ABCMdb

PMID: 12508051

Vergani P, Nairn AC, Gadsby DC
On the mechanism of MgATP-dependent gating of CFTR Cl- channels.
J Gen Physiol. 2003 Jan;121(1):17-36., [PubMed]

Sentences

No. Mutations Sentence Comment

4 ABCC7 p.Lys1250Ala ABCC7 p.Lys464Ala ABCC7 p.Asp1370Asn 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 ABCC7 p.Lys1250Ala ABCC7 p.Asp1370Asn ABCC7 p.Glu1371Ser NBD2 catalytic site mutations K1250A, D1370N, and E1371S were found to prolong open bursts. Login to comment 9 ABCC7 p.Lys464Ala However, when hydrolysis at NBD2 was impaired, the NBD1 mutation K464A shortened the prolonged open bursts. Login to comment 32 ABCC7 p.Lys1250Ala ABCC7 p.Lys464Ala ABCC7 p.Asp1370Asn ABCC7 p.Asp572Asn 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 34 ABCC7 p.Lys1250Ala ABCC7 p.Lys464Ala ABCC7 p.Gln552His ABCC7 p.Gln552Ala Thus, the K1250A mutation dramatically prolonged burst duration, suggesting that hydrolysis at NBD2 might be coupled to burst termination (Carson et al., 1995; Gunderson and Kopito, 1995), whereas the NBD1 mutations K464A, Q552A, and Q552H somewhat slowed channel opening to a burst, suggesting that NBD1 might be a site of ATP interactions governing opening (Carson et al., 1995; Carson and Welsh 1995). Login to comment 41 ABCC7 p.Lys1250Ala ABCC7 p.Lys464Ala ABCC7 p.Asp1370Asn 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 ABCC7 p.Lys1250Ala ABCC7 p.Lys1250Ala ABCC7 p.Lys464Ala ABCC7 p.Lys464Ala ABCC7 p.Asp1370Asn 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 85 ABCC7 p.Lys1250Ala We do not report absolute values for ␶ib and rCO in these cases, but only values relative to some other experimental condition applied to the same patch, and these should be relatively insensitive to N. Thus, for MgATP dose-response curves (Fig. 2), rates were normalized to those in bracketing segments at 5 mM MgATP; for the poorly hydrolyzable nucleotides, rates were normalized to those obtained in the same patches at 10 ␮M (Figs. 7 and 11) or 50 ␮M MgATP (Fig. 8); for K1250A mutant openings in 10 ␮M MgATP, rates were normalized to those in nominally MgATP-free bath solution. Login to comment 89 ABCC7 p.Lys464Ala ABCC7 p.Asp1370Asn 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 ABCC7 p.Asp1370Asn 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 ABCC7 p.Lys464Ala ABCC7 p.Asp1370Asn (B and C) Representative traces for prephosphorylated K464A and D1370N channels. Login to comment 114 ABCC7 p.Lys464Ala ABCC7 p.Asp1370Asn 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 ABCC7 p.Lys464Ala ABCC7 p.Asp1370Asn 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 ABCC7 p.Lys464Ala ABCC7 p.Lys464Ala ABCC7 p.Asp1370Asn ABCC7 p.Asp1370Asn 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 ABCC7 p.Lys464Ala ABCC7 p.Asp1370Asn 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 126 ABCC7 p.Lys1250Ala Similar kinetic analysis of patches containing few channels proved technically difficult for K1250A CFTR (NBD2 Walker A lysine mutant) due to the extremely prolonged bursts (see Fig. 6 C, below), which precluded collection of enough events to reliably estimate absolute values of rCO or Po. Login to comment 127 ABCC7 p.Lys1250Ala So we recorded macroscopic current in patches with hundreds or thousands of WT or K1250A channels (Fig. 3, A and B), and determined relative Po as a function of [MgATP] (Fig. 3 C) by normalizing current amplitude at each test [MgATP] to that during bracketing exposures at 5 mM MgATP (Fig. 3, A and B). Login to comment 128 ABCC7 p.Lys1250Ala The curve for K1250A was strongly shifted to higher [MgATP] and was still not saturated at 10 mM MgATP. Login to comment 132 ABCC7 p.Lys1250Ala Therefore, in K1250A, as