,500 19.2&#b1;3.3 E1371Q (No FSK) 6 - 25.5&#b1;3.5 E1371Q (FSK) 8 -28&#b1;9 22.3&#b1;4.0 E1371Q (FSK, No ATP, No GTP) 8 270&#b1;130 19.4&#b1;4.5 E1371Q + AMP-PNP (No FSK) 4 - 24.7&#b1;6.5 E1371Q + AMP-PNP (FSK) 8 180&#b1;170 17.4&#b1;4.0 Vector Control 4 15&#b1;38 - FSK stimulation was calculated as the percentage increase in current density at -60 mV from the Erev, after 5-min exposure to 10 bc;M FSK. Login to comment
121 We tested this prediction by using DeltaR-CFTR with the E1371S mutation, which like E1371Q CFTR is hydrolysis defective [8] and found, unexpectedly, that the double-mutant CFTR channels did not respond to changes in [Cl- ]o (external Cl- stimulation; no FSK/genistein: 0.0&#b1;7.0 %, n=9; with FSK/genistein: 16.7&#b1; 7.7 %, n=4) (Fig. 4c-e). Login to comment
140 Figure 6 shows that when total [Cl- ] was 3 nA 100 ms DeltaR (No Stim) (i) (ii) (iii) DeltaR (No Stim) DeltaR-E1371S (No Stim) A B D C E 3 nA 100 ms DeltaR-E1371S (No Stim) (i) (ii) (iii) Fig. 4 Role of the R domain in [Cl- ]o sensing by CFTR. Login to comment
141 a, c Representative fWCR current recordings measured between &#b1;100 mV in 20 mV steps from HEK cells transfected with deltaR-CFTR or deltaR-E1371S CFTR as indicated. Login to comment
177 The one caveat to this interpretation is that [Cl- ]o sensing by DeltaR-CFTR was abolished when ATP hydrolysis at NBD2 was blocked by the E1371S mutation. Login to comment

[hide] Full-open and closed CFTR channels, with lateral t... Cell Mol Life Sci. 2015 Apr;72(7):1377-403. doi: 10.1007/s00018-014-1749-2. Epub 2014 Oct 7. Mornon JP, Hoffmann B, Jonic S, Lehn P, Callebaut I
Full-open and closed CFTR channels, with lateral tunnels from the cytoplasm and an alternative position of the F508 region, as revealed by molecular dynamics.
Cell Mol Life Sci. 2015 Apr;72(7):1377-403. doi: 10.1007/s00018-014-1749-2. Epub 2014 Oct 7., [PMID:25287046]

Abstract [show]
In absence of experimental 3D structures, several homology models, based on ABC exporter 3D structures, have provided significant insights into the molecular mechanisms underlying the function of the cystic fibrosis transmembrane conductance regulator (CFTR) protein, a chloride channel whose defects are associated with cystic fibrosis (CF). Until now, these models, however, did not furnished much insights into the continuous way that ions could follow from the cytosol to the extracellular milieu in the open form of the channel. Here, we have built a refined model of CFTR, based on the outward-facing Sav1866 experimental 3D structure and integrating the evolutionary and structural information available today. Molecular dynamics simulations revealed significant conformational changes, resulting in a full-open channel, accessible from the cytosol through lateral tunnels displayed in the long intracellular loops (ICLs). At the same time, the region of nucleotide-binding domain 1 in contact with one of the ICLs and carrying amino acid F508, the deletion of which is the most common CF-causing mutation, was found to adopt an alternative but stable position. Then, in a second step, this first stable full-open conformation evolved toward another stable state, in which only a limited displacement of the upper part of the transmembrane helices leads to a closure of the channel, in a conformation very close to that adopted by the Atm1 ABC exporter, in an inward-facing conformation. These models, supported by experimental data, provide significant new insights into the CFTR structure-function relationships and into the possible impact of CF-causing mutations.

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309 The existence of the alternative position of F508 was further supported by the fact that the modification of F508C by benzyl-methanethiosulfonate (MTSBn), conserving the F508 aromatic character and restoring gating activity (lost for the F508C mutation in the open state-locked E1371S variant) [76], can be accommodated in both the initial and MD-generated models of CFTR [Online Resource 21 (A)]. Login to comment

[hide] Modulation of CFTR gating by permeant ions. J Gen Physiol. 2015 Jan;145(1):47-60. doi: 10.1085/jgp.201411272. Epub 2014 Dec 15. Yeh HI, Yeh JT, Hwang TC
Modulation of CFTR gating by permeant ions.
J Gen Physiol. 2015 Jan;145(1):47-60. doi: 10.1085/jgp.201411272. Epub 2014 Dec 15., [PMID:25512598]

Abstract [show]
Cystic fibrosis transmembrane conductance regulator (CFTR) is unique among ion channels in that after its phosphorylation by protein kinase A (PKA), its ATP-dependent gating violates microscopic reversibility caused by the intimate involvement of ATP hydrolysis in controlling channel closure. Recent studies suggest a gating model featuring an energetic coupling between opening and closing of the gate in CFTR's transmembrane domains and association and dissociation of its two nucleotide-binding domains (NBDs). We found that permeant ions such as nitrate can increase the open probability (Po) of wild-type (WT) CFTR by increasing the opening rate and decreasing the closing rate. Nearly identical effects were seen with a construct in which activity does not require phosphorylation of the regulatory domain, indicating that nitrate primarily affects ATP-dependent gating steps rather than PKA-dependent phosphorylation. Surprisingly, the effects of nitrate on CFTR gating are remarkably similar to those of VX-770 (N-(2,4-Di-tert-butyl-5-hydroxyphenyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide), a potent CFTR potentiator used in clinics. These include effects on single-channel kinetics of WT CFTR, deceleration of the nonhydrolytic closing rate, and potentiation of the Po of the disease-associated mutant G551D. In addition, both VX-770 and nitrate increased the activity of a CFTR construct lacking NBD2 (DeltaNBD2), indicating that these gating effects are independent of NBD dimerization. Nonetheless, whereas VX-770 is equally effective when applied from either side of the membrane, nitrate potentiates gating mainly from the cytoplasmic side, implicating a common mechanism for gating modulation mediated through two separate sites of action.

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124 The lengthening of the relaxation time constant by NO3 &#e032; was also seen with a hydrolysis-deficient mutant, E1371S-CFTR (n = 5). Login to comment