ABCMdb

ABCC7 p.Lys1250Arg

Predicted by SNAP2:	A: D (95%), C: D (95%), D: D (95%), E: D (95%), F: D (95%), G: D (95%), H: D (95%), I: D (95%), L: D (95%), M: D (95%), N: D (95%), P: D (95%), Q: D (95%), R: D (95%), S: D (95%), T: D (95%), V: D (95%), W: D (95%), Y: D (95%),
Predicted by PROVEAN:	A: D, C: D, D: D, E: D, F: D, G: D, H: D, I: 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] CFTR channel opening by ATP-driven tight dimerizat... Nature. 2005 Feb 24;433(7028):876-80. Vergani P, Lockless SW, Nairn AC, Gadsby DC
CFTR channel opening by ATP-driven tight dimerization of its nucleotide-binding domains.
Nature. 2005 Feb 24;433(7028):876-80., 2005-02-24 [PMID:15729345]

Abstract [show]
ABC (ATP-binding cassette) proteins constitute a large family of membrane proteins that actively transport a broad range of substrates. Cystic fibrosis transmembrane conductance regulator (CFTR), the protein dysfunctional in cystic fibrosis, is unique among ABC proteins in that its transmembrane domains comprise an ion channel. Opening and closing of the pore have been linked to ATP binding and hydrolysis at CFTR's two nucleotide-binding domains, NBD1 and NBD2 (see, for example, refs 1, 2). Isolated NBDs of prokaryotic ABC proteins dimerize upon binding ATP, and hydrolysis of the ATP causes dimer dissociation. Here, using single-channel recording methods on intact CFTR molecules, we directly follow opening and closing of the channel gates, and relate these occurrences to ATP-mediated events in the NBDs. We find that energetic coupling between two CFTR residues, expected to lie on opposite sides of its predicted NBD1-NBD2 dimer interface, changes in concert with channel gating status. The two monitored side chains are independent of each other in closed channels but become coupled as the channels open. The results directly link ATP-driven tight dimerization of CFTR's cytoplasmic nucleotide-binding domains to opening of the ion channel in the transmembrane domains. This establishes a molecular mechanism, involving dynamic restructuring of the NBD dimer interface, that is probably common to all members of the ABC protein superfamily.

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112 Current levels of the triple mutant R555K T1246N K1250R did not change when [ATP] was increased to 10 mM, indicating that 5 mM [ATP] was saturating. Login to comment
132 In other ABC-ATPases, mutating the key lysine in the phosphate-binding loop to arginine drastically reduces or abolishes hydrolysis (see, for example, ref. 24) and, as would be predicted if hydrolysis at CFTR`s NBD2 catalytic site were markedly slowed, CFTR channels carrying the corresponding mutation (K1250R) have prolonged open burst durations (Fig. 4e; mean time constant of current decay upon ATP removal t ¼ 9.3 ^ 0.5 s; n ¼ 49). Login to comment
133 Introducing the T1246N mutation into the K1250R background decreased Po, corresponding to destabilization of the open burst state by 2.5 ^ 1.0kT with respect to the closed state. However, adding the R555K mutation to T1246N-K1250R channels restored high stability of the open state (Fig. 4e, f). 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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78 Introducing the T1246N mutation in a non-hydrolytic background (mutated at a crucial lysine, K1250R [39]) strongly destabilized the open state with respect to the 3 Mutant cycle analysis using activation free energies for the opening reaction Coupling between Arg555 and Thr1246 increases as the channel approaches the open state. Login to comment

[hide] Thermodynamics of CFTR channel gating: a spreading... J Gen Physiol. 2006 Nov;128(5):523-33. Epub 2006 Oct 16. Csanady L, Nairn AC, Gadsby DC
Thermodynamics of CFTR channel gating: a spreading conformational change initiates an irreversible gating cycle.
J Gen Physiol. 2006 Nov;128(5):523-33. Epub 2006 Oct 16., [PMID:17043148]

Abstract [show]
CFTR is the only ABC (ATP-binding cassette) ATPase known to be an ion channel. Studies of CFTR channel function, feasible with single-molecule resolution, therefore provide a unique glimpse of ABC transporter mechanism. CFTR channel opening and closing (after regulatory-domain phosphorylation) follows an irreversible cycle, driven by ATP binding/hydrolysis at the nucleotide-binding domains (NBD1, NBD2). Recent work suggests that formation of an NBD1/NBD2 dimer drives channel opening, and disruption of the dimer after ATP hydrolysis drives closure, but how NBD events are translated into gate movements is unclear. To elucidate conformational properties of channels on their way to opening or closing, we performed non-equilibrium thermodynamic analysis. Human CFTR channel currents were recorded at temperatures from 15 to 35 degrees C in inside-out patches excised from Xenopus oocytes. Activation enthalpies(DeltaH(double dagger)) were determined from Eyring plots. DeltaH(double dagger) was 117 +/- 6 and 69 +/- 4 kJ/mol, respectively, for opening and closure of partially phosphorylated, and 96 +/- 6 and 73 +/- 5 kJ/mol for opening and closure of highly phosphorylated wild-type (WT) channels. DeltaH(double dagger) for reversal of the channel opening step, estimated from closure of ATP hydrolysis-deficient NBD2 mutant K1250R and K1250A channels, and from unlocking of WT channels locked open with ATP+AMPPNP, was 43 +/- 2, 39 +/- 4, and 37 +/- 6 kJ/mol, respectively. Calculated upper estimates of activation free energies yielded minimum estimates of activation entropies (DeltaS(double dagger)), allowing reconstruction of the thermodynamic profile of gating, which was qualitatively similar for partially and highly phosphorylated CFTR. DeltaS(double dagger) appears large for opening but small for normal closure. The large DeltaH(double dagger) and DeltaS(double dagger) (TDeltaS(double dagger) >/= 41 kJ/mol) for opening suggest that the transition state is a strained channel molecule in which the NBDs have already dimerized, while the pore is still closed. The small DeltaS(double dagger) for normal closure is appropriate for cleavage of a single bond (ATP's beta-gamma phosphate bond), and suggests that this transition state does not require large-scale protein motion and hence precedes rehydration (disruption) of the dimer interface.

