ABCMdb

ABCC7 p.Asp1370Asn

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

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[hide] Insight in eukaryotic ABC transporter function by ... FEBS Lett. 2006 Feb 13;580(4):1064-84. Epub 2006 Jan 19. Frelet A, Klein M
Insight in eukaryotic ABC transporter function by mutation analysis.
FEBS Lett. 2006 Feb 13;580(4):1064-84. Epub 2006 Jan 19., 2006-02-13 [PMID:16442101]

Abstract [show]
With regard to structure-function relations of ATP-binding cassette (ABC) transporters several intriguing questions are in the spotlight of active research: Why do functional ABC transporters possess two ATP binding and hydrolysis domains together with two ABC signatures and to what extent are the individual nucleotide-binding domains independent or interacting? Where is the substrate-binding site and how is ATP hydrolysis functionally coupled to the transport process itself? Although much progress has been made in the elucidation of the three-dimensional structures of ABC transporters in the last years by several crystallographic studies including novel models for the nucleotide hydrolysis and translocation catalysis, site-directed mutagenesis as well as the identification of natural mutations is still a major tool to evaluate effects of individual amino acids on the overall function of ABC transporters. Apart from alterations in characteristic sequence such as Walker A, Walker B and the ABC signature other parts of ABC proteins were subject to detailed mutagenesis studies including the substrate-binding site or the regulatory domain of CFTR. In this review, we will give a detailed overview of the mutation analysis reported for selected ABC transporters of the ABCB and ABCC subfamilies, namely HsCFTR/ABCC7, HsSUR/ABCC8,9, HsMRP1/ABCC1, HsMRP2/ABCC2, ScYCF1 and P-glycoprotein (Pgp)/MDR1/ABCB1 and their effects on the function of each protein.

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149 D572N resulted in a marked decrease in sensitivity to channel activation while D1370N resulted in an increase in sensitivity [60,96]. Login to comment

[hide] Regulation of CFTR Cl- channel gating by ATP bindi... Proc Natl Acad Sci U S A. 2000 Jul 18;97(15):8675-80. Ikuma M, Welsh MJ
Regulation of CFTR Cl- channel gating by ATP binding and hydrolysis.
Proc Natl Acad Sci U S A. 2000 Jul 18;97(15):8675-80., 2000-07-18 [PMID:10880569]

Abstract [show]
Opening and closing of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel is regulated by the interaction of ATP with its two cytoplasmic nucleotide-binding domains (NBD). Although ATP hydrolysis by the NBDs is required for normal gating, the influence of ATP binding versus hydrolysis on specific steps in the gating cycle remains uncertain. Earlier work showed that the absence of Mg(2+) prevents hydrolysis. We found that even in the absence of Mg(2+), ATP could support channel activity, albeit at a reduced level compared with the presence of Mg(2+). Application of ATP with a divalent cation, including the poorly hydrolyzed CaATP complex, increased the rate of opening. Moreover, in CFTR variants with mutations that disrupt hydrolysis, ATP alone opened the channel and Mg(2+) further enhanced ATP-dependent opening. These data suggest that ATP alone can open the channel and that divalent cations increase ATP binding. Consistent with this conclusion, when we mutated an aspartate thought to bind Mg(2+), divalent cations failed to increase activity compared with ATP alone. Two observations suggested that divalent cations also stabilize the open state. In wild-type CFTR, CaATP generated a long duration open state, whereas ATP alone did not. With a CFTR variant in which hydrolysis was disrupted, MgATP, but not ATP alone, produced long openings. These results suggest a gating cycle for CFTR in which ATP binding opens the channel and either hydrolysis or dissociation leads to channel closure. In addition, the data suggest that ATP binding and hydrolysis by either NBD can gate the channel.

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127 We studied the CFTR-D1370N mutant; this channel has reduced activity (11, 39). Login to comment
129 We predicted that CFTR-D1370N would not discriminate between ATP, MgATP, and CaATP. Login to comment
148 Effect of MgATP, ATP alone, and CaATP on Cl- current of CFTR-D1370N. Login to comment
194 It is also noteworthy that MgATP did not stimulate current to a greater extent than ATP alone when CFTR had a mutation in NBD2 (D1370N) that disrupts divalent cation binding (Fig. 2). Login to comment

[hide] ATP hydrolysis-coupled gating of CFTR chloride cha... Biochemistry. 2001 May 15;40(19):5579-86. Zou X, Hwang TC
ATP hydrolysis-coupled gating of CFTR chloride channels: structure and function.
Biochemistry. 2001 May 15;40(19):5579-86., 2001-05-15 [PMID:11341822]

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186 Gunderson and Kopito (31) demonstrated that the mean open time for the D1370N mutant is longer than that of the wild-type CFTR. Login to comment

[hide] Down-regulation of volume-sensitive Cl- channels b... Pflugers Arch. 2002 Nov;445(2):177-86. Epub 2002 Sep 7. Ando-Akatsuka Y, Abdullaev IF, Lee EL, Okada Y, Sabirov RZ
Down-regulation of volume-sensitive Cl- channels by CFTR is mediated by the second nucleotide-binding domain.
Pflugers Arch. 2002 Nov;445(2):177-86. Epub 2002 Sep 7., [PMID:12457238]

Abstract [show]
Transient expression of wild-type human cystic fibrosis transmembrane conductance regulator (CFTR) in HEK293T cells resulted in a profound decrease in the amplitude of volume-sensitive outwardly rectifying Cl- channel (VSOR) current without changing the single-channel amplitude. This effect was not mimicked by expression of the DeltaF508 mutant of CFTR, which did not reach the plasma membrane. The VSOR regulation by CFTR was not affected by G551D mutation at first nucleotide-binding domain (NBD1), which is known to impair CFTR interaction with the outwardly rectifying chloride channel, ORCC, epithelial amiloride-sensitive Na-channel, ENaC, and renal potassium channel, ROMK2. The CFTR-VSOR interaction was insensitive to the deletion mutation, DeltaTRL, which is known to impair CFTR-PDZ domain binding. In contrast, the G1349D mutant, which impairs ATP binding at NBD2, effectively abolished the down-regulatory effect of CFTR. Furthermore, the K1250M mutation at the Walker A motif and the D1370N mutation at the Walker B motif, both known to impair ATP hydrolysis at NBD2, completely abolished the VSOR regulation by CFTR. Thus, we conclude that an ATP-hydrolysable conformation of NBD2 is essential for the regulation of the VSOR by the CFTR protein, and that VSOR is a first channel regulated by CFTR through its NBD2.

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6 Furthermore, the K1250M mutation at the Walker A motif and the D1370N mutation at the Walker B motif, both known to impair ATP hydrolysis at NBD2, completely abolished the VSOR regulation by CFTR. Login to comment
123 To check whether ATP hydrolysis at NBD2 is required for CFTR`s regulatory function, we generated two other NBD2-mutants of CFTR, K1250M and D1370N. Login to comment
125 The plasmalemmal distribution of K1250M and D1370N CFTR was confirmed by immunofluorescence microscopy (Fig. 6A) and immunoblotting (Fig. 6B). Login to comment
131 Relative integrated optical densities of the mature bands are shown as percentages of the respective b-actin bands (taken as 100%) In whole-cell patch-clamp experiments, cells expressing the K1250M or D1370N mutant exhibited VSOR currents (Fig. 7A) as large as those observed in mock-transfected cells (Fig. 3A). Login to comment
137 Since VSOR-non-regulating NBD2 mutants (G1349D, K1250M and D1370N) were expressed to approximately the same level as VSOR-regulating WT and G551D CFTR (as seen from immunostaining and Western blotting data), we may exclude the possibility that the down-regulation is simply a side-effect of overexpression of a foreign protein. Login to comment
155 This was, in fact, confirmed by a yeast two- Fig. 7A, B Effects of expression of Walker A or Walker B mutants on VSOR current densities in HEK293T cells. A Time course of VSOR current activation by hypotonic stimulation of cells transfected with the K1250M (top) or D1370N (bottom) mutant, taken during application of alternating pulses from 0 to €40 mV every 15 s. B VSOR current densities from mock-transfected, K1250M mutant-transfected and D1370N mutant-transfected cells, recorded at +40 mV after reaching a steady-state level. Login to comment
175 In our study, the K1250M and D1370N mutations effectively abolished the down-regulatory effect of CFTR on VSOR currents. Login to comment