in WT CFTR, it is only the dependence of channel opening rate on [MgATP] that underlies the [MgATP] dependence of Po. Login to comment 134 ABCC7 p.Lys1250Ala ABCC7 p.Lys1250Ala In fact, this relationship implies that the effective dissociation constant for MgATP activation of opening of K1250A channels is likely even larger than is apparent in Fig. 3 C because the other effect of the K1250A mutation, marked slowing of channel closure from bursts, would by itself shift the Po versus [MgATP] curve to lower [MgATP], opposite to our experimental observation. Login to comment 137 ABCC7 p.Lys1250Ala The K1250A mutation strongly shifts the [MgATP] dependence of Po to higher [MgATP]. Login to comment 140 ABCC7 p.Lys1250Ala (B) Macroscopic current of K1250A channels was reduced Ն2-fold on lowering [MgATP] from 5 to 1 mM. Login to comment 144 ABCC7 p.Lys1250Ala ABCC7 p.Lys1250Ala Michaelis fit parameters for WT: Po max ϭ 1.04 Ϯ 0.01, K0.5 ϭ 57 Ϯ 2 ␮M; for K1250A: Po max ϭ 2.45 Ϯ 0.88, K0.5 ϭ 6.5 Ϯ 4.8 mM; for display, WT (circles) and K1250A (inverted triangles) data (mean Ϯ SD, 3 Յ n Յ9) were renormalized to these Po max values. Login to comment 145 ABCC7 p.Lys1250Ala Because 10 mM, the highest [MgATP] used, was still far from saturating for K1250A channels, the fit for this mutant is less accurate, evident from large errors on fit parameters. Login to comment 151 ABCC7 p.Lys464Ala Catalytic Site Mutations at NBD1 Do Not Alter Channel Closing from Normal, MgATP-elicited Bursts The average rate of closure of K464A mutant CFTR channels from open bursts was closely similar to that of WT CFTR under comparable conditions (Figs. 4, A-F, and 5, A, B, and E); it was likewise approximately independent of [MgATP] (Fig. 2 E, red triangles) and it was similarly reduced roughly twofold by strong phosphorylation (Fig. 4, D and E; Table I). Login to comment 152 ABCC7 p.Lys464Ala Also like WT, the burst duration distributions of K464A mutant channels were well described by single exponential functions (Fig. 4, D-F). Login to comment 154 ABCC7 p.Asp572Asn ABCC7 p.Ser573Glu The mean closing rate from bursts was not substantially altered by these NBD1 mutations (compare Fig. 5 E and Table I): for D572N, rOC(5 mM MgATP ϩ PKA) ϭ 1.4 Ϯ 0.2 s-1 (n ϭ 9), and rOC(5 mM MgATP) ϭ 3.1 Ϯ 0.6 s-1 (n ϭ 3); for S573E, rOC(5 mM MgATP ϩ PKA) ϭ 2.2 Ϯ 0.3 s-1 (n ϭ 7). Login to comment 156 ABCC7 p.Asp572Asn Thus, for D572N CFTR, rCO(5 mM MgATP ϩ PKA) ϭ 0.34 Ϯ 0.1 s-1 (n ϭ 9), and rCO(5 mM MgATP) ϭ 0.35 Ϯ 0.1 s-1 (n ϭ 3), although these values ("total" estimates, see materials and methods) likely overestimate true opening rate, as the somewhat lower maximal Po (0.18 vs. 0.29 for WT) of this mutant precluded Figure 4. Login to comment 157 ABCC7 p.Lys464Ala Burst duration distributions are similar for WT (A-C) and K464A (D-F) channels under comparable conditions, as indicated (PKA present for left column only). Login to comment 160 ABCC7 p.Lys464Ala Only for K464A at ␮M MgATP (F) could the likelihood be significantly increased by including a second component, though with a shorter (but not longer; Ikuma and Welsh, 2000) mean: ␶1 ϭ 30 ms, a1 ϭ 0.17; ␶2 ϭ 263 ms, a2 ϭ 0.83; increase in log likelihood, ⌬LL ϭ 8.3; number of bursts fitted, M ϭ 263; giving (⌬LL - ln(2M) ϭ 2.0). Login to comment 161 ABCC7 p.Lys464Ala The small differences between means at mM and ␮M MgATP (B vs. C, E vs. F) may be only apparent, as the mean ␶b, estimated by multichannel kinetic fits, from these same stretches of record at ␮M MgATP is not significantly different from that during intervening stretches in 5 mM MgATP (for WT: ␶b␮M/␶b5mM ϭ 1.03 Ϯ 0.07, n ϭ 9; for K464A: ␶b␮M/␶b5mM ϭ 0.95 Ϯ 0.13, n ϭ 7). Login to comment 162 