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9 ∆H‡ for reversal of the channel opening step, estimated from closure of ATP hydrolysis-deficient NBD2 mutant K1250R and K1250A channels, and from unlocking of WT channels locked open with ATP+AMPPNP, was 43 ± 2, 39 ± 4, and 37 ± 6 kJ/mol, respectively. Login to comment
41 M AT E R I A L S A N D M E T H O D S Molecular Biology pGEMHE-WT was constructed as previously described (Chan et al., 2000), and the K1250R and K1250A mutations introduced using QuikChange (Stratagene) as previously described (Vergani et al., 2003, 2005). Login to comment
88 S2-S4 show parallel macroscopic current and temperature records illustrating temperature dependence of closure of partially phosphorylated K1250R and K1250A, and of AMPPNP-locked WT, CFTR, respectively, recorded at -80 to -20 mV between 25°C and 31°C. Fig. S5 demonstrates that exposure to millimolar levels of the hydrolysis products ADP+Pi does not cause opening of prephosphorylated WT CFTR channels. Login to comment
89 Fig. S6 shows the predicted energetic profile of CFTR gating obtained using K1250A, not K1250R (as in Fig. 6), as a model for nonhydrolytic channel closure. Login to comment
105 Temperature Dependence of Closing Rate of Hydrolysis-deficient Mutant K1250R and K1250A CFTR Channels To estimate ∆H‡ for channel closing when the normal route for channel closure via ATP hydrolysis was unavailable, we studied the temperature dependence of the closing rate of two channel constructs in which the composite NBD2 site was made catalytically inactive by mutation of the conserved NBD2 Walker A lysine, K1250. Login to comment
107 In another ABC ATPase, P-glycoprotein, the Lys-to-Arg mutation of either Walker A lysine abolishes ATP hydrolysis (Lerner-Marmarosh et al., 1999), and in CFTR, the K1250R mutation prolongs open burst durations by >20-fold (compare Vergani et al., 2005), consistent with abolished, or greatly diminished, ATP hydrolysis. Login to comment
108 Closure of K1250R or of K1250A mutant CFTR channels is too slow to allow kinetic analysis of individual gating events and so it was assayed as current decay after sudden removal of ATP. Login to comment
110 Prephosphorylated K1250R CFTR channels in macropatches were repeatedly opened by brief exposures to 2 mM MgATP at temperatures alternating between 25°C and ‫°51ف‬C (Fig. 3 A), or 25°C and ‫°13ف‬C (Fig. S2). Login to comment
114 The Eyring plot (Fig. 4 B) of the normalized closing rates obtained from single exponential fits (Fig. 4 A, smooth lines) to the decaying currents after ATP removal yielded a ∆H‡ for nonhydrolytic closure of 39 ± 4 kJ/mol, similar to that obtained for K1250R channels (Fig. 3 B). Login to comment
115 Interestingly, whereas open burst duration of WT channels was greater than twofold longer in the presence of PKA than shortly after its withdrawal, the average closing time constant of K1250R at 25°C, equivalent to its mean open burst duration, was only slightly longer (Fig. 3 A; Fig. S2) upon removal of PKA+ATP (7.3 ± 0.8 s, n = 12) than upon removal of just ATP (5.7 ± 0.4 s, n = 30). Login to comment
116 But, because the fall in Po that signals partial dephosphorylation of WT channels upon PKA removal occurs so rapidly (i.e., in 2-3 s; Csanády et al., 2000), the phosphorylation status of K1250R channels after removal of ATP+PKA, during their gradual closure, which takes tens of seconds (compare Fig. 3 A, current segments fitted by magenta lines), is uncertain. Login to comment
130 Temperature dependence of gating of partially phosphorylated K1250R CFTR. Login to comment
131 (A) Macroscopic current trace (top) from ‫000,2ف‬ K1250R CFTR channels at -20 mV, with simultaneously recorded temperature (bottom). Login to comment
135 (B) Eyring plot of normalized closing rates ( ˆk ) of K1250R CFTR channels upon ATP removal, fitted by a straight line to obtain ∆H‡ value shown; closing rates, obtained as 1/τ from single-exponential fits, as in A, were normalized to their average values in bracketing control segments at 25°C. Figure 4. Login to comment
145 From an Eyring plot of normalized unlocking rates (Fig. 5 B), the rough estimate of ∆H‡ for unlocking from AMPPNP of partially phosphorylated WT CFTR was 37 ± 6 kJ/ mol, similar to the value obtained above for closure of partially phosphorylated K1250R and K1250A channels opened by just ATP (Fig. 3 B and Fig. 4 B). Login to comment
166 We chose NBD2 catalytic site mutant K1250R as one model for nonhydrolytic closure because the mutation conserves charge in the anticipated catalytic interface, and, in P-glycoprotein, the corresponding K-to-R mutation essentially abolishes ATP hydrolysis (Lerner-Marmarosh et al., 1999). Login to comment
167 We selected the mutant K1250A as a second model for nonhydrolytic closure because it displays much slower closure than K1250R (e.g., Vergani et al., 2003, 2005) and because the K1250A mutation abolishes ATP hydrolysis by CFTR (Ramjeesingh et al., 1999). Login to comment
179 Second, because ∆H‡ is high (117 kJ/mol) for opening, but small for nonhydrolytic closure (e.g., 43 kJ/mol using K1250R as a model), α Δ O-CH must be large (+74 kJ/mol; Fig. 6 A, red line). Login to comment
180 Third, in contrast, α Δ O-CG is estimated to be rather small (e.g., -1 kJ/mol using K1250R as a model; Fig. 6 A, blue line), which in turn suggests that α Δ O-CS is large ( α Δ O-CT S ≈ 75 kJ/mol; Fig. 6 A, green line). Login to comment
182 Also, independent evidence that α Δ O-CG ≈ 0 is provided by the observation that steady-state Po ≈ 0.5 for the nonhydrolytic K1250R mutant, which is presumed to gate at thermodynamic equilibrium (Vergani et al., 2005). Login to comment
187 The paucity of ATPase measurements for CFTR mutants means that we cannot be certain that the charge-sparing mutation K1250R abolishes ATP hydrolysis in CFTR, even though the equivalent mutation in P-glycoprotein abolishes ATP hydrolysis (Lerner-Marmarosh et al., 1999). Login to comment
188 The charge-neutralizing mutation K1250A, on the other hand,doesabrogateATPhydrolysisinCFTR(Ramjeesingh et al., 1999) and yields an open burst state more stable than that of K1250R (Vergani et al., 2003, 2005). Login to comment
189 But using the closing rate of K1250A (instead of K1250R) channels as a model for nonhydrolytic closure, and hence for reversal of channel opening, yields barrier values for this step (∆H‡ = 39 kJ/mol and Δ maxG‡ = 81 kJ/mol) that are onlyslightlydifferentfromthoseestimatedusingK1250R. Login to comment
202 Forward (left to right) ∆H‡ values were obtained from slopes of Eyring plots for WT opening and closing rates (Fig. 1 C), ∆H‡ for the reversal of opening reflects the slope of the Eyring plot for K1250R closing rate (Fig. 3B). Login to comment
203 Corresponding Δ maxG‡ values were computed as RT ln(kBT/(kh)), by substituting the rates of WT opening (0.3 s-1) and closure (3.9 s-1), and of K1250R closure (0.2 s-1), for k. 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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115 Introducing the T1246N mutation in a non-hydrolytic background (mutated at a crucial lysine, K1250R; Lerner-Marmarosh et al. 1999) strongly destabilized the open state with respect to the closed one. Login to comment
116 However, the same T to N mutation, when introduced in the R555K K1250R non-hydrolytic background, did not significantly alter the closed- to-open equilibrium. Login to comment
113 Introducing the T1246N mutation in a non-hydrolytic background (mutated at a crucial lysine, K1250R; Lerner-Marmarosh et al. 1999) strongly destabilized the open state with respect to the closed one. Login to comment
114 However, the same T to N mutation, when introduced in the R555K K1250R non-hydrolytic background, did not significantly alter the closed- to-open equilibrium. Login to comment

