ABCMdb

PMID: 19966305

Csanady L, Vergani P, Gadsby DC
Strict coupling between CFTR's catalytic cycle and gating of its Cl- ion pore revealed by distributions of open channel burst durations.
Proc Natl Acad Sci U S A. 2010 Jan 19;107(3):1241-6. Epub 2009 Dec 4., 2010-01-19 [PubMed]

Sentences

No. Mutations Sentence Comment

7 ABCC7 p.Lys464Ala ABCC7 p.Asp1370Asn 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 ABCC7 p.Asp1370Asn Burst Duration Distribution of Nonhydrolytic Mutant D1370N Further Supports Irreversible Mechanism for WT. Login to comment 50 ABCC7 p.Asp1370Asn 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 ABCC7 p.Asp1370Asn 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 ABCC7 p.Asp1370Asn 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 ABCC7 p.Lys464Ala ABCC7 p.Asp1370Asn (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 ABCC7 p.Lys464Ala ABCC7 p.Asp1370Asn ATP was 2 mM for WT and D1370N, but 5 mM for K464A. Login to comment 66 ABCC7 p.Lys464Ala Burst Duration Distribution of K464A Mutant Reveals Profoundly Altered Gating Mechanism. Login to comment 67 ABCC7 p.Lys464Ala Although the K464A mutation lowers CFTR ATPase turnover rate ~10-fold (21), τb was essentially unaffected by this mutation (Fig. 1D Inset). Login to comment 68 ABCC7 p.Lys464Ala However, the shape of the distribution of K464A burst durations (Fig. 1D; reconstructed from 2,327 events) clearly differed from that of WT CFTR. Login to comment 70 ABCC7 p.Lys464Ala However, because the K464A mutant does carry out some ATP hydrolysis, albeit slowly (21), we also evaluated a partially hydrolytic mechanism by leaving k-1 in scheme 2 as a free parameter. Login to comment 73 ABCC7 p.Lys464Ala The rate estimates from this fit suggest that in K464A CFTR, the rate of the ATP hydrolysis step (k1) is slowed by only ~4-fold compared with WT, consistent with the fact that this mutation is not in the composite NBD2 site, where ATP hydrolysis occurs. Login to comment 74 ABCC7 p.Lys464Ala But this analysis also indicates that the K464A mutation greatly destabilizes the prehydrolytic dimer (k-1 is increased). Login to comment 78 ABCC7 p.Lys1250Ala ABCC7 p.Glu1371Ser ABCC7 p.Lys1250Arg As a three-parameter fit of scheme 2 to the data in Fig. 1B (and also Fig. 3) did not provide a reliable estimate of this small rate (SI Text), to estimate k-1 we measured the macroscopic closing rates of prephosphorylated K1250A, K1250R, and E1371S mutant channels (e.g., Fig. 2A) upon sudden removal of ATP. Login to comment 79 ABCC7 p.Lys1250Ala ABCC7 p.Glu1371Ser ABCC7 p.Lys1250Arg These rates, obtained as the reciprocals of the time constants of fitted single exponentials (e.g., Fig. 2A, blue line), were 0.044 ± 0.004 s-1 (n = 9) for K1250A (Fig. 2C, blue bar), 0.22 ± 0.01 s-1 (n = 17) for K1250R, and 0.036 ± 0.002 s-1 (n = 16) for E1371S. Login to comment 81 ABCC7 p.Lys1250Arg As we cannot be certain which of these mutant channels, when open, most closely resembles the O1 state of a WT CFTR channel gating in ATP, we tentatively chose the closing rate of K1250R as an estimate of k-1 for WT CFTR (Fig. 4E Right, blue bar) on the grounds that the lysine-to- arginine mutation at least conserves charge in the vicinity of the ATP bound within the NBD2 composite site. Login to comment 82 ABCC7 p.Asp1370Asn 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 84 ABCC7 p.Lys464Ala Acceleration of Nonhydrolytic Channel Closure by the K464A Mutation Supports Microscopic Burst Duration Analysis. Login to comment 85 ABCC7 p.Lys464Ala ABCC7 p.Lys464Ala Assuming k-1 = 0.22 s-1 for WT, the ML fit of scheme 2 to the K464A burst distribution (Fig. 1D) suggests that rate k-1 is increased by ~15-fold in K464A CFTR. Login to comment 86 ABCC7 p.Lys464Ala This conclusion from the distribution of microscopic burst durations is corroborated by the fact that closure of nonhydrolytic CFTR mutants and of WT channels "locked" in open bursts by nonhydrolyzable ATP analogs or by orthovanadate is