ABCMdb

ABCC7 p.Asp1370Asn

None has been submitted yet.

Publications

PMID: 16442101 [PubMed] Frelet A et al: "Insight in eukaryotic ABC transporter function by mutation analysis."

No. Sentence Comment

149 D572N resulted in a marked decrease in sensitivity to channel activation while D1370N resulted in an increase in sensitivity [60,96]. Login to comment

PMID: 10880569 [PubMed] Ikuma M et al: "Regulation of CFTR Cl- channel gating by ATP binding and hydrolysis."

No. Sentence Comment

127 We studied the CFTR-D1370N mutant; this channel has reduced activity (11, 39). Login to comment
129 We predicted that CFTR-D1370N would not discriminate between ATP, MgATP, and CaATP. Login to comment
148 Effect of MgATP, ATP alone, and CaATP on Cl- current of CFTR-D1370N. Login to comment
194 It is also noteworthy that MgATP did not stimulate current to a greater extent than ATP alone when CFTR had a mutation in NBD2 (D1370N) that disrupts divalent cation binding (Fig. 2). Login to comment

PMID: 11341822 [PubMed] Zou X et al: "ATP hydrolysis-coupled gating of CFTR chloride channels: structure and function."

No. Sentence Comment

186 Gunderson and Kopito (31) demonstrated that the mean open time for the D1370N mutant is longer than that of the wild-type CFTR. Login to comment

PMID: 12457238 [PubMed] Ando-Akatsuka Y et al: "Down-regulation of volume-sensitive Cl- channels by CFTR is mediated by the second nucleotide-binding domain."

No. Sentence Comment

6 Furthermore, the K1250M mutation at the Walker A motif and the D1370N mutation at the Walker B motif, both known to impair ATP hydrolysis at NBD2, completely abolished the VSOR regulation by CFTR. Login to comment
123 To check whether ATP hydrolysis at NBD2 is required for CFTR`s regulatory function, we generated two other NBD2-mutants of CFTR, K1250M and D1370N. Login to comment
125 The plasmalemmal distribution of K1250M and D1370N CFTR was confirmed by immunofluorescence microscopy (Fig. 6A) and immunoblotting (Fig. 6B). Login to comment
131 Relative integrated optical densities of the mature bands are shown as percentages of the respective b-actin bands (taken as 100%) In whole-cell patch-clamp experiments, cells expressing the K1250M or D1370N mutant exhibited VSOR currents (Fig. 7A) as large as those observed in mock-transfected cells (Fig. 3A). Login to comment
137 Since VSOR-non-regulating NBD2 mutants (G1349D, K1250M and D1370N) were expressed to approximately the same level as VSOR-regulating WT and G551D CFTR (as seen from immunostaining and Western blotting data), we may exclude the possibility that the down-regulation is simply a side-effect of overexpression of a foreign protein. Login to comment
155 This was, in fact, confirmed by a yeast two- Fig. 7A, B Effects of expression of Walker A or Walker B mutants on VSOR current densities in HEK293T cells. A Time course of VSOR current activation by hypotonic stimulation of cells transfected with the K1250M (top) or D1370N (bottom) mutant, taken during application of alternating pulses from 0 to €40 mV every 15 s. B VSOR current densities from mock-transfected, K1250M mutant-transfected and D1370N mutant-transfected cells, recorded at +40 mV after reaching a steady-state level. Login to comment
175 In our study, the K1250M and D1370N mutations effectively abolished the down-regulatory effect of CFTR on VSOR currents. Login to comment

PMID: 12508051 [PubMed] Vergani P et al: "On the mechanism of MgATP-dependent gating of CFTR Cl- channels."

