ABCMdb

ABCC7 p.Tyr1219Gly

Predicted by SNAP2:	A: D (85%), C: D (85%), D: D (95%), E: D (91%), F: D (80%), G: D (91%), H: D (85%), I: D (85%), K: D (91%), L: D (91%), M: D (91%), N: D (91%), P: D (95%), Q: D (91%), R: D (91%), S: D (91%), T: D (91%), V: D (85%), W: D (80%),
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, W: 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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78 However, for the Y1219G mutant, 0.1 mM hardly induces any current different from the basal activity. 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
100 the current for Y1219G (unpublished data). Login to comment
102 Fig. 1 D summarizes normalized macroscopic ATP dose-response relationships of WT, W401G, and Y1219G. Login to comment
106 The ATP dose-response relationships of Y1219F and Y1219I mutants lie between those of WT and Y1219G. Login to comment
109 To test this hypothesis, we examined single-channel kinetics of WT, W401G, and Y1219G. Login to comment
112 However, even at 20 mM ATP, most of the closed events for Y1219G last for several seconds. Login to comment
113 Since the opening rate of Y1219G is not saturated at 20 mM ATP, we also studied single-channel kinetics of Y1219I, which shows a smaller shift in the ATP dose-response relationship (Fig. 2 A). 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
122 (A) Normalized ATP dose-response relationships of WT (black line, Michaelis-Menten fit from Fig. 1 D), Y1219W (brown), Y1219F (pink), Y1219I (blue), and Y1219G (green line, Michaelis-Menten fit from Fig. 1 D). 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
129 (D) Relationships between channel opening rates and [ATP] for ∆R-CFTR (black), ∆R-Y1219I (blue), and ∆R-Y1219G (green). Login to comment
134 Fig. 2 D shows a similar rightward shift of the ATP dose-response relationships for Y1219I and Y1219G mutants under the ∆R-CFTR background. Login to comment
137 Fig. 3 A summarizes the mean open time for WT, W401G, and Y1219G. Login to comment
138 Although the Y1219G mutation causes a dramatic change of the relationship between [ATP] and the opening rate, it has negligible effect on the mean open time. Login to comment
156 The mean open time of Y1219G in the presence of 20 mM ATP is 399.1 ± 40.4 ms (n = 5). 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
166 In contrast, although the Y1219G mutation greatly decreases the apparent affinity of ATP (Fig. 2 C), introducing this mutation into the E1371S background has little effect on the relaxation time constant (107.6 ± 12.4 s, n = 7) (Fig. 3, B and C). Login to comment
167 In addition, W401G/Y1219G/E1371S has a relaxation time constant of 49.0 ± 5.3 s (Fig. 3, B and C), which is similar to that of W401G/E1371S, indicating that W401, but not Y1219, plays a dominant role in modulating the open time. Login to comment
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
188 P-ATP also increases the relaxation time constant of Y1219G/E1371S by approximately twofold. Login to comment
206 Interestingly, although the Y1219G mutation causes a drastic shift of the ATP dose-response relationship (Figs. 1 and 2), it does not affect the mean open time. 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] G551D and G1349D, two CF-associated mutations in t... J Gen Physiol. 2007 Apr;129(4):285-98. Epub 2007 Mar 12. Bompadre SG, Sohma Y, Li M, Hwang TC
G551D and G1349D, two CF-associated mutations in the signature sequences of CFTR, exhibit distinct gating defects.