[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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4 The rate of opening to a burst (1/␶ib) 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. Login to comment
7 NBD2 catalytic site mutations K1250A, D1370N, and E1371S were found to prolong open bursts. Login to comment
32 However, in CFTR the Walker A NBD2 mutation K1250A abolished ATP hydrolysis, whereas the NBD1 mutation K464A simply reduced overall hydrolytic activity (Ramjeesingh et al., 1999); and biochemical studies of Walker B aspartate mutations in CFTR (D572N in NBD1, D1370N in NBD2) have not yet been performed. Login to comment
41 We studied in detail the dependence of channel gating on [MgATP], gating in the presence of poorly hydrolyzable nucleotide analogs, as well as the effects of mutating residues in the Walker A (K464A and K1250A) and Walker B motifs (in particular, D1370N in NBD2). Login to comment
52 Amounts of cRNA injected were adjusted to vary the level of expression: up to 40 ng/oocyte was required for high expression of K1250A or K464A/K1250A mutant channels, whereas 0.1-0.25 ng/oocyte sufficed for single channel recordings of WT, K464A, or D1370N channels. Login to comment
89 T A B L E I Kinetic Parameters of WT and Mutant CFTR Channels WT K464A D1370N mean Ϯ SEM n mean Ϯ SEM n mean Ϯ SEM n (A) 5 mM MgATP ϩ 300 nM PKA ␶b 644 Ϯ 63 30 620 Ϯ 58 21 3,768 Ϯ 499 21 ␶ib 1,671 Ϯ 172 30 2,760 Ϯ 439 21 3,588 Ϯ 414 21 1,552 Ϯ 170 19 2,438 Ϯ 483 12 2,849 Ϯ 491 12 ␶F 19.3 Ϯ 2.0 30 20.8 Ϯ 2.1 21 49.9 Ϯ 4.3 21 nF 0.57 Ϯ 0.06 30 0.50 Ϯ 0.06 21 2.76 Ϯ 0.25 21 rCO 0.75 Ϯ 0.06 30 0.50 Ϯ 0.05 21 0.38 Ϯ 0.05 21 0.77 Ϯ 0.08 19 0.54 Ϯ 0.08 12 0.47 Ϯ 0.07 12 rOC 1.95 Ϯ 0.15 30 1.92 Ϯ 0.15 21 0.43 Ϯ 0.07 21 (B) 5 mM MgATP ␶b 338 Ϯ 22 18 309 Ϯ 23 8 1,748 Ϯ 215 17 ␶ib 4,506 Ϯ 497 18 6,752 Ϯ 1314 8 9,503 Ϯ 1440 17 4,454 Ϯ 1382 5 6,928 Ϯ 1666 6 7,584 Ϯ 1967 9 ␶F 23.5 Ϯ 3.2 18 16.1 Ϯ 2.2 8 51.5 Ϯ 6.0 17 nF 0.42 Ϯ 0.05 18 0.39 Ϯ 0.06 8 1.40 Ϯ 0.13 17 rCO 0.27 Ϯ 0.03 18 0.18 Ϯ 0.03 8 0.16 Ϯ 0.03 17 0.33 Ϯ 0.09 5 0.18 Ϯ 0.03 6 0.22 Ϯ 0.05 9 rOC 3.28 Ϯ 0.21 18 3.43 Ϯ 0.25 8 0.75 Ϯ 0.09 17 (C) 50 ␮M MgATP ␶b 355 Ϯ 44 12 323 Ϯ 136 4 1,433 Ϯ 381 4 ␶F 27.3 Ϯ 5.2 12 22.1 Ϯ 4.4 4 46.2 Ϯ 10.8 4 nF 0.38 Ϯ 0.05 12 0.45 Ϯ 0.11 4 1.91 Ϯ 0.34 4 (D) 5 mM MgAMPPNP ␶b 1,619 Ϯ 232 32 271 Ϯ 52 8 ␶F 59.5 Ϯ 6.6 32 26.8 Ϯ 7.7 8 nF 2.40 Ϯ 0.26 32 0.38 Ϯ 0.10 8 Kinetic parameters were obtained using a maximum likelihood simultaneous fit to dwell-time histograms at all conductance levels (Csanády, 2000). Login to comment
94 The significance of the slight prolongation of ␶F for the D1370N mutant and for WT in 5 mM MgAMPPNP is unknown, but the rate rOF remained 1-2 s-1 for all conditions and mutants tested. Login to comment
113 (B and C) Representative traces for prephosphorylated K464A and D1370N channels. Login to comment
114 Relative opening (D) and closing (E) rates (mean Ϯ SEM, 2 Յ n Յ 7) from analysis of records as in A-C for WT (blue circles), K464A (red triangles), and D1370N (green squares) channels at 10 ␮M Յ [MgATP] Յ 5 mM, plotted on semilogarithmic axes. Login to comment
116 Curves in D show Michaelis-Menten fits, yielding K0.5 of 56 Ϯ 5, 807 Ϯ 185, 391 Ϯ 118 ␮M, and rCOmax of 1.02, 1.16, and 1.08, for WT, K464A, and D1370N, respectively. Login to comment
123 Compared with WT, both K464A (Walker A lysine in NBD1) and D1370N (Walker B aspartate in NBD2) mutant CFTR channels opened less frequently at low [MgATP] (e.g., 50 ␮M; Figs. 2, A-D), and this defect could be largely overcome by raising the [MgATP], so that, at saturating [MgATP], opening rates of WT, K464A, and D1370N channels differed by less than a factor of two (Table I). Login to comment
125 As expected (see below) for channels in which opening rate, but not closing rate, is sensitive to [MgATP], the dependence of Po on [MgATP] was not very different from that of rCO, shown in Fig. 2 D, for WT (see Fig. 3 C), K464A, or D1370N channels. Login to comment
162 (G and H) Representative traces showing gating of K464A and D1370N channels at 15 ␮M MgATP (after PKA removal). Login to comment
164 Though variability among the four patches containing sufficiently few D1370N channels precluded pooling the data for burst distribution analysis, in none of those patches (analyzed separately) did introduction of a second component significantly improve the maximum likelihood fit. Login to comment
168 D1370N channels closed 4-5-fold more slowly than WT CFTR (Figs. 2 E and 6, A vs. B; Table I), and this reduced closing rate was constant at all [MgATP] tested (Figs. 2 E and 4 H), although, as for WT CFTR, strong phosphorylation slowed closing of D1370N channels roughly twofold (Table I). Login to comment
171 Moreover, for both K1250A and D1370N mutants, this macroscopic current decay followed a single exponential time course, implying the presence of a single population of open bursts (for K1250A, see Figs. 3 B and 10 E; for D1370N, decay time constants were: ␶[after 5 mM MgATP ϩ PKA] ϭ 6.4 Ϯ 1.6 s, n ϭ 6; ␶[after 5 mM MgATP] ϭ 2.2 Ϯ 0.5 s, n ϭ 7; ␶[after 300 ␮M MgATP] ϭ 1.9 Ϯ 0.3 s, n ϭ 8; cf. Table I). 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
266 Our results show that mutations within the Walker motifs of either NBD1 (K464A) or NBD2 (D1370N, Figure 11. Login to comment
270 K1250A) reduce the apparent affinity of the MgATP binding site(s) involved in channel opening (Figs. 2 and 3), but (at least for K464A and D1370N) affect the maximal opening rate little (Table I). Login to comment
280 Therefore, the simplest interpretation of the reduced apparent affinity with which MgATP elicits opening of K464A and D1370N (and K1250A) mutants compared with WT is that the mutations impair nucleotide binding at two different sites, such that at subsaturating [MgATP] channel opening is limited by MgATP binding at NBD1 in K464A, but at NBD2 in D1370N (and K1250A). Login to comment
286 Allosteric interactions between CFTR`s two NBDs (compare Powe et al., 2002) could, therefore, permit the K464A, D1370N, and K1250A mutations to all affect the same binding site. Login to comment
291 Moreover, covalent modification of the NBD2 Walker A sequence (Cotten and Welsh, 1998), and the K1250A (Fig. 3 C) and the D1370N (Fig. 2 D) mutations (‫9-8ف‬ Å apart; e.g., Hung et al., 1998), all reduce apparent affinity for MgATP activation of opening. Login to comment
293 Most likely, therefore, the rightward shift in [MgATP] dependence of D1370N (and K1250A) open- ing rate reflects the lower affinity of a binding step, required for channel opening, at NBD2 itself. Login to comment
298 In fact, although opening rates for WT and D1370N mutant CFTR channels (Fig. 2 D, blue and green symbols) are satisfactorily described by the Michaelis equation (i.e., opening limited by binding to a single site) the opening rates of K464A channels (Fig. 2 D, red symbols) at low (Յ50 ␮M) [MgATP] are slightly higher than expected. Login to comment
300 Therefore, present evidence suggests that nucleotide normally binds to both of WT CFTR`s NBDs before the channel opens, and that opening is limited by nucleotide binding at NBD2 in WT, D1370N, and K1250A CFTR channels, but probably by nucleotide binding at NBD1 in K464A CFTR channels. Login to comment
316 For example, our measurements of D1370N CFTR gating show a twofold reduction in maximal opening rate (Table I), but no ATPase measurements are available for D1370N CFTR. Login to comment
324 We found no clear dependence of burst duration on [MgATP] (10 ␮M to 5 mM) in WT CFTR (Figs. 2 E, 3 A, and 4, B and C) or in K464A, D1370N, or K1250A mutant channels (Figs. 2 E, 3 B, and 4, E-H), indicating that all ATP binding events precede channel opening and no further binding to the open channel is needed to complete the gating cycle. Login to comment
329 Moreover, our finding that D1370N channels at low (15 ␮M) [MgATP] both enter and exit bursts more slowly on average than WT channels (Figs. 2, D-E, and 4 H) demonstrates that this single NBD2 mutation impacts every gating cycle, regardless of the fact that D1370N channels have an intact WT NBD1 sequence. Login to comment
366 Even higher levels of steady state phosphorylation could prolong normal hydrolytic bursts (Table I, WT and K464A), as well as nonhydrolytic locked-open bursts (Table I, D1370N; Fig. 10 A vs. Fig. 9; Fig. 3 B vs. Fig. 10 E), by stabilizing the open burst states more than the transition states for both possible pathways (forward or backward; Fig. 12 A) for terminating the burst. Login to comment
379 Unfortunately, the difficulty of collecting adequate numbers of CFTR`s relatively infrequent gating events, combined with the lack of biochemical information on CFTR mutants (whether D1370N is capable of ATP hydrolysis, for instance), precludes extraction of the many (Ն7) rate constants from fits to data, even for a scheme as simple as the one in Fig. 12 A. Login to comment
382 Similarly, the effect of the D1370N mutation seen in Fig. 2 D (and on Po) can be mimicked by an ‫-01ف‬fold acceleration of the MgATP dissociation rate from NBD2 with the Ͻ2-fold observed reduction in maximal opening rate (Table IB), and (assuming that hydrolysis is abolished) by appropriate speeding of nonhydrolytic closing. 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] ADP inhibits function of the ABC transporter cysti... Proc Natl Acad Sci U S A. 2005 Feb 8;102(6):2216-20. Epub 2005 Jan 31. Randak CO, Welsh MJ
ADP inhibits function of the ABC transporter cystic fibrosis transmembrane conductance regulator via its adenylate kinase activity.
Proc Natl Acad Sci U S A. 2005 Feb 8;102(6):2216-20. Epub 2005 Jan 31., 2005-02-08 [PMID:15684079]

Abstract [show]
ADP interacts with the nucleotide-binding domains (NBDs) of the cystic fibrosis transmembrane conductance regulator (CFTR) to inhibit its Cl- channel activity. Because CFTR NBD2 has reversible adenylate kinase activity (ATP + AMP<==> ADP + ADP) that gates the channel, we asked whether ADP might inhibit current through this enzymatic activity. In adenylate kinases, binding of the two ADP molecules is cooperative. Consistent with this hypothesis, CFTR current inhibition showed positive cooperativity for ADP. We also found that ADP inhibition of current was attenuated when we prevented adenylate kinase activity with P1,P5-di(adenosine-5') pentaphosphate. Additional studies suggested that adenylate kinase-dependent inhibition involved phosphotransfer between two nucleotide diphosphates. These data indicate that the adenylate kinase reaction at NBD2 contributed to the inhibitory effect of ADP. Finding that ADP inhibits function via an adenylate kinase activity also helps explain the earlier observation that mutations that disrupt adenylate kinase activity also disrupt ADP inhibition. Thus, the results reveal a previously unrecognized mechanism by which ADP inhibits an ABC transporter.

