ABCMdb

ABCC7 p.Trp401Gly

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

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

Abstract [show]
Cystic fibrosis transmembrane conductance regulator (CFTR), a member of the ABC (ATP binding cassette) transporter family, is a chloride channel whose activity is controlled by protein kinase-dependent phosphorylation. Opening and closing (gating) of the phosphorylated CFTR is coupled to ATP binding and hydrolysis at CFTR's two nucleotide binding domains (NBD1 and NBD2). Recent studies present evidence that the open channel conformation reflects a head-to-tail dimerization of CFTR's two NBDs as seen in the NBDs of other ABC transporters (Vergani et al., 2005). Whether these two ATP binding sites play an equivalent role in the dynamics of NBD dimerization, and thus in gating CFTR channels, remains unsettled. Based on the crystal structures of NBDs, sequence alignment, and homology modeling, we have identified two critical aromatic amino acids (W401 in NBD1 and Y1219 in NBD2) that coordinate the adenine ring of the bound ATP. Conversion of the W401 residue to glycine (W401G) has little effect on the sensitivity of the opening rate to [ATP], but the same mutation at the Y1219 residue dramatically lowers the apparent affinity for ATP by >50-fold, suggesting distinct roles of these two ATP binding sites in channel opening. The W401G mutation, however, shortens the open time constant. Energetic analysis of our data suggests that the free energy of ATP binding at NBD1, but not at NBD2, contributes significantly to the energetics of the open state. This kinetic and energetic asymmetry of CFTR's two NBDs suggests an asymmetric motion of the NBDs during channel gating. Opening of the channel is initiated by ATP binding at the NBD2 site, whereas separation of the NBD dimer at the NBD1 site constitutes the rate-limiting step in channel closing.

