ABCMdb

ABCC7 p.Tyr1219Gly

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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

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100 the current for Y1219G (unpublished data).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

PMID: 17353351 [PubMed] Bompadre SG et al: "G551D and G1349D, two CF-associated mutations in the signature sequences of CFTR, exhibit distinct gating defects."

No. Sentence Comment

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

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

No. Sentence Comment

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

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
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

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

No. Sentence Comment

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

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
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

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

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

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

No. Sentence 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
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

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

No. Sentence Comment

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

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

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

PMID: 24876383 [PubMed] Wei S et al: "Conserved allosteric hot spots in the transmembrane domains of cystic fibrosis transmembrane conductance regulator (CFTR) channels and multidrug resistance protein (MRP) pumps."

No. Sentence Comment

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

PMID: 26606940 [PubMed] Wei S et al: "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."

No. Sentence Comment

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

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

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

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

No. Sentence Comment

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

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
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