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31 For example, structural studies predict that the K1250A and D1370N mutations alter the ATP-binding sites, and these mutations disrupted both ATPase activity and adenylate kinase activities, as well as ADP-dependent inhibition. Login to comment

[hide] CFTR gating I: Characterization of the ATP-depende... J Gen Physiol. 2005 Apr;125(4):361-75. Epub 2005 Mar 14. Bompadre SG, Ai T, Cho JH, Wang X, Sohma Y, Li M, Hwang TC
CFTR gating I: Characterization of the ATP-dependent gating of a phosphorylation-independent CFTR channel (DeltaR-CFTR).
J Gen Physiol. 2005 Apr;125(4):361-75. Epub 2005 Mar 14., [PMID:15767295]

Abstract [show]
The CFTR chloride channel is activated by phosphorylation of serine residues in the regulatory (R) domain and then gated by ATP binding and hydrolysis at the nucleotide binding domains (NBDs). Studies of the ATP-dependent gating process in excised inside-out patches are very often hampered by channel rundown partly caused by membrane-associated phosphatases. Since the severed DeltaR-CFTR, whose R domain is completely removed, can bypass the phosphorylation-dependent regulation, this mutant channel might be a useful tool to explore the gating mechanisms of CFTR. To this end, we investigated the regulation and gating of the DeltaR-CFTR expressed in Chinese hamster ovary cells. In the cell-attached mode, basal DeltaR-CFTR currents were always obtained in the absence of cAMP agonists. Application of cAMP agonists or PMA, a PKC activator, failed to affect the activity, indicating that the activity of DeltaR-CFTR channels is indeed phosphorylation independent. Consistent with this conclusion, in excised inside-out patches, application of the catalytic subunit of PKA did not affect ATP-induced currents. Similarities of ATP-dependent gating between wild type and DeltaR-CFTR make this phosphorylation-independent mutant a useful system to explore more extensively the gating mechanisms of CFTR. Using the DeltaR-CFTR construct, we studied the inhibitory effect of ADP on CFTR gating. The Ki for ADP increases as the [ATP] is increased, suggesting a competitive mechanism of inhibition. Single channel kinetic analysis reveals a new closed state in the presence of ADP, consistent with a kinetic mechanism by which ADP binds at the same site as ATP for channel opening. Moreover, we found that the open time of the channel is shortened by as much as 54% in the presence of ADP. This unexpected result suggests another ADP binding site that modulates channel closing.

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327 The same paper shows that the D1370N mutation at the Walker B motif in NBD2 also decreases the apparent affinity of ATP (compare Bompadre et al., 2005). 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
4 Single-channel kinetic analysis of ⌬R/D1370N-CFTR shows unequivocally that the open time of this mutant channel is decreased by ADP. 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
47 Mutation of this aspartate to asparagine (D1370N) results in channels that exhibit longer open and closed times (Gunderson and Kopito, 1995; Vergani et al., 2003), resembling the elusive "slow gating" mode described in our previous paper (Bompadre et al., 2005). Login to comment
51 Single-channel dwell-time analysis of ⌬R/D1370N-CFTR channels shows unequivocally that the open time of the channel is decreased in the presence of ADP. 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
81 Fig. S1 shows the ATP dose response for the D1370N mutant. Login to comment
82 R E S U L T S Effect of ADP on ⌬R/D1370N-CFTR In the accompanying paper (Bompadre et al., 2005), we demonstrated that ADP can shorten the open time of ⌬R-CFTR and this effect is more prominent when the channel is in the slow gating mode with longer Figure 1. Login to comment
83 ADP shortens the open time of ⌬R/D1370N-CFTR channels. Login to comment
89 Since the D1370N mutants exhibit open and closed times on the order of seconds (i.e., they mimic the slow gating mode described in Bompadre et al., 2005), we decided to use this mutant to further study the effect of ADP on the open time. Login to comment
90 Vergani et al. (2003) found that the ATP dose response for the D1370N mutant shifted to the right compared with that of WT channel. Login to comment
92 To study the effect of ADP on single-channel kinetics, we introduced the D1370N mutation into the ⌬R-CFTR background because ⌬R-CFTR is insensitive to dephosphorylation-induced rundown, and we can more easily obtain patches with fewer channels for kinetic analysis (Bompadre et al., 2005). Login to comment
93 In excised inside-out patches, ⌬R/D1370N-CFTR channels were opened with 1 mM ATP, and subsequent application of 1 mM ATP plus 1 mM ADP inhibited the currents by an average of 63 Ϯ 5% (n ϭ 5). Login to comment
102 Furthermore, the open time constant for D1370N-CFTR may still be too short for isolating the putative ADP-bound open state. Login to comment
321 From single-channel analysis, all we could observe was a shortening of the mean open time for ⌬R- (Bompadre et al., 2005) or ⌬R/ D1370N-CFTR (Fig. 1). Login to comment

[hide] Control of the CFTR channel's gates. Biochem Soc Trans. 2005 Nov;33(Pt 5):1003-7. Vergani P, Basso C, Mense M, Nairn AC, Gadsby DC
Control of the CFTR channel's gates.
Biochem Soc Trans. 2005 Nov;33(Pt 5):1003-7., [PMID:16246032]

Abstract [show]
Unique among ABC (ATP-binding cassette) protein family members, CFTR (cystic fibrosis transmembrane conductance regulator), also termed ABCC7, encoded by the gene mutated in cystic fibrosis patients, functions as an ion channel. Opening and closing of its anion-selective pore are linked to ATP binding and hydrolysis at CFTR's two NBDs (nucleotide-binding domains), NBD1 and NBD2. Isolated NBDs of prokaryotic ABC proteins form homodimers upon binding ATP, but separate after hydrolysis of the ATP. By combining mutagenesis with single-channel recording and nucleotide photolabelling on intact CFTR molecules, we relate opening and closing of the channel gates to ATP-mediated events in the NBDs. In particular, we demonstrate that two CFTR residues, predicted to lie on opposite sides of its anticipated NBD1-NBD2 heterodimer interface, are energetically coupled when the channels open but are independent of each other in closed channels. This directly links ATP-driven tight dimerization of CFTR's cytoplasmic NBDs to opening of the ion channel in the transmembrane domains. Evolutionary conservation of the energetically coupled residues in a manner that preserves their ability to form a hydrogen bond argues that this molecular mechanism, involving dynamic restructuring of the NBD dimer interface, is shared by all members of the ABC protein superfamily.

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39 To determine which of the two composite sites in the NBD1-NBD2 dimer is involved in channel opening, we introduced mutations at residues seen to interact directly with the bound nucleotide in the solved crystal structures, in the head of either NBD1 [K464A (Lys464 → Ala)] or NBD2 (D1370N). 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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160 This conclusion was reached after finding that the ATP dose-response relationships of the Walker A mutants K464A and K1250A and the Walker B mutant D1370N were shifted towards higher [ATP] com- paredto theATPdose-response curvefor wild-typechannels. Login to comment
181 Single channel analysis indicates that theY1219G mutation reduces the opening rate of the channel while not affecting the open time (i.e. this mutation probably does not affect ATP hydrolysis in ABP2 like K1250A or D1370N). Login to comment

[hide] Review. ATP hydrolysis-driven gating in cystic fib... Philos Trans R Soc Lond B Biol Sci. 2009 Jan 27;364(1514):247-55. Muallem D, Vergani P
Review. ATP hydrolysis-driven gating in cystic fibrosis transmembrane conductance regulator.
Philos Trans R Soc Lond B Biol Sci. 2009 Jan 27;364(1514):247-55., 2009-01-27 [PMID:18957373]

Abstract [show]
Proteins belonging to the ATP-binding cassette superfamily couple ATP binding and hydrolysis at conserved nucleotide-binding domains (NBDs) to diverse cellular functions. Most superfamily members are transporters, while cystic fibrosis transmembrane conductance regulator (CFTR), alone, is an ion channel. Despite this functional difference, recent results have suggested that CFTR shares a common molecular mechanism with other members. ATP binds to partial binding sites on the surface of the two NBDs, which then associate to form a NBD dimer, with complete composite catalytic sites now buried at the interface. ATP hydrolysis and gamma-phosphate dissociation, with the loss of molecular contacts linking the two sides of the composite site, trigger dimer dissociation. The conformational signals generated by NBD dimer formation and dissociation are transmitted to the transmembrane domains where, in transporters, they drive the cycle of conformational changes that translocate the substrate across the membrane; in CFTR, they result in opening and closing (gating) of the ion-permeation pathway.

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61 For both mutants (K464A in site 1 and D1370N in site 2), opening rate was reduced at low [ATP], but fast opening could be restored by increasing [ATP] (figure 2b, open symbols). Login to comment
86 (b) Hyperbolic relationship between [ATP] and opening rates (apparent dissociation constants are 56G5, 807G185, 391G118 mM for WT (filled circles), K464A (open triangles) and D1370N (open squares), respectively). Login to comment
59 For both mutants (K464A in site 1 and D1370N in site 2), opening rate was reduced at low [ATP], but fast opening could be restored by increasing [ATP] (figure 2b, open symbols). Login to comment
84 (b) Hyperbolic relationship between [ATP] and opening rates (apparent dissociation constants are 56G5, 807G185, 391G118 mM for WT (filled circles), K464A (open triangles) and D1370N (open squares), respectively). Login to comment

[hide] Direct sensing of intracellular pH by the cystic f... J Biol Chem. 2009 Dec 18;284(51):35495-506. Epub . Chen JH, Cai Z, Sheppard DN
Direct sensing of intracellular pH by the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel.
J Biol Chem. 2009 Dec 18;284(51):35495-506. Epub ., 2009-12-18 [PMID:19837660]

Abstract [show]
In cystic fibrosis (CF), dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel disrupts epithelial ion transport and perturbs the regulation of intracellular pH (pH(i)). CFTR modulates pH(i) through its role as an ion channel and by regulating transport proteins. However, it is unknown how CFTR senses pH(i). Here, we investigate the direct effects of pH(i) on recombinant CFTR using excised membrane patches. By altering channel gating, acidic pH(i) increased the open probability (P(o)) of wild-type CFTR, whereas alkaline pH(i) decreased P(o) and inhibited Cl(-) flow through the channel. Acidic pH(i) potentiated the MgATP dependence of wild-type CFTR by increasing MgATP affinity and enhancing channel activity, whereas alkaline pH(i) inhibited the MgATP dependence of wild-type CFTR by decreasing channel activity. Because these data suggest that pH(i) modulates the interaction of MgATP with the nucleotide-binding domains (NBDs) of CFTR, we examined the pH(i) dependence of site-directed mutations in the two ATP-binding sites of CFTR that are located at the NBD1:NBD2 dimer interface (site 1: K464A-, D572N-, and G1349D-CFTR; site 2: G551D-, K1250M-, and D1370N-CFTR). Site 2 mutants, but not site 1 mutants, perturbed both potentiation by acidic pH(i) and inhibition by alkaline pH(i), suggesting that site 2 is a critical determinant of the pH(i) sensitivity of CFTR. The effects of pH(i) also suggest that site 2 might employ substrate-assisted catalysis to ensure that ATP hydrolysis follows NBD dimerization. We conclude that the CFTR Cl(-) channel senses directly pH(i). The direct regulation of CFTR by pH(i) has important implications for the regulation of epithelial ion transport.