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6 Conversion of the W401 residue to glycine (W401G) has little effect on the sensitivity of the opening rate to [ATP], but the same mutation at the Y1219 residue dramatically lowers the apparent affinity for ATP by >50-fold, suggesting distinct roles of these two ATP binding sites in channel opening. Login to comment
7 The W401G mutation, however, shortens the open time constant. Login to comment
35 Although conversion of W401 to glycine (i.e., W401G) has little effect on the sensitivity of the channel opening rate to [ATP], this mutation shortens the mean open time. Login to comment
36 The shortening of the open time by the W401G mutation is also seen with the hydrolysis-deficient mutant background (i.e., E1371S), suggesting that the effect of W401G mutation is not through a perturbation of ATP hydrolysis. Login to comment
77 For the W401G mutant, 0.1 mM ATP elicits a similar level of activity. Login to comment
79 Unlike WT and W401G, 2.75 mM ATP does not saturate Figure 1. Login to comment
94 Current induced by various [ATP] was normalized to 2.75 mM ATP in the case of WT and W401G and to 20 mM ATP in the case of Y1219G. Login to comment
96 (D) ATP dose-response relationships of WT (black), W401G (red), and Y1219G (green). Login to comment
98 The K1/2 values are 0.09 ± 0.02 mM, 0.11 ± 0.02 mM, and 4.72 ± 1.12 mM for WT, W401G, and Y1219G, respectively. Login to comment
102 Fig. 1 D summarizes normalized macroscopic ATP dose-response relationships of WT, W401G, and Y1219G. Login to comment
103 Although the W401G mutation does not affect the apparent affinity, converting Y1219 to glycine causes a dramatic rightward shift of the ATP dose-response curve with a K1/2 of 4.72 ± 1.12 mM, >50-fold higher than that of WT (0.09 ± 0.02 mM). Login to comment
109 To test this hypothesis, we examined single-channel kinetics of WT, W401G, and Y1219G. Login to comment
111 At 2.75 mM ATP, WT and W401G channels close for hundreds of milliseconds between opening bursts. Login to comment
116 Fig. 2 C shows the relationship between the opening rate and [ATP] for WT, W401G, Y1219I, and Y1219G. Login to comment
125 (B) Representative single-channel current traces of WT, W401G, Y1219G, and Y1219I in response to [ATP] as marked. Login to comment
126 (C) Relationships between channel opening rates and [ATP] for WT (black), W401G (red), Y1219I (blue), and Y1219G (green). Login to comment
136 Although mutations of the W401 residue at the NBD1 site had minimal effect on the relationship between [ATP] and the opening rate (Fig. 2, B and C), close inspection of the single-channel current trace reveals that the channel open time is shorter for the W401G mutant (Fig. 2 B). Login to comment
137 Fig. 3 A summarizes the mean open time for WT, W401G, and Y1219G. Login to comment
139 In contrast, W401G exhibits ‫%04ف‬ decrease of the mean open time, suggesting that mutations that decrease the ATP binding affinity at the NBD1 site destabilize the open channel conformation (i.e., increase the closing rate). Login to comment
140 It should be noted that this shortening of the mean open time by W401G mutant is not readily reflected in the ATP dose-response relationship (Fig. 1 D). Login to comment
142 We considered three possibilities for the shortened open time seen with the W401G mutation at NBD1. Login to comment
143 First, since it is established that ATP hydrolysis at NBD2 leads to channel closing during the ATP hydrolysis-driven gating cycle, it is possible that the W401G mutation accelerates the ATP hydrolysis rate of NBD2. Login to comment
147 Third, the shortened open time of the W401G could be due to an allosteric effect. Login to comment
148 To examine the potential effect of mutation in NBD1 on ATP hydrolysis at NBD2, we introduced the W401G mutation into the E1371S background, a mutant CFTR whose ATP hydrolysis is abolished (Moody et al., 2002; Tombline et al., 2004; Vergani et al., 2005). Login to comment
149 If the shortened open time seen with the W401G mutant is indeed due to an increased ATP hydrolysis rate, the effect on the open time should be at least reduced once the ATP hydrolysis is eliminated. Login to comment
155 (A) Mean open times of WT and W401G in the presence of 2.75 mM ATP are 441.3 ± 49.4 ms (n = 13) and 248.7 ± 11.3 ms (n = 22), respectively. Login to comment
157 *** indicates P < 0.001 between WT and W401G. Login to comment
158 (B) Representative current relaxation traces upon withdrawal of 1 mM ATP plus PKA for E1371S, W401G/E1371S, triple/ E1371S, Y1219G/E1371S, and W401G/Y1219G/E1371S. Login to comment
164 Fig. 3 B shows experiments using current relaxation analysis to estimate the open time constants for E1371S and W401G/E1371S. Login to comment
165 Our results show that the relaxation time constant for W401G/E1371S (59.1 ± 4.6 s, n = 8) is shortened by ‫,%05ف‬ compared with that of E1371S (111.7 ± 12.1 s, n = 15) (Fig. 3 C), suggesting that the shorter open time of W401G is not secondary to an altered ATP hydrolysis rate. Login to comment
167 In addition, W401G/Y1219G/E1371S has a relaxation time constant of 49.0 ± 5.3 s (Fig. 3, B and C), which is similar to that of W401G/E1371S, indicating that W401, but not Y1219, plays a dominant role in modulating the open time. Login to comment
172 Third, the shortened relaxation time constant due to the W401G mutation can be prolonged by P-ATP (see below). Login to comment
178 We converted all three aromatic amino acids, including W401, F409, and F430 to glycine in the E1371S background and examined current relaxations of the W401G/F409G/F430G/E1371S (or triple/E1371S). Login to comment
179 Compared with the current relaxation of W401G/ E1371S (Fig. 3 B), the triple/E1371S mutation further shortens the time course of current decay. Login to comment
186 Fig. 4 B shows experiments examining current relaxations upon removal of ATP or P-ATP for E1371S, W401G/E1371S, Y1219G/ E1371S, and triple/E1371S. Login to comment
189 However, this prolongation effect of P-ATP is significantly larger for W401G/E1371S (2.7-fold) and triple/E1371S (greater than fourfold; Fig. 4 C). Login to comment
217 (B) Representative current relaxation traces of E1371S, W401G/E1371S, triple/ E1371S, and Y1219G/E1371S after withdrawal of 1 mM ATP plus PKA or 50 μM P-ATP plus PKA. Horizontal scale bars represent 200 s. Login to comment

[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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182 Interestingly, the equivalent mutation in ABP1, W401G, does not change the opening rate of the channel but instead increases the closing rate (Fig.4). Login to comment
193 A: τo for wild-type (WT) and W401G-CFTR in the presence of 2.75 mmol/L ATP. Login to comment
194 B: Representative current relaxation traces for E1371S, W401G/E1371S and W401G/ F409G/F430G/E1371S (or triple/E1371S). Login to comment
219 However, the mutation W401G/G551D (W401G is located at ABP1) decreases remarkably the effect of P-ATP, suggesting that it is at this binding site (ABP1) where P-ATP binds to increase the G551D-CFTR activity[66] . Login to comment

[hide] Mechanism of G551D-CFTR (cystic fibrosis transmemb... J Biol Chem. 2008 Feb 29;283(9):5364-9. Epub 2007 Dec 30. Bompadre SG, Li M, Hwang TC
Mechanism of G551D-CFTR (cystic fibrosis transmembrane conductance regulator) potentiation by a high affinity ATP analog.
J Biol Chem. 2008 Feb 29;283(9):5364-9. Epub 2007 Dec 30., 2008-02-29 [PMID:18167357]