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

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

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78 As a three-parameter fit of scheme 2 to the data in Fig. 1B (and also Fig. 3) did not provide a reliable estimate of this small rate (SI Text), to estimate k-1 we measured the macroscopic closing rates of prephosphorylated K1250A, K1250R, and E1371S mutant channels (e.g., Fig. 2A) upon sudden removal of ATP. Login to comment
79 These rates, obtained as the reciprocals of the time constants of fitted single exponentials (e.g., Fig. 2A, blue line), were 0.044 ± 0.004 s-1 (n = 9) for K1250A (Fig. 2C, blue bar), 0.22 ± 0.01 s-1 (n = 17) for K1250R, and 0.036 ± 0.002 s-1 (n = 16) for E1371S. Login to comment
81 As we cannot be certain which of these mutant channels, when open, most closely resembles the O1 state of a WT CFTR channel gating in ATP, we tentatively chose the closing rate of K1250R as an estimate of k-1 for WT CFTR (Fig. 4E Right, blue bar) on the grounds that the lysine-to- arginine mutation at least conserves charge in the vicinity of the ATP bound within the NBD2 composite site. Login to comment
166 (a) k-1 for partially phosphorylated WT is modeled by the closing rate of partially phosphorylated K1250R. 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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19 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. Login to comment
28 M AT E R I A L S A N D M E T H O D S Molecular biology CFTR mutants were constructed by using either pGEMHE-WT (Chan et al., 2000) or pGEMHE-K1250R (Vergani et al., 2005) as template. Login to comment
72 In this study, the average durations of stationary segments of record used for estimating Po;max were 40-50 s for the wild-type (WT), F1296S, N1303Q, and F1296S/N1303Q constructs (estimated single-channel cycle times 1.25 s in saturating ATP; Fig. 8 A), but 100-130 s for K1250R, F1296S/K1250R, and N1303Q/K1250R, and 220 s for F1296S/ N1303Q/K1250R (estimated single-channel cycle times 13 s in saturating ATP; Fig. 8 A). Login to comment
109 Figs. S3 and S4 show verification of Po;max estimates in single-channel patches for WT, F1296S, N1303Q, and F1296S/N1303Q (Fig. S3), as well as for K1250R, F1296S/K1250R, N1303Q/K1250R, and F1296S/N1303Q/K1250R (Fig. S4). Login to comment
111 Fig. S6 illustrates apparent affinities for ATP in the K1250R background. Login to comment
113 Fig. S8 depicts predicted Po time courses in response to the addition/removal of ATP for WT, F1296S/N1303Q, K1250R, and F1296S/N1303Q/K1250R, calculated using Scheme 2. Login to comment
140 Because openings in the absence of ATP must necessarily be nonhydrolytic, the NBD2 Walker A mutation K1250R, known to abolish ATP hydrolysis in ABC proteins (Lerner-Marmarosh et al., 1999; Payen et al., 2005), is not expected to affect this spontaneous gating. Login to comment
141 To test this idea, we studied the same mutant cycle also in a K1250R background (Fig. 4). Login to comment
142 Indeed, although K1250R, F1296S/K1250R, and N1303Q/K1250R ATP removal rapidly abolished currents for both single mutants just as for WT (Fig. 2, A-C), in the case of the double mutant, a constitutive basal activity persisted even after ATP removal (Fig. 2 D, magnified in inset) and did not vanish even over the time course of several minutes. Login to comment
146 Although Figure 4. The stabilizing site-1-site-2 interaction that facilitates channel opening in the absence of ATP is preserved in the ATP hydrolysis-deficient K1250R mutant. Login to comment
147 (A) Representative traces of K1250R, F1296S/K1250R, N1303Q/K1250R, and F1296S/N1303Q/K1250R currents illustrating segments in 0 mM ATP and bracketing segments in 2 mM ATP. Dotted lines show zero current level (determined for the triple mutant similarly to that in Fig. S2). Login to comment
148 (B) Estimation of Po;max for K1250R (black), F1296S/K1250R (red), N1303Q/K1250R (blue), and F1296S/N1303Q/K1250R (green) by stationary noise analysis. Login to comment
150 (D) Thermodynamic mutant cycle built on Po;bas/(1Po;bas) values; notation as in Fig. 3 D. analogous constructs in the K1250R background (Fig. S4, A and B). Login to comment
155 constructs showed hardly detectable basal activity, a markedly elevated spontaneous activity was observed for the F1296S/N1303Q/K1250R triple mutant (Fig. 4 A), persisting even minutes after ATP washout. Login to comment
