ABCMdb

ABCC7 p.Trp401Phe

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Publications

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

No. Sentence 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

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

4 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). Login to comment
34 Here, guided by accumulated structural and functional understanding of NBDs, we first identified W401Y,W401F mutations that strengthened ligand binding in the NBD1 head subdomain. Login to comment
36 The presence of the Asp-551 residue, which disfavors NBD dimer formation, however, raised the question of whether ATP molecule potentiates W401Y,W401F/G551D channels by binding at the NBD interface (i.e. site 1) or interacting only with the monomeric NBD1. Login to comment
37 Our data suggested that the former is likely the case as the effects of ATP on W401F/G551D channels can be further enhanced by the H1348G mutation in the NBD2 tail subdomain. Login to comment
54 For the W401F/H1348G/G551D channel, the steady-state open probability (Po) in the presence of ATP was estimated by stationary noise analysis of macroscopic currents using the equation ␴2 /i ϭ ͑1 - Po͒I (Eq. 1) where ␴2 is the variance, i is the unitary current amplitude, and I is the amplitude of the steady-state currents. Login to comment
62 This can be readily done for WT and W401F/ H1348G/⌬F508 channels (as seen in Fig. 5). Login to comment
100 Interestingly, we found that phenylalanine (W401F) appeared better than tyrosine (W401Y), which was in turn superior to tryptophan, in stabilizing the lock-open state, suggesting that W401Y and W401F mutations might enhance nucleotide-NBD1 interactions. Login to comment
103 We then incorporated W401Y or W401F mutation into G551D channels, intending to help G551D-NBD1 to bind nucleotide more tightly. Login to comment
106 It can be seen that W401F, which yielded a more stable lock-open state in the WT background (Fig. 1D), was also more effective than W401Y in conferring ATP-dependent activation of G551D channels. Login to comment
110 First, the response to ATP seen in W401Y,W401F/G551D channels could be due to an altered ligand-NBD1 interaction as proposed, or alternatively, is a result of a restored ability for ATP to gate CFTR through binding to site 2. Login to 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
113 Second, as PPi fails to lock open G551D-containing channels (data not shown), presumably due to the negatively charged Asp-551 side chain in site 2, it is desirable to have another parameter in support of the conjecture that W401Y,W401F mutations do tighten nucleotide binding in G551D-NBD1. Login to comment
114 One possible approach is to fit the current relaxation traces of W401Y,W401F/G551D channels upon removal of the nucleotide (Fig. 2A, inset) as the resulting time constants could potentially reflect the mean nucleotide dwell time in NBD1 of these channels (see also under "Discussion"). Login to comment
118 Does the ATP or PATP molecule that potentiates W401Y,W401F/G551D channels bind in the head subdomain of the monomeric NBD1 or NBD1-NBD2 dimer interface (i.e. site 1)? Login to comment
120 Interestingly, we found that non-conservative mutations L1346Q and S1347G (Fig. 3A) and G1349I greatly reduced the nucleotide-dependent activation of W401F/G551D channels. Login to comment
121 For example, ATP and PATP, which increased the basal activity of W401F/G551D-CFTR by ϳ12-fold (Fig. 3D, blue dashed line) and ϳ30-fold (green dashed line), respectively, led to only ϳ2.5-and ϳ6-fold current increases when the S1347G mutation was present (Fig. 3D). Login to comment
122 These results thus suggest that ATP or PATP resides in site 1 and interacts with both NBD1 head and NBD2 tail subdomains to support gating of W401Y,W401F/G551D channels. Login to comment
123 The idea that both the head of NBD1 and the tail of NBD2 are involved in ATP-dependent activation of W401Y,W401F/ G551D channels implicates that optimizing the interactions of ATP with the NBD2 tail subdomain may also improve the func- FIGURE 2. Login to comment