Abstract [show]
Cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel gated by ATP binding and hydrolysis at its nucleotide binding domains (NBD). The NBDs dimerize in a head-to-tail configuration, forming two ATP binding pockets (ABP) with the ATP molecules buried at the dimer interface. Previous studies have indicated that ABP2, formed by the Walker A and B motifs of NBD2 and the signature sequence of NBD1, is the site critical for the ATP-dependent opening of CFTR. The G551D mutation in ABP2, the third most common cystic fibrosis-associated mutation, abolishes ATP-dependent gating, resulting in an open probability that is approximately 100-fold lower than that of wild-type channels. Interestingly, we found that the ATP analog N6-(2-phenylethyl)-ATP (P-ATP) increases G551D currents mainly by increasing the open time of the channel. This effect is reduced when P-ATP is applied together with ATP, suggesting a competition between ATP and P-ATP for a common binding site. Introducing mutations that lower the nucleotide binding affinity at ABP2 did not alter significantly the effects of P-ATP on G551D-CFTR, whereas an equivalent mutation at ABP1 (consisting of the Walker A and B motifs of NBD1 and the signature sequence of NBD2) dramatically decreased the potency of P-ATP, indicating that ABP1 is the site where P-ATP binds to increase the activity of G551D-CFTR. These results substantiate the idea that nucleotide binding at ABP1 stabilizes the open channel conformation. Our observation that P-ATP enhances the G551D activity by binding at ABP1 implicates that ABP1 can potentially be a target for drugs to bind and increase the channel activity.

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40 EXPERIMENTAL PROCEDURES Site-directed Mutagenesis-The constructs containing single mutations (G551D, W401G, and Y1219G) have been described previously (6, 7). Login to comment
93 As demonstrated previously, W401G and Y1219G are two mutations that can serve thispurpose.Trp-401wasshowninteractingdirectlywiththeade- nine ring of ATP via a stacking mechanism in the crystal structure of NBD1 from human CFTR (Ref. 19, Protein Data Bank (PDB) code 1XMI). Login to comment
95 It was found that, under the WT-CFTR background, the Y1219G mutation, but not the W401G mutation, causes a right- wardshiftoftheATPdose-responsecurve(6).Asimilarresultwas observed for P-ATP (see supplemental materials). Login to comment
110 In contrast, introducing W401G in the G551D background significantly reduced the effect of P-ATP. Login to comment
111 The activity of W401G/G551D-CFTR channels in the presence of 10 ␮M P-ATP is smaller than the activity of G551D channels under the same conditions. Login to comment
112 The mean current -fold increase is 1.9 Ϯ 0.2 for W401G/G551D-CFTR when compared with 6.2 Ϯ 0.7 for G551D-CFTR. Login to comment
115 The rightward shift of the P-ATP dose-response relationship suggests that mutating Trp-401 to glycine at ABP1 lowers the P-ATP binding affinity (Fig. 6). Login to comment
135 P-ATP dose-response relationships for G551D/Y1219G-(Œ) and W401G/G551D-CFTR (f). Login to comment
138 The K1/2 values are 13 Ϯ 5 and 79 Ϯ 30 ␮M for G551D/ Y1219G-CFTR and W401G/G551D-CFTR, respectively. Login to comment
172 P-ATP effect on W401G/G551D-CFTR. Login to comment
173 A, single-channel traces of W401G/G551D-CFTR in the presence or absence of P-ATP. Login to comment

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

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

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No. Sentence Comment
793 In addition, whether ATP binding at ABP1 is essential for channel opening by ATP binding at ABP2 is questioned since, unlike the Y1219G mutation, the W401G mutation in the ABP1 has little effect on the apparent affinity for ATP in both macroscopic and microscopic measurements (360). Login to comment
794 Although the mutations that presumably decrease ATP affinity at the ABP1 (K464A and W401G) have questionable effects on the ability of ATP to increase the opening rate of CFTR, both mutants show a shortened open time, suggesting a destabilization of the open state by the mutations (237, 360). Login to comment
804 Thus a reduction of the free energy of ATP binding could be reported as a decreased open-time constant as seen with the K464A and W401G mutants. 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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65 The dose-response curve of channel opening rate as a function of [ATP] was seen to shift dramatically to the right when the consensus site 2 was mutated ( Y1219G or Y1219I) but not when the degenerate site 1 was altered (W401G). Login to comment
68 Possibly W401G channels open frequently as monoliganded, while WT, with its high-affinity site 1, only rarely would. Login to comment
82 For example, the W401G mutant, with a weakened contact with the adenine of the ATP molecule bound at site 1, presented a less than twofold reduction in mean open time, interpreted by the 10-2 0 0 2 4 6 relativeopeningrate closingrate(s-1) 0.5 1.0 10-1 1 10 10-2 10-1 1 10 [MgATP] (mM)[MgATP] (mM) (b) (a) 10s 5mM 5mM [ATP] 50µM2s0.4 pA WT CFTR (c) Figure 2. Login to comment
63 The dose-response curve of channel opening rate as a function of [ATP] was seen to shift dramatically to the right when the consensus site 2 was mutated ( Y1219G or Y1219I) but not when the degenerate site 1 was altered (W401G). Login to comment
66 Possibly W401G channels open frequently as monoliganded, while WT, with its high-affinity site 1, only rarely would. Login to comment
80 For example, the W401G mutant, with a weakened contact with the adenine of the ATP molecule bound at site 1, presented a less than twofold reduction in mean open time, interpreted by the 10-2 0 0 2 4 6 relativeopeningrate closingrate(s-1) 0.5 1.0 10-1 1 10 10-2 10-1 1 10 [MgATP] (mM)[MgATP] (mM) (b) (a) 10s 5mM 5mM [ATP] 50µM2s0.4 pA WT CFTR (c) Figure 2. Login to comment