No. Sentence Comment

4 The rate of opening to a burst (1/␶ib) was a saturable function of [MgATP], but apparent affinity was reduced by mutations in either of CFTR`s nucleotide binding domains (NBDs): K464A in NBD1, and K1250A or D1370N in NBD2. Login to comment
7 NBD2 catalytic site mutations K1250A, D1370N, and E1371S were found to prolong open bursts. Login to comment
32 However, in CFTR the Walker A NBD2 mutation K1250A abolished ATP hydrolysis, whereas the NBD1 mutation K464A simply reduced overall hydrolytic activity (Ramjeesingh et al., 1999); and biochemical studies of Walker B aspartate mutations in CFTR (D572N in NBD1, D1370N in NBD2) have not yet been performed. Login to comment
41 We studied in detail the dependence of channel gating on [MgATP], gating in the presence of poorly hydrolyzable nucleotide analogs, as well as the effects of mutating residues in the Walker A (K464A and K1250A) and Walker B motifs (in particular, D1370N in NBD2). Login to comment
52 Amounts of cRNA injected were adjusted to vary the level of expression: up to 40 ng/oocyte was required for high expression of K1250A or K464A/K1250A mutant channels, whereas 0.1-0.25 ng/oocyte sufficed for single channel recordings of WT, K464A, or D1370N channels. Login to comment
89 T A B L E I Kinetic Parameters of WT and Mutant CFTR Channels WT K464A D1370N mean Ϯ SEM n mean Ϯ SEM n mean Ϯ SEM n (A) 5 mM MgATP ϩ 300 nM PKA ␶b 644 Ϯ 63 30 620 Ϯ 58 21 3,768 Ϯ 499 21 ␶ib 1,671 Ϯ 172 30 2,760 Ϯ 439 21 3,588 Ϯ 414 21 1,552 Ϯ 170 19 2,438 Ϯ 483 12 2,849 Ϯ 491 12 ␶F 19.3 Ϯ 2.0 30 20.8 Ϯ 2.1 21 49.9 Ϯ 4.3 21 nF 0.57 Ϯ 0.06 30 0.50 Ϯ 0.06 21 2.76 Ϯ 0.25 21 rCO 0.75 Ϯ 0.06 30 0.50 Ϯ 0.05 21 0.38 Ϯ 0.05 21 0.77 Ϯ 0.08 19 0.54 Ϯ 0.08 12 0.47 Ϯ 0.07 12 rOC 1.95 Ϯ 0.15 30 1.92 Ϯ 0.15 21 0.43 Ϯ 0.07 21 (B) 5 mM MgATP ␶b 338 Ϯ 22 18 309 Ϯ 23 8 1,748 Ϯ 215 17 ␶ib 4,506 Ϯ 497 18 6,752 Ϯ 1314 8 9,503 Ϯ 1440 17 4,454 Ϯ 1382 5 6,928 Ϯ 1666 6 7,584 Ϯ 1967 9 ␶F 23.5 Ϯ 3.2 18 16.1 Ϯ 2.2 8 51.5 Ϯ 6.0 17 nF 0.42 Ϯ 0.05 18 0.39 Ϯ 0.06 8 1.40 Ϯ 0.13 17 rCO 0.27 Ϯ 0.03 18 0.18 Ϯ 0.03 8 0.16 Ϯ 0.03 17 0.33 Ϯ 0.09 5 0.18 Ϯ 0.03 6 0.22 Ϯ 0.05 9 rOC 3.28 Ϯ 0.21 18 3.43 Ϯ 0.25 8 0.75 Ϯ 0.09 17 (C) 50 ␮M MgATP ␶b 355 Ϯ 44 12 323 Ϯ 136 4 1,433 Ϯ 381 4 ␶F 27.3 Ϯ 5.2 12 22.1 Ϯ 4.4 4 46.2 Ϯ 10.8 4 nF 0.38 Ϯ 0.05 12 0.45 Ϯ 0.11 4 1.91 Ϯ 0.34 4 (D) 5 mM MgAMPPNP ␶b 1,619 Ϯ 232 32 271 Ϯ 52 8 ␶F 59.5 Ϯ 6.6 32 26.8 Ϯ 7.7 8 nF 2.40 Ϯ 0.26 32 0.38 Ϯ 0.10 8 Kinetic parameters were obtained using a maximum likelihood simultaneous fit to dwell-time histograms at all conductance levels (Csanády, 2000). Login to comment
94 The significance of the slight prolongation of ␶F for the D1370N mutant and for WT in 5 mM MgAMPPNP is unknown, but the rate rOF remained 1-2 s-1 for all conditions and mutants tested. Login to comment
113 (B and C) Representative traces for prephosphorylated K464A and D1370N channels. Login to comment
114 Relative opening (D) and closing (E) rates (mean Ϯ SEM, 2 Յ n Յ 7) from analysis of records as in A-C for WT (blue circles), K464A (red triangles), and D1370N (green squares) channels at 10 ␮M Յ [MgATP] Յ 5 mM, plotted on semilogarithmic axes. Login to comment
116 Curves in D show Michaelis-Menten fits, yielding K0.5 of 56 Ϯ 5, 807 Ϯ 185, 391 Ϯ 118 ␮M, and rCOmax of 1.02, 1.16, and 1.08, for WT, K464A, and D1370N, respectively. Login to comment
123 Compared with WT, both K464A (Walker A lysine in NBD1) and D1370N (Walker B aspartate in NBD2) mutant CFTR channels opened less frequently at low [MgATP] (e.g., 50 ␮M; Figs. 2, A-D), and this defect could be largely overcome by raising the [MgATP], so that, at saturating [MgATP], opening rates of WT, K464A, and D1370N channels differed by less than a factor of two (Table I). Login to comment
125 As expected (see below) for channels in which opening rate, but not closing rate, is sensitive to [MgATP], the dependence of Po on [MgATP] was not very different from that of rCO, shown in Fig. 2 D, for WT (see Fig. 3 C), K464A, or D1370N channels. Login to comment
162 (G and H) Representative traces showing gating of K464A and D1370N channels at 15 ␮M MgATP (after PKA removal). Login to comment
164 Though variability among the four patches containing sufficiently few D1370N channels precluded pooling the data for burst distribution analysis, in none of those patches (analyzed separately) did introduction of a second component significantly improve the maximum likelihood fit. Login to comment
168 D1370N channels closed 4-5-fold more slowly than WT CFTR (Figs. 2 E and 6, A vs. B; Table I), and this reduced closing rate was constant at all [MgATP] tested (Figs. 2 E and 4 H), although, as for WT CFTR, strong phosphorylation slowed closing of D1370N channels roughly twofold (Table I). Login to comment
171 Moreover, for both K1250A and D1370N mutants, this macroscopic current decay followed a single exponential time course, implying the presence of a single population of open bursts (for K1250A, see Figs. 3 B and 10 E; for D1370N, decay time constants were: ␶[after 5 mM MgATP ϩ PKA] ϭ 6.4 Ϯ 1.6 s, n ϭ 6; ␶[after 5 mM MgATP] ϭ 2.2 Ϯ 0.5 s, n ϭ 7; ␶[after 300 ␮M MgATP] ϭ 1.9 Ϯ 0.3 s, n ϭ 8; cf. Table I). Login to comment
179 WT (A), D1370N (B), K1250A (C), and E1371S (D) CFTR channels were activated by 5 mM MgATP plus PKA as indicated: burst termination (‫-4.0ف‬pA downward steps) after nucleotide washout was slowed for NBD2 mutants, relative to WT. Login to comment
266 Our results show that mutations within the Walker motifs of either NBD1 (K464A) or NBD2 (D1370N, Figure 11. Login to comment
270 K1250A) reduce the apparent affinity of the MgATP binding site(s) involved in channel opening (Figs. 2 and 3), but (at least for K464A and D1370N) affect the maximal opening rate little (Table I). Login to comment
280 Therefore, the simplest interpretation of the reduced apparent affinity with which MgATP elicits opening of K464A and D1370N (and K1250A) mutants compared with WT is that the mutations impair nucleotide binding at two different sites, such that at subsaturating [MgATP] channel opening is limited by MgATP binding at NBD1 in K464A, but at NBD2 in D1370N (and K1250A). Login to comment
286 Allosteric interactions between CFTR`s two NBDs (compare Powe et al., 2002) could, therefore, permit the K464A, D1370N, and K1250A mutations to all affect the same binding site. Login to comment
291 Moreover, covalent modification of the NBD2 Walker A sequence (Cotten and Welsh, 1998), and the K1250A (Fig. 3 C) and the D1370N (Fig. 2 D) mutations (‫9-8ف‬ Å apart; e.g., Hung et al., 1998), all reduce apparent affinity for MgATP activation of opening. Login to comment
293 Most likely, therefore, the rightward shift in [MgATP] dependence of D1370N (and K1250A) open- ing rate reflects the lower affinity of a binding step, required for channel opening, at NBD2 itself. Login to comment
298 In fact, although opening rates for WT and D1370N mutant CFTR channels (Fig. 2 D, blue and green symbols) are satisfactorily described by the Michaelis equation (i.e., opening limited by binding to a single site) the opening rates of K464A channels (Fig. 2 D, red symbols) at low (Յ50 ␮M) [MgATP] are slightly higher than expected. Login to comment
300 Therefore, present evidence suggests that nucleotide normally binds to both of WT CFTR`s NBDs before the channel opens, and that opening is limited by nucleotide binding at NBD2 in WT, D1370N, and K1250A CFTR channels, but probably by nucleotide binding at NBD1 in K464A CFTR channels. Login to comment
316 For example, our measurements of D1370N CFTR gating show a twofold reduction in maximal opening rate (Table I), but no ATPase measurements are available for D1370N CFTR. Login to comment
324 We found no clear dependence of burst duration on [MgATP] (10 ␮M to 5 mM) in WT CFTR (Figs. 2 E, 3 A, and 4, B and C) or in K464A, D1370N, or K1250A mutant channels (Figs. 2 E, 3 B, and 4, E-H), indicating that all ATP binding events precede channel opening and no further binding to the open channel is needed to complete the gating cycle. Login to comment
329 Moreover, our finding that D1370N channels at low (15 ␮M) [MgATP] both enter and exit bursts more slowly on average than WT channels (Figs. 2, D-E, and 4 H) demonstrates that this single NBD2 mutation impacts every gating cycle, regardless of the fact that D1370N channels have an intact WT NBD1 sequence. Login to comment
366 Even higher levels of steady state phosphorylation could prolong normal hydrolytic bursts (Table I, WT and K464A), as well as nonhydrolytic locked-open bursts (Table I, D1370N; Fig. 10 A vs. Fig. 9; Fig. 3 B vs. Fig. 10 E), by stabilizing the open burst states more than the transition states for both possible pathways (forward or backward; Fig. 12 A) for terminating the burst. Login to comment
379 Unfortunately, the difficulty of collecting adequate numbers of CFTR`s relatively infrequent gating events, combined with the lack of biochemical information on CFTR mutants (whether D1370N is capable of ATP hydrolysis, for instance), precludes extraction of the many (Ն7) rate constants from fits to data, even for a scheme as simple as the one in Fig. 12 A. Login to comment
382 Similarly, the effect of the D1370N mutation seen in Fig. 2 D (and on Po) can be mimicked by an ‫-01ف‬fold acceleration of the MgATP dissociation rate from NBD2 with the Ͻ2-fold observed reduction in maximal opening rate (Table IB), and (assuming that hydrolysis is abolished) by appropriate speeding of nonhydrolytic closing. Login to comment