ABCC7 p.Lys464Ala ABCC7 p.Asp1370Asn (G and H) Representative traces showing gating of K464A and D1370N channels at 15 ␮M MgATP (after PKA removal). Login to comment 163 ABCC7 p.Lys464Ala Prolonged bursts of K464A channels (Ikuma and Welsh, 2000) are not evident. Login to comment 164 ABCC7 p.Asp1370Asn 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 166 ABCC7 p.Ser573Glu For S573E channels, rCO(5 mM MgATP ϩ PKA) ϭ 0.9 Ϯ 0.2 s-1 (n ϭ 7, total estimate) and 1 Ϯ 0.2 s-1 (n ϭ 4, best estimate). Login to comment 168 ABCC7 p.Asp1370Asn ABCC7 p.Asp1370Asn 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 169 ABCC7 p.Lys1250Ala The K1250A mutation more dramatically slowed channel closing from bursts, resulting in prolonged bursts lasting tens of seconds (Fig. 6 C; cf. Carson et al., 1995; Gunderson and Kopito, 1995; Ramjeesingh et al., 1999; Zeltwanger et al., 1999). Login to comment 170 ABCC7 p.Lys1250Ala Analysis of the macroscopic current relaxation upon nucleotide withdrawal in patches containing many K1250A channels indicates that their average burst duration was ‫08ف‬ s in the presence of PKA (see below, Fig. 10, E and G) but ‫04ف‬ s after PKA had been removed (Fig. 3 B), at least two orders of magnitude longer than bursts of WT channels under the same conditions (Fig. 3, A vs. B; Fig. 6, A vs. C). Login to comment 171 ABCC7 p.Lys1250Ala ABCC7 p.Lys1250Ala ABCC7 p.Asp1370Asn ABCC7 p.Asp1370Asn 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 ABCC7 p.Lys1250Ala ABCC7 p.Asp1370Asn ABCC7 p.Glu1371Ser 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 180 ABCC7 p.Lys1250Ala ABCC7 p.Glu1371Ser Note persistence of brief (intraburst) closures during K1250A and E1371S bursts, long after nucleotide withdrawal. Login to comment 183 ABCC7 p.Lys464Ala ABCC7 p.Asp572Asn ABCC7 p.Ser573Glu Patches contained one WT (A), K464A (B), or S573E (D) channel, or more than one D572N (C) channel. Login to comment 184 ABCC7 p.Lys464Ala ABCC7 p.Asp572Asn ABCC7 p.Ser573Glu (E) Summary of mean (ϮSEM) ␶b values at 5 mM MgATP and 300 nM PKA (n ϭ 30, 21, 9, and 7 for WT, K464A, D572N, and S573E, respectively). Login to comment 218 ABCC7 p.Lys464Ala ABCC7 p.Lys464Ala Closing from Locked-open Bursts Is Faster for K464A Mutants than for WT Channels Although the K464A mutation did not alter open burst duration of channels exposed to MgATP (Figs. 2 E, 4, and 5), regardless of phosphorylation status (Fig. 4; Table I), it did significantly reduce the duration of certain unusually prolonged bursts. Login to comment 222 ABCC7 p.Lys464Ala For K464A channels, on the other hand (Fig. 10 B), the slow component comprised a somewhat smaller fraction (as ϭ 0.63 Ϯ 0.04, n ϭ 16, Fig. 10 C) of the current Figure 8. Login to comment 227 ABCC7 p.Lys464Ala The smaller fractional amplitude of the slow component for K464A channels can be explained by this observed shortening of their locked-open bursts without the mutation markedly altering the frequency of entry into such bursts. Login to comment 232 ABCC7 p.Lys464Ala The analogous estimate for K464A channels gives an average of 1 locking in every ‫6ف‬ openings. Login to comment 234 ABCC7 p.Lys1250Ala ABCC7 p.Lys464Ala The K464A mutation also shortened (Fig. 10, E-G) the similarly prolonged bursts of NBD2 mutant K1250A channels exposed to MgATP alone (Fig. 6 C). Login to comment 235 ABCC7 p.Lys1250Ala The control record (Fig. 10 E) illustrates the slow decay of macroscopic current after washout of MgATP and PKA from a patch containing hundreds of K1250A CFTR Figure 9. Login to comment 242 ABCC7 p.Lys464Ala The K464A mutation speeds exit from locked open burst states. Login to comment 244 ABCC7 p.Lys464Ala (B) Current decay is much faster