J Gen Physiol. 2007 Apr;129(4):285-98. Epub 2007 Mar 12., [PMID:17353351]

Abstract [show]
Mutations in the gene encoding cystic fibrosis transmembrane conductance regulator (CFTR) result in cystic fibrosis (CF). CFTR is a chloride channel that is regulated by phosphorylation and gated by ATP binding and hydrolysis at its nucleotide binding domains (NBDs). G551D-CFTR, the third most common CF-associated mutation, has been characterized as having a lower open probability (Po) than wild-type (WT) channels. Patients carrying the G551D mutation present a severe clinical phenotype. On the other hand, G1349D, also a mutant with gating dysfunction, is associated with a milder clinical phenotype. Residues G551 and G1349 are located at equivalent positions in the highly conserved signature sequence of each NBD. The physiological importance of these residues lies in the fact that the signature sequence of one NBD and the Walker A and B motifs from the other NBD form the ATP-binding pocket (ABP1 and ABP2, named after the location of the Walker A motif) once the two NBDs dimerize. Our studies show distinct gating characteristics for these mutants. The G551D mutation completely eliminates the ability of ATP to increase the channel activity, and the observed activity is approximately 100-fold smaller than WT-CFTR. G551D-CFTR does not respond to ADP, AMP-PNP, or changes in [Mg(2+)]. The low activity of G551D-CFTR likely represents the rare ATP-independent gating events seen with WT channels long after the removal of ATP. G1349D-CFTR maintains ATP dependence, albeit with a Po approximately 10-fold lower than WT. Interestingly, compared to WT results, the ATP dose-response relationship of G1349D-CFTR is less steep and shows a higher apparent affinity for ATP. G1349D data could be well described by a gating model that predicts that binding of ATP at ABP1 hinders channel opening. Thus, our data provide a quantitative explanation at the single-channel level for different phenotypes presented by patients carrying these two mutations. In addition, these results support the idea that CFTR's two ABPs play distinct functional roles in gating.

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200 It remains possible that this mutation lowers ATP binding affinity as other mutations in ABP2 (e.g., Y1219G in Zhou et al., 2006; K1250A in Vergani et al., 2003). 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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180 The mutation Y1219G shows an ATP dose-response relationship shifted more than 50-fold towards higher [ATP], however more conservative mutations (Y1219I, Y1219F) show smaller shifts, indicating the importance of the nature of the side chain in the interaction with the ATP molecule. 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
96 Although the Y1219G mutation decreases the apparent affinity of ATP and P-ATP in WT background, introducing this mutation into the G551D background has little influence on the FIGURE 2. Login to comment
106 Fig. 4A shows a representative trace of G551D/ Y1219G-CFTR channels in the absence and presence of 10 ␮M P-ATP. Login to comment
109 The G551D/Y1219G-CFTR P-ATP dose response is very similar to the G551D-CFTR dose response, suggesting that lowering the binding affinity at the ABP2 site does not alter the effect of P-ATP. Login to comment
130 P-ATP effect on G551D/Y1219G-CFTR. Login to comment
131 A, single-channel traces of Y1219G/G551D-CFTR in the presence or absence of P-ATP. Login to comment
132 The mutation Y1219G (located in ABP2) decreases the apparent P-ATP binding affinity under the WT background (see supplemental materials), but when introduced into the G551D background, the P-ATP potentiation is not affected. Login to comment
133 B, comparison of the mean currents and mean open times for G551D and G551D/Y1219G in the presence of 10 ␮M P-ATP (n ϭ 5-11). 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

[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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785 When Y1219 is mutated to a glycine (Y1219G), the ATP dose-response relationship shows a dramatic rightward shift with a K0.5 Ͼ50-fold higher than that of wild-type channels. Login to comment