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6 Because these data suggest that pHi modulates the interaction of MgATP with the nucleotide-binding domains (NBDs) of CFTR, we examined the pHi dependence of site-directed mutations in the two ATP-binding sites of CFTR that are located at the NBD1:NBD2 dimer interface (site 1: K464A-, D572N-, and G1349D-CFTR; site 2: G551D-, K1250M-, and D1370N-CFTR). Login to comment
47 To study the CFTR variants K464A, D572N, and D1370N, we employed the vaccinia virus/bacteriophage T7 hybrid expression system to transiently express CFTR variants in HeLa cells as described previously (17, 18). Login to comment
204 Previous studies have demonstrated that the mutations D572N- and D1370N-CFTR abolish Mg2ϩ binding to the NBDs (21, 22). Login to comment
205 Figs. 6C and 8 and supplemental Fig. 3, B and C, demonstrate that the gating behavior of D572N- and D1370N-CFTR Cl-channels at different pHi diverges from that of wild-type CFTR in several important respects. Login to comment
206 First, at pHi 7.3, the Po of D572N-CFTR was the same as wild-type CFTR, whereas that of D1370N-CFTR was reduced (Fig. 6C). Login to comment
208 By contrast, for D1370N-CFTR at pHi 6.3, gating behavior and, hence, Po were unchanged (Fig. 6C and supplemental Fig. 3C). Login to comment
209 Third, in striking contrast to wild-type CFTR, at pHi 8.3 D572N-CFTR channel gating was enhanced because MBD was increased 0.6-fold and IBI decreased 0.3-fold, whereas that of D1370N-CFTR was unaltered (Figs. 2 and 8 and supplemental Fig. 3, B and C). Login to comment
210 As a result, at pHi 8.3, the Po of wild-type CFTR decreased, that of D1370N-CFTR was unchanged, whereas that of D572N-CFTR increased (Fig. 6C). Login to comment
213 Second the lack of effect of pHi on D1370N argues that the pHi sensitivity of CFTR channel gating is dependent on Mg2ϩ binding to site 2. Login to comment
218 B and C, effects of pHi on the Po of wild-type (WT), D572N-, and D1370N-CFTR in the presence of ATP (3 mM in B or 1 mM in C). Login to comment
219 In B, wild-type CFTR data were acquired in the presence (circles) and absence (columns) of Mg2ϩ (3 mM), whereas in C, wild-type, D572N-, and D1370N-CFTR data were acquired in the continuous presence of Mg2ϩ (3 mM). Login to comment
312 By contrast, Hϩ ions are either without effect (D1370N-CFTR) or inhibit (K1250M-CFTR) the gating behavior of site-directed mutations in ATP-binding site 2. 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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7 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. Login to comment
49 Burst Duration Distribution of Nonhydrolytic Mutant D1370N Further Supports Irreversible Mechanism for WT. Login to comment
50 As a control, we chose to study the NBD2 Walker B mutant D1370N because the analogous mutation completely abolished ATP hydrolysis in other ABC proteins (27-29). Login to comment
51 The distribution of burst durations of D1370N channels gating in 2 mM ATP (Fig. 1C), reconstructed from 530 bursts, indeed differs qualitatively from that of WT channels (Fig. 1B) in that it decays monotonically. Login to comment
54 Interestingly, a combination of two positive-amplitude exponential components slightly improved the fit (ΔLL = 3.24; P = 0.03; Fig. 1C, red solid line; see SI Text for more fitting results), suggesting a mixture of two types of open bursts for D1370N; the major population, with an average lifetime of ~2 s, seems interspersed with a few brief bursts of ~200 ms. Login to comment
60 (B-D) Histograms of open burst durations for prephosphorylated WT (B), D1370N (C), and K464A (D) CFTR channels; 30-s segments of representative single-channel current recordings are shown above each panel. Login to comment
65 ATP was 2 mM for WT and D1370N, but 5 mM for K464A. Login to comment
82 Insofar as ATP hydrolysis is also absent in D1370N (k1 = 0), for this mutant the rate of channel closure from an open burst reflects the rate of dissociation of the prehydrolytic NBD dimer (k-1; Fig. 4E Right, green bar). Login to comment
104 Fig. 4 A-C compares such average parameters for fully (navy blue) and partially (royal blue) phosphorylated WT, and partially phosphorylated D1370N (green) and K464A (red) CFTR channels. Login to comment
105 Consistent with previous reports, for D1370N CFTR channels gating in near-saturating ATP (2 mM) (16), τb is ~4-fold longer than, but τib is like, that of WT (Fig. 4 B and C, green bars) (cf. refs. 9, 16, 30), whereas for prephosphorylated K464A CFTR channels in saturating ATP (5 mM) (16), τb is comparable to, but τib is at least ~2-fold longer than, that of WT (Fig. 4 B and C, red bars) (9, 14, 16 but cf. ref. 32). Login to comment
111 The interpretation that this irreversible cycle is driven by ATP hydrolysis is validated by the lack of any such fit improvement for the presumed nonhydrolytic D1370N mutant (Fig. 1C). Login to comment
126 The nonhydrolytic NBD2 mutant D1370N is a more extreme case, with an estimated coupling ratio of 0% (green bars, Fig. 4E Left and Right and Fig. 4F). Login to comment
141 Our analysis of the D1370N mutant, for example, implies that this mutation does not prevent cycling between inward- (closed channel) and outward-facing (open channel) conformations of CFTR (Fig. 4D, green; cf. Fig. 4F). Login to comment
161 (A-D) Open probabilities (A), mean burst (B), and interburst (C) durations obtained from multichannel fits, and calculated channel cycle times (D) for fully (navy blue) and partially (royal blue) phosphorylated WT, and partially phosphorylated D1370N (green) and K464A (red) CFTR. Login to comment
162 [ATP] was 2 mM for WT and D1370N, but 5 mM for K464A. Login to comment
164 (E) ML estimates of rates k1 (Left), k2 (Center), and k-1 (Right) for fully (navy blue) and partially (royal blue) phosphorylated WT, and partially phosphorylated D1370N (green) and K464A (red) CFTR channels. Login to comment
169 Probabilities for exiting state O1 (Top Right) in either of two possible directions are printed in color for partially phosphorylated WT (blue), K464A (red), and D1370N (green). Login to comment
184 Therefore, although both methods yielded qualitatively similar results, we used the distributions obtained using method (i) for WT and K464A (Figs. 1 B and D, 3B, and 4), and that obtained using method (ii) for D1370N (Fig. 1C). 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] State-dependent regulation of cystic fibrosis tran... J Biol Chem. 2010 Dec 24;285(52):40438-47. Epub 2010 Oct 15. Wang G
State-dependent regulation of cystic fibrosis transmembrane conductance regulator (CFTR) gating by a high affinity Fe3+ bridge between the regulatory domain and cytoplasmic loop 3.
J Biol Chem. 2010 Dec 24;285(52):40438-47. Epub 2010 Oct 15., 2010-12-24 [PMID:20952391]

Abstract [show]
The unique regulatory (R) domain differentiates the human CFTR channel from other ATP-binding cassette transporters and exerts multiple effects on channel function. However, the underlying mechanisms are unclear. Here, an intracellular high affinity (2.3 x 10(-19) M) Fe(3+) bridge is reported as a novel approach to regulating channel gating. It inhibited CFTR activity by primarily reducing an open probability and an opening rate, and inhibition was reversed by EDTA and phenanthroline. His-950, His-954, Cys-832, His-775, and Asp-836 were found essential for inhibition and phosphorylated Ser-768 may enhance Fe(3+) binding. More importantly, inhibition by Fe(3+) was state-dependent. Sensitivity to Fe(3+) was reduced when the channel was locked in an open state by AMP-PNP. Similarly, a K978C mutation from cytoplasmic loop 3 (CL3), which promotes ATP-independent channel opening, greatly weakened inhibition by Fe(3+) no matter whether NBD2 was present or not. Therefore, although ATP binding-induced dimerization of NBD1-NBD2 is required for channel gating, regulation of CFTR activity by Fe(3+) may involve an interaction between the R domain and CL3. These findings may support proximity of the R domain to the cytoplasmic loops. They also suggest that Fe(3+) homeostasis may play a critical role in regulating pathophysiological CFTR activity because dysregulation of this protein causes cystic fibrosis, secretary diarrhea, and infertility.

