ABCMdb

ABCC7 p.Trp401Gly

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Publications

PMID: 16966475 [PubMed] Zhou Z et al: "The two ATP binding sites of cystic fibrosis transmembrane conductance regulator (CFTR) play distinct roles in gating kinetics and energetics."

No. Sentence Comment

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

PMID: 17700963 [PubMed] Bompadre SG et al: "Cystic fibrosis transmembrane conductance regulator: a chloride channel gated by ATP binding and hydrolysis."

No. Sentence Comment

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

PMID: 18167357 [PubMed] Bompadre SG et al: "Mechanism of G551D-CFTR (cystic fibrosis transmembrane conductance regulator) potentiation by a high affinity ATP analog."

No. Sentence Comment

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

PMID: 18391167 [PubMed] Chen TY et al: "CLC-0 and CFTR: chloride channels evolved from transporters."

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

PMID: 18957373 [PubMed] Muallem D et al: "Review. ATP hydrolysis-driven gating in cystic fibrosis transmembrane conductance regulator."

No. Sentence Comment

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

PMID: 19332488 [PubMed] Hwang TC et al: "Gating of the CFTR Cl- channel by ATP-driven nucleotide-binding domain dimerisation."

No. Sentence Comment

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

PMID: 19332621 [PubMed] Tsai MF et al: "State-dependent modulation of CFTR gating by pyrophosphate."

No. Sentence Comment

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

PMID: 20406820 [PubMed] Miki H et al: "Potentiation of disease-associated cystic fibrosis transmembrane conductance regulator mutants by hydrolyzable ATP analogs."

No. Sentence Comment

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

PMID: 20421370 [PubMed] Tsai MF et al: "Stable ATP binding mediated by a partial NBD dimer of the CFTR chloride channel."

No. Sentence Comment

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

PMID: 20861014 [PubMed] Tsai MF et al: "Optimization of the degenerated interfacial ATP binding site improves the function of disease-related mutant cystic fibrosis transmembrane conductance regulator (CFTR) channels."

No. Sentence Comment

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

PMID: 20628841 [PubMed] Shimizu H et al: "A stable ATP binding to the nucleotide binding domain is important for reliable gating cycle in an ABC transporter CFTR."

No. Sentence Comment

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

PMID: 25225552 [PubMed] Lin WY et al: "A single amino acid substitution in CFTR converts ATP to an inhibitory ligand."

No. Sentence Comment

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

PMID: 25277268 [PubMed] Broadbent SD et al: "The cystic fibrosis transmembrane conductance regulator is an extracellular chloride sensor."

No. Sentence Comment

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