ABCMdb

PMID: 8741733

Wilkinson DJ, Mansoura MK, Watson PY, Smit LS, Collins FS, Dawson DC
CFTR: the nucleotide binding folds regulate the accessibility and stability of the activated state.
J Gen Physiol. 1996 Jan;107(1):103-19., [PubMed]

Sentences

No. Mutations Sentence Comment

11 ABCC7 p.Asp1370Asn 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 ABCC7 p.Asp1370Asn ABCC7 p.Asp572Asn ABCC7 p.Lys464Gln 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 60 ABCC7 p.Lys1250Gln ABCC7 p.Lys464Gln Representative time courses are shown for wild type CFTR and two variants, K464Q and K1250Q. Login to comment 61 ABCC7 p.Lys1250Gln ABCC7 p.Lys464Gln Representative time courses are shown for wild type CFTR and two variants, K464Q and K1250Q. Login to comment 64 ABCC7 p.Lys1250Gln The transient increase in gel was most evident for wild-type CFTR and mutants such as K1250Q, which remained fully activated for several minutes after washout of IBMX. Login to comment 65 ABCC7 p.Lys1250Gln ABCC7 p.Lys464Gln In contrast, the decline in gel for mutants such as K464Q was rapid, showing only a slight increase after IBMX withdrawal. Login to comment 66 ABCC7 p.Lys464Gln In contrast, the decline in gel for mutants such as K464Q was rapid, showing only a slight increase after IBMX withdrawal. Login to comment 75 ABCC7 p.Lys464Gln In previous studies (Drumm et al., 1991; Snfit et al., 1993), we estimated the apparent KAfor dose-dependent activation from loga- WILKINSON ET AL. A B A 100 v 80 o 4O 2O - 0 A K464Q ~ - ~ 9 wild type I , , , ,i , ,, , i , ,, , i , , , ,l ,, , , i , , , , 0 10 20 30 40 50 60 minutes .37 O .14 I I 0 10 2o I I I I I I I I I i lO 20 minutes 60 120 FmURE 2. Login to comment 76 ABCC7 p.Lys1250Gln ABCC7 p.Lys464Gln ABCC7 p.Lys464Gln (A) Representative time courses for deactivation of wild type CFTR (0) and the analogous lysine to glutamine mutants in NBF1 (K464Q, A) and NBF2 (K1250Q, ~). Login to comment 77 ABCC7 p.Lys1250Gln ABCC7 p.Lys464Gln (A) Representative time courses for deactivation of wild type CFTR (0) and the analogous lysine to glutamine mutants in NBF1 (K464Q, A) and NBF2 (K1250Q, ~). Login to comment 79 ABCC7 p.Lys1250Gln ABCC7 p.Lys464Gln In the experiments shown, the values of gel(max) for wild-type CFTR, K464Q, and K1250Q were 27.9, 36.3, and 30.3 I,S, and the corresponding minimum membrane conductances alter deactivation of CFTR were 2.1, 0.7, and 1.0 ~S, respectively. Login to comment 80 ABCC7 p.Lys1250Gln ABCC7 p.Lys464Gln In the experiments shown, the values of gel(max) for wild-type CFTR, K464Q, and K1250Q were 27.9, 36.3, and 30.3 I,S, and the corresponding minimum membrane conductances alter deactivation of CFTR were 2.1, 0.7, and 1.0 ~S, respectively. Login to comment 82 ABCC7 p.Lys464Gln For wild-type (0), K464Q (A), and KI250Q (~), the values of *k,,a determined from linear regressions (lines) were 0.134, 0.166, and 0.019 rain -l, and the corresponding regression coefficients (r e) were 0.957, 0.999, and 0.998, respectively. Login to comment 83 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp ABCC7 p.Asp572Asn ABCC7 p.Lys464Gln rithmic dose-response plots, but for comparison with rates of activation we sought a more unbiased estimate of Ka that took into account three factors: (1) the activation produced by forskolin alone, (2) the block of CFTR by high concentrations of IBMX, and (3) the fact that for insensitive mutants such as G551D, D572N, and G1349D the dose-response showed no tendency toward saturation at the highest concentrations of IBMX. Login to comment 84 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp ABCC7 p.Asp572Asn rithmic dose-response plots, but for comparison with rates of activation we sought a more unbiased estimate of Ka that took into