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83 More importantly, a D1370N mutant, which removes the Mg2ϩ binding site (32), was also inhibited by Fe3ϩ (Fig. 1C), and inhibition was reversed by 5 mM EDTA (Fig. 1E). Login to comment
84 However, unlike the WT channel, the D1370N mutant exhibited less inhibition by Fe3ϩ but more reversal of inhibition by EDTA. Login to comment
108 The arrows indicate the final concentrations. C, effects of Fe3ϩ (5 ϫ 10-19 M) and Fe2ϩ (5 ϫ 10-13 M) on the WT hCFTR and the D1370N mutant currents (n ϭ 4-8). 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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113 To further explore this relationship between the mean open time and the potency of CFTRinh-172, we tested CFTRinh-172 on a CFTR mutant, D1370N-CFTR, which exhibits an open time of 1 s (Bompadre et al., 2005). Login to comment
116 Fig. 4 B demonstrates the effects of 1 µM CFTRinh-172 on the macroscopic current of D1370N-CFTR. Login to comment
142 For D1370N-CFTR with an open time of 1 s, the IC50 is already in the nanomolar range. Login to comment
160 (A) Representative single-channel trace for D1370N-CFTR, a hydrolysis-deficient mutant with a mean open time of 1 s. Login to comment
161 (B) A continuous current recording showing a reversible inhibition of macroscopic D1370N-CFTR currents by 1 µM CFTRinh-172. Login to comment
162 (C) Dose-response relationships of CFTRinh-172 for WT-CFTR gated by ATP or P-ATP and D1370N-CFTR gated by ATP. Login to comment
165 Fitting parameters: for P-ATP-gated WT channels: IC50 = 0.37 ± 0.22 µM and Hill coefficient, n = 0.69 ± 0.38; for D1370N-CFTR: IC50 = 0.064 ± 0.007 µM and n = 1.26 ± 0.17. Login to comment

[hide] Regulation of Cl-/ HCO3- exchange by cystic fibros... J Biol Chem. 1999 Feb 5;274(6):3414-21. Lee MG, Wigley WC, Zeng W, Noel LE, Marino CR, Thomas PJ, Muallem S
Regulation of Cl-/ HCO3- exchange by cystic fibrosis transmembrane conductance regulator expressed in NIH 3T3 and HEK 293 cells.
J Biol Chem. 1999 Feb 5;274(6):3414-21., 1999-02-05 [PMID:9920885]

Abstract [show]
A central function of cystic fibrosis transmembrane conductance regulator (CFTR)-expressing tissues is the secretion of fluid containing 100-140 mM HCO3-. High levels of HCO3- maintain secreted proteins such as mucins (all tissues) and digestive enzymes (pancreas) in a soluble and/or inactive state. HCO3- secretion is impaired in CF in all CFTR-expressing, HCO3--secreting tissues examined. The mechanism responsible for this critical problem in CF is unknown. Since a major component of HCO3- secretion in CFTR-expressing cells is mediated by the action of a Cl-/HCO3- exchanger (AE), in the present work we examined the regulation of AE activity by CFTR. In NIH 3T3 cells stably transfected with wild type CFTR and in HEK 293 cells expressing WT and several mutant CFTR, activation of CFTR by cAMP stimulated AE activity. Pharmacological and mutagenesis studies indicated that expression of CFTR in the plasma membrane, but not the Cl- conductive function of CFTR was required for activation of AE. Furthermore, mutations in NBD2 altered regulation of AE activity by CFTR independent of their effect on Cl- channel activity. At very high expression levels CFTR modified the sensitivity of AE to 4,4'-diisothiocyanatostilbene-2, 2'-disulfonate. The novel finding of regulation of Cl-/HCO3- exchange by CFTR reported here may have important physiological implications and explain, at least in part, the impaired HCO3- secretion in CF.

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52 The mutagenesis primers were as follows: P205S primer, 5Ј-CGT GTG GAT CGC TTC TTT GCA AGT GGC-3Ј; W846term, 5Ј-GAG CAT ACC AGC AGT GAC TAC ATA GAA CAC ATA CCT TCG ATA TAT TAC-3Ј; G1247D/G1249E, 5Ј-GTG GGC CTC TTG GGA AGA ACT GAT TCA GAG AAG AGT ACT TTG TTA TCA GC-3Ј; K1250M, 5Ј-CTT GGG AAG AAC TGG ATC AGG GAT GAG TAC TTT GTT ATC AGC-3Ј; D1370N, 5Ј-GTA AGG CGA AGA TCT TGC TGC TTA ATG AAC CCA GTG CTC ATT TGG ATC-3Ј. Login to comment
163 Fig. 6 (i-k) shows the plasma membrane localization of K1250M CFTR, D1370N CFTR, and the double mutant G1247D/ G1249E CFTR, respectively. Login to comment
241 However, D1370N CFTR had nearly normal Cl-channel activity (32) and was expressed in the plasma membrane (Fig. 6j), but was unable to activate AE (Fig. 12c). Login to comment
266 The D1370N mutant had minimal effect on AE activity (c). Login to comment
273 In this respect the results obtained with D1370N CFTR are of particular interest since this mutation in NBD2 did not ablate channel activity (32) but eliminated regulation of AE activity by CFTR. Login to comment
274 Recently, it was reported that the D1506A mutation of the sulfonylurea receptor 1 protein, which corresponds to D1370N of human CFTR, similarly failed to stimulate the KATP channel (33). Login to comment

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

Abstract [show]
Cystic fibrosis transmembrane conductance regulator (CFTR) is a membrane-spanning adenosine 5'-triphosphate (ATP)-binding cassette (ABC) transporter. ABC transporters and other nuclear and cytoplasmic ABC proteins have ATPase activity that is coupled to their biological function. Recent studies with CFTR and two nonmembrane-bound ABC proteins, the DNA repair enzyme Rad50 and a structural maintenance of chromosome (SMC) protein, challenge the model that the function of all ABC proteins depends solely on their associated ATPase activity. Patch clamp studies indicated that in the presence of physiologically relevant concentrations of adenosine 5'-monophosphate (AMP), CFTR Cl(-) channel function is coupled to adenylate kinase activity (ATP+AMP &lrarr2; 2 ADP). Work with Rad50 and SMC showed that these enzymes catalyze both ATPase and adenylate kinase reactions. However, despite the supportive electrophysiological results with CFTR, there are no biochemical data demonstrating intrinsic adenylate kinase activity of a membrane-bound ABC transporter. We developed a biochemical assay for adenylate kinase activity, in which the radioactive gamma-phosphate of a nucleotide triphosphate could transfer to a photoactivatable AMP analog. UV irradiation could then trap the (32)P on the adenylate kinase. With this assay, we discovered phosphoryl group transfer that labeled CFTR, thereby demonstrating its adenylate kinase activity. Our results also suggested that the interaction of nucleotide triphosphate with CFTR at ATP-binding site 2 is required for adenylate kinase activity. These biochemical data complement earlier biophysical studies of CFTR and indicate that the ABC transporter CFTR can function as an adenylate kinase.

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183 2) Patch clamp studies showed that mutations K1250A and D1370N, located within conserved motifs of ATP-binding site 2, abolished the effects of Ap5A and AMP on CFTR current. Login to comment

[hide] A mutation in CFTR modifies the effects of the ade... Biophys J. 2008 Dec;95(11):5178-85. Epub 2008 Sep 19. Dong Q, Randak CO, Welsh MJ
A mutation in CFTR modifies the effects of the adenylate kinase inhibitor Ap5A on channel gating.
Biophys J. 2008 Dec;95(11):5178-85. Epub 2008 Sep 19., [PMID:18805924]

Abstract [show]
Mutations in the gene that encodes the cystic fibrosis transmembrane conductance regulator (CFTR) cause cystic fibrosis. The CFTR anion channel is controlled by ATP binding and enzymatic activity at the two nucleotide-binding domains. CFTR exhibits two types of enzymatic activity: 1), ATPase activity in the presence of ATP and 2), adenylate kinase activity in the presence of ATP plus physiologic concentrations of AMP or ADP. Previous work showed that P(1),P(5)-di(adenosine-5')pentaphosphate (Ap(5)A), a specific adenylate kinases inhibitor, inhibited wild-type CFTR. In this study, we report that Ap(5)A increased activity of CFTR with an L1254A mutation. This mutation increased the EC50 for ATP by >10-fold and reduced channel activity by prolonging the closed state. Ap(5)A did not elicit current on its own nor did it alter ATP EC50 or maximal current. However, it changed the relationship between ATP concentration and current. At submaximal ATP concentrations, Ap(5)A stimulated current by stabilizing the channel open state. Whereas previous work indicated that adenylate kinase activity regulated channel opening, our data suggest that Ap(5)A binding may also influence channel closing. These results also suggest that a better understanding of the adenylate kinase activity of CFTR may be of value in developing new therapeutic strategies for cystic fibrosis.

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171 For example, the K1250A and D1370N mutations also have a reduced opening rate, prolonged burst duration, and increased ATP EC50 (11,12,14,15,29,32,39,43). Login to comment

[hide] Cystic fibrosis: a multiple exocrinopathy caused b... Am J Med. 1998 Jun;104(6):576-90. Schwiebert EM, Benos DJ, Fuller CM
Cystic fibrosis: a multiple exocrinopathy caused by dysfunctions in a multifunctional transport protein.
Am J Med. 1998 Jun;104(6):576-90., [PMID:9674722]

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224 In NBD2, a few key mutations have been found that include missense mutations (G1349D, D1370N, K1250M, K1250Q, G1244E, S1255P) and several nonsense mutations (W1282X, S1255X, W1316X). Login to comment

[hide] ClC and CFTR chloride channel gating. Annu Rev Physiol. 1998;60:689-717. Foskett JK
ClC and CFTR chloride channel gating.
Annu Rev Physiol. 1998;60:689-717., [PMID:9558482]

Abstract [show]
Chloride channels are widely expressed and play important roles in cell volume regulation, transepithelial transport, intracellular pH regulation, and membrane excitability. Most chloride channels have yet to be identified at a molecular level. The ClC gene family and the cystic fibrosis transmembrane conductance regulator (CFTR) are distinct chloride channels expressed in many cell types, and mutations in their genes are the cause of several diseases including myotonias, cystic fibrosis, and kidney stones. Because of their molecular definition and roles in disease, these channels have been studied intensively over the past several years. The focus of this review is on recent studies that have provided new insights into the mechanisms governing the opening and closing, i.e. gating, of the ClC and CFTR chloride channels.