787 The ATP dose-response relationships of Y1219F and Y1219I mutants lie between those of wild type and Y1219G, suggesting a correlation between changes of the ATP sensitivity and the chemical natures of the side chain at this position. Login to comment
788 Single-channel kinetic analysis indicates that the shifts of the ATP dose-response relationships in Y1219G and Y1219I mutants are mainly due to changes of the opening rate (360). Login to comment
790 Unlike the mutation at the Walker A lysine residue, the Y1219G mutation does not affect the open-time constant significantly, suggesting that the mutation does not alter ATP hydrolysis at ABP2. Login to 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

[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
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

[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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20 Taking advantage of the slow opening rate of Y1219G-CFTR, we are able to identify a C2-equivalent state (C2*), which exists before the channel in the C1 state is opened by ATP. 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
198 (C) Effects of PPi on Y1219G-CFTR. Login to comment
207 Fitting the data points (red curve) by a single-exponential function estimates the lifetime of the C2 state to be 27.4 s. the data for WT-CFTR (Fig. 5 A), the current relaxation upon the removal of ATP and MgPPi follows a monotonic decay with a time constant of ␶ = 30.7 ± 4.5 s (n = 5), indicating that almost all Y1219G-CFTR channels have been locked open under this experimental condition. Login to comment
232 Fig. 5 C shows that 2 mM MgPPi, when added to 2 mM ATP solution, dramatically increased the Y1219G-CFTR macroscopic current. Login to comment
252 To address this question, we used Y1219G-CFTR to test whether MgPPi has the same effect on these two account for the long-lasting memory assumed by the C2 state. Login to comment
291 Treating the channels that have been closed for a long time (thus in the C1 state) with a low concentration of ATP should favor an accumulation of the C2* state because the transition rate from C2* to O is significantly decreased by the Y1219G mutation, whereas a high concentration of ATP opens the channel more frequently and thus brings more channels to the C2 state. Login to comment
293 After PKA and ATP activation and a 1-min washout, we first treated Y1219G channels with 500 μM ATP, which only elicited minimal current. Login to comment
302 tition between ATP and MgAMP-PNP, suggesting that the Y1219G mutation decreases the binding affinity of ATP to a similar extent as it lowers the affinity for MgAMP-PNP. Login to comment
309 As seen in Fig. 10 (B and C), when [ATP] = [MgAMP-PNP] = 2 mM, the fractional amplitudes of the slow component upon current relaxation are 73 ± 3% (n = 6) and 71 ± 4% (n = 6) for WT-CFTR and Y1219G-CFTR, respectively. Login to comment
310 Thus, although the Y1219G mutation alters the competition between ATP and MgPPi for the NBD2 site (Fig. 5), the same mutation does not significantly affect the compe- Figure 9. Login to comment
312 (A) A continuous current trace of Y1219G-CFTR. Login to comment
326 (C) Y1219G channels opened by ATP plus PKA were locked open by 2 mM MgAMP-PNP plus 2 mM ATP. Login to comment
335 Zhou et al. (2006) reported that Y1219G-CFTR, which presumably loses the ␲-electron- stacking interaction between the aromatic side chain of the tyrosine residue and the adenine ring of ATP, has a far lower ATP binding affinity compared with WT-CFTR. Login to comment
340 Although we cannot rule out the possibility that MgPPi has a low binding affinity simply because it does not bind to the Walker A domain as well as ATP, the observation that MgAMPPNP elicits a maximal effect on CFTR at low millimolar concentration (Vergani et al., 2003) (Fig. 10 A), and that the binding affinity of MgAMP-PNP is weakened by Y1219G mutation (Fig. 10 C), suggests that a lack of the ring-ring interaction may have a greater impact on the ligand binding affinity than a slight structural alteration of the phosphate group. Login to comment
442 Here, using the Y1219G mutation to slow down the channel opening rate, we identify another closed state (C2*) that exists before the channel is opened by ATP from the C1 state (Figs. 8 B and 9). 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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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