PMID: 15284228 [PubMed] Kidd JF et al: "A heteromeric complex of the two nucleotide binding domains of cystic fibrosis transmembrane conductance regulator (CFTR) mediates ATPase activity."

No. Sentence Comment

190 Unfortunately, there are no ATPase data available for the NBD2 Walker B mutations D1370N and E1371S, which would be expected to severely impair hydrolysis. Login to comment

PMID: 15684079 [PubMed] Randak CO et al: "ADP inhibits function of the ABC transporter cystic fibrosis transmembrane conductance regulator via its adenylate kinase activity."

No. Sentence Comment

31 For example, structural studies predict that the K1250A and D1370N mutations alter the ATP-binding sites, and these mutations disrupted both ATPase activity and adenylate kinase activities, as well as ADP-dependent inhibition. Login to comment

PMID: 15767295 [PubMed] Bompadre SG et al: "CFTR gating I: Characterization of the ATP-dependent gating of a phosphorylation-independent CFTR channel (DeltaR-CFTR)."

No. Sentence Comment

327 The same paper shows that the D1370N mutation at the Walker B motif in NBD2 also decreases the apparent affinity of ATP (compare Bompadre et al., 2005). Login to comment

PMID: 15767296 [PubMed] Bompadre SG et al: "CFTR gating II: Effects of nucleotide binding on the stability of open states."

No. Sentence Comment

3 To further study this effect of ADP on the open state, we have used two CFTR mutants (D1370N and E1371S); both have longer open times because of impaired ATP hydrolysis at NBD2. Login to comment
4 Single-channel kinetic analysis of ⌬R/D1370N-CFTR shows unequivocally that the open time of this mutant channel is decreased by ADP. Login to comment
45 To study in more detail the effect of nucleotide binding on the open time of CFTR, we used both macroscopic current relaxation and single-channel kinetic analysis for CFTR mutants with impaired ATP hydrolysis: specifically, the D1370N and E1371S mutations in NBD2. Login to comment
47 Mutation of this aspartate to asparagine (D1370N) results in channels that exhibit longer open and closed times (Gunderson and Kopito, 1995; Vergani et al., 2003), resembling the elusive "slow gating" mode described in our previous paper (Bompadre et al., 2005). Login to comment
51 Single-channel dwell-time analysis of ⌬R/D1370N-CFTR channels shows unequivocally that the open time of the channel is decreased in the presence of ADP. Login to comment
56 M A T E R I A L S A N D M E T H O D S Construction of CFTR Mutants CFTR mutations E1371S and D1370N were introduced into the plasmid pBQ4.7 WT-CFTR (Powe et al., 2002) by using the QuikChange kit (Stratagene). Login to comment
57 The 0.9-kb PflMI-XhoI fragments containing the mutations from the pBQ4.7 were used to substitute the corresponding regions in pBudCE4.1 split ⌬R CFTR (Ai et al., 2004) to obtain pBudCE4.1 ⌬R/D1370N and pBudCE4.1 ⌬R/E1371S CFTR. Login to comment
58 Similarly, the 0.9-kb PflMI-EcoRV fragments were used to replace the corresponding ones in pcDNA 3.1 WT-CFTR or pcDNA 3.1 K464A-CFTR (Powe et al., 2002) to generate pcDNA3.1 D1370N, pcDNA 3.1 E1371S, and pcDNA3.1 K464A/ E1371S CFTR constructs. Login to comment
81 Fig. S1 shows the ATP dose response for the D1370N mutant. Login to comment
82 R E S U L T S Effect of ADP on ⌬R/D1370N-CFTR In the accompanying paper (Bompadre et al., 2005), we demonstrated that ADP can shorten the open time of ⌬R-CFTR and this effect is more prominent when the channel is in the slow gating mode with longer Figure 1. Login to comment
83 ADP shortens the open time of ⌬R/D1370N-CFTR channels. Login to comment
89 Since the D1370N mutants exhibit open and closed times on the order of seconds (i.e., they mimic the slow gating mode described in Bompadre et al., 2005), we decided to use this mutant to further study the effect of ADP on the open time. Login to comment
90 Vergani et al. (2003) found that the ATP dose response for the D1370N mutant shifted to the right compared with that of WT channel. Login to comment
92 To study the effect of ADP on single-channel kinetics, we introduced the D1370N mutation into the ⌬R-CFTR background because ⌬R-CFTR is insensitive to dephosphorylation-induced rundown, and we can more easily obtain patches with fewer channels for kinetic analysis (Bompadre et al., 2005). Login to comment
93 In excised inside-out patches, ⌬R/D1370N-CFTR channels were opened with 1 mM ATP, and subsequent application of 1 mM ATP plus 1 mM ADP inhibited the currents by an average of 63 Ϯ 5% (n ϭ 5). Login to comment
102 Furthermore, the open time constant for D1370N-CFTR may still be too short for isolating the putative ADP-bound open state. Login to comment
321 From single-channel analysis, all we could observe was a shortening of the mean open time for ⌬R- (Bompadre et al., 2005) or ⌬R/ D1370N-CFTR (Fig. 1). Login to comment

PMID: 16246032 [PubMed] Vergani P et al: "Control of the CFTR channel's gates."