124 W401Y and W401F mutations confer ATP-dependent activa- tionofG551Dchannels.A,applicationofATPorPATPsignificantlyincreased currents of W401Y,W401F/G551D channels. Login to comment
125 Insets, current relaxation traces recorded after the removal of ATP (blue box) or PATP (green box) for W401F/ G551D-CFTR. Login to comment
127 C, effects of W401Y,W401F mutations on the relaxation time constants upon removal of ATP or PATP as shown in panel A. Note that for G551D-CFTR (Trp-401), no current decay was seen because the effect of ATP was negligible. Login to comment
133 Moreover, the H1348G mutation further improved the function of W401F/G551D channels so that the application of ATP and PATP increased the basal activity by ϳ25- and ϳ75-fold, respectively (Fig. 3, C and D, and supplemental Fig. S5), further supporting the notion that optimizing ATP binding in site 1 enhances the function of G551D channels. Login to comment
139 A, the S1347G mutation diminished the response of W401F/G551D channels to ATP or PATP. B, incorporating the H1348G mutation into G551D-CFTR conferred responsiveness to ATP. Login to comment
140 C, H1348G enhanced the response of W401F/G551D-CFTR to ATP. Login to comment
141 D, histogram summarizing the ratio of ATP (blue bars)- or PATP (green bars)-induced current over basal current for W401F/G551D channels combined with a mutation in the NBD2 signature motif as marked. Login to comment
142 Dashed lines, the ratio of IATP/IBasal (blue) and IPATP/IBasal (green) for W401F/G551D channels. Login to comment
145 Single-channel kinetics of G551D channels with W401Y,W401F or H1348G mutations. Login to comment
156 W401F and H1348G Mutations Improve the Function of WT and ⌬F508 Channels-To this point, we have demonstrated that optimizing the interactions of ATP with site 1 components, NBD1 head (W401Y and W401F) and NBD2 tail (H1348G), ameliorates the gating defects of G551D channels, which hold a non-functional site 2. Login to comment
160 Indeed, when W401F and H1348G mutations were engineered into WT channels (Fig. 5A), the mean open time of WT-CFTR was more than quadrupled (Fig. 5C) with the already high Po (ϳ0.4) nearly doubled (ϳ0.78, Fig. 5D). Login to comment
162 In either case, W401F/H1348G mutations did not significantly alter the opening rate (Fig. 5E). Login to comment
165 First, ATP-site 1 interactions of the CFTR channel can be strengthened by introducing mutations in both the head domain of NBD1 (i.e. W401Y,W401F) and the tail domain of NBD2 (i.e. H1348G). Login to comment
180 Effects of W401F/H1348G mutations on WT and ⌬F508 channels. Login to comment
181 A, 30-s single-channel recordings of WT and W401F/H1348G channels exposed to 2.75 mM ATP. Login to comment
182 B, current recordings of ⌬F508 and ⌬F508/W401F/ H1348G channels in the presence of 2.75 mM ATP. Login to comment
191 It is this correlation between the chemical nature of mutations and the stability of the lock-open state that grants us the confidence that W401Y,W401F and H1348G mutations, which prolonged the lock-open duration of WT-CFTR, indeed tighten ATP binding in site 1. Login to comment
195 The results shown in Fig. 2C did suggest that W401Y,W401F mutations enhance nucleotide binding in G551D site 1, like in the case of WT site 1. Login to comment
199 Unlike WT and ⌬F508 channels, whose Po in the presence or absence of W401F/H1348G mutations (Fig. 5) can be measured with reasonable accuracy, the G551D-containing channels exhibit a Po too low to be derived from single-channel analysis. Login to comment
204 For instance, that the W401F/H1348G/G551D channel has an IATP/IBasal of ϳ25 indicates that the Po for this mutant is ϳ0.1. Login to comment
205 To verify this value, we performed stationary noise analysis for the W401F/ H1348G/G551D mutant, and the resulting Po of 0.09 Ϯ 0.01 (supplemental Fig. S7) did provide some reassurance. Login to comment
209 We have observed that W401F/H1348G mutations in site 1 prolonged the mean open time of WT-CFTR (Fig. 5C). Login to comment
211 Thus, it appears that a stronger ATP binding in site 1, due to the presence of W401F/H1348G mutations, can allosterically tighten the connection between two NBDs around site 2, thereby slowing down channel closure. Login to comment