157 Similarly to their hy-drolytic counterparts, Po;bas was 10-fold higher in F1296S/N1303Q/K1250R compared with the other three constructs, and the mutant cycle built on the closed-open equilibrium constant Po;bas/(1Po;bas) yielded a Gint of 2.36 ± 0.58 kT (Fig. 4 D)-again, significantly different from zero (P < 0.01). Login to comment
161 (A) Summary of Po;max values for K1250R (black), F1296S/K1250R (red), N1303Q/K1250R (blue), and F1296S/N1303Q/K1250R (green) obtained from the data presented in Fig. 4 B. Login to comment
168 Interestingly, Po;max of K1250R (0.68 ± 0.06; n = 6) was only slightly affected by the substitutions at sites 1 and 2, and even these small changes were mostly additive (Fig. 5 A; compare Fig. S4 B), such that a mutant cycle built on Po;max/(1Po;max) yielded a Gint of zero (0.03 ± 0.14 kT; Fig. 5 B; compare Fig. S4 C). Login to comment
171 The K1250R mutation itself is known to prolong open-channel burst durations by 20-30-fold (Vergani et al., 2005; Csanády et al., 2006) due to the slow rate of dissociation of the ATP-bound NBD dimer in the absence of ATP hydrolysis. Login to comment
172 Consistent with those reports, upon the sudden removal of ATP, we saw macroscopic K1250R currents decline with a time constant of 8 s (Fig. 5 C, black single-exponential fit In a nonhydrolytic background, interaction between sites 1 and 2 is similar for ATP-bound closed and open states, but changes after ATP removal Because the interaction between sites 1 and 2 changes during ATP-independent spontaneous openings (Figs. 3 D and 4 D), we wondered whether the same interaction also plays a role in normal, ATP-dependent channel opening. Login to comment
175 Thus, to test for a possible change in interaction between sites 1 and 2 during ATP-driven reversible opening and closure, we repeated the mutant cycle analysis in the nonhydrolytic K1250R background, comparing Po;max values for K1250R, F1296S/K1250R, N1303Q/ K1250R, and F1296S/N1303Q/K1250R (Fig. 5 A), Figure 6. ATP binding affects energetic coupling between sites 1 and 2 in closed channels. Login to comment
184 Although neither the F1296S nor the N1303Q mutation, when introduced one at a time, affected the time constant of current relaxation of K1250R upon ATP removal (Fig. 5 C, red and blue fit lines and bars), this relaxation time constant (relax) was prolonged by approximately fourfold, to 31 ± 5 s (n = 10), in the triple mutant F1296S/ N1303Q/K1250R (Fig. 5 C, green fit line and bar). Login to comment
213 Thus, in the K1250R background, apparent ATP affinities were three- to fourfold decreased (Fig. S6, A and B), corresponding to six- to ninefold increased values of KrCO (Fig. S6 C), but a mutant cycle built on the latter values yielded a Gint not significantly different from zero (Fig. S6 D). Login to comment
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
268 The two rates assumed to be changed by the F1296S/N1303Q double mutation, and by the K1250R mutation, are shown in red and magenta, respectively, belowtheWTrates. Login to comment
269 (B)Tablesummarizingparam- eters Po;bas and KPo predicted by Scheme 2 for WT (using the rates in black in A) and F1296S/ N1303Q (using the two rates in red in A), as well as Po;max and relax for K1250R and F1296S/ N1303Q/K1250R (using the rates printed in magenta for steps C4→O2 and O2→C1). Login to comment
272 in an unchanged Po;max (Fig. 8 B), just as we have observed for F1296S/N1303Q/K1250R (Fig. 5 A). Login to comment
274 Such predicted time courses are summarized in Fig. S8 for WT, F1296S/N1303Q, K1250R, and F1296S/N1303Q/K1250R. Login to comment
286 nonhydrolytic mutant K1250R (Fig. 5 C, black). Login to comment
287 The Po;max values of 0.6 measured for the long-burst nonhydrolytic mutants (Fig. 5 A) suggest that the K1250R mutation, in addition to abrogating ATP hydrolysis, also slows maximal opening rate. Login to comment
288 We therefore modeled the effect of the K1250R mutation by simultaneously setting rate O2→C1 to zero and rate C4→O2 to 0.2 s1 (Fig. 8 A, magenta). Login to comment
291 Parameters Po;bas and KPo calculated for Scheme 2 using the rates plotted in black in Fig. 8 A, as well as Po;max and relax calculated using the two rates adjusted for K1250R (Fig. 8 A, magenta), are in good agreement with the measured values (Fig. 8 B, left column, measured values are shown in parentheses below each calculated parameter). Login to comment
298 A comparable (100-fold) decrease in rate C4→C3 (Fig. 8 A, red) reproduces the approximately fourfold prolonged relax (Fig. 8 B) we have observed for F1296S/N1303Q/K1250R (Fig. 5 C). Login to comment