No. Sentence Comment

39 To determine which of the two composite sites in the NBD1-NBD2 dimer is involved in channel opening, we introduced mutations at residues seen to interact directly with the bound nucleotide in the solved crystal structures, in the head of either NBD1 [K464A (Lys464 → Ala)] or NBD2 (D1370N). 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

160 This conclusion was reached after finding that the ATP dose-response relationships of the Walker A mutants K464A and K1250A and the Walker B mutant D1370N were shifted towards higher [ATP] com- paredto theATPdose-response curvefor wild-typechannels. Login to comment
181 Single channel analysis indicates that theY1219G mutation reduces the opening rate of the channel while not affecting the open time (i.e. this mutation probably does not affect ATP hydrolysis in ABP2 like K1250A or D1370N). Login to comment

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

No. Sentence Comment

61 For both mutants (K464A in site 1 and D1370N in site 2), opening rate was reduced at low [ATP], but fast opening could be restored by increasing [ATP] (figure 2b, open symbols). Login to comment
86 (b) Hyperbolic relationship between [ATP] and opening rates (apparent dissociation constants are 56G5, 807G185, 391G118 mM for WT (filled circles), K464A (open triangles) and D1370N (open squares), respectively). Login to comment
59 For both mutants (K464A in site 1 and D1370N in site 2), opening rate was reduced at low [ATP], but fast opening could be restored by increasing [ATP] (figure 2b, open symbols). Login to comment
84 (b) Hyperbolic relationship between [ATP] and opening rates (apparent dissociation constants are 56G5, 807G185, 391G118 mM for WT (filled circles), K464A (open triangles) and D1370N (open squares), respectively). Login to comment

PMID: 19837660 [PubMed] Chen JH et al: "Direct sensing of intracellular pH by the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel."

No. Sentence Comment

6 Because these data suggest that pHi modulates the interaction of MgATP with the nucleotide-binding domains (NBDs) of CFTR, we examined the pHi dependence of site-directed mutations in the two ATP-binding sites of CFTR that are located at the NBD1:NBD2 dimer interface (site 1: K464A-, D572N-, and G1349D-CFTR; site 2: G551D-, K1250M-, and D1370N-CFTR). Login to comment
47 To study the CFTR variants K464A, D572N, and D1370N, we employed the vaccinia virus/bacteriophage T7 hybrid expression system to transiently express CFTR variants in HeLa cells as described previously (17, 18). Login to comment
204 Previous studies have demonstrated that the mutations D572N- and D1370N-CFTR abolish Mg2ϩ binding to the NBDs (21, 22). Login to comment
205 Figs. 6C and 8 and supplemental Fig. 3, B and C, demonstrate that the gating behavior of D572N- and D1370N-CFTR Cl-channels at different pHi diverges from that of wild-type CFTR in several important respects. Login to comment
206 First, at pHi 7.3, the Po of D572N-CFTR was the same as wild-type CFTR, whereas that of D1370N-CFTR was reduced (Fig. 6C). Login to comment
208 By contrast, for D1370N-CFTR at pHi 6.3, gating behavior and, hence, Po were unchanged (Fig. 6C and supplemental Fig. 3C). Login to comment
209 Third, in striking contrast to wild-type CFTR, at pHi 8.3 D572N-CFTR channel gating was enhanced because MBD was increased 0.6-fold and IBI decreased 0.3-fold, whereas that of D1370N-CFTR was unaltered (Figs. 2 and 8 and supplemental Fig. 3, B and C). Login to comment
210 As a result, at pHi 8.3, the Po of wild-type CFTR decreased, that of D1370N-CFTR was unchanged, whereas that of D572N-CFTR increased (Fig. 6C). Login to comment
213 Second the lack of effect of pHi on D1370N argues that the pHi sensitivity of CFTR channel gating is dependent on Mg2ϩ binding to site 2. Login to comment
218 B and C, effects of pHi on the Po of wild-type (WT), D572N-, and D1370N-CFTR in the presence of ATP (3 mM in B or 1 mM in C). Login to comment
219 In B, wild-type CFTR data were acquired in the presence (circles) and absence (columns) of Mg2ϩ (3 mM), whereas in C, wild-type, D572N-, and D1370N-CFTR data were acquired in the continuous presence of Mg2ϩ (3 mM). Login to comment
312 By contrast, Hϩ ions are either without effect (D1370N-CFTR) or inhibit (K1250M-CFTR) the gating behavior of site-directed mutations in ATP-binding site 2. Login to comment

PMID: 19966305 [PubMed] Csanady L et al: "Strict coupling between CFTR's catalytic cycle and gating of its Cl- ion pore revealed by distributions of open channel burst durations."

No. Sentence Comment

7 We show that the wild-type CFTR channel gating cycle is essentially irreversible and tightly coupled to the ATPase cycle, and that this coupling is completely destroyed by the NBD2 Walker B mutation D1370N but only partially disrupted by the NBD1 Walker A mutation K464A. Login to comment
49 Burst Duration Distribution of Nonhydrolytic Mutant D1370N Further Supports Irreversible Mechanism for WT. Login to comment
50 As a control, we chose to study the NBD2 Walker B mutant D1370N because the analogous mutation completely abolished ATP hydrolysis in other ABC proteins (27-29). Login to comment
51 The distribution of burst durations of D1370N channels gating in 2 mM ATP (Fig. 1C), reconstructed from 530 bursts, indeed differs qualitatively from that of WT channels (Fig. 1B) in that it decays monotonically. Login to comment
54 Interestingly, a combination of two positive-amplitude exponential components slightly improved the fit (ΔLL = 3.24; P = 0.03; Fig. 1C, red solid line; see SI Text for more fitting results), suggesting a mixture of two types of open bursts for D1370N; the major population, with an average lifetime of ~2 s, seems interspersed with a few brief bursts of ~200 ms. Login to comment
60 (B-D) Histograms of open burst durations for prephosphorylated WT (B), D1370N (C), and K464A (D) CFTR channels; 30-s segments of representative single-channel current recordings are shown above each panel. Login to comment
65 ATP was 2 mM for WT and D1370N, but 5 mM for K464A. Login to comment
82 Insofar as ATP hydrolysis is also absent in D1370N (k1 = 0), for this mutant the rate of channel closure from an open burst reflects the rate of dissociation of the prehydrolytic NBD dimer (k-1; Fig. 4E Right, green bar). Login to comment
104 Fig. 4 A-C compares such average parameters for fully (navy blue) and partially (royal blue) phosphorylated WT, and partially phosphorylated D1370N (green) and K464A (red) CFTR channels. Login to comment
105 Consistent with previous reports, for D1370N CFTR channels gating in near-saturating ATP (2 mM) (16), τb is ~4-fold longer than, but τib is like, that of WT (Fig. 4 B and C, green bars) (cf. refs. 9, 16, 30), whereas for prephosphorylated K464A CFTR channels in saturating ATP (5 mM) (16), τb is comparable to, but τib is at least ~2-fold longer than, that of WT (Fig. 4 B and C, red bars) (9, 14, 16 but cf. ref. 32). Login to comment
111 The interpretation that this irreversible cycle is driven by ATP hydrolysis is validated by the lack of any such fit improvement for the presumed nonhydrolytic D1370N mutant (Fig. 1C). Login to comment
126 The nonhydrolytic NBD2 mutant D1370N is a more extreme case, with an estimated coupling ratio of 0% (green bars, Fig. 4E Left and Right and Fig. 4F). Login to comment
141 Our analysis of the D1370N mutant, for example, implies that this mutation does not prevent cycling between inward- (closed channel) and outward-facing (open channel) conformations of CFTR (Fig. 4D, green; cf. Fig. 4F). Login to comment
161 (A-D) Open probabilities (A), mean burst (B), and interburst (C) durations obtained from multichannel fits, and calculated channel cycle times (D) for fully (navy blue) and partially (royal blue) phosphorylated WT, and partially phosphorylated D1370N (green) and K464A (red) CFTR. Login to comment
162 [ATP] was 2 mM for WT and D1370N, but 5 mM for K464A. Login to comment
164 (E) ML estimates of rates k1 (Left), k2 (Center), and k-1 (Right) for fully (navy blue) and partially (royal blue) phosphorylated WT, and partially phosphorylated D1370N (green) and K464A (red) CFTR channels. Login to comment
169 Probabilities for exiting state O1 (Top Right) in either of two possible directions are printed in color for partially phosphorylated WT (blue), K464A (red), and D1370N (green). Login to comment
184 Therefore, although both methods yielded qualitatively similar results, we used the distributions obtained using method (i) for WT and K464A (Figs. 1 B and D, 3B, and 4), and that obtained using method (ii) for D1370N (Fig. 1C). Login to comment