account three factors: (1) the activation produced by forskolin alone, (2) the block of CFTR by high concentrations of IBMX, and (3) the fact that for insensitive mutants such as G551D, D572N, and G1349D the dose-response showed no tendency toward saturation at the highest concentrations of IBMX. Login to comment 88 ABCC7 p.Lys1250Ala The Kj fbr IBMX block was estimated from the response of the hypersensitive mutant, K1250A. Login to comment 89 ABCC7 p.Lys1250Ala ABCC7 p.Lys1250Ala Because K1250A is maximally activated at an IBMX concentration of,'-,1 raM, the block by higher concentrations of IBMX is readily apparent. Login to comment 90 ABCC7 p.Lys1250Ala Because K1250A is maximally activated at an IBMX concentration of,'-,1 raM, the block by higher concentrations of IBMX is readily apparent. Login to comment 94 ABCC7 p.Lys1250Ala The value of N'!I/N'~I~ estimated for the mutant K1250A indicated that the maximum gcJ actually observed, g~'!i~'X,was nearly equal to the maximum possible activation, N'~l, presumably because this conductance is achieved at an IBMX concentration of 1 raM, where block is minimal. Login to comment 95 ABCC7 p.Lys1250Ala ABCC7 p.Lys1250Ala The calculated curves for activation and block of K1250A are shown in Fig. 3 A, where the values of gel are normalized to the theoretical maximum, N'!]. Login to comment 96 ABCC7 p.Lys1250Ala The calculated curves for activation and block of K1250A are shown in Fig. 3 A, where the values of gel are normalized to the theoretical maximum, N'!]. Login to comment 97 ABCC7 p.Lys1250Ala The magnitude of the transient increase in gel tor K1250A after removal of 5 mM IBMX in the rate experiments (cf. Fig. 5 B) was consistent with this interpretation. Login to comment 98 ABCC7 p.Lys1250Ala The magnitude of the transient increase in gel tor K1250A after removal of 5 mM IBMX in the rate experiments (cf. Fig. 5 B) was consistent with this interpretation. Login to comment 99 ABCC7 p.Lys464Gln For wild-wpe CFTR and the mutant K464Q, the observed values of ~'!i'• were ~60% and 27% of the theoretical maximum g'cl, respectively. Login to comment 100 ABCC7 p.Lys464Gln For wild-wpe CFTR and the mutant K464Q, the observed values of ~'!i'ߦ were ~60% and 27% of the theoretical maximum g'cl, respectively. Login to comment 125 ABCC7 p.Lys1250Ala The fit of Eq. 2 to the combined data for the hypersensitive mutant K1250A yielded a value of K1for the block by IBMX, as described in the text. Login to comment 126 ABCC7 p.Lys1250Ala (A) The plotted points (O) are means -+ SEM for 10 oocytes expressing the mutant K1250A. Login to comment 127 ABCC7 p.Lys1250Ala The fit of Eq. 2 to the combined data for the hypersensitive mutant K1250A yielded a value of K1for the block by IBMX, as described in the text. Login to comment 128 ABCC7 p.Lys1250Ala (A) The plotted points (O) are means -+ SEM for 10 oocytes expressing the mutant K1250A. Login to comment 129 ABCC7 p.Lys1250Ala ABCC7 p.Lys464Gln (B) IBMX dose-response relations for steady state activation of K1250A (~), wild-type CFTR (Q, n = 26), and K464Q (A, n = 5). Login to comment 131 ABCC7 p.Lys1250Ala ABCC7 p.Lys464Gln (B) IBMX dose-response relations for steady state activation of K1250A (~), wild-type CFTR (Q, n = 26), and K464Q (A, n = 5). Login to comment 133 ABCC7 p.Lys1250Ala ABCC7 p.Lys464Gln In Fig. 3 B, the activation components for wild-type CFTR and the mutants K1250A and K464Q were simulated by adding back the blocked component and plotting the adjusted data points along with the curves calculated using the estimated KAvalues. Login to comment 135 ABCC7 p.Lys1250Ala ABCC7 p.Lys464Gln In Fig. 3 B, the activation components for wild-type CFTR and the mutants K1250A and K464Q were simulated by adding back the blocked