[hide] Mutant cycles at CFTR's non-canonical ATP-binding ... J Gen Physiol. 2011 Jun;137(6):549-62. doi: 10.1085/jgp.201110608. Epub 2011 May 16. Szollosi A, Muallem DR, Csanady L, Vergani P
Mutant cycles at CFTR's non-canonical ATP-binding site support little interface separation during gating.
J Gen Physiol. 2011 Jun;137(6):549-62. doi: 10.1085/jgp.201110608. Epub 2011 May 16., [PMID:21576373]

Abstract [show]
Cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel belonging to the adenosine triphosphate (ATP)-binding cassette (ABC) superfamily. ABC proteins share a common molecular mechanism that couples ATP binding and hydrolysis at two nucleotide-binding domains (NBDs) to diverse functions. This involves formation of NBD dimers, with ATP bound at two composite interfacial sites. In CFTR, intramolecular NBD dimerization is coupled to channel opening. Channel closing is triggered by hydrolysis of the ATP molecule bound at composite site 2. Site 1, which is non-canonical, binds nucleotide tightly but is not hydrolytic. Recently, based on kinetic arguments, it was suggested that this site remains closed for several gating cycles. To investigate movements at site 1 by an independent technique, we studied changes in thermodynamic coupling between pairs of residues on opposite sides of this site. The chosen targets are likely to interact based on both phylogenetic analysis and closeness on structural models. First, we mutated T460 in NBD1 and L1353 in NBD2 (the corresponding site-2 residues become energetically coupled as channels open). Mutation T460S accelerated closure in hydrolytic conditions and in the nonhydrolytic K1250R background; mutation L1353M did not affect these rates. Analysis of the double mutant showed additive effects of mutations, suggesting that energetic coupling between the two residues remains unchanged during the gating cycle. We next investigated pairs 460-1348 and 460-1375. Although both mutations H1348A and H1375A produced dramatic changes in hydrolytic and nonhydrolytic channel closing rates, in the corresponding double mutants these changes proved mostly additive with those caused by mutation T460S, suggesting little change in energetic coupling between either positions 460-1348 or positions 460-1375 during gating. These results provide independent support for a gating model in which ATP-bound composite site 1 remains closed throughout the gating cycle.