for the K464A mutant in the same conditions. Login to comment 245 ABCC7 p.Lys464Ala Blue fit lines in A and B show only the slow components of double exponential fits, with ␶s ϭ 67.8s, as ϭ 0.92 for WT, and ␶s ϭ 8.7s, as ϭ 0.79 for K464A. Login to comment 246 ABCC7 p.Lys464Ala (C and D) Summaries of fractional amplitude, as (C), and time constant, ␶s (D), of the slow component from 18 WT and 16 K464A experiments. Login to comment 247 ABCC7 p.Lys464Ala ABCC7 p.Lys464Ala In controls with no MgAMPPNP, closure after exposure to MgATP and PKA yielded ␶ ϭ 1.9 Ϯ 0.2 s (n ϭ 35) for WT and ␶ ϭ 1.0 Ϯ 0.1 s (n ϭ 34) for K464A, and both constructs sometimes showed a small amplitude slower component: for WT, ␶s ϭ 7.6 Ϯ 1.7 s, as ϭ 0.1 Ϯ 0.03 (in 13/35 patches); for K464A, ␶s ϭ 5.9 Ϯ 0.8 s, as ϭ 0.24 Ϯ 0.04 (20/24 patches). Login to comment 248 ABCC7 p.Lys1250Ala (E) Macroscopic K1250A currents, activated by 5 mM MgATP ϩ PKA, decay slowly on nucleotide withdrawal. Login to comment 249 ABCC7 p.Lys1250Ala ABCC7 p.Lys1250Ala ABCC7 p.Lys464Ala ABCC7 p.Lys464Ala (F) The additional K464A mutation accelerates channel closure from bursts: for the traces shown, ␶ ϭ 71.7s (K1250A) and ␶ ϭ 29.7s (K464A/K1250A). Login to comment 250 ABCC7 p.Lys1250Ala ABCC7 p.Lys1250Ala ABCC7 p.Lys464Ala (G) Mean time constants of all 9 K1250A and 9 K464A/K1250A relaxations, each well fit by a single exponential. Login to comment 253 ABCC7 p.Lys1250Ala ABCC7 p.Lys1250Ala ABCC7 p.Lys464Ala However, in double mutant K464A/K1250A CFTR channels (Fig. 10 F) the current relaxation time constant (␶ ϭ 36 Ϯ 4 s, n ϭ 9), and hence the mean open-burst dwell time, was less than half that of channels bearing the K1250A mutation alone (Fig. 10 G). Login to comment 254 ABCC7 p.Lys464Ala ABCC7 p.Lys464Ala Closing from Bursts During Activation by Poorly Hydrolyzable Analogs Alone Is Faster for K464A Mutants than for WT Channels Like WT CFTR (Fig. 7), mutant K464A channels could be opened by millimolar concentrations of the analogs MgAMPPNP or MgATP␥S alone (Fig. 11), with rates of opening to bursts of 1.5 Ϯ 0.2% (n ϭ 4) at 0.5 mM and 2.9 Ϯ 0.3% (n ϭ 4) at 5 mM MgAMPPNP, and 5.0 Ϯ 0.6% (n ϭ 8) at 2 mM MgATP␥S, of the maximal rate at saturating [MgATP], values not very different from those for WT channels under the same conditions. Login to comment 255 ABCC7 p.Lys464Ala ABCC7 p.Lys464Ala However, the 5-10-fold prolongation of WT bursts by these analogs (Fig. 7 A) was not evident in K464A channels (Fig. 11): for K464A channels opened by MgAMPPNP or MgATP␥S alone, the mean ␶b values were only 1.1 Ϯ 0.2 (n ϭ 8) or 2.2 Ϯ 0.5 (n ϭ 8) times larger, respectively, than at 10 ␮M MgATP. Login to comment 256 ABCC7 p.Lys464Ala Because this consequence of the K464A mutation is manifest during exposure to essentially nonhydrolyzable ATP analogs it cannot be ascribed to any failure of the mutant channel to hydrolyze nucleotide at the NBD1 catalytic site, but instead must be attributed to the alteration of NBD1 structure per se. D I S C U S S I O N We may draw several conclusions from these analyses of gating kinetics of WT and of NBD mutant CFTR channels, in the presence of MgATP and/or of poorly-hydrolyzable analogs: (a) nucleotide binds at both NBD1 and NBD2 catalytic sites before channel opening; (b) the slow opening transition, after nucleotide binding, is highly sensitive to the structures of the beta-␥ phosphate bridging group and of the ␥ phosphate; (c) no further nucleotide binding is required to terminate an open burst; (d) hydrolysis of the nucleotide at NBD2 precedes normal, rapid closing from bursts; (e) if that hydrolysis is prevented, the structure of the NBD1 catalytic site and of the nucleotide bound there can