component and plotting the adjusted data points along with the curves calculated using the estimated KAvalues. Login to comment 150 ABCC7 p.Gly551Asp ABCC7 p.Gly551Ser ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Ser ABCC7 p.Gly551Ala ABCC7 p.Gly1349Ala The values listed in Table I show that NBF mutations generally reduced the value of (ko,, + ko~), in some cases by more TABLE I Summary ofActivation and DeactivationDatafor Wild-typeCFFR and Mutants of theInvariant Glycinein NBFI ((;,551)orNBF2 (G1349) CFTR Activation Deactivation klon KA (k,,n+ k,,n) (10 ~miu-1 k,,n kos latency *k,,t~ (raM) n (10-~min l) raM-l) (10-3min 1) (10 ~rain-j) n (min) (10-s min-I) wt 0.65 + 0.08 26 664 _+51 118 _+9 588 +-45 76 + 6 20 6.0 _+0.3 88 -+6 16 G551A 3.0 -+0.5*r 6 104 _+5"r 13 _+0.6*r 65 + 3*z 39 -+2* 5 7.7 +_0.5: 70 -+13: 4 G551S 4.7 +-0.5* 5 82 _+6*r 8 -+0.6*: 42 -+3*: 40 -+3*r 10 3.9 +_0.3*** 88 +-6: 6 G551D 9.3 -+0.01" 6 57 _+9*r 4 -+0.6*: 20 -+3*: 37 -+6"r 5 1.8 _+0.2"~ 84 -+10~ 6 G1349A 1.1 + 0.07*: 5 210 _+24"~ 35 -+4*: 172 -+20*: 38 +-4* 4 1.7 _+0.3"~ 184 + 20*: 5 G1349S 3.5 +-0.3* 4 199 _+46*: 23 -+5*: 117 -+27*r 82 -+19+ 6 2.3 _+0.5*+ 144 -+15": 6 G1349D 9.3 + 0.01" 8 114 _+16*++ 8 -+1": 40 +-6*r 74 -+11~ 5 0.6 -+0.1*++ 286 -+37*: 4 Valuesweredetermined as describedin Methods.The symbols(*) and (~) indicatesignificantdifferencesfrom wild-typeCFFRand the analogousmu- tant, respectively(P< 0.05). Login to comment 152 ABCC7 p.Gly551Asp ABCC7 p.Gly551Ser ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Ser ABCC7 p.Gly551Ala ABCC7 p.Gly1349Ala The values listed in Table I show that NBF mutations generally reduced the value of (ko,, + ko~), in some cases by more TABLE I Summary ofActivation and DeactivationDatafor Wild-typeCFFR and Mutants of theInvariant Glycinein NBFI ((;,551)orNBF2 (G1349) CFTR Activation Deactivation klon KA (k,,n+ k,,n) (10 ~miu-1 k,,n kos latency *k,,t~ (raM) n (10-~min l) raM-l) (10-3min 1) (10 ~rain-j) n (min) (10-s min-I) wt 0.65 + 0.08 26 664 _+51 118 _+9 588 +-45 76 + 6 20 6.0 _+0.3 88 -+6 16 G551A 3.0 -+0.5*r 6 104 _+5"r 13 _+0.6*r 65 + 3*z 39 -+2* 5 7.7 +_0.5: 70 -+13: 4 G551S 4.7 +-0.5* 5 82 _+6*r 8 -+0.6*: 42 -+3*: 40 -+3*r 10 3.9 +_0.3*** 88 +-6: 6 G551D 9.3 -+0.01" 6 57 _+9*r 4 -+0.6*: 20 -+3*: 37 -+6"r 5 1.8 _+0.2"~ 84 -+10~ 6 G1349A 1.1 + 0.07*: 5 210 _+24"~ 35 -+4*: 172 -+20*: 38 +-4* 4 1.7 _+0.3"~ 184 + 20*: 5 G1349S 3.5 +-0.3* 4 199 _+46*: 23 -+5*: 117 -+27*r 82 -+19+ 6 2.3 _+0.5*+ 144 -+15": 6 G1349D 9.3 + 0.01" 8 114 _+16* + + 8 -+1": 40 +-6*r 74 -+11~ 5 0.6 -+0.1* + + 286 -+37*: 4 Valuesweredetermined as describedin Methods.The symbols(*) and (~) indicatesignificantdifferencesfrom wild-typeCFFRand the analogousmu- tant, respectively(P< 0.05). Login to comment 170 ABCC7 p.Gly551Ala ABCC7 p.Gly1349Ala Substitutions for the invariant glycine in either NBF produced similar increases in KA, with the exception of the mutation that CFFR Activation: Roles of NBF1 and NBF2 was conservative with respect to polarity and size, alanine for glycine, which in NBF1 (G551A) produced a nearly fivefold increase in KA but in NBF2 (G1349A) produced less than a twofold increase. Login to comment 172 ABCC7 p.Gly551Ala ABCC7 p.Gly1349Ala Substitutions for the invariant glycine in either NBF produced similar increases in KA, with the exception of the mutation that CFFR Activation: Roles of NBF1 and NBF2 was conservative with respect to polarity and size, alanine for glycine, which in NBF1 (G551A) produced a nearly fivefold increase in KA but in NBF2 (G1349A) produced less than a twofold increase. Login to comment 176 ABCC7 p.Gly551Asp ABCC7 p.Gly551Ser ABCC7 p.Gly551Ala In NBF1, substitution to alanine (G551A), the most conservative change possible, reduced the relaxation rate by more