126 The dose-response relationships of P-dATP for G551D/Y1219G-CFTR (Fig. 4C) show that P-dATP increases the current of the G551D/Y1219G mutant to a somewhat similar extent as for G551D channels. 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
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
249 In fact the mutation Y1219G in ABP2, which greatly decreases the apparent nucleotide-binding affinity at this site, barely diminishes the effect of P-dATP. Login to comment
269 The decrease in the effect of P-dATP in ⌬F508 channels that contain the Y1219G mutation is easier to understand, because this mutation decreases the ATP-binding affinity at ABP2, the site that controls the ATP-dependent opening of the channel, by Ͼ50-fold. 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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272 However, in Figs. S5 and S6, we show that this is unlikely the case because introducing the Y1219G mutation, which greatly disrupts ATP or PATP binding in NBD2 (Fig. S5), into the G551D background does not significantly (P = 0.78) alter the time constant of current increase upon ATP/PATP switch (Fig. S6). 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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112 Moreover, we also found that mutating the Trp-401-equivalent residue in NBD2 (Y1219G), which greatly decreases the NBD2 ATP affinity (19), had little effect on ATP-mediated activation of W401Y,W401F/ G551D channels (supplemental Fig. S4). Login to comment
221 However, this NBD dimer state, if it exists, may have an unoccupied site 2 to avoid a possible steric clash between ATP and the Asp-551 residue, an idea resonant with our third conclusion that NBD2 could remain vacant during ATP-dependent gating of optimized G551D channels as the Y1219G mutation, which disrupts ATP binding in NBD2, posed no functional impact on these channels (supplemental Fig. S4). Login to comment

[hide] Conserved allosteric hot spots in the transmembran... J Biol Chem. 2014 Jul 18;289(29):19942-57. doi: 10.1074/jbc.M114.562116. Epub 2014 May 29. Wei S, Roessler BC, Chauvet S, Guo J, Hartman JL 4th, Kirk KL
Conserved allosteric hot spots in the transmembrane domains of cystic fibrosis transmembrane conductance regulator (CFTR) channels and multidrug resistance protein (MRP) pumps.
J Biol Chem. 2014 Jul 18;289(29):19942-57. doi: 10.1074/jbc.M114.562116. Epub 2014 May 29., [PMID:24876383]

Abstract [show]
ATP-binding cassette (ABC) transporters are an ancient family of transmembrane proteins that utilize ATPase activity to move substrates across cell membranes. The ABCC subfamily of the ABC transporters includes active drug exporters (the multidrug resistance proteins (MRPs)) and a unique ATP-gated ion channel (cystic fibrosis transmembrane conductance regulator (CFTR)). The CFTR channel shares gating principles with conventional ligand-gated ion channels, but the allosteric network that couples ATP binding at its nucleotide binding domains (NBDs) with conformational changes in its transmembrane helices (TMs) is poorly defined. It is also unclear whether the mechanisms that govern CFTR gating are conserved with the thermodynamically distinct MRPs. Here we report a new class of gain of function (GOF) mutation of a conserved proline at the base of the pore-lining TM6. Multiple substitutions of this proline promoted ATP-free CFTR activity and activation by the weak agonist, 5'-adenylyl-beta,gamma-imidodiphosphate (AMP-PNP). TM6 proline mutations exhibited additive GOF effects when combined with a previously reported GOF mutation located in an outer collar of TMs that surrounds the pore-lining TMs. Each TM substitution allosterically rescued the ATP sensitivity of CFTR gating when introduced into an NBD mutant with defective ATP binding. Both classes of GOF mutations also rescued defective drug export by a yeast MRP (Yor1p) with ATP binding defects in its NBDs. We conclude that the conserved TM6 proline helps set the energy barrier to both CFTR channel opening and MRP-mediated drug efflux and that CFTR channels and MRP pumps utilize similar allosteric mechanisms for coupling conformational changes in their translocation pathways to ATP binding at their NBDs.