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24 Mutation T460S accelerated closure in hydrolytic conditions and in the nonhydrolytic K1250R background; mutation L1353M did not affect these rates. Login to comment
37 pGEMHE-K1250R (Vergani et al., 2005) was used as a template for the corresponding nonhydrolytic mutants. Login to comment
102 Fitting the relaxation time course after ATP removal for the nonhydrolytic H1375A/K1250R and T460S/H1375A/K1250R constructs consistently required a double exponential with two slow time constants (each in the seconds range), suggesting two populations of open-channel bursts (see Fig. 9 A). Login to comment
151 In CFTR, the equivalent mutation, K1250R, caused an increase in open burst duration (Vergani et al., 2005; Csanády et al., 2006), consistent with blocking of the fast hydrolytic closure pathway. Login to comment
152 To determine if the mutations T460S, L1353M, and T460S/L1353M increased the rate of nonhydrolytic closure from an open state with ATP bound at both composite sites, we introduced the above site-1 mutations in a K1250R background. Login to comment
154 The fitted time constant for current decay, relaxation (Fig. 5 A, inset), provided an estimate for the average lifetime of the open state, which was 5.9 ± 0.5 s (n = 13) for K1250R (black bar) and unchanged in L1353M/ K1250R (7.2 ± 0.8 s; n = 10; P = 0.11; blue bar), but significantly reduced in T460S/K1250R (4.2 ± 0.3 s; n = 13; P < 0.01; red bar). Login to comment
155 Because relaxation was additively affected in T460S/L1353M/K1250R (4.5 ± 0.6 s; n = 10; P < 0.05; green bar), G‡ int(closing) was not significantly different from zero (Fig. 5 B), indicating that the coupling between the two residues on opposite sides of composite site 1 was not changed along the nonhydrolytic closure pathway between the ATP-bound open state and the transition state. Login to comment
182 However, in constructs carrying the K1250R mutation, the hydrolytic pathway is effectively blocked, and the gating cycle, in saturating [ATP], is reduced to a simple equilibrium between the open and closed states. Login to comment
183 Thus, we can use Po in the K1250R background to determine the free energy difference between the open and closed states for each of the constructs (Gopen-closed) and analyze the results using mutant cycle formalism. Login to comment
185 Consistent with changes in closing rate, Po was significantly reduced for T460S/K1250R (0.28 ± 0.06; n = 6; P < 0.01; Fig. 5 C, red bar) and T460S/L1353M/ K1250R (0.26 ± 0.03; n = 8; P < 0.01; green bar) compared with K1250R (0.55 ± 0.07; n = 9; black bar), but not for L1353M/K1250R (0.55 ± 0.05; n = 8; blue bar). Login to comment
186 Here too, the coupling energy, Gint(open-closed), was not significantly different from zero (Fig. 5 D), indicating that there was Figure 5. The T460S mutation destabilizes the open state of CFTR in the nonhydrolytic K1250R background. Login to comment
194 nonhydrolytic relaxation nor the reduction in nonhydrolytic Po by the T460S mutation (compare red vs. black bars in Fig. 5, A and C) was apparent when this mutation was introduced into an H1348A/K1250R background (compare green vs. blue bars in Fig. 7, A and D, left), these deviations from additivity resulted in a small change in T460-H1348 interaction energy only between the transition state for nonhydrolytic closure and the open ground state (Gint(closing) = 0.43 ± 0.14 kT; P = 0.01; Fig. 7 B), but not between open and closed ground states (Fig. 7 D, right; P = 0.1). Login to comment
202 To look for changes in interactions between positions 460 and 1348 during nonhydrolytic closure, we created nonhydrolytic H1348A/K1250R and T460S/H1348A/ K1250R channels and compared their closing rates by studying macroscopic current relaxations after ATP removal (Fig. 7 A). Login to comment
203 Interestingly, mutation H1348A prolonged the time constant of the current relaxation (to 20 ± 2 s; n = 8; Fig. 7 A, blue bar) to a similar extent as it did normal burst durations; and noise analysis (Fig. 7 C) attested to the fact that the prolonged open time of H1348A/K1250R is associated with an unusually high open probability (Po = 0.83 ± 0.03 s; n = 6; Fig. 7 D, left, blue bar). Login to comment
214 We next studied the effect of the mutations at our target pair T460-H1375 on nonhydrolytic closing rate, in a K1250R background. Login to comment
215 Because for both H1375A/K1250R green vs. blue bar) attested to an increased opening rate in the double mutant (Fig. 8 D, left, green vs. blue bar). Login to comment
216 However, because a similar tendency (although to a lesser extent) was also apparent in the WT background (see Fig. 3 C, red vs. black bar), the mutant cycle built Figure 7. The H1348A mutation stabilizes the open state of CFTR in the nonhydrolytic K1250R background. Login to comment
217 (A) Representative normalized decay time courses of macroscopic currents for H1348A/K1250R and T460S/ H1348A/K1250R CFTR after the removal of 2 mM ATP (gray). Login to comment
241 An alteration and T460S/H1375A/K1250R adequate fitting of the relaxation time course after ATP removal consistently required a double exponential with two slow time constants (each in the seconds range; Fig. 9 A), average steady-state closing rate was estimated from a double-exponential fit as described in Materials and methods. Login to comment
242 Unexpectedly, when the H1375A mutation was introduced into the K1250R background, average nonhydrolytic closing rate was not slowed, but rather slightly accelerated (Fig. 9 A, blue bar). Login to comment
244 Finally, by noise analysis (Fig. 9 C), mutation T460S reduced Po in the H1375A/K1250R background (compare green vs. blue bars in Fig. 9 D, left) to a similar extent as it did in the single-mutant K1250R background (compare red vs. black bars in Fig. 5 C), yielding a Gint(open-closed) not significantly different from zero (Fig. 9 D; P = 0.15). Login to comment
247 To validate this proposal using an independent approach, we resorted to thermodynamic Figure 9. Effects of mutations at positions 460 and 1375 on nonhydrolytic gating in the K1250R background. Login to comment
248 (A) Representative normalized decay time courses of macroscopic currents for H1375A/K1250R and T460S/H1375A/K1250R CFTR after the removal of 2 mM ATP. Solid blue and green lines are fitted bi-exponentials. Login to comment
249 Fitted parameters were 1 = 2.8 s, 2 = 11 s, A1 = 0.77, and A2 = 0.23 for the H1375A/ K1250R trace, and 1 = 2.8 s, 2 = 15 s, A1 = 0.82, and A2 = 0.18 for the T460S/H1375A/ K1250R trace. Login to comment
274 Our results on T460S/K1250R and H1348A/K1250R indeed show a respective decrease and increase in Po (Figs. 5 C and 7 D), confirming that the open ground state is destabilized in T460S/K1250R, but stabilized in H1348A/K1250R, with respect to the closed ground state. Login to comment
278 This mutation affected the rates of hydrolytic (Fig. 8 B) and nonhydrolytic (Fig. 9 A) closure in opposite ways, and in the K1250R background, the double-exponential relaxation after ATP removal suggested a mixture of two types of nonhydrolytic bursts. Login to comment

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

Abstract [show]
Cystic fibrosis transmembrane conductance regulator (CFTR) is a unique member of the ATP-binding cassette (ABC) protein superfamily. Unlike most other ABC proteins that function as active transporters, CFTR is an ATP-gated chloride channel. The opening of CFTR's gate is associated with ATP-induced dimerization of its two nucleotide-binding domains (NBD1 and NBD2), whereas gate closure is facilitated by ATP hydrolysis-triggered partial separation of the NBDs. This generally held theme of CFTR gating-a strict coupling between the ATP hydrolysis cycle and the gating cycle-is put to the test by our recent finding of a short-lived, post-hydrolytic state that can bind ATP and reenter the ATP-induced original open state. We accidentally found a mutant CFTR channel that exhibits two distinct open conductance states, the smaller O1 state and the larger O2 state. In the presence of ATP, the transition between the two states follows a preferred O1-->O2 order, a telltale sign of a violation of microscopic reversibility, hence demanding an external energy input likely from ATP hydrolysis, as such preferred gating transition was abolished in a hydrolysis-deficient mutant. Interestingly, we also observed a considerable amount of opening events that contain more than one O1-->O2 transition, indicating that more than one ATP molecule may be hydrolyzed within an opening burst. We thus conclude a nonintegral stoichiometry between the gating cycle and ATP consumption. Our results lead to a six-state gating model conforming to the classical allosteric mechanism: both NBDs and transmembrane domains hold a certain degree of autonomy, whereas the conformational change in one domain will facilitate the conformational change in the other domain.