than sixfold, and the less conservative substitutions to serine (G551S) and aspartic acid (G551D) progressively reduced the relaxation rate. Login to comment 177 ABCC7 p.Gly1349Ala At the analogous site in NBF2, the most conservative substitution (G1349A) also reduced the relaxation rate, but by only about threefold. Login to comment 178 ABCC7 p.Gly551Asp ABCC7 p.Gly551Ser ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Ser ABCC7 p.Gly551Ala Substitutions to serine (G1349S) and aspartic acid (G1349D) produced progressive reductions such that the relaxation rate for the least conservative mutation, G1349D, was about twice that for the comparable mutation in NBF1. Login to comment 179 ABCC7 p.Gly1349Ala At the analogous site in NBF2, the most conservative substitution (G1349A) also reduced the relaxation rate, but by only about threefold. Login to comment 180 ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Ser Substitutions to serine (G1349S) and aspartic acid (G1349D) produced progressive reductions such that the relaxation rate for the least conservative mutation, G1349D, was about twice that for the comparable mutation in NBF1. Login to comment 194 ABCC7 p.Gly551Ser ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Ser ""'"~"NBF1 NBF2 ,~j:~ ,pit'-" 9 G551S o G1349S 20 jl~ 9 G551 D o G1349D o i, ,,,i,0, ,i,,,,i,,, ,i,, ,,i , ~ ,, B o lO 20 30 40 50 60 ,~ 100 80 E 60 or 40 2o ~ 0 c I0o- 80 " 6o - 40 .z- 20 - o- ictOOO'~ .D-O*'Q / ;~ / Eof 9 . Login to comment 195 ABCC7 p.Lys1250Ala ABCC7 p.Asp1370Asn ABCC7 p.Asp572Asn ABCC7 p.Lys464Gln ABCC7 p.Lys1250Cys 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 197 ABCC7 p.Gly551Ser ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Ser ""'"~" NBF1 NBF2 ,~j:~ ,pit'-" 9 G551S o G1349S 20 jl~ 9 G551 D o G1349D o i, ,,,i,0, ,i,,,,i,,, ,i,, ,,i , ~ ,, B o lO 20 30 40 50 60 ,~ 100 80 E 60 o r 40 2o ~ 0 c I0o- 80 " 6o - 40 .z20 - o- ictOOO'~ .D-O*'Q / ;~ / Eof 9 . Login to comment 198 ABCC7 p.Lys1250Ala ABCC7 p.Asp1370Asn ABCC7 p.Asp572Asn ABCC7 p.Lys464Gln ABCC7 p.Lys1250Cys 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 201 ABCC7 p.Asp1370Asn ABCC7 p.Asp572Asn (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 204 ABCC7 p.Asp1370Asn ABCC7 p.Asp572Asn (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 218 ABCC7 p.Gly1349Ala Here, even the most conservative replacement (G1349A) dramatically shortened the latency and significantly increased the rate of exponential decline. Login to comment 219 ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Ser This destabilization of the activated state is clearly evident in the representative time courses for deactivation of the mutants G1349S and G1349D (Fig. 5 A). Login to comment 220 ABCC7 p.Gly1349Ala Here, even the most conservative replacement (G1349A) dramatically shortened the latency and significantly increased the rate of exponential decline. Login to comment 221 ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Ser ABCC7 p.Gly551Ala The most conservative substitution (G551A) did not appreciably alter the latency. Login to comment 222 ABCC7 p.Gly551Asp ABCC7 p.Gly551Ser ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Ser The less conservative substitutions (G551S, G551D) progressively decreased the latency, but the reductions were always less than those induced by the corresponding mutations (G1349S, G1349D) in NBF2. Login to comment 223 ABCC7 p.Gly551Asp ABCC7 p.Gly551Ser ABCC7 p.Gly551Ala The progressive destabilization of the active state suggested by the decreased latency seen with the G551S and G551D substitutions was not evident, however, in the subsequent phase of exponential decline. Login to comment 224 ABCC7 p.Gly551Asp ABCC7 p.Gly551Ser ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Ser