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324 An interesting contrast to the effects of the K1250A mutation in the Walker A motif was revealed in a D1370N mutant in the Walker B motif. Login to comment

[hide] CFTR: the nucleotide binding folds regulate the ac... J Gen Physiol. 1996 Jan;107(1):103-19. Wilkinson DJ, Mansoura MK, Watson PY, Smit LS, Collins FS, Dawson DC
CFTR: the nucleotide binding folds regulate the accessibility and stability of the activated state.
J Gen Physiol. 1996 Jan;107(1):103-19., [PMID:8741733]

Abstract [show]
The functional roles of the two nucleotide binding folds, NBF1 and NBF2, in the activation of the cystic fibrosis transmembrane conductance regulator (CFTR) were investigated by measuring the rates of activation and deactivation of CFTR Cl- conductance in Xenopus oocytes. Activation of wild-type CFTR in response to application of forskolin and 3-isobutyl-1-methylxanthine (IBMX) was described by a single exponential. Deactivation after washout of the cocktail consisted of two phases: an initial slow phase, described by a latency, and an exponential decline. Rate analysis of CFTR variants bearing analogous mutations in NBF1 and NBF2 permitted us to characterize amino acid substitutions according to their effects on the accessibility and stability of the active state. Access to the active state was very sensitive to substitutions for the invariant glycine (G551) in NBF1, where mutations to alanine (A), serine (S), or aspartic acid (D) reduced the apparent on rate by more than tenfold. The analogous substitutions in NBF2 (G1349) also reduced the on rate, by twofold to 10-fold, but substantially destabilized the active state as well, as judged by increased deactivation rates. In the putative ATP-binding pocket of either NBF, substitution of alanine, glutamine (Q), or arginine (R) for the invariant lysine (K464 or K1250) reduced the on rate similarly, by two- to fourfold. In contrast, these analogous substitutions produced opposite effects on the deactivation rate. NBF1 mutations destabilized the active state, whereas the analogous substitutions in NBF2 stabilized the active state such that activation was prolonged compared with that seen with wild-type CFTR. Substitution of asparagine (N) for a highly conserved aspartic acid (D572) in the ATP-binding pocket of NBF1 dramatically slowed the on rate and destabilized the active state. In contrast, the analogous substitution in NBF2 (D1370N) did not appreciably affect the on rate and markedly stabilized the active state. These results are consistent with a hypothesis for CFTR activation that invokes the binding and hydrolysis of ATP at NBF1 as a crucial step in activation, while at NBF2, ATP binding enhances access to the active state, but the rate of ATP hydrolysis controls the duration of the active state. The relatively slow time courses for activation and deactivation suggest that slow processes modulate ATP-dependent gating.

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11 In contrast, the analogous substitution in NBF2 (D1370N) did not appreciably affect the on rate and markedly stabilized the active state. These results are consistent with a hypothesis for CFTR activation that invokes the binding and hydrolysis of ATP at NBF1 as a crucial step in activation, while at NBF2, ATP binding enhances access to the active state, but the rate of ATP hydrolysis controls the duration of the active state. Login to comment
19 In contrast, mutations in the putative ATP-binding pockets of the two NBFs produced opposite results, a reduction in sensitivity for mutations in NBF1 (K464Q, D572N) and an increase in sensitivity for the analogous mutations in NBF2 (K12500~ D1370N). Login to comment
195 a~ODiap- 9 K464Q a I ' ' ' ' I ' ' ' ' I ' ' 0 10 20 ~ O -0 0, 9 -0~176176176o ....... 9.... -o*- -o*- 9 i~ e'~176176176176 9 D572N o i , , , , i , , , , i , , , , I , , , , i , , , , i , , , , 0 I0 20 30 40 50 minutes K1250A K1250C I i 30 D1370N 6O FIGURE4. Login to comment
201 (C) Substitutions of asparagine for the Walker consensus B aspartic acid in the ATP-binding pocket of NBFI (D572N, 0) or NBF2 (D1370N,0). Login to comment
247 0 30 60 90 12O 15O i \ 9 DsZ2No D1370N iX',, - ~ o ~ ***************************** I ' ' ' I ' ' ' I ' ' ' I ' ' ' I ' ' ' 0 20 40 60 80 100 minutes FIGURE 6. Login to comment
256 In contrast, the analogous substitution in NBF2 (D1370N) produced only a modest decrease in (ko, + kor0, evident in Fig. 4 C. The values of the derived parameters (k'o,, ko~) for this slightly hypersensitive mutant, however, suggest that the decrease in KAwas a reflection of a fourfold decrease in ko~, whereas the apparent kon was not significantly different from that of wild-type CFTR. Login to comment
261 Particularly noteworthy in this regard is the D1370N mutation, which produced no significant effect on the apparent on rate but markedly delayed deactivation. Login to comment
277 Similarly, although the K1250R and D1370N mutants exhibited an increased latency, the values of *ko~ were not significantly different from that of wild type CFTR. Login to comment
281 + kott) (10-3 min-l kon kofr latency *k~m CFTR (mM) n (10-3min-]) mM-1) (10-3min 1) (10-3min-l) n (min) (10 3min i) n wt 0.65 • 0.08 26 664 • 51 118 • 9 558 • 45 76-+ 6 20 6.0 • 0.3 88 • 6 16 K464R 2.6 • 0.1": 4 153 + 20**+ 20 • 3*** 101 • 13''` 52 • 7*: 5 1.3 • 0.2*++ 174 • 14"** 7 K464Q 3.3 • 0.5"* 5 331 • 56*** 40 -+ 7* 199 • 34* 132 • 22*'` 5 1.9 • 0.3"I 142 -+ 19''` 5 K464A 4.6 • 0.7** 6 289 • 49* 30 • 5** 151 • 26*** 139 • 24*: 7 1.1 • 0.1"** 133 • 14"** 8 D572N 9.3 + 0.02*: 6 106 • 7*: 7-+0.5*: 37-+3*** 69 • 5+* 4 0.9 • 0.2*** 245 • 32*: 3 K1250R 0.17 • 0.07*: 5 239 •33*** 46 -+ 6"+* 231 • 32*: 8 • 1": 10 10.4 • 0.8"~ 100 • 7** 6 K1250Q 0.12 • 0.04*** 5 150 • 18''` 29 • 4* 146 -+ 18" 4 + 0.4"I 5 22.3 • 2.4*: 30 •5": 5 K1250A 0.07 + 0.02*: 10 218 • 18" 43 • 4*'` 215 • 18": 3 -+0.3*~* 5 15.6-+ 1.0"** 43 -+5** 5 D1370N 0.16 + 0.04*'` 7 449 - 79*: 87 • 15: 435 +76** 14 - 2*: 5 16.3-4-1.2"" 69-+ 6** 5 The symbols (*) and ('`) indicate significant differences from wild-type CFTR and the analogous mutant, respectively (P < 0.05). Login to comment
321 Of particular interest in this regard was the effect of the NBF2 mutation, D1370N. Login to comment
339 Although the structural and functional studies of these other proteins do not offer a completely coherent picture of the role of the Walker B aspartic acid, the results of the rate analysis for the CFTR mutant D1370N are consistent with a role for this aspartic acid in ATP hydrolysis. Login to comment
198 a~ODiap- 9 K464Q a I ' ' ' ' I ' ' ' ' I ' ' 0 10 20 ~ O -0 0, 9 -0 ~176176176 o ....... 9.... -o*- -o*- 9 i~ e'~176176176176 9 D572N o i , , , , i , , , , i , , , , I , , , , i , , , , i , , , , 0 I0 20 30 40 50 minutes K1250A K1250C I i 30 D1370N 6O FIGURE4. Login to comment
204 (C) Substitutions of asparagine for the Walker consensus B aspartic acid in the ATP-binding pocket of NBFI (D572N, 0) or NBF2 (D1370N,0). Login to comment
249 0 30 60 90 12O 15O i \ 9 DsZ2No D1370N iX',, - ~ o ~ ***************************** I ' ' ' I ' ' ' I ' ' ' I ' ' ' I ' ' ' 0 20 40 60 80 100 minutes FIGURE 6. Login to comment
258 In contrast, the analogous substitution in NBF2 (D1370N) produced only a modest decrease in (ko, + kor0, evident in Fig. 4 C. The values of the derived parameters (k'o,, ko~) for this slightly hypersensitive mutant, however, suggest that the decrease in KAwas a reflection of a fourfold decrease in ko~, whereas the apparent kon was not significantly different from that of wild-type CFTR. Login to comment
263 Particularly noteworthy in this regard is the D1370N mutation, which produced no significant effect on the apparent on rate but markedly delayed deactivation. Login to comment
279 Similarly, although the K1250R and D1370N mutants exhibited an increased latency, the values of *ko~ were not significantly different from that of wild type CFTR. Login to comment
283 + kott) (10-3 min-l kon kofr latency *k~m CFTR (mM) n (10-3 min-]) mM-1) (10-3 min 1) (10-3min-l) n (min) (10 3min i) n wt 0.65 ߦ 0.08 26 664 ߦ 51 118 ߦ 9 558 ߦ 45 76 -+ 6 20 6.0 ߦ 0.3 88 ߦ 6 16 K464R 2.6 ߦ 0.1": 4 153 + 20**+ 20 ߦ 3*** 101 ߦ 13''` 52 ߦ 7*: 5 1.3 ߦ 0.2*++ 174 ߦ 14"** 7 K464Q 3.3 ߦ 0.5"* 5 331 ߦ 56*** 40 -+ 7* 199 ߦ 34* 132 ߦ 22*'` 5 1.9 ߦ 0.3"I 142 -+ 19''` 5 K464A 4.6 ߦ 0.7** 6 289 ߦ 49* 30 ߦ 5** 151 ߦ 26*** 139 ߦ 24*: 7 1.1 ߦ 0.1"** 133 ߦ 14"** 8 D572N 9.3 + 0.02*: 6 106 ߦ 7*: 7 -+0.5*: 37 -+3*** 69 ߦ 5+* 4 0.9 ߦ 0.2*** 245 ߦ 32*: 3 K1250R 0.17 ߦ 0.07*: 5 239 ߦ 33*** 46 -+ 6"+* 231 ߦ 32*: 8 ߦ 1": 10 10.4 ߦ 0.8"~ 100 ߦ 7** 6 K1250Q 0.12 ߦ 0.04*** 5 150 ߦ 18''` 29 ߦ 4* 146 -+ 18" 4 + 0.4"I 5 22.3 ߦ 2.4*: 30 ߦ 5": 5 K1250A 0.07 + 0.02*: 10 218 ߦ 18" 43 ߦ 4*'` 215 ߦ 18": 3 -+0.3*~* 5 15.6 -+ 1.0"** 43 -+5** 5 D1370N 0.16 + 0.04*'` 7 449 - 79*: 87 ߦ 15: 435 + 76** 14 - 2*: 5 16.3 -4-1.2"" 69 -+ 6** 5 The symbols (*) and ('`) indicate significant differences from wild-type CFTR and the analogous mutant, respectively (P < 0.05). Login to comment
323 Of particular interest in this regard was the effect of the NBF2 mutation, D1370N. Login to comment
341 Although the structural and functional studies of these other proteins do not offer a completely coherent picture of the role of the Walker B aspartic acid, the results of the rate analysis for the CFTR mutant D1370N are consistent with a role for this aspartic acid in ATP hydrolysis. Login to comment