PMID: 21486785 [PubMed] Jih KY et al: "The most common cystic fibrosis-associated mutation destabilizes the dimeric state of the nucleotide-binding domains of CFTR."

No. Sentence Comment

105 Tight binding of nucleotides in NBD1 prolongs the channel locked-open time In a previous report (Tsai et al. 2010a), we demonstrated that the locked-open time of WT-CFTR induced by PPi is prolonged by replacing ATP with the high affinity ATP analogue N6 -phenylethyl-ATP (P-ATP), or by introducing 'gain-of-function` mutations to the ATP-binding site 1 (mutations which increase the Po of CFTR, such as W401F and H1348G) as the locked-open state reflects an NBD dimer with ATP-binding site 1 occupied by ATP and ATP-binding site 2 by PPi (Tsai et al. 2009). Login to comment
106 In Fig. 3, we show that the gain-of-function mutations W401F and H1348G (Fig. 3A) and P-ATP (Fig. 3B) also prolong the locked-open time of F508-CFTR channels. Login to comment
107 Compared to F508-CFTR, the double mutant W401F/ F508-CFTR ( F508/DM)prolongedthelocked-opentimeby~2-fold, and the triple mutant W401F/H1348G/ F508-CFTR ( F508/TM) by ~4-fold. Login to comment
127 C, summary of PPi locked-open times for each construct ( F508/DM: W401F/ F508-CFTR, F508/TM: W401F/H1348G/ F508-CFTR). Login to comment

PMID: 22966014 [PubMed] Jih KY et al: "Nonintegral stoichiometry in CFTR gating revealed by a pore-lining mutation."

No. Sentence Comment

71 Fig. S1 shows the closed- time distribution for R352C-CFTR. Fig. S2 presents dwell-time distributions for O1 and O2 states. Login to comment
72 Fig. S3 demonstrates the effects of the W401F mutation on the single-channel kinetics of R352C-CFTR. Login to comment
130 ATP hydrolysis drives the O1→O2 transition Although our initial observations were made with Cysless/R352C-CFTR, this unique pattern of gating transitions was also seen when we introduced the R352C mutation into the WT background (Fig. 3 A and Table 1). Login to comment
131 One notable difference between R352C- and Cysless/ Ta b l e 1 Summary of opening events by different gating patterns in three CFTR mutants Gating topology O1-O2 O1 O2 O2-O1 (O1-O2) n # Total Cysless/R352C 2.75 mM ATP 100 µM ATP 720 (45%) 663 (56%) 290 (18%) 216 (18%) 175 (11%) 137 (12%) 42 (3%) 32 (3%) 375 (23%) 128 (11%) 1,602 (100%) 1,176 (100%) R352C 2.75 mM ATP 100 µM ATP 834 (55%) 1,246 (59%) 301 (20%) 406 (19%) 173 (11%) 281 (13%) 39 (3%) 45 (2%) 169 (11%) 121 (6%) 1,516 (100%) 2,099 (100%) R352C/W401F 2.75 mM ATP 100 µM ATP 733 (44%) 1,189 (54%) 326 (19%) 367 (17%) 122 (7%) 337 (15%) 28 (2%) 60 (3%) 474 (28%) 232 (11%) 1,683 (100%) 2,185 (100%) Five different gating patterns are illustrated on the top of the table. Login to comment
182 Quantitative analysis of gating events indeed demonstrates a higher percentage of gating events with reentry transitions in W401F/R352C-CFTR (Table 1). Login to comment
186 Prolonged O1 and O2 dwell times were also found in W401F/R352C-CFTR, but to a lesser extent. Login to comment
187 The effect of Cysless and W401F mutations in prolonging the open time of R352C mutant channels is consistent with the observations made for the same mutants in the WT background (Bai et al., 2010; Tsai et al., 2010a). Login to comment
188 D I S C U S S I O N An accidental discovery of the R352C mutation grants us the opportunity to actually "see"-in real time-ATP hydrolysis taking place during CFTR gating as the ordered transition between two distinct levels of open channel conductance (O1 and O2) indicates an input of the free energy from ATP hydrolysis. Login to comment
206 W401F mutation promotes O2→O1 transition In our latest paper (Jih et al., 2012), using nonhydrolyzable ATP analogues (pyrophosphate or adenylyl-imidodi- phosphate) as baits, we captured a post-hydrolytic state (state X), which, like the O2 state in this paper, can reenter the prehydrolytic open state (O1) upon ATP binding to the vacant ABP2. Login to comment
208 Interestingly, a conservative mutation in NBD1 (W401F, both W and F are aromatic amino acids) can enhance the responsiveness of state X to ATP as well as nonhydrolyzable ATP analogues for reasons yet to be elucidated. Login to comment
209 Nevertheless, the striking functional similarities between state X in Jih et al. (2012) and the O2 state in this paper prompt us to test the effect of the W401F mutation on R352C-CFTR. Fig. S3 shows a representative single-channel trace of W401F/R352C-CFTR. Login to comment
210 Compared state C is nearly 1 s for R352C-CFTR. Login to comment
183 Quantitative analysis of gating events indeed demonstrates a higher percentage of gating events with reentry transitions in W401F/R352C-CFTR (Table 1). Login to comment
211 Nevertheless, the striking functional similarities between state X in Jih et al. (2012) and the O2 state in this paper prompt us to test the effect of the W401F mutation on R352C-CFTR. Fig. S3 shows a representative single-channel trace of W401F/R352C-CFTR. Login to comment