The less conservative substitutions (G551S, G551D) progressively decreased the latency, but the reductions were always less than those induced by the corresponding mutations (G1349S, G1349D) in NBF2. Login to comment 225 ABCC7 p.Gly551Asp ABCC7 p.Gly551Ser The progressive destabilization of the active state suggested by the decreased latency seen with the G551S and G551D substitutions was not evident, however, in the subsequent phase of exponential decline. Login to comment 229 ABCC7 p.Lys464Ala ABCC7 p.Lys464Gln The calculated values of k,,~.for K464Q and K464A indicated that the substitutions to alanine or glutamine also increased the off rate under activating conditions, which contributed to the increase in Ka and compensated somewhat for the reduction in relaxation rate caused by the reduced on rate. Login to comment 231 ABCC7 p.Lys464Ala ABCC7 p.Lys464Gln The calculated values of k,,~.for K464Q and K464A indicated that the substitutions to alanine or glutamine also increased the off rate under activating conditions, which contributed to the increase in Ka and compensated somewhat for the reduction in relaxation rate caused by the reduced on rate. Login to comment 236 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp 2O 0 ao•-•lo o - .~ 8o- 0 40-- 2o- =o _: 0-- C lOO- 8o --: 6o --: 40 - 2o - o--: NBF1 NBF2 9 Qs ls o a s49s i ~'~'-,,"".~ 9 G551D = G1349D ~. Login to comment 237 ABCC7 p.Lys464Gln O *o ~ q. v~ ,,~,,s =~ ..... ===o~ _ -~-...-0 ------=._.a ~..~..-9:...e..o.~ * ............. "" "o~'"(~ -~ 9o O ~ - -oO- - - - ,u -- * ~- - - Z~, I ' ' ' ' [ .... ' I ' ' ' ' I ' ' ' ' I ' ' ' ' 0 10 20 30 40 50 \ "0,'~-.= " K464Q o K1250( " " ::'~:.-. Login to comment 238 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp 2O 0 aoߦ-ߦl o o - .~ 8o- 0 40-- 2o- =o _: 0-- C lOO- 8o --: 6o --: 40 - 2o - o--: NBF1 NBF2 9 Qs ls o a s49s i ~'~'-,,"".~ 9 G551D = G1349D ~. Login to comment 239 ABCC7 p.Lys464Gln O *o ~ q. v~ ,,~,,s =~ ..... ===o~ _ -~-... -0 ------=._.a ~..~..-9:...e..o.~ * ..... ........ "" "o~'"(~ -~ 9o O ~ - -oO- - - - ,u -- * ~- - - Z~, I ' ' ' ' [ .... ' I ' ' ' ' I ' ' ' ' I ' ' ' ' 0 10 20 30 40 50 \ "0,'~-.= " K464Q o K1250( " " ::'~:.-. Login to comment 247 ABCC7 p.Asp1370Asn 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 249 ABCC7 p.Asp1370Asn 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 254 ABCC7 p.Asp572Asn ABCC7 p.Gly551Ala sensus B aspartic acid in NBF1 (D572N) produced a profound reduction in the rate of approach to steady state activation (Fig. 4 C); the value of (kon + kof0 was comparable with that seen with the G551A mutation (cf. Tables I and II). Login to comment 256 ABCC7 p.Asp1370Asn ABCC7 p.Asp572Asn ABCC7 p.Gly551Ala 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 258 ABCC7 p.Asp1370Asn 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 260 ABCC7 p.Lys1250Arg In contrast, substitutions at the analogous sites in NBF2 (K1250 or D1370) actually increased the latency by two- to threefold, and, with the exception of the most conservative substitution, K1250R, the values of *koff were decreased compared with that of wild-type CFTR, indicating stabilization of the active state. These results suggest that the consensus A lysine and consensus B aspartic acid in the ATP binding pocket of NBF1 contribute to stabilization of the active state, whereas their analogues in NBF2 are involved in terminating the active state. Login to comment 261 ABCC7 p.Asp1370Asn 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 262 ABCC7 p.Lys1250Arg In contrast, substitutions at the analogous sites in NBF2 (K1250 or D1370) actually increased the latency by two- to threefold, and, with the exception of the most conservative substitution, K1250R, the