[hide] Epitope tagging permits cell surface detection of ... Am J Physiol. 1995 Dec;269(6 Pt 1):C1565-76. Howard M, DuVall MD, Devor DC, Dong JY, Henze K, Frizzell RA
Epitope tagging permits cell surface detection of functional CFTR.
Am J Physiol. 1995 Dec;269(6 Pt 1):C1565-76., [PMID:8572187]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) is a phosphorylation-activated Cl channel responsible for adenosine 3',5'-cyclic monophosphate (cAMP)-induced Cl secretion across the apical membranes of epithelial cells. To optimize its detection for membrane localization studies, we tagged CFTR with epitope sequences at the carboxy terminus or in the fourth external loop. When epitopes were added to the fourth external loop, the N-linked glycosylation sites in that loop were either preserved or they were mutated to produce a deglycosylated CFTR (dgCFTR). Tagged CFTRs were expressed in HeLa cells, and their cAMP-sensitive Cl permeability was assayed using the halide-sensitive fluorophore SPQ. CFTRs containing the M2 epitope showed halide permeability responses to cAMP, whereas cells expressing CFTR with the hemagglutinin (HA) tag showed little or no cAMP response. Xenopus oocytes expressing dgCFTR, with or without the M2 epitope, showed Cl conductance responses that were 20% of the wild-type response, whereas M2-tagged constructs retaining the glycosylation sites responded like wild-type CFTR. External M2-tagged CFTR was detected in the surface membrane of nonpermeabilized cells. The surface expression of the mutant M2-tagged CFTRs correlated with processing of these mutants (Gregory et al. Mol. Cell. Biol. 11:3886-3893, 1991). M2-901/CFTR is a useful reporter for the trafficking of wild-type and mutant CFTRs to the cell surface.

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251 M2-901 CFIR 5 0 -1 mV C constructed two dgCFTR mutants that are not associated with disease, K1250M and D1370N, also bearing This is a technical study that identifies sites appropri- the M2 epitope at position 901. Login to comment
255 Cell surface expression of M2-901/CFTR has been observed in a expression of M2-901-labeled G551D, G1349D, K1250M, and D1370N dgCFTRs correlates with carbohydrate variety of cell types including HEp-2, BSC40, and addition (Fig &A-II). Login to comment
265 A: G551D; B: G1349D; C: K1250M; D: D1370N; E: AF508; F: N1303K. Login to comment

[hide] Conformational states of CFTR associated with chan... Cell. 1995 Jul 28;82(2):231-9. Gunderson KL, Kopito RR
Conformational states of CFTR associated with channel gating: the role ATP binding and hydrolysis.
Cell. 1995 Jul 28;82(2):231-9., [PMID:7543023]

Abstract [show]
CFTR is a member of the traffic ATPase superfamily and a Cl- ion channel that appears to require ATP hydrolysis for gating. Analysis of single CFTR Cl- channels reconstituted into planar lipid bilayers revealed the presence of two open conductance states that are connected to each other and to the closed state by an asymmetric cycle of gating events. We show here that the transition between the two open conductance states is directly coupled to ATP hydrolysis by one of the consensus nucleotide-binding folds, designated NBF2. Moreover, the transition between the closed state and one of the open states is linked to the binding of ATP. This analysis permits real-time visualization of conformational changes associated with a single cycle of ATP hydrolysis by a single protein molecule and suggests a model describing a role for ATP in CFTR gating.

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92 0 2 B low MI 2* C D1370N (high Mg2+) 2sec -----]0,5 pA Figure 4. Login to comment
109 Gating of CFTR channels harboring a D1370N mutation closely resembled that of the wild-type channel in EDTA (Figure 4C). Login to comment
111 Moreover, even in the presence of Mg2+,we were unable to induce locking of the D1370N channels with PPi (data not shown). Login to comment
114 By contrast, D1370N mutants, which can be mimicked by applying EDTA to wild-type channels, are also blocked from completing the full (C~O1~O2~C) gating cycle through 02, but are neither locked open in O1 nor capable of becoming locked by PP~.These two classes of mutants thus define two kinetic states of the O1 conductance level: an initial open state that is reversible (back to C) and independent of Mg2÷, and a subsequent state that requires Mg2÷ and is effectively irreversible. Login to comment
121 The simplest interpretation of these data is that ATP binding ConformationalStatesof CFTR 09 08 07 06 Oo 05 040.3 * 32 wildtype K464A K1250A G1247D/ D1370N 01249E 6~se' B 2500 20O0 5 1500 1OO0 500 o~ 0 w[Id{ype K464A K1250A C1247D/ Ol 370N G1249E Figure5. Login to comment
241 Acknowledgments Correspondence should be addressed to R. R. K. We thank M. Welsh for kindly providing the K1250M and D1370N mutants, P. Quinton for his insightful discussions and critical reading of the manuscript, C. Login to comment
110 Gating of CFTR channels harboring a D1370N mutation closely resembled that of the wild-type channel in EDTA (Figure 4C). Login to comment
112 Moreover, even in the presence of Mg2+,we were unable to induce locking of the D1370N channels with PPi (data not shown). Login to comment
115 By contrast, D1370N mutants, which can be mimicked by applying EDTA to wild-type channels, are also blocked from completing the full (C~O1~O2~C) gating cycle through 02, but are neither locked open in O1 nor capable of becoming locked by PP~.These two classes of mutants thus define two kinetic states of the O1 conductance level: an initial open state that is reversible (back to C) and independent of Mg2&#f7;, and a subsequent state that requires Mg2&#f7; and is effectively irreversible. Login to comment
122 The simplest interpretation of these data is that ATP binding ConformationalStatesof CFTR 09 08 07 06 Oo 05 040.3 * 32 wildtype K464A K1250A G1247D/ D1370N 01249E 6~se' B 2500 20O0 5 1500 1OO0 500 o ~ 0 w[Id{ype K464A K1250A C1247D/ Ol 370N G1249E Figure5. Login to comment
242 Acknowledgments Correspondence should be addressed to R. R. K. We thank M. Welsh for kindly providing the K1250M and D1370N mutants, P. Quinton for his insightful discussions and critical reading of the manuscript, C. Login to comment

[hide] Functional roles of the nucleotide-binding folds i... Proc Natl Acad Sci U S A. 1993 Nov 1;90(21):9963-7. Smit LS, Wilkinson DJ, Mansoura MK, Collins FS, Dawson DC
Functional roles of the nucleotide-binding folds in the activation of the cystic fibrosis transmembrane conductance regulator.
Proc Natl Acad Sci U S A. 1993 Nov 1;90(21):9963-7., [PMID:7694298]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR), a member of the traffic ATPase superfamily, possesses two putative nucleotide-binding folds (NBFs). The NBFs are sufficiently similar that sequence alignment of highly conserved regions can be used to identify analogous residues in the two domains. To determine whether this structural homology is paralleled in function, we compared the activation of chloride conductance by forskolin and 3-isobutyl-1-methylxanthine in Xenopus oocytes expressing CFTRs bearing mutations in NBF1 or NBF2. Mutation of a conserved glycine in the putative linker domain in either NBF produced virtually identical changes in the sensitivity of chloride conductance to activating conditions, and mutation of this site in both NBFs produced additive effects, suggesting that in the two NBFs this region plays a similar and critical role in the activation process. In contrast, amino acid substitutions in the Walker A and B motifs, thought to form an integral part of the nucleotide-binding pockets, produced strikingly different effects in NBF1 and NBF2. Substitutions for the conserved lysine (Walker A) or aspartate (Walker B) in NBF1 resulted in a marked decrease in sensitivity to activation, whereas the same changes in NBF2 produced an increase in sensitivity. These results are consistent with a model for the activation of CFTR in which both NBF1 and NBF2 are required for normal function but in which either the nature or the exact consequences of nucleotide binding differ for the two domains.

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60 For this reason, expression levels for easily activated constructs (wild type, G551A, G1349A, K1250Q, and D1370N) were adjusted by reducing the amount of injected RNA so that the maximum Cl- conductance was similar to that attained by less sensitive constructs. Login to comment
108 A 80+ 60 + 40+- 20 + wt rl% - 7 1 k (12) t rd:\z-Za uab/ZN k b) A-- K A D1370N ( 5) T/ z T A T I I 0 f I~0A I~~~~~~ T t u A~~~~ 0.2. ,0.02 0.05 0.2 0.5 IBMX, mM 2 45 FIG. 5. Login to comment

[hide] An intrinsic adenylate kinase activity regulates g... Cell. 2003 Dec 26;115(7):837-50. Randak C, Welsh MJ
An intrinsic adenylate kinase activity regulates gating of the ABC transporter CFTR.
Cell. 2003 Dec 26;115(7):837-50., [PMID:14697202]

Abstract [show]
Cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel in the ATP binding cassette (ABC) transporter family. Like other ABC transporters, it can hydrolyze ATP. Yet while ATP hydrolysis influences channel gating, it has long seemed puzzling that CFTR would require this reaction because anions flow passively through CFTR. Moreover, no other ion channel is known to require the large energy of ATP hydrolysis to gate. We found that CFTR also has adenylate kinase activity (ATP + AMP <=> ADP + ADP) that regulates gating. When functioning as an adenylate kinase, CFTR showed positive cooperativity for ATP suggesting its two nucleotide binding domains may dimerize. Thus, channel activity could be regulated by two different enzymatic reactions, ATPase and adenylate kinase, that share a common ATP binding site in the second nucleotide binding domain. At physiologic nucleotide concentrations, adenylate kinase activity, rather than ATPase activity may control gating, and therefore involve little energy consumption.