PMID: 22508846 [PubMed] Jih KY et al: "Identification of a novel post-hydrolytic state in CFTR gating."

No. Sentence Comment

89 Fig. S3 shows the open dwell-time histograms of WT- and W401F-CFTR. Login to comment
118 On the other hand, mutating the residue Trp401 (W401), which forms ring-ring stacking interactions with ATP in ABP1 (Lewis et al., 2004; Zhou et al., 2006), to phenylalanine significantly increased the propensity of state X in response to PPi (i.e., a higher percentage of channels locked open from state X; WT, 49 ± 2% and n = 13; W401F, 59 ± 2% and n = 13; P < 0.05). Login to comment
120 Therefore, opposite to P-ATP, the W401F mutation enhances the responsiveness of state X to PPi but may destabilize the C2 state. Login to comment
135 It is noteworthy that the success rate of this line of experiments is lower for WT channels (see supplemental Discussion for more details), but similar observations were made for WT-CFTR (Fig. S1), indicating that such open X-locked-open transitions are not just idiosyncratic for W401F mutant channels. Login to comment
147 However, the conserved W401F mutation (see below), which significantly prolongs the open time (Tsai et al., 2010a), makes such experiments more feasible. Login to comment
148 In the presence of ATP, the W401F-CFTR channel exhibits similar behavior as WT-CFTR: the channel opens into bursts that last for hundreds of milliseconds to seconds, and each opening burst is separated by a long interburst that also closes in the range of hundreds of milliseconds (Fig. 6). Login to comment
157 (B) A similar protocol was conducted with W401F-CFTR channels. Login to comment
163 For reasons unclear at this moment, the W401F mutation could somehow improve the responsiveness of state X to PPi (Fig. 5) and therefore presumably to ATP as well. Login to comment
164 We therefore quantified the open burst time of W401F-CFTR at different [ATP]. Login to comment
166 Furthermore, the notion that a prolonged open burst time seen with the W401F mutation is caused by reentry of the channel from state X predicts a disparity between the mean open time estimated from microscopic analysis and the relaxation time constant obtained from macroscopic current decay upon ATP removal, which prohibits reentry to occur. Login to comment
167 Fig. 7 (B and C) shows that when fitting the current relaxation of W401F mac roscopic current after washout of 10 mM ATP, the time constant is almost identical to the mean open time of W401F channels in the presence of micromolar ATP. Login to comment
168 Figure 6. Single-channel ligand exchange for W401F-CFTR. Login to comment
169 (A and C) A single W401F-CFTR was activated by ATP, and the ligand was switched to a 1-s PPi (A) or AMP-PNP (C) pulse in the open state. Login to comment
188 As shown in Fig. 5, the W401F mutation and the high-affinity ATP analogue P-ATP, both acting on ABP1, affect state X and the C2 state very differently. Login to comment
193 Figure 7. The mean open time of W401F-CFTR is [ATP] dependent. Login to comment
194 (A) Microscopic current traces of W401F-CFTR in the presence of 10 mM (top), 1 mM (middle), or 100 µM (bottom) ATP. Login to comment
195 (B) Mean open time of W401F-CFTR at different [ATP] (closed circles). Login to comment
197 (C)Macroscopic current of W401F-CFTR was activated by 10 mM ATP to a steady state. Login to comment
205 Furthermore, the prolonged open burst time in the presence of higher [ATP] (>1 mM; P < 0.05 when comparing 1 and 10 mM) for W401F-CFTR indicates that the lifetime of state X as a closed state must be indistinguishable from that of the flickers so that closed events corresponding to state X are excluded by the analysis. Login to comment
223 By comparing WT-CFTR and W401F-CFTR, the increasing reentry events (X→X→O1) results in differences only in the mean open time and the tail of the fitted curve (Fig. S3), which is supplanted with an increased number of long opening bursts (>1,000 ms). Login to comment