greatly accelerated by the K464A mutation (16, 18, 30, 32). Login to comment 87 ABCC7 p.Lys1250Ala ABCC7 p.Lys1250Ala ABCC7 p.Lys464Ala For instance, closure of the catalytically incompetent NBD2 Walker A mutant K1250A is accelerated ~10-fold in the double-mutant K464A/K1250A, as reported by the rate of macroscopic current decay upon ATP removal (Fig. 2B; red line is a single-exponential fit; Fig. 2C, red bar). Login to comment 97 ABCC7 p.Lys464Ala Slow nonhydrolytic closing rate and its acceleration by the K464A mutation. Login to comment 98 ABCC7 p.Lys1250Ala ABCC7 p.Lys1250Ala ABCC7 p.Lys464Ala (A and B) Macroscopic currents of prephosphorylated K1250A (A) and K464A/K1250A (B) CFTR channels were activated by application of 10 mM ATP. Login to comment 100 ABCC7 p.Lys1250Ala ABCC7 p.Lys1250Ala ABCC7 p.Lys464Ala (C) Mean (±SEM) closing rates estimated as the inverses of the current relaxation time constants (τrelax), for K1250A (blue) and K464A/K1250A (red). Login to comment 104 ABCC7 p.Lys464Ala ABCC7 p.Asp1370Asn 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 ABCC7 p.Lys464Ala ABCC7 p.Asp1370Asn 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 ABCC7 p.Asp1370Asn 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 115 ABCC7 p.Lys464Ala However, the fit for K464A provides additional support for this assignment. Login to comment 117 ABCC7 p.Lys464Ala Thus, the set k1' = 15.4 s-1 , k2' = 4.30 s-1 , k-1' = 3.39 s-1 fits the observed pdf for K464A just as well as the set displayed in Fig. 4E (k1 = 0.91 s-1 , k2 = 18.8 s-1 , k-1 = 3.39 s-1 ). Login to comment 118 ABCC7 p.Lys464Ala But this alternative set can be ruled out as it would yield an essentially hydrolytic mechanism for K464A (with a coupling ratio of ~82%) in contradiction of the observed severe defect in ATPase turnover rate (21). Login to comment 120 ABCC7 p.Lys464Ala The NBD1 Walker A mutant K464A has received much previous attention. Login to comment 121 ABCC7 p.Lys464Ala That this mutation, in a catalytically inactive binding site (17, 18), affects channel gating only slightly (Fig. 4D; cf. refs. 16, 21, 32) contrasts with its substantial suppression of the rate of ATP hydrolysis of purified K464A CFTR protein (to <10% of WT) (21). Login to comment 122 ABCC7 p.Lys464Ala The altered shape of the distribution of K464A burst durations (Fig. 1D) now provides a satisfying explanation for this dissociation between channel cycle time and ATPase rate. Login to comment 123 ABCC7 p.Lys464Ala The ratio k1/(k1 + k-1) estimated from the scheme 2 fit (Fig.4E Left and Right, red bars) suggests that in K464A approximately one out of every five bursts proceeds through the normal irreversible hydrolytic pathway (Fig. 4F). Login to comment 124 ABCC7 p.Lys464Ala This "coupling ratio" between channel opening and ATP hydrolysis of ~21% for K464A CFTR contrasts with that of ≥95% for WT channels (blue bars, Fig. 4E Left and Right and Fig. 4F). Login to comment 125 ABCC7 p.Lys464Ala Because the cycle time of K464A channels is prolonged ~2-fold compared with WT (Fig. 4D), the predicted overall ATPase turnover rate is on the order of 10% of WT, which is in very reasonable agreement with the measured value (21). Login to comment 126 ABCC7 p.Asp1370Asn 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 ABCC7 p.Asp1370Asn 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 ABCC7 p.Lys464Ala ABCC7 p.Asp1370Asn (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 ABCC7 p.Lys464Ala ABCC7 p.Asp1370Asn [ATP] was 2 mM for WT and D1370N, but 5 mM for K464A. Login to comment 164 ABCC7 p.Lys464Ala ABCC7 p.Asp1370Asn (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 166 ABCC7 p.Lys1250Arg (a) k-1 for partially phosphorylated WT is modeled by the closing rate of partially phosphorylated K1250R. Login to comment 169 ABCC7 p.Lys464Ala ABCC7 p.Asp1370Asn 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 ABCC7 p.Lys464Ala ABCC7 p.Asp1370Asn 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