modulate rate of exit from the resulting prolonged (locked) open burst. Login to comment 266 ABCC7 p.Lys464Ala ABCC7 p.Asp1370Asn Our results show that mutations within the Walker motifs of either NBD1 (K464A) or NBD2 (D1370N, Figure 11. Login to comment 267 ABCC7 p.Lys464Ala Gating of prephosphorylated K464A channels by poorly hydrolyzable ATP analogs, as indicated. Login to comment 268 ABCC7 p.Lys464Ala Unlike WT (Fig. 7 A), K464A burst duration was not increased during exposure to MgAMPPNP (A and B, ␶b ϭ 270 Ϯ 50 ms, n ϭ 8), and was only slightly increased during exposure to ATP␥S (C, ␶b ϭ 655 Ϯ 170 ms, n ϭ 8), compared with bursts in MgATP (␶b ϭ 276 Ϯ 21 ms, n ϭ 16) in the same patches. Login to comment 269 ABCC7 p.Lys464Ala ABCC7 p.Lys464Ala Note that, due to the lower apparent affinity of K464A for MgATP (Fig. 2), the relative opening rate of mutant channels at 10 ␮M MgATP averaged only 2.3 Ϯ 0.8% (n ϭ 3) of that in saturating MgATP (compared with ‫%11ف‬ for WT), so the opening rate of K464A channels was similar in the presence of millimolar concentrations of the poorly hydrolyzable analogs or of 10 ␮M MgATP. Login to comment 270 ABCC7 p.Lys1250Ala ABCC7 p.Lys464Ala ABCC7 p.Asp1370Asn 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 275 ABCC7 p.Lys1250Ala ABCC7 p.Lys464Ala ABCC7 p.Lys464Ala ABCC7 p.Lys464Ala Accordingly, although no major difference in [␣32P]8-azidoATP photolabeling at 0ЊC was detected between WT and K464A/K1250A (Carson et al., 1995) or K464A CFTR (Vergani et al., 2002), the K464A mutation alone greatly reduced photolabeling of NBD1 by ␮M [␣32P]8-azidoATP at 37ЊC (Aleksandrov et al., 2002) and virtually abolished stable (i.e., surviving extensive post-incubation washing) photolabeling at 30ЊC (unpublished data). Login to comment 280 ABCC7 p.Lys1250Ala ABCC7 p.Lys1250Ala ABCC7 p.Lys464Ala ABCC7 p.Lys464Ala ABCC7 p.Asp1370Asn ABCC7 p.Asp1370Asn 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 ABCC7 p.Lys1250Ala ABCC7 p.Lys464Ala ABCC7 p.Asp1370Asn 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 ABCC7 p.Lys1250Ala ABCC7 p.Asp1370Asn 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 ABCC7 p.Lys1250Ala ABCC7 p.Asp1370Asn 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 297 ABCC7 p.Lys464Ala However, we cannot rule out that, at low [MgATP], mutant K464A CFTR channels might open to bursts with only NBD2 occupied by nucleotide. Login to comment 298 ABCC7 p.Lys464Ala ABCC7 p.Asp1370Asn 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 299 ABCC7 p.Lys464Ala ABCC7 p.Lys464Ala If confirmed, these results would be consistent with the right-shifted K464A [MgATP]-rCO curve reflecting principally a reduced nucleotide affinity at NBD1 (now lower than the affinity at NBD2) and a low, but nonzero, opening rate of K464A mutant CFTR channels with nucleotide bound only at the unmodified NBD2 site. Login to comment 300 ABCC7 p.Lys1250Ala ABCC7 p.Lys464Ala ABCC7 p.Asp1370Asn 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 305 ABCC7 p.Lys464Ala But, by directly comparing gating of the same channels, in the same patch, during exposure to MgAMPPNP, MgAMPPCP, or MgATP␥S, and to MgATP, we find that at concentrations of these analogs expected to be saturating (Figs. 7 and 8; see also Weinreich et al., 1999; Aleksandrov et al., 2001, 2002) the opening rates of WT (Figs. 7-9) and K464A (Fig. 11) channels are only ‫%5ف‬ of that reached at saturating [MgATP]. Login to comment 311 ABCC7 p.Lys464Ala Whereas the K464A mutation in NBD1 has been reported to reduce ‫02ف‬ fold the ATPase activity of purified CFTR (Ramjeesingh et al., 1999), we find that the same mutation diminishes maximal opening rate by only Ͻ50%, similar to effects