[hide] Gating of the CFTR Cl- channel by ATP-driven nucle... J Physiol. 2009 May 15;587(Pt 10):2151-61. Epub 2009 Mar 30. Hwang TC, Sheppard DN
Gating of the CFTR Cl- channel by ATP-driven nucleotide-binding domain dimerisation.
J Physiol. 2009 May 15;587(Pt 10):2151-61. Epub 2009 Mar 30., 2009-05-15 [PMID:19332488]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) plays a fundamental role in fluid and electrolyte transport across epithelial tissues. Based on its structure, function and regulation, CFTR is an ATP-binding cassette (ABC) transporter. These transporters are assembled from two membrane-spanning domains (MSDs) and two nucleotide-binding domains (NBDs). In the vast majority of ABC transporters, the NBDs form a common engine that utilises the energy of ATP hydrolysis to pump a wide spectrum of substrates through diverse transmembrane pathways formed by the MSDs. By contrast, in CFTR the MSDs form a pathway for passive anion flow that is gated by cycles of ATP binding and hydrolysis by the NBDs. Here, we consider how the interaction of ATP with two ATP-binding sites, formed by the NBDs, powers conformational changes in CFTR structure to gate the channel pore. We explore how conserved sequences from both NBDs form ATP-binding sites at the interface of an NBD dimer and highlight the distinct roles that each binding site plays during the gating cycle. Knowledge of how ATP gates the CFTR Cl- channel is critical for understanding CFTR's physiological role, its malfunction in disease and the mechanism of action of small molecules that modulate CFTR channel gating.

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66 The mutant Y1219G-CFTR, but not W401G-CFTR, exhibited a dramatic rightward shift of the relationship between [ATP] and the opening rate of the CFTR Cl-channel. Login to comment

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

Abstract [show]
Cystic fibrosis transmembrane conductance regulator (CFTR) is an adenosine triphosphate (ATP)-gated chloride channel. ATP-induced dimerization of CFTR's two nucleotide-binding domains (NBDs) has been shown to reflect the channel open state, whereas hydrolysis of ATP is associated with channel closure. Pyrophosphate (PPi), like nonhydrolytic ATP analogues, is known to lock open the CFTR channel for tens of seconds when applied with ATP. Here, we demonstrate that PPi by itself opens the CFTR channel in a Mg(2+)-dependent manner long after ATP is removed from the cytoplasmic side of excised membrane patches. However, the short-lived open state (tau approximately 1.5 s) induced by MgPPi suggests that MgPPi alone does not support a stable NBD dimer configuration. Surprisingly, MgPPi elicits long-lasting opening events (tau approximately 30 s) when administrated shortly after the closure of ATP-opened channels. These results indicate the presence of two different closed states (C(1) and C(2)) upon channel closure and a state-dependent effect of MgPPi on CFTR gating. The relative amount of channels entering MgPPi-induced long-open bursts during the ATP washout phase decreases over time, indicating a time-dependent dissipation of the closed state (C(2)) that can be locked open by MgPPi. The stability of the C(2) state is enhanced when the channel is initially opened by N(6)-phenylethyl-ATP, a high affinity ATP analogue, but attenuated by W401G mutation, which likely weakens ATP binding to NBD1, suggesting that an ATP molecule remains bound to the NBD1 site in the C(2) state. Taking advantage of the slow opening rate of Y1219G-CFTR, we are able to identify a C(2)-equivalent state (C(2)*), which exists before the channel in the C(1) state is opened by ATP. This closed state responds to MgPPi much more inefficiently than the C(2) state. Finally, we show that MgAMP-PNP exerts its effects on CFTR gating via a similar mechanism as MgPPi. The structural and functional significance of our findings is discussed.