values of *koff were decreased compared with that of wild-type CFTR, indicating stabilization of the active state. These results suggest that the consensus A lysine and consensus B aspartic acid in the ATP binding pocket of NBF1 contribute to stabilization of the active state, whereas their analogues in NBF2 are involved in terminating the active state. Login to comment 263 ABCC7 p.Asp1370Asn 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 270 ABCC7 p.Gly551Asp ABCC7 p.Gly551Ser Here, however, mutations in the binding pocket clearly hastened deactivation, evidenced by decreases in the latency and increases in *kom while the mutations of glycine 551 moderately decreased *kom The latency was also decreased progressively by the less conservative mutations, G551S and G551D. Login to comment 272 ABCC7 p.Gly551Asp ABCC7 p.Gly551Ser Here, however, mutations in the binding pocket clearly hastened deactivation, evidenced by decreases in the latency and increases in *kom while the mutations of glycine 551 moderately decreased *kom The latency was also decreased progressively by the less conservative mutations, G551S and G551D. Login to comment 275 ABCC7 p.Gly551Asp ABCC7 p.Gly551Ser For mutants of the NBF1 glycine (G551S and G551D), the values of *kon. Login to comment 277 ABCC7 p.Gly551Asp ABCC7 p.Gly551Ser ABCC7 p.Asp1370Asn ABCC7 p.Lys1250Arg 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 279 ABCC7 p.Asp1370Asn ABCC7 p.Lys1250Arg 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 ABCC7 p.Lys1250Ala ABCC7 p.Lys464Ala ABCC7 p.Asp1370Asn ABCC7 p.Asp572Asn ABCC7 p.Lys1250Arg ABCC7 p.Lys1250Gln ABCC7 p.Lys464Arg ABCC7 p.Lys464Gln + 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 283 ABCC7 p.Lys1250Ala ABCC7 p.Lys464Ala ABCC7 p.Asp1370Asn ABCC7 p.Asp572Asn ABCC7 p.Lys1250Arg ABCC7 p.Lys1250Gln ABCC7 p.Lys464Arg ABCC7 p.Lys464Gln + 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 321 ABCC7 p.Asp1370Asn Of particular interest in this regard was the effect of the NBF2 mutation, D1370N. Login to comment 323 ABCC7 p.Asp1370Asn Of particular interest in this regard was the effect of the NBF2 mutation, D1370N. Login to comment 339 ABCC7 p.Asp1370Asn 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 340 ABCC7 p.Gly551Asp ABCC7 p.Gly551Ser ABCC7 p.Gly1349Asp Role of the Invariant Glycine The functional importance of the invariant glycine in NBF1 (G551) or NBF2 (G1349) is evident from the existence of mutations (G551S, G551D, and G1349D) that are associated with cystic fibrosis in humans (Cutting et al., 1990; Kerem et al., 1990; Strong et al., 1991). Login to comment 341 ABCC7 p.Asp1370Asn 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 342 ABCC7 p.Gly551Asp ABCC7 p.Gly551Ser ABCC7 p.Gly1349Asp Role of the Invariant Glycine The functional importance of the invariant glycine in NBF1 (G551) or NBF2 (G1349) is evident from the existence of mutations (G551S, G551D, and G1349D) that are associated with cystic fibrosis in humans (Cutting et al., 1990; Kerem et al., 1990; Strong et al., 1991). Login to comment 345 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp Support for the latter view is provided by the observation that the mutations G551D and G1349D impair ATP binding in isolated fusion proteins of NBF1 and NBF2, respectively (Logan et al., 1994). Login to comment 346 ABCC7 p.Gly551Ser ABCC7 p.Gly1349Asp On the other hand, the results of Anderson and Welsh (1992) suggested that the mutations G551S and G1349D reduced the open probability of CFTR C1- channels without changing the K~/2 for the effect of ATP concentration on open probability. Login to comment 347 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp Support for the latter view is provided by the observation that the mutations