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294 Notably, this result is somewhat different from that shown in Vergani et al. (2005) in which the current relaxation is prolonged in T1246N/K1250R- CFTR. Login to comment
296 Nonetheless, the locked-open time is further shortened when combining R352C and T1246N mutations together (Fig. 7, D and E), suggesting that the two mutations affects two different kinetic steps as described above. 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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260 In contrast, substitutions at the analogous sites in NBF2 (K1250 or D1370) actually increased the latency by two- to threefold, and, with the exception of the most conservative substitution, K1250R, the values of *koff were decreased compared with that of wild-type CFTR, indicating stabilization of the active state. These results suggest that the consensus A lysine and consensus B aspartic acid in the ATP binding pocket of NBF1 contribute to stabilization of the active state, whereas their analogues in NBF2 are involved in terminating the active state. 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
262 In contrast, substitutions at the analogous sites in NBF2 (K1250 or D1370) actually increased the latency by two- to threefold, and, with the exception of the most conservative substitution, K1250R, the values of *koff were decreased compared with that of wild-type CFTR, indicating stabilization of the active state. These results suggest that the consensus A lysine and consensus B aspartic acid in the ATP binding pocket of NBF1 contribute to stabilization of the active state, whereas their analogues in NBF2 are involved in terminating the active state. 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

[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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89 Alanine and arginine substitutions at lysine-464 and -1250 were associated with sensitivities similar to those observed with the glutamine substitutions (K464A or K464R, Kil2 = 0.8 mM; K1250A or K1250R, K,12 < 0.02 mM). Login to comment

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

Abstract [show]
Cystic fibrosis transmembrane conductance regulator (CFTR) channel opening and closing are driven by cycles of adenosine triphosphate (ATP) binding-induced formation and hydrolysis-triggered disruption of a heterodimer of its cytoplasmic nucleotide-binding domains (NBDs). Although both composite sites enclosed within the heterodimer interface contain ATP in an open CFTR channel, ATP hydrolysis in the sole catalytically competent site causes channel closure. Opening of the NBD interface at that site then allows ADP-ATP exchange. But how frequently, and how far, the NBD surfaces separate at the other, inactive composite site remains unclear. We assessed separation at each composite site by monitoring access of nucleotide-sized hydrophilic, thiol-specific methanothiosulfonate (MTS) reagents to interfacial target cysteines introduced into either LSGGQ-like ATP-binding cassette signature sequence (replacing equivalent conserved serines: S549 and S1347). Covalent MTS-dependent modification of either cysteine while channels were kept closed by the absence of ATP impaired subsequent opening upon ATP readdition. Modification while channels were opening and closing in the presence of ATP caused macroscopic CFTR current to decline at the same speed as when the unmodified channels shut upon sudden ATP withdrawal. These results suggest that the target cysteines can be modified only in closed channels; that after modification the attached MTS adduct interferes with ATP-mediated opening; and that modification in the presence of ATP occurs rapidly once channels close, before they can reopen. This interpretation was corroborated by the finding that, for either cysteine target, the addition of the hydrolysis-impairing mutation K1250R (catalytic site Walker A Lys) similarly slowed, by an order of magnitude, channel closing on ATP removal and the speed of modification by MTS reagent in ATP. We conclude that, in every CFTR channel gating cycle, the NBD dimer interface separates simultaneously at both composite sites sufficiently to allow MTS reagents to access both signature-sequence serines. Relatively rapid modification of S1347C channels by larger reagents-MTS-glucose, MTS-biotin, and MTS-rhodamine-demonstrates that, at the noncatalytic composite site, this separation must exceed 8 A.