PMID: 20876359 [PubMed] Szollosi A et al: "Involvement of F1296 and N1303 of CFTR in induced-fit conformational change in response to ATP binding at NBD2."

No. Sentence Comment

242 Although for WT CFTR and for the nonhydrolytic mutant D1370N these two parameters are in rough agreement (Csanády et al., 2010), such comparisons have not yet been done for several other NBD2mutantsdefectiveinATPhydrolysis(e.g.,K1250R, K1250A, E1371S, and E1371Q). Login to comment

PMID: 20952391 [PubMed] Wang G et al: "State-dependent regulation of cystic fibrosis transmembrane conductance regulator (CFTR) gating by a high affinity Fe3+ bridge between the regulatory domain and cytoplasmic loop 3."

No. Sentence Comment

83 More importantly, a D1370N mutant, which removes the Mg2ϩ binding site (32), was also inhibited by Fe3ϩ (Fig. 1C), and inhibition was reversed by 5 mM EDTA (Fig. 1E). Login to comment
84 However, unlike the WT channel, the D1370N mutant exhibited less inhibition by Fe3ϩ but more reversal of inhibition by EDTA. Login to comment
108 The arrows indicate the final concentrations. C, effects of Fe3ϩ (5 ϫ 10-19 M) and Fe2ϩ (5 ϫ 10-13 M) on the WT hCFTR and the D1370N mutant currents (n ϭ 4-8). Login to comment

PMID: 21078867 [PubMed] Kopeikin Z et al: "On the mechanism of CFTR inhibition by a thiazolidinone derivative."

No. Sentence Comment

113 To further explore this relationship between the mean open time and the potency of CFTRinh-172, we tested CFTRinh-172 on a CFTR mutant, D1370N-CFTR, which exhibits an open time of 1 s (Bompadre et al., 2005). Login to comment
116 Fig. 4 B demonstrates the effects of 1 µM CFTRinh-172 on the macroscopic current of D1370N-CFTR. Login to comment
142 For D1370N-CFTR with an open time of 1 s, the IC50 is already in the nanomolar range. Login to comment
160 (A) Representative single-channel trace for D1370N-CFTR, a hydrolysis-deficient mutant with a mean open time of 1 s. Login to comment
161 (B) A continuous current recording showing a reversible inhibition of macroscopic D1370N-CFTR currents by 1 µM CFTRinh-172. Login to comment
162 (C) Dose-response relationships of CFTRinh-172 for WT-CFTR gated by ATP or P-ATP and D1370N-CFTR gated by ATP. Login to comment
165 Fitting parameters: for P-ATP-gated WT channels: IC50 = 0.37 ± 0.22 µM and Hill coefficient, n = 0.69 ± 0.38; for D1370N-CFTR: IC50 = 0.064 ± 0.007 µM and n = 1.26 ± 0.17. Login to comment

PMID: 9920885 [PubMed] Lee MG et al: "Regulation of Cl-/ HCO3- exchange by cystic fibrosis transmembrane conductance regulator expressed in NIH 3T3 and HEK 293 cells."

No. Sentence Comment

52 The mutagenesis primers were as follows: P205S primer, 5Ј-CGT GTG GAT CGC TTC TTT GCA AGT GGC-3Ј; W846term, 5Ј-GAG CAT ACC AGC AGT GAC TAC ATA GAA CAC ATA CCT TCG ATA TAT TAC-3Ј; G1247D/G1249E, 5Ј-GTG GGC CTC TTG GGA AGA ACT GAT TCA GAG AAG AGT ACT TTG TTA TCA GC-3Ј; K1250M, 5Ј-CTT GGG AAG AAC TGG ATC AGG GAT GAG TAC TTT GTT ATC AGC-3Ј; D1370N, 5Ј-GTA AGG CGA AGA TCT TGC TGC TTA ATG AAC CCA GTG CTC ATT TGG ATC-3Ј. Login to comment
163 Fig. 6 (i-k) shows the plasma membrane localization of K1250M CFTR, D1370N CFTR, and the double mutant G1247D/ G1249E CFTR, respectively. Login to comment
241 However, D1370N CFTR had nearly normal Cl-channel activity (32) and was expressed in the plasma membrane (Fig. 6j), but was unable to activate AE (Fig. 12c). Login to comment
266 The D1370N mutant had minimal effect on AE activity (c). Login to comment
273 In this respect the results obtained with D1370N CFTR are of particular interest since this mutation in NBD2 did not ablate channel activity (32) but eliminated regulation of AE activity by CFTR. Login to comment
274 Recently, it was reported that the D1506A mutation of the sulfonylurea receptor 1 protein, which corresponds to D1370N of human CFTR, similarly failed to stimulate the KATP channel (33). Login to comment

PMID: 22948143 [PubMed] Randak CO et al: "Demonstration of Phosphoryl Group Transfer Indicates That the ATP-binding Cassette (ABC) Transporter Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Exhibits Adenylate Kinase Activity."

No. Sentence Comment

183 2) Patch clamp studies showed that mutations K1250A and D1370N, located within conserved motifs of ATP-binding site 2, abolished the effects of Ap5A and AMP on CFTR current. Login to comment

PMID: 18805924 [PubMed] Dong Q et al: "A mutation in CFTR modifies the effects of the adenylate kinase inhibitor Ap5A on channel gating."

No. Sentence Comment

171 For example, the K1250A and D1370N mutations also have a reduced opening rate, prolonged burst duration, and increased ATP EC50 (11,12,14,15,29,32,39,43). Login to comment

PMID: 9674722 [PubMed] Schwiebert EM et al: "Cystic fibrosis: a multiple exocrinopathy caused by dysfunctions in a multifunctional transport protein."

No. Sentence Comment

224 In NBD2, a few key mutations have been found that include missense mutations (G1349D, D1370N, K1250M, K1250Q, G1244E, S1255P) and several nonsense mutations (W1282X, S1255X, W1316X). Login to comment

PMID: 9558482 [PubMed] Foskett JK et al: "ClC and CFTR chloride channel gating."