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228 In addition, we Walker A and B motif mutations (K1250A and D1370N) abolished both ATPase and adenylate kinase activities studied a relatively common CF-associated mutation, N1303K (Osborne et al., 1992); interestingly, two other (Figures 7A and 7B). Login to comment
272 Results with the Walker B Asp Discussion mutations (D572N in NBD1 and D1370N in NBD2) exactly paralleled the P loop mutations (Figures 7D-7F), further Earlier work indicated that CFTR can function as an ATPase and that hydrolysis contributes to channel gat- suggesting that adenylate kinase activity resides in NBD2. Login to comment
280 NBD2 was wild-type or contained K1250A, D1370N, or N1303K mutations. Login to comment
288 Data are from 5 (wild-type, K464A, D572N), 9 (K1250A), 10 (D1370N), and 3 (N1303K) membrane patches. Asterisks indicate p b0d; 0.05 compared to wild-type by ANOVA followed by Dunnett`s multiple comparison test. Login to comment

[hide] ATP and AMP mutually influence their interaction w... J Biol Chem. 2013 Sep 20;288(38):27692-701. doi: 10.1074/jbc.M113.479675. Epub 2013 Aug 6. Randak CO, Dong Q, Ver Heul AR, Elcock AH, Welsh MJ
ATP and AMP mutually influence their interaction with the ATP-binding cassette (ABC) adenylate kinase cystic fibrosis transmembrane conductance regulator (CFTR) at separate binding sites.
J Biol Chem. 2013 Sep 20;288(38):27692-701. doi: 10.1074/jbc.M113.479675. Epub 2013 Aug 6., [PMID:23921386]

Abstract [show]
Cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel in the ATP-binding cassette (ABC) transporter protein family. In the presence of ATP and physiologically relevant concentrations of AMP, CFTR exhibits adenylate kinase activity (ATP + AMP &lrarr2; 2 ADP). Previous studies suggested that the interaction of nucleotide triphosphate with CFTR at ATP-binding site 2 is required for this activity. Two other ABC proteins, Rad50 and a structural maintenance of chromosome protein, also have adenylate kinase activity. All three ABC adenylate kinases bind and hydrolyze ATP in the absence of other nucleotides. However, little is known about how an ABC adenylate kinase interacts with ATP and AMP when both are present. Based on data from non-ABC adenylate kinases, we hypothesized that ATP and AMP mutually influence their interaction with CFTR at separate binding sites. We further hypothesized that only one of the two CFTR ATP-binding sites is involved in the adenylate kinase reaction. We found that 8-azidoadenosine 5'-triphosphate (8-N3-ATP) and 8-azidoadenosine 5'-monophosphate (8-N3-AMP) photolabeled separate sites in CFTR. Labeling of the AMP-binding site with 8-N3-AMP required the presence of ATP. Conversely, AMP enhanced photolabeling with 8-N3-ATP at ATP-binding site 2. The adenylate kinase active center probe P(1),P(5)-di(adenosine-5') pentaphosphate interacted simultaneously with an AMP-binding site and ATP-binding site 2. These results show that ATP and AMP interact with separate binding sites but mutually influence their interaction with the ABC adenylate kinase CFTR. They further indicate that the active center of the adenylate kinase comprises ATP-binding site 2.

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275 Error bars, S.E. Nucleotide Interactions with the ABC Adenylate Kinase CFTR 27698 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 288ߦNUMBER 38ߦSEPTEMBER 20, 2013 at SEMMELWEIS UNIV OF MEDICINE on December , D1370N, abolished Ap5A inhibition of current, whereas the homologous mutations in ATP-binding site 1, K464A and D572N, did not. Login to comment
302 (b) Patch clamp studies showed that CFTR mutations K1250A and D1370N, located within the conserved Walker A and B motifs of ATP-binding site 2, abolished the effects of Ap5A and AMP on CFTR current. 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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163 To this end, we introduced the D1370N mutation into the &#e044;R background and characterized gating kinetics in the presence of NO3 &#e032; or Cl&#e032; (Fig. 5 C). Login to comment
166 Comparing the single-channel kinetic parameters of D1370N-CFTR further confirms that the effect of NO3 &#e032; on both the Figure 5.ߓ Effects of NO3 &#e032; on single-channel kinetics of &#e044;R-CFTR and D1370N/&#e044;R-CFTR. Login to comment
171 (C) NO3 &#e032; increases the Po of a hydrolysis-deficient mutant CFTR (D1370N). Login to comment
172 (D) Microscopic kinetic parameters for D1370N/ &#e044;R-CFTR in the presence of bath Cl&#e032; or NO3 &#e032; . Login to comment

[hide] Timing of CFTR Pore Opening and Structure of Its T... Cell. 2015 Oct 22;163(3):724-33. doi: 10.1016/j.cell.2015.09.052. Epub 2015 Oct 22. Sorum B, Czege D, Csanady L
Timing of CFTR Pore Opening and Structure of Its Transition State.
Cell. 2015 Oct 22;163(3):724-33. doi: 10.1016/j.cell.2015.09.052. Epub 2015 Oct 22., [PMID:26496611]

Abstract [show]
In CFTR, the chloride ion channel mutated in cystic fibrosis (CF) patients, pore opening is coupled to ATP-binding-induced dimerization of two cytosolic nucleotide binding domains (NBDs) and closure to dimer disruption following ATP hydrolysis. CFTR opening rate, unusually slow because of its high-energy transition state, is further slowed by CF mutation DeltaF508. Here, we exploit equilibrium gating of hydrolysis-deficient CFTR mutant D1370N and apply rate-equilibrium free-energy relationship analysis to estimate relative timing of opening movements in distinct protein regions. We find clear directionality of motion along the longitudinal protein axis and identify an opening transition-state structure with the NBD dimer formed but the pore still closed. Thus, strain at the NBD/pore-domain interface, the DeltaF508 mutation locus, underlies the energetic barrier for opening. Our findings suggest a therapeutic opportunity to stabilize this transition-state structure pharmacologically in DeltaF508-CFTR to correct its opening defect, an essential step toward restoring CFTR function.

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4 Here, we exploit equilibrium gating of hydrolysis-deficient CFTR mutant D1370N and apply rate-equilibrium free-energy relationship analysis to estimate relative timing of opening movements in distinct protein regions. Login to comment
37 To make CFTR gate at equilibrium, we introduced the NBD2 Walker-B mutation D1370N (Figure 1A, bottom, red star) because, among several hydrolysis-disrupting mutations tested, D1370N only slightly reduces the apparent affinity for ATP, and does not prolong open bursts to an extent incompatible with single-channel gating analysis (Csana &#b4; dy et al., 2010). Login to comment
40 Choice of a Suitable Background Construct for REFER Analysis (A) Domain organizations of WT (top) and cut-DR(D1370N) (bottom) CFTR: TMDs (gray), intracellular loops containing coupling helices (light violet), NBD1 (green), NBD2 (blue), R domain (magenta), membrane (yellow). Login to comment
42 The D1370N mutation in NBD2 is depicted by a red star. Login to comment
48 The D1370N mutation abrogates ATP hydrolysis (red cross) and confines gating in saturating ATP to a simple C1 4 O1 equilibrium (red box). Login to comment
53 Thus, we chose cut-DR(D1370N) as the background construct for our REFER study (Figure 1A, bottom). Login to comment
54 Gating of cut-DR(D1370N) indeed proved PKA-independent but remained strictly ATP-dependent with an apparent affinity for ATP of 288 &#b1; 27 mM (Figures S1A and S1B). Login to comment
55 Just as for WT (Winter et al., 1994; Zeltwanger et al., 1999; Csana &#b4; dy et al., 2000; Vergani et al., 2003), cut-DR (Csana &#b4; dy et al., 2000; Bompadre et al., 2005), and D1370N (Vergani et al., 2003) CFTR channels, mean open burst duration (tb) of cut-DR(D1370N) proved largely Figure 2. Login to comment
56 Timing of Motion at Position 1246 of the NBD1-NBD2 Interface (A) Inward single-channel currents of the cut-DR(D1370N) CFTR background construct (top trace) and of channels bearing mutations T1246V, T1246P, T1246C, T1246N, and T1246A, respectively, in the same background. Login to comment
63 Timing of Motion at Position 275 of the NBD2-TMD Interface (A) Inward single-channel currents of the cut-DR(D1370N) CFTR background construct (top trace) and of channels bearing mutations Y275F, Y275E, Y275K, Y275L, and Y275S, respectively, in the same background. Currents were recorded at 80 mV, in symmetrical 140 mM Cl ; dashes on the left mark zero-current level. Login to comment
74 Timing of Motion at Position 348 in the Pore Region (A) Inward single-channel currents of the cut-DR(D1370N) CFTR background construct (top trace) and of channels bearing mutations M348I, M348K, M348C, M348N, and M348A, respectively, in the same background. Currents were recorded at 80 mV, in symmetrical 140 mM Cl ; dashes on the left mark zero-current level. Login to comment
79 Timing of Motion in the Narrow Region of the Pore Studied by Anion Replacement (A) Pairs of segments of inward single-channel current from three patches containing single cut-DR(D1370N) CFTR channels. Login to comment
108 Thus, replacement of chloride with other permeant anions might be viewed as a structural perturbation of the ''selectivity filter.`` We therefore studied changes in the pattern of single-channel gating of our background construct cut-DR(D1370N) in response to sudden replacement of cytosolic chloride with nitrate, bromide, or formate. Login to comment
130 The point representing the cut-DR(D1370N) background construct in chloride is highlighted by a black circle. Login to comment
153 To study the pore opening step, we therefore employed the D1370N background mutation that truncates the gating cycle to an equilibrium scheme (Figure 1B, red frame). Login to comment
162 Because gating of cut-DR(D1370N), like that of WT CFTR, is strictly ATP-dependent (Figures S1 and S2), our conclusions do not necessarily apply to the mechanism of the extremely infrequent spontaneous openings observable in the absence of ATP that are promoted by certain mutations (Wang et al., 2010) and drugs (Jih and Hwang, 2013). Login to comment
165 EXPERIMENTAL PROCEDURES pGEMHE-CFTR(837-1480(D1370N)) was constructed from pGEMHE-CFTR(837-1480), mutations at positions 275 and 348 were introduced into pGEMHE-CFTR(1-633) (Csana &#b4;dy et al., 2000), and mutations at position 1246 into pGEMHE-CFTR(837-1480(D1370N)) using Stratagene QuickChange. Login to comment