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22 This interpretation was corroborated by the finding that, for either cysteine target, the addition of the hydrolysis-impairing mutation K1250R (catalytic site Walker A Lys) similarly slowed, by an order of magnitude, channel closing on ATP removal and the speed of modification by MTS reagent in ATP. Login to comment
80 This template served for subsequent individual Ser- to-Cys mutations at positions 549, 605, and 1,347; Ala-to-Cys at 1,374 (Fig. S1); and the hydrolysis-impairing mutation K1250R, all introduced using QuikChange (Agilent Technologies). Login to comment
181 In CFTR channels mutated at NBD2 Walker A K1250, open burst durations are prolonged by about one (K1250R) or two (K1250A) orders of magnitude, in oocyte patches at room temperature (Vergani et al., 2003, 2005; Csan&#e1;dy et al., 2006). Login to comment
182 Accordingly, the current decay time constant on ATP withdrawal from S549C or S1347C CFTR channels bearing the K1250R mutation was slowed approximately 10-fold, to &#e07a;15 s (Fig. 7, A-C, gray fit curves and bars). Login to comment
184 The ratios of the current decay time constants upon MTS modification in the presence of ATP (Fig. 7 C, red and green bars) to those upon ATP washout (Fig. 7 C, gray bars) therefore remained near unity, averaging 1.2 &#b1; 0.2 (n = 3) for MTSET+ action on S549C-K1250R, and 1.2 &#b1; 0.1 (n = 7) for MTSACE action on S1347C-K1250R (Fig. 7 D, red and green open bars). Login to comment
185 The matching time courses of current decline caused by ATP removal or to MTS modification of either target cysteine, S549C or S1347C, despite over an order of Figure 7.ߓ Hydrolysis-impairing mutation, K1250R, of the conserved Walker A lysine in the active composite site similarly slows current decay after ATP washout and upon MTS modification of both S549C and S1347C channels. Login to comment
186 (A and B) ATP-activated (3 mM, black bars below records) currents of S549C-K1250R (A) and S1347C-K1250R (B) CFTR channels with single-exponential fits to current decline upon ATP removal (gray, &#e074;ATP w/o) or modification (&#e074;MTS) by 50 &#b5;M MTSET+ (red) or MTSACE (green); 20 mM DTT (black bars above records) restored activation of currents by ATP; asterisks above the records mark brief activations of Ca2+ - dependent Cl&#e032; currents to monitor speed of solution exchange (0.3 s in A and 0.2 s in B). Login to comment
187 (C) Average &#e074;ATPw/o (gray bars) with corresponding average &#e074;MTS from the same patches (left, S549C-K1250R: gray bar, w/o, 17 &#b1; 3.8 s; red bar, MTSET+ , 20.9 &#b1; 7.6 s; n = 3 measurements in three patches; right, S1347C-K1250R: gray bar, w/o, 15.4 &#b1; 2.0 s; green bar, MTSACE, 18.6 &#b1; 3.5 s; n = 9 and 7 measurements, respectively, in three patches). Login to comment
188 (D) Averages of individual ratios of washout and modification time constants determined for each pair of measurements (from experiments of C; red open bar, S549C-K1250R, &#e074;MTSET/&#e074;ATPw/o, 1.2 &#b1; 0.2; green open bar, S1347C-K1250R, &#e074;MTSACE/&#e074;ATPw/o, 1.2 &#b1; 0.1). Login to comment
331 That closure of (C832S-C1458S) channels containing S1347C or S549C target cysteines is indeed rate-limited by ATP hydrolysis (like wild type) is confirmed by the order of magnitude slowing of closure caused by the addition of the hydrolysis-impairing K1250R mutation (Figs. 7 and S4). Login to comment
332 Moreover, the &#e07a;15-s time constants for nonhydrolytic closure of those S1347C-K1250R-(C832S-C1458S) and S549C-K1250R-(C832S-C1458S) channels upon ATP washout are no shorter than those, 6-9 s, of K1250R CFTR channels bearing no other mutation (Vergani et al., 2005; Csan&#e1;dy et al., 2006; Szollosi et al., 2010, 2011). Login to comment
398 Closure of these cysteine-depleted channels containing a target cysteine is slowed at least an order of magnitude after the addition of the K1250R mutation (Figs. 7 and S4), arguing that closure of the unmodified channels, like that of wild-type CFTR, is normally rate-limited by hydrolysis of of hydrolysis appears sensitive to perturbations in and around the dead site, including mutation of the NBD1 Walker A motif (Powe et al., 2002; Vergani et al., 2003; Csan&#e1;dy et al., 2010, 2013) or NBD2 signature sequence (Tsai et al., 2010; Csan&#e1;dy et al., 2013), or replacement of the ATP bound there with an unnatural nucleotide (Tsai et al., 2010; Csan&#e1;dy et al., 2013). Login to comment
407 The fact that ATP-dependent current persisted after MTS modification means that CFTR channels continued to open and close, despite the presence of an &#e07a;8-&#c5; long, 6-&#c5; wide adduct covalently attached slowing of opening (C࢐O1; see above) and inferred severalfold speeding of nonhydrolytic closure (O1࢐C), could together explain the absence of measurable residual current of MTSACE-modified S1347C-K1250R channels. Login to comment
408 Assuming the K1250R mutation makes the rate k1 of the O1࢐O2 ATP hydrolysis step zero, the ATP washout time constant for unmodified S1347C-K1250R channels (Fig. 7 C) suggests that k&#e032;1 is &#e07a;0.06 s&#e032;1 , reflecting the considerable stability of the prehydrolytic NBD dimer. Login to comment
412 Finally, if the MTS adduct does destabilize the prehydrolytic dimer once S1347C-K1250R CFTR channels are modified, as the above analysis suggests, then our conclusion of strict state dependence of modification is further strengthened, particularly for nonhydrolytic channels. Login to comment
413 Otherwise, modification in the presence of ATP while S1347C-K1250R channels were open should have caused current to decay more rapidly than upon ATP washout before modification, contrary to observation (Figs. 7 and S4). Login to comment
418 Because no phosphate is released from the dead site, MTS access to S1347C requires dimer separation and hence channel closure, as for K1250R mutants. Login to comment
425 That observation is the apparent absence of residual current after MTSACE modification of S1374C-K1250R channels (Fig. 7 B) that are believed to close by nonhydrolytic dissociation of the NBD dimer (Vergani et al., 2005; Csan&#e1;dy et al., 2006, 2010), in contrast to the &#e07a;20% residual current after MTSACE observed (Figs. 5 B and 6 B) for S1347C channels that are closed by ATP hydrolysis. Login to comment
426 Opening rate of K1250R CFTR channels is maximal at 3 mM ATP (half-maximal [ATP] is &#e07a;150 &#b5;M; Szollosi et al., 2010) and comparable to that of wild-type CFTR (within a factor of 2 given the 10-fold longer open bursts, and Po of &#e07a;0.5, for K1250R; Vergani et al., 2005; Csan&#e1;dy et al., 2006, 2010; Szollosi et al., 2010). Login to comment
427 If the MTSACE adduct in the dead site influenced only channel opening, and exerted a similar approximate fivefold slowing effect on the opening of modified S1374C-K1250R channels as estimated above for modified S1347C channels, the residual current amplitude (percentage of control) of S1374C-K1250R ought to have been no smaller than that of S1347; in fact, it should be larger (ࣙ30%) because of their expected higher Po that results from slower closing. Login to comment
428 The lack of measurable (ࣙ5%) residual current in modified S1347C-K1250R channels, therefore, implies that the MTSACE adduct in the dead site exerted an additional effect, acceleration of nonhydrolytic closure; i.e., an increased rate k&#e032;1 of the step O1࢐C, the reversal of CFTR channel opening in the gating cycle C &#f083; O1࢐O2࢐C (Csan&#e1;dy et al., 2010). Login to comment

[hide] New structural insights into the gating movements ... J Gen Physiol. 2015 May;145(5):365-9. doi: 10.1085/jgp.201511399. Puljung MC
New structural insights into the gating movements of CFTR.
J Gen Physiol. 2015 May;145(5):365-9. doi: 10.1085/jgp.201511399., [PMID:25918357]

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79 To provide further evidence that the modification rates were limited by separation of the NBD dimer upon channel closure, the authors took advantage of a mutation (K1250R) in the Walker A motif of CS2. Login to comment
82 In the K1250R background, modification of cysteines in the signature sequences of both composite sites occurred at a much slower rate. Login to comment