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203 The data in Fig. 6 confirm this prediction both for the single P355A mutant (Fig. 6A) and for a double mutant in which the Pro-355 mutation was introduced into an NBD2 mutant that has a markedly reduced ATP affinity (Y1219G-CFTR; Fig. 6B). Login to comment
206 As reported previously by the latter authors, the Y1219G mutant of CFTR exhibited a marked rightward shift in the ATP dose-response curve relative to wild type CFTR with an EC50 of 1.5-2 mM. Login to comment
207 Introducing the P355A mutation increased the apparent ATP affinity of the Y1219G mutant (leftward shift in Fig. 6B). Login to comment
208 The K978C mutation in the outer TM collar (TM9) also increased the apparent ATP affinity of the Y1219G mutant and to a greater degree than the Pro-355 substitution, as would be expected if the former is a stronger GOF mutation (see also Fig. 5). Login to comment
210 These results also indicate that both classes of GOF mutations (Pro-355 in TM6; K978C in the outer TM collar) can compensate for a partial defect in ATP binding by allosteric coupling between the TMs and the NBDs (i.e. by "allosteric rescue" of the ATP binding defect). Login to comment
211 Homologous TM Mutations Rescue Defective Substrate Export by ATP Binding Mutants of a Yeast MRP-Our finding that an ATP binding mutant of CFTR (Y1219G in NBD2) was rescued by GOF mutations in the TMs motivated us to determine whether such allosteric coupling between the TMs and NBDs could also be observed for an MRP drug exporter. Login to comment
220 Curves, best fits to Hill equation with K values of 1522, 683, and 180 òe;M and Hill coefficients of 1.69, 1.93, and 1.72 for Y1219G (n afd; 6 patches), P355A/Y1219G (n afd; 5), and K978C/Y1219G (n afd; 4), respectively. Login to comment
221 *, p b0d; 0.05 compared with Y1219G by unpaired t test. Login to comment
233 Yor1p mutations at positions Pro-485 (TM6) and Lys-997 (TM9) that are homologous to the CFTR GOF mutations described above were assayed both as single mutants and as double mutants when combined with one of two NBD mutations that are expected to inhibit Mg-ATP binding to Yor1p: (i) Y1222G, an A-loop mutation homologous to the Y1219G mutation of CFTR, and (ii) D734N, a Walker B mutation that is predicted to reduce Mg-ATP binding because the conserved aspartate helps coordinate the metal cofactor in ABC exporters (13, 46, 47). Login to comment
304 This is supported by the strong effects of the two classes of GOF mutations on the ATP sensitivity of Y1219G-CFTR activity. Login to comment
305 The Y1219G mutation is located within a conserved aromatic region known as the A-loop that lies just upstream of the Walker A motif in NBD2. Login to comment
308 Both the P355A and K978C GOF mutations increased the ATP sensitivity of the Y1219G mutant with the K978C substitution restoring the ATP sensitivity of channel gating to nearly wild type levels. Login to comment
315 The other ATP binding mutant (Y1222G-Yor1p) was homologous to the A-loop Y1219G mutant in CFTR NBD2 that exhibits markedly reduced ATP sensitivity. Login to comment
316 As discussed above, the ATP sensitivity of Y1219G-CFTR was enhanced by both classes of GOF mutation, an effect we interpreted as allosteric rescue of an ATP binding defect by such TM mutations. Login to comment
202 The data in Fig. 6 confirm this prediction both for the single P355A mutant (Fig. 6A) and for a double mutant in which the Pro-355 mutation was introduced into an NBD2 mutant that has a markedly reduced ATP affinity (Y1219G-CFTR; Fig. 6B). Login to comment
205 As reported previously by the latter authors, the Y1219G mutant of CFTR exhibited a marked rightward shift in the ATP dose-response curve relative to wild type CFTR with an EC50 of 1.5-2 mM. Login to comment
219 Curves, best fits to Hill equation with K values of 1522, 683, and 180 òe;M and Hill coefficients of 1.69, 1.93, and 1.72 for Y1219G (n afd; 6 patches), P355A/Y1219G (n afd; 5), and K978C/Y1219G (n afd; 4), respectively. Login to comment
232 Yor1p mutations at positions Pro-485 (TM6) and Lys-997 (TM9) that are homologous to the CFTR GOF mutations described above were assayed both as single mutants and as double mutants when combined with one of two NBD mutations that are expected to inhibit Mg-ATP binding to Yor1p: (i) Y1222G, an A-loop mutation homologous to the Y1219G mutation of CFTR, and (ii) D734N, a Walker B mutation that is predicted to reduce Mg-ATP binding because the conserved aspartate helps coordinate the metal cofactor in ABC exporters (13, 46, 47). Login to comment
303 This is supported by the strong effects of the two classes of GOF mutations on the ATP sensitivity of Y1219G-CFTR activity. Login to comment
307 Both the P355A and K978C GOF mutations increased the ATP sensitivity of the Y1219G mutant with the K978C substitution restoring the ATP sensitivity of channel gating to nearly wild type levels. Login to comment
314 The other ATP binding mutant (Y1222G-Yor1p) was homologous to the A-loop Y1219G mutant in CFTR NBD2 that exhibits markedly reduced ATP sensitivity. Login to comment

[hide] Long-range coupling between the extracellular gate... FASEB J. 2015 Nov 25. pii: fj.15-278382. Wei S, Roessler BC, Icyuz M, Chauvet S, Tao B, Hartman JL 4th, Kirk KL
Long-range coupling between the extracellular gates and the intracellular ATP binding domains of multidrug resistance protein pumps and cystic fibrosis transmembrane conductance regulator channels.