of other NBD1 catalytic site mutations (Figs. 2 and 5). Login to comment 316 ABCC7 p.Asp1370Asn ABCC7 p.Asp1370Asn 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 318 ABCC7 p.Lys1250Ala ABCC7 p.Lys1250Ala ABCC7 p.Lys1250Ala On the other hand, ATPase measurements on purified CFTR have shown that the K1250A mutation abolished ATP hydrolysis (Ramjeesingh et al., 1999), whereas opening of K1250A channels was impaired, but not abolished, at normal [MgATP] (Carson et al., 1995; Gunderson and Kopito, 1995; Ramjeesingh et al., 1999; Powe et al., 2002); indeed, the greatly reduced apparent affinity for MgATP we observed (Fig. 3 C) implies that the maximal opening rate of K1250A may be several-fold greater than that measured at 1-2 mM MgATP. Login to comment 324 ABCC7 p.Lys1250Ala ABCC7 p.Lys464Ala ABCC7 p.Asp1370Asn 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 326 ABCC7 p.Lys1250Ala This [MgATP] dependence of burst duration was reported to be exaggerated in K1250A mutant channels, in which brief bursts were observed at 10 ␮M MgATP and only at higher concentrations did the characteristic (e.g., Fig. 6 C, above) prolonged bursts appear (Zeltwanger et al., 1999; Ikuma and Welsh, 2000; Powe et al., 2002). Login to comment 327 ABCC7 p.Lys1250Ala Though we occasionally observed brief bursts in K1250A channels at 10 ␮M MgATP (not illustrated), these were very rare, with a frequency of occurrence not demonstrably different from that in nominally MgATP-free bath solution (rCO10 ␮M/rCObath soln ϭ 0.72 Ϯ 0.12, n ϭ 6). Login to comment 328 ABCC7 p.Lys1250Ala Thus, brief bursts of K1250A channels might reflect infrequent nucleotide-independent events, unrelated to the physiological gating cycle of WT channels, an interpretation consistent with those brief bursts surviving mutation of the Walker A lysine in either, or both, NBDs (Zeltwanger et al., 1999; Ikuma and Welsh, 2000; Powe et al., 2002). Login to comment 329 ABCC7 p.Asp1370Asn ABCC7 p.Asp1370Asn 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 362 ABCC7 p.Lys464Ala However, the scheme as drawn suggests tight coupling between channel gating and ATP hydrolysis, which is inconsistent with the largely unaltered gating of the catalytically impaired K464A mutant (with ATPase Vmax apparently reduced ‫-02ف‬fold; Ramjeesingh et al., 1999). Login to comment 366 ABCC7 p.Lys464Ala ABCC7 p.Asp1370Asn 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 369 ABCC7 p.Lys1250Ala ABCC7 p.Lys464Ala But when hydrolysis (at NBD2) was prevented, by supplying nucleotide resistant to hydrolysis (Figs. 9, and 10, A-D; Fig. 7 vs. Fig. 11), by adding VO4 (Vergani et al., 2002), or by mutating the NBD2 Walker A lysine (K1250A; Fig. 10, E-G), the K464A mutation resulted in less prolonged bursts. Login to comment 370 ABCC7 p.Lys464Ala Very similar reduction of locked-open burst duration by the K464A mutation has been described recently in NIH3T3 and CHO cells (Powe et al., 2002). Login to comment 379 ABCC7 p.Asp1370Asn 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 381 ABCC7 p.Lys464Ala The influence of the K464A mutation seen in Fig. 2 D (also on Po) is then mimicked simply by an ‫-05ف‬fold acceleration of the MgATP dissociation rate from NBD1, together with the Ͻ2-fold observed reduction in maximal opening rate (Table IB). Login to comment 382 ABCC7 p.Asp1370Asn 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 639 ABCC7 p.Lys464Ala Effects on CFTR Cl- channel gating of Walker A lysine mutation K464A imply allosteric interaction between NBDs. Login to comment 646 ABCC7 p.Lys464Ala Effects on CFTR Cl-channel gating of Walker A lysine mutation K464A imply allosteric interaction between NBDs. Login to comment