No. Sentence Comment

324 An interesting contrast to the effects of the K1250A mutation in the Walker A motif was revealed in a D1370N mutant in the Walker B motif. Login to comment

PMID: 8741733 [PubMed] Wilkinson DJ et al: "CFTR: the nucleotide binding folds regulate the accessibility and stability of the activated state."

No. Sentence Comment

11 In contrast, the analogous substitution in NBF2 (D1370N) did not appreciably affect the on rate and markedly stabilized the active state. These results are consistent with a hypothesis for CFTR activation that invokes the binding and hydrolysis of ATP at NBF1 as a crucial step in activation, while at NBF2, ATP binding enhances access to the active state, but the rate of ATP hydrolysis controls the duration of the active state. Login to comment
19 In contrast, mutations in the putative ATP-binding pockets of the two NBFs produced opposite results, a reduction in sensitivity for mutations in NBF1 (K464Q, D572N) and an increase in sensitivity for the analogous mutations in NBF2 (K12500~ D1370N). Login to comment
195 a~ODiap- 9 K464Q a I ' ' ' ' I ' ' ' ' I ' ' 0 10 20 ~ O -0 0, 9 -0~176176176o ....... 9.... -o*- -o*- 9 i~ e'~176176176176 9 D572N o i , , , , i , , , , i , , , , I , , , , i , , , , i , , , , 0 I0 20 30 40 50 minutes K1250A K1250C I i 30 D1370N 6O FIGURE4. Login to comment
201 (C) Substitutions of asparagine for the Walker consensus B aspartic acid in the ATP-binding pocket of NBFI (D572N, 0) or NBF2 (D1370N,0). Login to comment
247 0 30 60 90 12O 15O i \ 9 DsZ2No D1370N iX',, - ~ o ~ ***************************** I ' ' ' I ' ' ' I ' ' ' I ' ' ' I ' ' ' 0 20 40 60 80 100 minutes FIGURE 6. Login to comment
256 In contrast, the analogous substitution in NBF2 (D1370N) produced only a modest decrease in (ko, + kor0, evident in Fig. 4 C. The values of the derived parameters (k'o,, ko~) for this slightly hypersensitive mutant, however, suggest that the decrease in KAwas a reflection of a fourfold decrease in ko~, whereas the apparent kon was not significantly different from that of wild-type CFTR. Login to comment
261 Particularly noteworthy in this regard is the D1370N mutation, which produced no significant effect on the apparent on rate but markedly delayed deactivation. Login to comment
277 Similarly, although the K1250R and D1370N mutants exhibited an increased latency, the values of *ko~ were not significantly different from that of wild type CFTR. Login to comment
281 + kott) (10-3 min-l kon kofr latency *k~m CFTR (mM) n (10-3min-]) mM-1) (10-3min 1) (10-3min-l) n (min) (10 3min i) n wt 0.65 • 0.08 26 664 • 51 118 • 9 558 • 45 76-+ 6 20 6.0 • 0.3 88 • 6 16 K464R 2.6 • 0.1": 4 153 + 20**+ 20 • 3*** 101 • 13''` 52 • 7*: 5 1.3 • 0.2*++ 174 • 14"** 7 K464Q 3.3 • 0.5"* 5 331 • 56*** 40 -+ 7* 199 • 34* 132 • 22*'` 5 1.9 • 0.3"I 142 -+ 19''` 5 K464A 4.6 • 0.7** 6 289 • 49* 30 • 5** 151 • 26*** 139 • 24*: 7 1.1 • 0.1"** 133 • 14"** 8 D572N 9.3 + 0.02*: 6 106 • 7*: 7-+0.5*: 37-+3*** 69 • 5+* 4 0.9 • 0.2*** 245 • 32*: 3 K1250R 0.17 • 0.07*: 5 239 •33*** 46 -+ 6"+* 231 • 32*: 8 • 1": 10 10.4 • 0.8"~ 100 • 7** 6 K1250Q 0.12 • 0.04*** 5 150 • 18''` 29 • 4* 146 -+ 18" 4 + 0.4"I 5 22.3 • 2.4*: 30 •5": 5 K1250A 0.07 + 0.02*: 10 218 • 18" 43 • 4*'` 215 • 18": 3 -+0.3*~* 5 15.6-+ 1.0"** 43 -+5** 5 D1370N 0.16 + 0.04*'` 7 449 - 79*: 87 • 15: 435 +76** 14 - 2*: 5 16.3-4-1.2"" 69-+ 6** 5 The symbols (*) and ('`) indicate significant differences from wild-type CFTR and the analogous mutant, respectively (P < 0.05). Login to comment
321 Of particular interest in this regard was the effect of the NBF2 mutation, D1370N. Login to comment
339 Although the structural and functional studies of these other proteins do not offer a completely coherent picture of the role of the Walker B aspartic acid, the results of the rate analysis for the CFTR mutant D1370N are consistent with a role for this aspartic acid in ATP hydrolysis. Login to comment
198 a~ODiap- 9 K464Q a I ' ' ' ' I ' ' ' ' I ' ' 0 10 20 ~ O -0 0, 9 -0 ~176176176 o ....... 9.... -o*- -o*- 9 i~ e'~176176176176 9 D572N o i , , , , i , , , , i , , , , I , , , , i , , , , i , , , , 0 I0 20 30 40 50 minutes K1250A K1250C I i 30 D1370N 6O FIGURE4. Login to comment
204 (C) Substitutions of asparagine for the Walker consensus B aspartic acid in the ATP-binding pocket of NBFI (D572N, 0) or NBF2 (D1370N,0). Login to comment
249 0 30 60 90 12O 15O i \ 9 DsZ2No D1370N iX',, - ~ o ~ ***************************** I ' ' ' I ' ' ' I ' ' ' I ' ' ' I ' ' ' 0 20 40 60 80 100 minutes FIGURE 6. Login to comment
258 In contrast, the analogous substitution in NBF2 (D1370N) produced only a modest decrease in (ko, + kor0, evident in Fig. 4 C. The values of the derived parameters (k'o,, ko~) for this slightly hypersensitive mutant, however, suggest that the decrease in KAwas a reflection of a fourfold decrease in ko~, whereas the apparent kon was not significantly different from that of wild-type CFTR. Login to comment
263 Particularly noteworthy in this regard is the D1370N mutation, which produced no significant effect on the apparent on rate but markedly delayed deactivation. Login to comment
279 Similarly, although the K1250R and D1370N mutants exhibited an increased latency, the values of *ko~ were not significantly different from that of wild type CFTR. Login to comment
283 + kott) (10-3 min-l kon kofr latency *k~m CFTR (mM) n (10-3 min-]) mM-1) (10-3 min 1) (10-3min-l) n (min) (10 3min i) n wt 0.65 ߦ 0.08 26 664 ߦ 51 118 ߦ 9 558 ߦ 45 76 -+ 6 20 6.0 ߦ 0.3 88 ߦ 6 16 K464R 2.6 ߦ 0.1": 4 153 + 20**+ 20 ߦ 3*** 101 ߦ 13''` 52 ߦ 7*: 5 1.3 ߦ 0.2*++ 174 ߦ 14"** 7 K464Q 3.3 ߦ 0.5"* 5 331 ߦ 56*** 40 -+ 7* 199 ߦ 34* 132 ߦ 22*'` 5 1.9 ߦ 0.3"I 142 -+ 19''` 5 K464A 4.6 ߦ 0.7** 6 289 ߦ 49* 30 ߦ 5** 151 ߦ 26*** 139 ߦ 24*: 7 1.1 ߦ 0.1"** 133 ߦ 14"** 8 D572N 9.3 + 0.02*: 6 106 ߦ 7*: 7 -+0.5*: 37 -+3*** 69 ߦ 5+* 4 0.9 ߦ 0.2*** 245 ߦ 32*: 3 K1250R 0.17 ߦ 0.07*: 5 239 ߦ 33*** 46 -+ 6"+* 231 ߦ 32*: 8 ߦ 1": 10 10.4 ߦ 0.8"~ 100 ߦ 7** 6 K1250Q 0.12 ߦ 0.04*** 5 150 ߦ 18''` 29 ߦ 4* 146 -+ 18" 4 + 0.4"I 5 22.3 ߦ 2.4*: 30 ߦ 5": 5 K1250A 0.07 + 0.02*: 10 218 ߦ 18" 43 ߦ 4*'` 215 ߦ 18": 3 -+0.3*~* 5 15.6 -+ 1.0"** 43 -+5** 5 D1370N 0.16 + 0.04*'` 7 449 - 79*: 87 ߦ 15: 435 + 76** 14 - 2*: 5 16.3 -4-1.2"" 69 -+ 6** 5 The symbols (*) and ('`) indicate significant differences from wild-type CFTR and the analogous mutant, respectively (P < 0.05). Login to comment
323 Of particular interest in this regard was the effect of the NBF2 mutation, D1370N. Login to comment
341 Although the structural and functional studies of these other proteins do not offer a completely coherent picture of the role of the Walker B aspartic acid, the results of the rate analysis for the CFTR mutant D1370N are consistent with a role for this aspartic acid in ATP hydrolysis. Login to comment