FASEB J. 2015 Nov 25. pii: fj.15-278382., [PMID:26606940]

Abstract [show]
The ABCC transporter subfamily includes pumps, the long and short multidrug resistance proteins (MRPs), and an ATP-gated anion channel, the cystic fibrosis transmembrane conductance regulator (CFTR). We show that despite their thermodynamic differences, these ABCC transporter subtypes use broadly similar mechanisms to couple their extracellular gates to the ATP occupancies of their cytosolic nucleotide binding domains. A conserved extracellular phenylalanine at this gate was a prime location for producing gain of function (GOF) mutants of a long MRP in yeast (Ycf1p cadmium transporter), a short yeast MRP (Yor1p oligomycin exporter), and human CFTR channels. Extracellular gate mutations rescued ATP binding mutants of the yeast MRPs and CFTR by increasing ATP sensitivity. Control ATPase-defective MRP mutants could not be rescued by this mechanism. A CFTR double mutant with an extracellular gate mutation plus a cytosolic GOF mutation was highly active (single-channel open probability >0.3) in the absence of ATP and protein kinase A, each normally required for CFTR activity. We conclude that: 1) all 3 ABCC transporter subtypes use similar mechanisms to couple their extracellular gates to ATP occupancy and 2) highly active CFTR channels that bypass defects in ATP binding or phosphorylation can be produced.-Wei, S., Roessler, B. C., Icyuz, M., Chauvet, S., Tao, B., Hartman, J. L., IV, Kirk, K. L. Long-range coupling between the extracellular gates and the intracellular ATP binding domains of multidrug resistance protein pumps and cystic fibrosis transmembrane conductance regulator channels.

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92 We chose this ATP binding mutant of Yor1p for detailed analysis because 1) our earlier results (15) showed that it was possible to rescue its oligomycin growth phenotype by introducing cytosolic GOF mutations that were predicted by our CFTR findings and 2) detailed ATP titrations can be performed for the analogous CFTR A loop mutant (Y1219G) to explore the mechanism underlying such GOF effects (15). Login to comment
166 The extracellular F337S substitution enhances the ATP sensitivities of CFTR channels including the Y1219G ATP binding mutant GOF mutations that increase the ligand-free activities of allosteric proteins such as hormone receptors and neurotransmitter-gated channels also reciprocally enhance ligand sensitivity by biasing the equilibrium toward those conformations with the higher ligand affinities (i.e., the activated receptor or open channel) (14, 15, 44-46). Login to comment
167 The ATP titration data in Fig. 6 indicate that such reciprocity is also apparent for the F337S mutation. Login to comment
168 This substitution substantially increased the ATP sensitivity of CFTR activation when introduced either into the WT backgroundor intotheNBD2A loop mutantthatlacks the conserved tyrosine that stacks against the adenine ring of ATP at site 2 (Y1219G-CFTR). Login to comment
208 Substantial F337S/K978C-CFTR-mediated currents could be detected for macropatches that were excised in the absence of both PKA and ATP in Figure 6. Login to comment
209 The extracellular F337S mutation increases the ATP sensitivity of CFTR activation either in the WT background or in the Y1219G ATP binding mutant. Login to comment
214 n = 4 patches for each construct except the Y1219G single mutant (n = 5). Login to comment
217 # P , 0.05 compared with Y1219G by unpaired Student`s t test. Login to comment
260 This in- terpretationisconsistentwiththestronginhibitoryeffectof the analogous mutation (Y1219G)on the ATP sensitivity of CFTR channel activation (15, 36), which could be reversed by introducing a second site GOF mutation of the corresponding phenylalanine. Login to comment