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19 The stability of the C2 state is enhanced when the channel is initially opened by N6 -phenylethyl-ATP, a high affinity ATP analogue, but attenuated by W401G mutation, which likely weakens ATP binding to NBD1, suggesting that an ATP molecule remains bound to the NBD1 site in the C2 state. Login to comment
35 Electrophysiological recordings Before inside-out patch clamp recordings, glass chips containing CHO cells transfected with various CFTR constructs, W401G, Y1219G, S1347G, E1371S, and WT-CFTR, were transferred to a continuously perfused chamber located on the stage of an inverted microscope (Olympus). Login to comment
208 As a control, when a similar experiment was performed for W401G, ATP still out-competes MgPPi as demonstrated by a large fraction of the fast component during current relaxation (not depicted). Login to comment
243 Previously, Zhou et al. (2006) showed that P-ATP may assume a tighter binding than ATP at NBD1 of W401G-CFTR. Login to comment
244 Indeed, opening of W401G-CFTR channels with P-ATP results in a higher fraction (24 ± 1.5%; n = 5) of lock-open channels (Fig. 7, B and C). Login to comment
265 We then used the same protocol to test the effect of MgPPi on the W401G mutation, which likely decreases the Figure 7. Login to comment
268 (B) The lock-open efficiency of MgPPi was reduced by the W401G mutation, and P-ATP can partially restore the effectiveness of MgPPi on W401g-CFTR. Login to comment
285 Similar experiments as shown in Fig. 7 were performed for MgAMP-PNP with W401G-CFTR and P-ATP, and virtually identical results were obtained (not depicted). Login to comment
392 Second, W401G mutation, which likely reduces ATP binding affinity in NBD1 (Zhou et al., 2006), decreases the stability of the C2 state. Login to comment
433 Echoing this observation, the lock-open duration of MgPPi is reduced by mutations that decrease the ATP binding affinity to NBD1, K464A, and W401G, but can be partially restored by a high affinity ATP analogue, P-ATP (unpublished data; compare Powe et al., 2002; Zhou et al., 2006). Login to comment
440 Supporting this hypothesis, we demonstrated that the C2 state dissipates much faster when residues at either NBD1 (W401G; Fig. 7 B) or the signature sequence of NBD2 were mutated (S1347G; Fig. S1). Login to comment

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

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

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8 We show that P-dATP binds to ABP1 to potentiate the activity of G551D, and mutations in both sides of ABP1 (W401G and S1347G) decrease its potentiation effect, suggesting that the action of P-dATP takes place at the interface of both NBDs. Login to comment
123 Structural Nature of the Interaction between P-dATP and G551D-CFTR-To further understand the effect of P-dATP on G551D channels, we made mutations that lower the apparent binding affinity of ATP in each ABP, W401G in ABP1, and Y1219G in ABP2 (2). Login to comment
127 On the other hand, the dose-response relationship of P-dATP for the W401G/G551D mutant (Fig. 4C) shows a significant rightward shift compared with that of G551D-CFTR accompanied with a considerable reduction of the maximal effect of P-dATP. Login to comment
150 In fact the mutation S1347G in NBD2 decreases the potentiation effect of P-dATP to a similar extent as the W401G mutation in NBD1 (Fig. 4C). Login to comment
163 Representative current traces of G551D/Y1219G (A), W401G/G551D (B), and G551D/S1347G (D) in the presence of 10 ␮M P-dATP. Login to comment
164 C, P-dATP dose-response relationships for G551D (red, F), W401G/G551D (blue, E), G551D/Y1219G (green, Œ), and G551D/S1347G (black, f). Login to comment
169 F, comparison between the -fold increase in the current for G551D (red), W401G/G551D (blue), and G551D/S1347G (black) channels in the presence of saturating concentrations of dATP, P-ATP, and P-dATP. Login to comment
192 Indeed, introducing the mutation W401G (ABP1) or the corresponding mutation Y1219G (ABP2) in ⌬F508-CFTR resulted in a reduction of the effect of 10 ␮M P-dATP (3.6 Ϯ 0.7 and 0.7 Ϯ 0.1 current -fold increase, respectively), suggesting that binding of P-dATP to both ABPs is involved in mediating the effect of P-dATP on ⌬F508-CFTR channels (Fig. 8). Login to comment
228 P-dATP effect on W401G/⌬F508-CFTR and ⌬F508/Y1219G-CFTR. Login to comment
229 Representative current traces of W401G/⌬F508 (A) and ⌬F508/Y1219G (B)inthepresenceof50␮M P-dATP.C,summaryofthemaximumcurrent-fold increase in activity induced by 10 ␮M and 50 ␮M P-dATP in W401F/⌬F508 and ⌬F508/Y1219G, and 10 ␮M P-dATP in ⌬F508. Login to comment
241 In support of this notion, the mutation W401G, which likely weakens nucleotide binding to ABP1 (2), decreases the potentiation effect of P-dATP in G551D channels (Fig. 4C). Login to comment
270 On the other hand, it was somewhat surprising to observe a reduction of the effect of P-dATP on W401G/⌬F508, because the W401G mutation alone does not affect much the effect of P-dATP on WT-CFTR (see supplemental information). Login to comment