PMID: 8572187 [PubMed] Howard M et al: "Epitope tagging permits cell surface detection of functional CFTR."

No. Sentence Comment

251 M2-901 CFIR 5 0 -1 mV C constructed two dgCFTR mutants that are not associated with disease, K1250M and D1370N, also bearing This is a technical study that identifies sites appropri- the M2 epitope at position 901. Login to comment
255 Cell surface expression of M2-901/CFTR has been observed in a expression of M2-901-labeled G551D, G1349D, K1250M, and D1370N dgCFTRs correlates with carbohydrate variety of cell types including HEp-2, BSC40, and addition (Fig &A-II). Login to comment
265 A: G551D; B: G1349D; C: K1250M; D: D1370N; E: AF508; F: N1303K. Login to comment

PMID: 7543023 [PubMed] Gunderson KL et al: "Conformational states of CFTR associated with channel gating: the role ATP binding and hydrolysis."

No. Sentence Comment

92 0 2 B low MI 2* C D1370N (high Mg2+) 2sec -----]0,5 pA Figure 4. Login to comment
109 Gating of CFTR channels harboring a D1370N mutation closely resembled that of the wild-type channel in EDTA (Figure 4C). Login to comment
111 Moreover, even in the presence of Mg2+,we were unable to induce locking of the D1370N channels with PPi (data not shown). Login to comment
114 By contrast, D1370N mutants, which can be mimicked by applying EDTA to wild-type channels, are also blocked from completing the full (C~O1~O2~C) gating cycle through 02, but are neither locked open in O1 nor capable of becoming locked by PP~.These two classes of mutants thus define two kinetic states of the O1 conductance level: an initial open state that is reversible (back to C) and independent of Mg2÷, and a subsequent state that requires Mg2÷ and is effectively irreversible. Login to comment
121 The simplest interpretation of these data is that ATP binding ConformationalStatesof CFTR 09 08 07 06 Oo 05 040.3 * 32 wildtype K464A K1250A G1247D/ D1370N 01249E 6~se' B 2500 20O0 5 1500 1OO0 500 o~ 0 w[Id{ype K464A K1250A C1247D/ Ol 370N G1249E Figure5. Login to comment
241 Acknowledgments Correspondence should be addressed to R. R. K. We thank M. Welsh for kindly providing the K1250M and D1370N mutants, P. Quinton for his insightful discussions and critical reading of the manuscript, C. Login to comment
110 Gating of CFTR channels harboring a D1370N mutation closely resembled that of the wild-type channel in EDTA (Figure 4C). Login to comment
112 Moreover, even in the presence of Mg2+,we were unable to induce locking of the D1370N channels with PPi (data not shown). Login to comment
115 By contrast, D1370N mutants, which can be mimicked by applying EDTA to wild-type channels, are also blocked from completing the full (C~O1~O2~C) gating cycle through 02, but are neither locked open in O1 nor capable of becoming locked by PP~.These two classes of mutants thus define two kinetic states of the O1 conductance level: an initial open state that is reversible (back to C) and independent of Mg2&#f7;, and a subsequent state that requires Mg2&#f7; and is effectively irreversible. Login to comment
122 The simplest interpretation of these data is that ATP binding ConformationalStatesof CFTR 09 08 07 06 Oo 05 040.3 * 32 wildtype K464A K1250A G1247D/ D1370N 01249E 6~se' B 2500 20O0 5 1500 1OO0 500 o ~ 0 w[Id{ype K464A K1250A C1247D/ Ol 370N G1249E Figure5. Login to comment
242 Acknowledgments Correspondence should be addressed to R. R. K. We thank M. Welsh for kindly providing the K1250M and D1370N mutants, P. Quinton for his insightful discussions and critical reading of the manuscript, C. Login to comment

PMID: 7694298 [PubMed] Smit LS et al: "Functional roles of the nucleotide-binding folds in the activation of the cystic fibrosis transmembrane conductance regulator."

No. Sentence Comment

60 For this reason, expression levels for easily activated constructs (wild type, G551A, G1349A, K1250Q, and D1370N) were adjusted by reducing the amount of injected RNA so that the maximum Cl- conductance was similar to that attained by less sensitive constructs. Login to comment
108 A 80+ 60 + 40+- 20 + wt rl% - 7 1 k (12) t rd:\z-Za uab/ZN k b) A-- K A D1370N ( 5) T/ z T A T I I 0 f I~0A I~~~~~~ T t u A~~~~ 0.2. ,0.02 0.05 0.2 0.5 IBMX, mM 2 45 FIG. 5. Login to comment

PMID: 14697202 [PubMed] Randak C et al: "An intrinsic adenylate kinase activity regulates gating of the ABC transporter CFTR."