G551D and G1349D impair ATP binding in isolated fusion proteins of NBF1 and NBF2, respectively (Logan et al., 1994). Login to comment 348 ABCC7 p.Gly551Ser ABCC7 p.Gly1349Asp On the other hand, the results of Anderson and Welsh (1992) suggested that the mutations G551S and G1349D reduced the open probability of CFTR C1-channels without changing the K~/2 for the effect of ATP concentration on open probability. Login to comment 355 ABCC7 p.Gly551Ala The most conservative substitution for the glycine (G551A) dramatically attenuated the on rate but tended to stabilize the active state. Login to comment 357 ABCC7 p.Gly551Ala The most conservative substitution for the glycine (G551A) dramatically attenuated the on rate but tended to stabilize the active state. Login to comment 362 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp ABCC7 p.Asp572Asn In addition, concentrations of IBMX above 1 mM are required to achieve significant activation of CFTR mutants such as G551D, D572N, and G1349D (cf. Smit et al., 1993). Login to comment 364 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp ABCC7 p.Asp572Asn In addition, concentrations of IBMX above 1 mM are required to achieve significant activation of CFTR mutants such as G551D, D572N, and G1349D (cf. Smit et al., 1993). Login to comment 371 ABCC7 p.Lys1250Ala After washout of 10 tzM forskolin, there was no transient increase in gc~, but the rate of decline was Nfourfold slower for the hypersensitive K1250A mutant, which is consistent with the results seen with 5 mM IBMX. Login to comment 373 ABCC7 p.Lys1250Ala After washout of 10 tzM forskolin, there was no transient increase in gc~, but the rate of decline was Nfourfold slower for the hypersensitive K1250A mutant, which is consistent with the results seen with 5 mM IBMX. Login to comment 382 ABCC7 p.Lys1250Ala For example, the mutation K1250A increased the burst duration of CFTR by four- to fivefold, and the analysis of CFTR deactivation in oocytes indicates that this substitution produced at least a threefold increase in the stability of the active state (Table II and Fig. 7). Login to comment 383 ABCC7 p.Lys464Ala Similarly, the mutation K464A increased the interburst interval by fivefold, whereas rate analysis revealed a nearly fourfold slowing of the activation rate (k'o,1). Login to comment 384 ABCC7 p.Lys1250Ala ABCC7 p.Lys1250Ala However, the open probability of K1250A was decreased by more than twofold, largely because of a 30-50-fold increase in the interburst interval, an effect that is not predicted by the present results. Login to comment 385 ABCC7 p.Lys464Ala Similarly, the mutation K464A increased the interburst interval by fivefold, whereas rate analysis revealed a nearly fourfold slowing of the activation rate (k'o,1). Login to comment 386 ABCC7 p.Lys1250Ala However, the open probability of K1250A was decreased by more than twofold, largely because of a 30-50-fold increase in the interburst interval, an effect that is not predicted by the present results. Login to comment 406 ABCC7 p.Gly551Asp ABCC7 p.Gly551Ser ABCC7 p.Gly1349Asp The conserved glycines in NBF1 (G551) and NBF2 (G1349) are both sites of mutations that cause either mild (G551S) or severe (G551D, G1349D) cystic fibrosis (Smitet al., 1993) but have not been associated with protein processing defects such as those that characterize the AF508 mutation. Login to comment 408 ABCC7 p.Gly551Asp ABCC7 p.Gly551Ser ABCC7 p.Gly1349Asp The conserved glycines in NBF1 (G551) and NBF2 (G1349) are both sites of mutations that cause either mild (G551S) or severe (G551D, G1349D) cystic fibrosis (Smitet al., 1993) but have not been associated with protein processing defects such as those that characterize the AF508 mutation. Login to comment