301 GOF effects on CFTR channel gating were operationally defined as 1) large fractional currents that persist following ATP removal, 2) robust activation by the normally weak agonist, AMP-PNP, and 3) substantial increases in channel activities when introduced into CFTR constructs that cannot be activated by ATP, namely, the most common CF regulation mutant (G551D-CFTR) or a truncation mutant lacking NBD2 (D1198-CFTR). Login to comment
302 The F337S substitution also increased the ATP sensitivity of CFTR channel activation when introduced either into the WT background or into the Y1219G-CFTR ATP binding mutant. Login to comment
91 We chose this ATP binding mutant of Yor1p for detailed analysis because 1) our earlier results (15) showed that it was possible to rescue its oligomycin growth phenotype by introducing cytosolic GOF mutations that were predicted by our CFTR findings and 2) detailed ATP titrations can be performed for the analogous CFTR A loop mutant (Y1219G) to explore the mechanism underlying such GOF effects (15). Login to comment
165 The extracellular F337S substitution enhances the ATP sensitivities of CFTR channels including the Y1219G ATP binding mutant GOF mutations that increase the ligand-free activities of allosteric proteins such as hormone receptors and neurotransmitter-gated channels also reciprocally enhance ligand sensitivity by biasing the equilibrium toward those conformations with the higher ligand affinities (i.e., the activated receptor or open channel) (14, 15, 44-46). Login to comment
213 n = 4 patches for each construct except the Y1219G single mutant (n = 5). Login to comment
216 # P , 0.05 compared with Y1219G by unpaired Student`s t test. Login to comment
259 This in- terpretationisconsistentwiththestronginhibitoryeffectof the analogous mutation (Y1219G)on the ATP sensitivity of CFTR channel activation (15, 36), which could be reversed by introducing a second site GOF mutation of the corresponding phenylalanine. 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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21 However, interestingly, in macroscopic currents obtained from inside-out patch expressing Y1219G-CFTR, we have observed a significant slow component in the current relaxation after ATP washout. Login to comment
22 This suggests that a fraction of Y1219G-CFTR channels in the patch exhibited long-lasting openings after ATP washout whereas the remaining majority of Y1219G-CFTR channels closed within hundreds of milliseconds as WT-CFTR. Login to comment
57 We next examined the macroscopic current relaxation in inside-out patches containing those mutants. Indeed, the current decay upon washout of ATP for the Y1219G mutants consistently followed a biphasic time course with a visible slow component (Fig. 2a). Login to comment
59 Figure 2b compares macroscopic current relaxations for Y1219G, Y1219I, Y1219F and Y1219W mutants. Login to comment
60 Although a slow phase of current decay can be seen with Y1219G and Y1219I, this second phase is hardly discernable for the Y1219W mutant. Login to comment
67 The time constants of the initial fast phase for all the Y1219 mutants are within hundreds of milliseconds (sfast: 504 &#b1; 76 ms, n = 5 for Y1219G; 450 &#b1; 44 ms, n = 7 for Y1219I; 571 &#b1; 142 ms, n = 4 for Y1219F; 513 &#b1; 66 ms, n = 5 for Y1219W) and there is not significant difference in the fast time constant among these Y1219 mutants (Fig. 2b). Login to comment
68 On the other hand, the time constants of the slow component for Y1219F and Y1219W are shorter than those of Y1219G and Y1219I (Fig. 3a). Login to comment
69 More importantly, the fraction of the slow component (Fig. 3b) shows a gradual decrease as the mutation becomes less 'preserved` (Y1219G [ Y1219I [ Y1219F [ Y1219W). 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
78 The extended trace marks part of the slow current decay of Y1219G-CFTR after ATP removal. Login to comment
80 b Macroscopic current relaxations upon ATP wash-out for Y1219G, Y1219I, Y1219F and Y1219W. Login to comment