No. Sentence Comment

228 In addition, we Walker A and B motif mutations (K1250A and D1370N) abolished both ATPase and adenylate kinase activities studied a relatively common CF-associated mutation, N1303K (Osborne et al., 1992); interestingly, two other (Figures 7A and 7B). Login to comment
272 Results with the Walker B Asp Discussion mutations (D572N in NBD1 and D1370N in NBD2) exactly paralleled the P loop mutations (Figures 7D-7F), further Earlier work indicated that CFTR can function as an ATPase and that hydrolysis contributes to channel gat- suggesting that adenylate kinase activity resides in NBD2. Login to comment
280 NBD2 was wild-type or contained K1250A, D1370N, or N1303K mutations. Login to comment
288 Data are from 5 (wild-type, K464A, D572N), 9 (K1250A), 10 (D1370N), and 3 (N1303K) membrane patches. Asterisks indicate p b0d; 0.05 compared to wild-type by ANOVA followed by Dunnett`s multiple comparison test. Login to comment

PMID: 23921386 [PubMed] Randak CO et al: "ATP and AMP mutually influence their interaction with the ATP-binding cassette (ABC) adenylate kinase cystic fibrosis transmembrane conductance regulator (CFTR) at separate binding sites."

No. Sentence Comment

275 Error bars, S.E. Nucleotide Interactions with the ABC Adenylate Kinase CFTR 27698 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 288ߦNUMBER 38ߦSEPTEMBER 20, 2013 at SEMMELWEIS UNIV OF MEDICINE on December , D1370N, abolished Ap5A inhibition of current, whereas the homologous mutations in ATP-binding site 1, K464A and D572N, did not. Login to comment
302 (b) Patch clamp studies showed that CFTR mutations K1250A and D1370N, located within the conserved Walker A and B motifs of ATP-binding site 2, abolished the effects of Ap5A and AMP on CFTR current. Login to comment

PMID: 25512598 [PubMed] Yeh HI et al: "Modulation of CFTR gating by permeant ions."

No. Sentence Comment

163 To this end, we introduced the D1370N mutation into the &#e044;R background and characterized gating kinetics in the presence of NO3 &#e032; or Cl&#e032; (Fig. 5 C). Login to comment
166 Comparing the single-channel kinetic parameters of D1370N-CFTR further confirms that the effect of NO3 &#e032; on both the Figure 5.ߓ Effects of NO3 &#e032; on single-channel kinetics of &#e044;R-CFTR and D1370N/&#e044;R-CFTR. Login to comment
171 (C) NO3 &#e032; increases the Po of a hydrolysis-deficient mutant CFTR (D1370N). Login to comment
172 (D) Microscopic kinetic parameters for D1370N/ &#e044;R-CFTR in the presence of bath Cl&#e032; or NO3 &#e032; . Login to comment

PMID: 26496611 [PubMed] Sorum B et al: "Timing of CFTR Pore Opening and Structure of Its Transition State."

No. Sentence Comment

4 Here, we exploit equilibrium gating of hydrolysis-deficient CFTR mutant D1370N and apply rate-equilibrium free-energy relationship analysis to estimate relative timing of opening movements in distinct protein regions. Login to comment
37 To make CFTR gate at equilibrium, we introduced the NBD2 Walker-B mutation D1370N (Figure 1A, bottom, red star) because, among several hydrolysis-disrupting mutations tested, D1370N only slightly reduces the apparent affinity for ATP, and does not prolong open bursts to an extent incompatible with single-channel gating analysis (Csana &#b4; dy et al., 2010). Login to comment
40 Choice of a Suitable Background Construct for REFER Analysis (A) Domain organizations of WT (top) and cut-DR(D1370N) (bottom) CFTR: TMDs (gray), intracellular loops containing coupling helices (light violet), NBD1 (green), NBD2 (blue), R domain (magenta), membrane (yellow). Login to comment
42 The D1370N mutation in NBD2 is depicted by a red star. Login to comment
48 The D1370N mutation abrogates ATP hydrolysis (red cross) and confines gating in saturating ATP to a simple C1 4 O1 equilibrium (red box). Login to comment
53 Thus, we chose cut-DR(D1370N) as the background construct for our REFER study (Figure 1A, bottom). Login to comment
54 Gating of cut-DR(D1370N) indeed proved PKA-independent but remained strictly ATP-dependent with an apparent affinity for ATP of 288 &#b1; 27 mM (Figures S1A and S1B). Login to comment
55 Just as for WT (Winter et al., 1994; Zeltwanger et al., 1999; Csana &#b4; dy et al., 2000; Vergani et al., 2003), cut-DR (Csana &#b4; dy et al., 2000; Bompadre et al., 2005), and D1370N (Vergani et al., 2003) CFTR channels, mean open burst duration (tb) of cut-DR(D1370N) proved largely Figure 2. Login to comment
56 Timing of Motion at Position 1246 of the NBD1-NBD2 Interface (A) Inward single-channel currents of the cut-DR(D1370N) CFTR background construct (top trace) and of channels bearing mutations T1246V, T1246P, T1246C, T1246N, and T1246A, respectively, in the same background. Login to comment
63 Timing of Motion at Position 275 of the NBD2-TMD Interface (A) Inward single-channel currents of the cut-DR(D1370N) CFTR background construct (top trace) and of channels bearing mutations Y275F, Y275E, Y275K, Y275L, and Y275S, respectively, in the same background. Currents were recorded at 80 mV, in symmetrical 140 mM Cl ; dashes on the left mark zero-current level. Login to comment
74 Timing of Motion at Position 348 in the Pore Region (A) Inward single-channel currents of the cut-DR(D1370N) CFTR background construct (top trace) and of channels bearing mutations M348I, M348K, M348C, M348N, and M348A, respectively, in the same background. Currents were recorded at 80 mV, in symmetrical 140 mM Cl ; dashes on the left mark zero-current level. Login to comment
79 Timing of Motion in the Narrow Region of the Pore Studied by Anion Replacement (A) Pairs of segments of inward single-channel current from three patches containing single cut-DR(D1370N) CFTR channels. Login to comment
108 Thus, replacement of chloride with other permeant anions might be viewed as a structural perturbation of the ''selectivity filter.`` We therefore studied changes in the pattern of single-channel gating of our background construct cut-DR(D1370N) in response to sudden replacement of cytosolic chloride with nitrate, bromide, or formate. Login to comment
130 The point representing the cut-DR(D1370N) background construct in chloride is highlighted by a black circle. Login to comment
153 To study the pore opening step, we therefore employed the D1370N background mutation that truncates the gating cycle to an equilibrium scheme (Figure 1B, red frame). Login to comment
162 Because gating of cut-DR(D1370N), like that of WT CFTR, is strictly ATP-dependent (Figures S1 and S2), our conclusions do not necessarily apply to the mechanism of the extremely infrequent spontaneous openings observable in the absence of ATP that are promoted by certain mutations (Wang et al., 2010) and drugs (Jih and Hwang, 2013). Login to comment
165 EXPERIMENTAL PROCEDURES pGEMHE-CFTR(837-1480(D1370N)) was constructed from pGEMHE-CFTR(837-1480), mutations at positions 275 and 348 were introduced into pGEMHE-CFTR(1-633) (Csana &#b4;dy et al., 2000), and mutations at position 1246 into pGEMHE-CFTR(837-1480(D1370N)) using Stratagene QuickChange. Login to comment