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

Abstract [show]
Cystic fibrosis transmembrane conductance regulator (CFTR), a member of the adenosine triphosphate (ATP) binding cassette (ABC) superfamily, is an ATP-gated chloride channel. Like other ABC proteins, CFTR encompasses two nucleotide binding domains (NBDs), NBD1 and NBD2, each accommodating an ATP binding site. It is generally accepted that CFTR's opening-closing cycles, each completed within 1 s, are driven by rapid ATP binding and hydrolysis events in NBD2. Here, by recording CFTR currents in real time with a ligand exchange protocol, we demonstrated that during many of these gating cycles, NBD1 is constantly occupied by a stably bound ATP or 8-N(3)-ATP molecule for tens of seconds. We provided evidence that this tightly bound ATP or 8-N(3)-ATP also interacts with residues in the signature sequence of NBD2, a telltale sign for an event occurring at the NBD1-NBD2 interface. The open state of CFTR has been shown to represent a two-ATP-bound NBD dimer. Our results indicate that upon ATP hydrolysis in NBD2, the channel closes into a "partial NBD dimer" state where the NBD interface remains partially closed, preventing ATP dissociation from NBD1 but allowing the release of hydrolytic products and binding of the next ATP to occur in NBD2. Opening and closing of CFTR can then be coupled to the formation and "partial" separation of the NBD dimer. The tightly bound ATP molecule in NBD1 can occasionally dissociate from the partial dimer state, resulting in a nucleotide-free monomeric state of NBDs. Our data, together with other structural/functional studies of CFTR's NBDs, suggest that this process is poorly reversible, implying that the channel in the partial dimer state or monomeric state enters the open state through different pathways. We therefore proposed a gating model for CFTR with two distinct cycles. The structural and functional significance of our results to other ABC proteins is discussed.

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98 When the ligand exchange experiment was performed with a single W401G channel (Fig. 3 A; similar results were seen in five other single-channel recordings), PATP (red trace) induced longer openings without an obvious delay observed with WT channels (compare Fig. 3 B with Fig. 1 B). Login to comment
100 We interpreted these results to mean that the W401G mutation in NBD1 significantly decreases the resident time of the stably bound ATP molecule (from 50 s for WT-CFTR to 2.5 s for W401G-CFTR) so that PATP can replace it more rapidly and exert its second effect: increasing channel open change in channel kinetics and shows that ligand switches from PATP back to ATP also cause an immediately changed closed time and a delayed alteration of the open time. Login to comment
117 Indeed, single S1347G channels, like W401G-CFTR (Fig. 3 A), opened into long bursts without a delay after changing the ligand from ATP to PATP (red trace in Fig. 4 B; channel kinetics summarized in Fig. 4 C), suggesting that the S1347G mutation in NBD2 dramatically shortens the dwell time of ATP in NBD1. Login to comment
132 (A) The response of a single-ATP-gated W401G channel to a sudden exposure of PATP (red trace). Login to comment
134 A similar observation was seen in five other patches containing a single W401G-CFTR channel. Login to comment
136 (C) Macroscopic currents recorded from hundreds of W401G channels. Login to comment

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

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

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82 This idea is recapitulated in Fig. 1B, which shows that the lock-open duration (i.e. current decay constant calculated from traces in Fig. 1C, see also "Experimental Procedures") of WT channels elicited by ATP and PPi (left) was shortened by the W401G mutation in NBD1 (middle) but was prolonged when PATP, instead of ATP, was used (right). Login to comment
88 As expected, non-conservative substitutions of Trp-401 with Ile or Gly (W401I and W401G), which are unable to stack FIGURE 1. Login to comment
93 P, PATP. B, current traces showing WTor W401G-CFTR channels locked open by PPi with ATP or PATP. Login to comment
96 Red, WT/ATP ϩ PPi; green, W401G/ATP ϩ PPi; light blue, WT/PATP ϩ PPi. Login to comment
189 These mutations include W401G,W401I (Fig. 1, B-D), which eliminate a ring-ring stacking interaction, S1347G (supplemental Fig. S6), which may break a hydrogen bond between ATP and the NBD2 signature motif, and G1349I (supplemental Fig. S6), whose side chain likely protrudes into site 1 and causes a steric clash with ATP (22-24). Login to comment

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

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

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58 In contrast, the current relaxation for the W401G mutants showed a major monotonic fast decay which was followed by some minimal residual channel activity (Fig. 2a). Login to comment
76 The high affinity of P-ATP was suggested to come from its slow dissociation rate from NBD1 and NBD2 whereas the closing rate in P-ATP was just a little slower than that in B A C Y1219G ATP 5 mM 10 s 1 pA 25 s 2 pA Y1219G 25 s 2 pA ATP 5 mM W401G W 9 1 2 1 Y I 9 1 2 1 Y Y1219F 50 s 50 s 20 pA 50 s 20 pA 50 s 20 pA 20 pA ATP 5 mM 10 s 0.4 pA Y1219G Fig. 2 Macroscopic current relaxations for Y1219 and W401 mutants. Login to comment
77 a Representative traces of Y1219G and W401G macroscopic currents responding to a rapid application and removal of 5 mM ATP. Login to comment
121 In contrast, decreasing ATP binding in NBD1 by the W401G mutation does not induce a significant slow component of current relaxations (Fig. 2a). Login to comment