82 The time constant and the fraction of the slow component are summarized in Fig. 3. c Single channel recording of Y1219G-CFTR showing a long opening even after ATP removal 35 30 25 20 15 10 5 0 Y1219G Y1219I Y1219F Y1219W 0.5 0.4 0.3 0.2 0.1 0.0 Y1219G Y1219I Y1219F Y1219W A slow / (A slow + A fast ) c4; slow (s) A B Fig. 3 Kinetic parameters of macroscopic current relaxations for Y1219G, Y1219I, Y1219F and Y1219W. Login to comment
83 a Time constants of the slow component of the current relaxation for the Y1219 mutants. Y1219G: n = 5; Y1219I: n = 7; Y1219F: n = 4; and Y1219W: n = 5. b Fraction of the slow component (Aslow/Aslow ? Login to comment
84 Afast) for the Y1219 mutants. Y1219G: n = 5; Y1219I: n = 7; Y1219F: n = 4; and Y1219W: n = 5 ATP because of their similar rate-limiting hydrolyzing rates [11]. Login to comment
132 Although the results with Y1219G and Y1219I mutations are quantified accurately, this may not be the case with the data for Y1219F and Y1219W. Login to comment
135 This effect is expected to be small when the probability of the stable open state is high such as in Y1219G or Y1219I. Login to comment
151 In fact, even when ATP affinity is drastically reduced by mutations such as Y1219G and Y1219I, the probability of the stable open state remains quite low compared to the short-lived open state. Login to comment
162 If different mutations at Y1219 can affect the entry rate and the exit rate differently as shown in Table 2, this hypothesis may explain most of the results Table 1 Parameter sets (s-1 ) for the ''kiss and run`` hypothesis kCO1 kO1C kO1O2 kO2C Y1219G 3 or 0a 3 2 9 10-2 3.8 9 10-2 Y1219I : : 1.3 9 10-2 : Y1219F : : 1.1 9 10-2 : Y1219W : : 4 9 10-3 : a kCO1 was set to 3 s-1 for simulating the ''with ATP`` condition and 0 s-1 for simulating the ''after the ATP washout`` condition. Login to comment
166 b Representative reproduced macroscopic current relaxations mimicking those in Y1219G (red), Y1219I (green), Y1219F (cyan) and Y1219W (blue). Login to comment
172 In addition, the slow time constant for Y1219G is not different from that for Y1219I despite the fraction of the slow phase is different between these two mutants. 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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58 We took a more conservative approach to analyze the single-channel open time for G551D and G551D/Y1219G channels as only current traces with up to two opening steps were used. Login to comment
126 Also consistent with our hypothesis, removing the entire side chain, i.e., the G551D/Y1219G mutation, completely obliterates the rapid current increasing phase (Fig. 4 C) upon ATP removal as if only minimal occupancy of site 2 takes place at 2 mM [ATP]. Login to comment
133 Y1219F, Y1219I, and Y1219G, mutations known to cause a graded change of the apparent affinity for Figure 3.ߓ Paradoxical [ATP] dependence of G551D-CFTR currents supports the hypothesis of two ATP-binding sites exerting opposite actions. Login to comment
154 Similar observations were made for G551D/Y1219G-CFTR, in which 20 &#b5;M ATP is expected to bear negligible ATP occupancy at site 2 (Fig. 7, C and D). Login to comment
159 Of note, the time constant of slow current decay phase in G551D/ Y1219G-CFTR was unchanged compared with G551D-CFTR (Figs. 4 C and 5 B), indicating that this slow current decay is not controlled by ATP dissociation from site 2. Login to comment
168 (A-C) Real-time current traces in response to ATP removal for G551D/Y1219F (A), G551D/Y1219I (B), and G551D/Y1219G (C). 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
214 (C) Single-channel recording of G551D/Y1219G-CFTR in the presence of 20 &#b5;M ATP. Login to comment
215 (D) Open time histogram 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
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
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