PMID: 23440202 [PubMed] Jih KY et al: "Vx-770 potentiates CFTR function by promoting decoupling between the gating cycle and ATP hydrolysis cycle."

No. Sentence Comment

157 Summary of the effects of Vx-770 on the gating patterns exhibited in R352C-CFTR and W401F/R352C-CFTR Total Experimental condition Without Vx-770* R352C (%) 834 (55) 301 (20) 173 (11) 39 (3) 169 (11) 1,516 (100) R352C/W401F (%) 733 (44) 326 (19) 122 (7) 28 (2) 474 (28) 1,683 (100) With 200 nM Vx-770 R352C (%) 578 (57) 126 (12) 73 (7) 20 (2) 217 (21) 1,014 (100) R352C/W401F (%) 411 (37) 162 (15) 79 (7) 31 (3) 425 (38) 1,108 (100) Five different categories of the gating pattern are illustrated. Login to comment
161 *Data for R352C-CFTR and R352C/W401F-CFTR in the absence of Vx-770 were taken from ref. 24. gate in the absence of ATP, C2 ࢒ O2) primarily affected by Vx-770 are supposed to take place in CFTR`s TMDs (24). Login to comment
178 Lately, we discovered that W401F, a conserved mutation in NBD1, increases the reentry frequency without significantly altering the O2 ࢐ C2 transition (24). Login to comment
179 Although how the W401F mutation modifies the function of NBDs is unclear, this finding does raise the possibility that manipulating NBD function can complement the action of Vx-770. Login to comment
180 As shown in Fig. S3 and Table 1, the W401F mutation almost doubles the reentry frequency of R352C-CFTR and also significantly enhances the effect of Vx-770 (Fig. 5, Table 1 and Fig. S3). Login to comment
208 W401F mutation and Vx-770 work additively on the reentry pathway. Login to comment
209 Representative single-channel traces for R352C/W401F-CFTR treated with 2.75 mM ATP in the absence (A) or presence (B) of 200 nM Vx-770. Login to comment

PMID: 23752332 [PubMed] Csanady L et al: "Conformational changes in the catalytically inactive nucleotide-binding site of CFTR."

No. Sentence Comment

49 In contrast, [ATP]-dependent prolongation of open burst durations for the W401F CFTR mutant (Jih et al., 2012b), or for WT CFTR gating in the presence of the potentiator compound Vx-770 (Jih and Hwang, 2013), has led to the suggestion that post-hydrolytic ADP/ATP exchange in site 2 might occur without intervening pore closure, thereby increasing the coupling ratio by allowing more than one ATP to be hydrolyzed in site 2 within a single gating cycle (Jih et al., 2012a). Login to comment
111 Channels opened by Because for another site-1 mutant (W401F) mean burst durations were shown to increase at high millimolar ATP concentrations (Jih et al., 2012b), we tested whether that was the case for H1348A CFTR. Login to comment
160 Because the W401F mutation also resides in site 1, we asked whether a reentry mechanism might explain the longer burst durations in P-ATP or in the presence of the H1348A mutation. Login to comment
164 Recent studies suggested a novel mechanism for a prolongation of steady-state CFTR burst durations for NBD1 mutant W401F (Jih et al., 2012a,b), by proposing the presence of a short time window at the end of each burst during which the hydrolysis products ADP and phosphate at site 2 can be replaced by a new ATP molecule without intervening channel gate closure. Login to comment
194 Because one such mutation (W401F) resides in site 1, we have evaluated the possibility that the H1348A mutation, or P-ATP bound in site 1, might also act by such a mechanism. Login to comment