[hide] A single amino acid substitution in CFTR converts ... J Gen Physiol. 2014 Oct;144(4):311-20. doi: 10.1085/jgp.201411247. Epub 2014 Sep 15. Lin WY, Jih KY, Hwang TC
A single amino acid substitution in CFTR converts ATP to an inhibitory ligand.
J Gen Physiol. 2014 Oct;144(4):311-20. doi: 10.1085/jgp.201411247. Epub 2014 Sep 15., [PMID:25225552]

Abstract [show]
Cystic fibrosis (CF), one of the most common lethal genetic diseases, is caused by loss-of-function mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encodes a chloride channel that, when phosphorylated, is gated by ATP. The third most common pathogenic mutation, a glycine-to-aspartate mutation at position 551 or G551D, shows a significantly decreased open probability (Po) caused by failure of the mutant channel to respond to ATP. Recently, a CFTR-targeted drug, VX-770 (Ivacaftor), which potentiates G551D-CFTR function in vitro by boosting its Po, has been approved by the FDA to treat CF patients carrying this mutation. Here, we show that, in the presence of VX-770, G551D-CFTR becomes responsive to ATP, albeit with an unusual time course. In marked contrast to wild-type channels, which are stimulated by ATP, sudden removal of ATP in excised inside-out patches elicits an initial increase in macroscopic G551D-CFTR current followed by a slow decrease. Furthermore, decreasing [ATP] from 2 mM to 20 microM resulted in a paradoxical increase in G551D-CFTR current. These results suggest that the two ATP-binding sites in the G551D mutant mediate opposite effects on channel gating. We introduced mutations that specifically alter ATP-binding affinity in either nucleotide-binding domain (NBD1 or NBD2) into the G551D background and determined that this disease-associated mutation converts site 2, formed by the head subdomain of NBD2 and the tail subdomain of NBD1, into an inhibitory site, whereas site 1 remains stimulatory. G551E, but not G551K or G551S, exhibits a similar phenotype, indicating that electrostatic repulsion between the negatively charged side chain of aspartate and the gamma-phosphate of ATP accounts for the observed mutational effects. Understanding the molecular mechanism of this gating defect lays a foundation for rational drug design for the treatment of CF.

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52 G551D- and G551D/W401G-CFTR and double exponential functions in WT-CFTR using a built-in Levenberg-Marquardt-based algorithm. Login to comment
184 (A) Acceleration of the slow-phase current decay by the W401G mutation. Login to comment
185 A continuous recording demonstrates an unequivocal biphasic response upon ATP washout for G551D/ W401G-CFTR. Login to comment
188 31.1 &#b1; 5.3 s (n = 12) for G551D-CFTR, 19.0 &#b1; 3.2 s (n = 8) for G551D/W401G-CFTR, and 31.7 &#b1; 5.9 s (n = 12) for G551D/Y1219G-CFTR. Login to comment

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

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

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112 To explore the role of phosphorylation further, we studied the effect of deleting the R domain from CFTR (residues 634-836) [12, 7], which removes all the major PKA/PKC Table 1 Summary of the FSK stimulation of whole cell currents and Erev shifts observed with the CFTR constructs used in this study CFTR Construct n FSK Stimulation (%&#b1;SEM) Erev shift (mV&#b1;SEM) WT (50 bc;M ATP) 5 180&#b1;96 15.0&#b1;3.6 WT (100 bc;M ATP) 6 12,000&#b1;6,000 15.2&#b1;3.0 WT (300 bc;M ATP) 8 1,200&#b1;600 17.0&#b1;3.0 WT (1 mM ATP) 24 13,000&#b1;6,000 23.7&#b1;1.8 WT (1.3 mM ATP) 9 1,400&#b1;900 16.7&#b1;2.6 WT (2 mM ATP) 24 6,100&#b1;5,300 16.7&#b1;1.6 WT (5 mM ATP) 7 1,600&#b1;1,000 20.1&#b1;4.4 WT (50 bc;M ATP + 50 bc;M P-ATP) 7 224&#b1;130 15.3&#b1;1.0 WT + Genistein 4 7,600&#b1;5,200 26.1&#b1;5.4 WT + AMP-PNP 5 2,800&#b1;2,500 21.8&#b1;5.5 WT (3 mM MgCl2) 7 28,000&#b1;17,000 18.3&#b1;3.1 R104Q 5 4,600&#b1;1,600 28.6&#b1;4.7 K114C 5 12,000&#b1;6,700 29.2&#b1;3.0 R117Q 4 33,000&#b1;20,000 30.1&#b1;3.4 K329A 5 13,000&#b1;10,000 33.7&#b1;2.1 R334Q 9 13,000&#b1;6,700 27.3&#b1;2.9 K335A 5 3,200&#b1;1,500 20.8&#b1;7.1 W401G 7 2,600&#b1;1,800 18.5&#b1;4.8 Delta-R (No Stim) 5 - 25.1&#b1;2.7 Delta-R (No FSK, Genistein) 5 140&#b1;13 22.7&#b1;3.0 Delta-R (FSK, No Genistein) 4 89&#b1;14 15.6&#b1;6.0 Delta-R (FSK + Genistein) 6 639&#b1;432 25.1&#b1;4.9 Delta-R-E1371S (No FSK) 9 - 21.4&#b1;4.8 Delta-R-E1371S (FSK) 4 2,600&#b1;1,400 15.3&#b1;4.7 K892Q 7 16,000&#b1;9,500 36.8&#b1;4.8 R899E 4 1,200&#b1;400 25.0&#b1;2.7 R899K 4 1,600&#b1;900 26.6&#b1;2.9 R899Q 7 5,400&#b1;2,800 30.0&#b1;1.3 R899Q + AMP-PNP 4 72,000&#b1;50,000 15.2&#b1;2.8 R899Q-E1371Q (No FSK) 4 - 18.4&#b1;5.9 R899Q-E1371Q (FSK) 6 107&#b1;48 15.6&#b1;3.0 R1128Q 6 14,000&#b1;6,100 41.1&#b1;4.2 Y1219G 6 3,200&#b1;2,500 19.2&#b1;3.3 E1371Q (No FSK) 6 - 25.5&#b1;3.5 E1371Q (FSK) 8 -28&#b1;9 22.3&#b1;4.0 E1371Q (FSK, No ATP, No GTP) 8 270&#b1;130 19.4&#b1;4.5 E1371Q + AMP-PNP (No FSK) 4 - 24.7&#b1;6.5 E1371Q + AMP-PNP (FSK) 8 180&#b1;170 17.4&#b1;4.0 Vector Control 4 15&#b1;38 - FSK stimulation was calculated as the percentage increase in current density at -60 mV from the Erev, after 5-min exposure to 10 bc;M FSK. Login to comment
131 Note that both of these mutations substantially reduce CFTR channel activity through mechanisms that are not secondary to changes in ATP hydrolysis rate [49], and in the case of W401G, not due to an apparent change in ATP binding affinity. Login to comment
132 Sub-panels a, b and e of Fig. 5 show that the FSK-stimulated W401G CFTR mutant exhibited significantly reduced [Cl- ]o sensing compared to WT CFTR, whereas the FSK-stimulated Y1219G CFTR mutant did not (Fig. 5c-e). Login to comment
133 We then reasoned that if events downstream of ATP binding to site 1, and/or NBD dimerisation, were altered by changes in [Cl- ]o (as the W401G data suggested), then the effect of [Cl- ]o on CFTR activity should be sensitive to the concentration of cytosolic ATP. Login to comment
149 Although neutralization of the positive charge at R899, as well as charge reversal, eliminated [Cl- ]o sensing by CFTR (Fig. 2), we have no direct evidence that Cl-ions W401G Y1219G 100 ms 5 nA W401G (i) (ii) (iii) (iv) 100 ms 4 nA (i) (ii) (iii) (iv) Y1219G A B E C D F Fig. 5 ATP binding to site 1, but not site 2, underlies [Cl- ]o sensing by CFTR. Login to comment
150 a, c Representative fWCR current recordings measured between &#b1;100 mVin 20 mV steps from HEK cells transfected with W401G CFTR or Y1219G CFTR, as indicated. The current traces are from the top down: (i) unstimulated in 155.5 mM [Cl- ]o, (ii) forskolin (FSK)-stimulated in 155.5 mM [Cl- ]o, (iii) FSK-stimulated in 35.5 mM [Cl- ]o and (iv) FSK-stimulated in 155.5 mM [Cl- ]o. Dotted line to the right of the current traces indicates zero current level. b, d Representative I-V plots for the data presented in a and c. e Percentage current stimulation by [Cl- ]o for WT CFTR (n=24) and for W401G (NBD1) and Y1219G (NBD2) mutants (see Fig. 1) (n=7-8). Login to comment
179 Thirdly, the fact that [Cl- ]o sensing is abolished by the W401G mutation in NBD1, but not by the corresponding NBD2 mutant, Y1219G (Fig. 5e), suggests that ATP binding at NBD1, and not NBD2, is mainly responsible for transducing the [Cl- ]o-dependent gating changes. Login to comment