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
X
ABCC7 p.Asp1370Asn 8741733:11:49
status: NEW
view ABCC7 p.Asp1370Asn details
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
X
ABCC7 p.Asp1370Asn 8741733:19:242
status: NEW
view ABCC7 p.Asp1370Asn details
ABCC7 p.Asp572Asn
X
ABCC7 p.Asp572Asn 8741733:19:159
status: NEW
view ABCC7 p.Asp572Asn details
ABCC7 p.Lys464Gln
X
ABCC7 p.Lys464Gln 8741733:19:152
status: NEW
view ABCC7 p.Lys464Gln details
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
X
ABCC7 p.Lys1250Gln 8741733:60:85
status: NEW
view ABCC7 p.Lys1250Gln details
ABCC7 p.Lys464Gln
X
ABCC7 p.Lys464Gln 8741733:60:75
status: NEW
view ABCC7 p.Lys464Gln details
Representative time courses are shown for wild type CFTR and two variants, K464Q and K1250Q. Login to comment
61 ABCC7 p.Lys1250Gln
X
ABCC7 p.Lys1250Gln 8741733:61:85
status: NEW
view ABCC7 p.Lys1250Gln details
ABCC7 p.Lys464Gln
X
ABCC7 p.Lys464Gln 8741733:61:75
status: NEW
view ABCC7 p.Lys464Gln details
Representative time courses are shown for wild type CFTR and two variants, K464Q and K1250Q. Login to comment
64 ABCC7 p.Lys1250Gln
X
ABCC7 p.Lys1250Gln 8741733:64:86
status: NEW
view ABCC7 p.Lys1250Gln details
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
X
ABCC7 p.Lys1250Gln 8741733:65:86
status: NEW
view ABCC7 p.Lys1250Gln details
ABCC7 p.Lys464Gln
X
ABCC7 p.Lys464Gln 8741733:65:52
status: NEW
view ABCC7 p.Lys464Gln details
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
X
ABCC7 p.Lys464Gln 8741733:66:52
status: NEW
view ABCC7 p.Lys464Gln details
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
X
ABCC7 p.Lys464Gln 8741733:75:179
status: NEW
view ABCC7 p.Lys464Gln details
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
X
ABCC7 p.Lys1250Gln 8741733:76:146
status: NEW
view ABCC7 p.Lys1250Gln details
ABCC7 p.Lys464Gln
X
ABCC7 p.Lys464Gln 8741733:76:126
status: NEW
view ABCC7 p.Lys464Gln details
ABCC7 p.Lys464Gln
X
ABCC7 p.Lys464Gln 8741733:76:179
status: NEW
view ABCC7 p.Lys464Gln details
(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
X
ABCC7 p.Lys1250Gln 8741733:77:146
status: NEW
view ABCC7 p.Lys1250Gln details
ABCC7 p.Lys464Gln
X
ABCC7 p.Lys464Gln 8741733:77:126
status: NEW
view ABCC7 p.Lys464Gln details
(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
X
ABCC7 p.Lys1250Gln 8741733:79:80
status: NEW
view ABCC7 p.Lys1250Gln details
ABCC7 p.Lys464Gln
X
ABCC7 p.Lys464Gln 8741733:79:69
status: NEW
view ABCC7 p.Lys464Gln details
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
X
ABCC7 p.Lys1250Gln 8741733:80:80
status: NEW
view ABCC7 p.Lys1250Gln details
ABCC7 p.Lys464Gln
X
ABCC7 p.Lys464Gln 8741733:80:69
status: NEW
view ABCC7 p.Lys464Gln details
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
X
ABCC7 p.Lys464Gln 8741733:82:19
status: NEW
view ABCC7 p.Lys464Gln details
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
X
ABCC7 p.Gly551Asp 8741733:83:308
status: NEW
view ABCC7 p.Gly551Asp details
ABCC7 p.Gly1349Asp
X
ABCC7 p.Gly1349Asp 8741733:83:326
status: NEW
view ABCC7 p.Gly1349Asp details
ABCC7 p.Asp572Asn
X
ABCC7 p.Asp572Asn 8741733:83:315
status: NEW
view ABCC7 p.Asp572Asn details
ABCC7 p.Lys464Gln
X
ABCC7 p.Lys464Gln 8741733:83:19
status: NEW
view ABCC7 p.Lys464Gln details
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
X
ABCC7 p.Gly551Asp 8741733:84:308
status: NEW
view ABCC7 p.Gly551Asp details
ABCC7 p.Gly1349Asp
X
ABCC7 p.Gly1349Asp 8741733:84:326
status: NEW
view ABCC7 p.Gly1349Asp details
ABCC7 p.Asp572Asn
X
ABCC7 p.Asp572Asn 8741733:84:315
status: NEW
view ABCC7 p.Asp572Asn details
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
X
ABCC7 p.Lys1250Ala 8741733:88:84
status: NEW
view ABCC7 p.Lys1250Ala details
The Kj fbr IBMX block was estimated from the response of the hypersensitive mutant, K1250A. Login to comment
89 ABCC7 p.Lys1250Ala
X
ABCC7 p.Lys1250Ala 8741733:89:8
status: NEW
view ABCC7 p.Lys1250Ala details
ABCC7 p.Lys1250Ala
X
ABCC7 p.Lys1250Ala 8741733:89:84
status: NEW
view ABCC7 p.Lys1250Ala details
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
X
ABCC7 p.Lys1250Ala 8741733:90:8
status: NEW
view ABCC7 p.Lys1250Ala details
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
X
ABCC7 p.Lys1250Ala 8741733:94:49
status: NEW
view ABCC7 p.Lys1250Ala details
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
X
ABCC7 p.Lys1250Ala 8741733:95:49
status: NEW
view ABCC7 p.Lys1250Ala details
ABCC7 p.Lys1250Ala
X
ABCC7 p.Lys1250Ala 8741733:95:50
status: NEW
view ABCC7 p.Lys1250Ala details
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
X
ABCC7 p.Lys1250Ala 8741733:96:50
status: NEW
view ABCC7 p.Lys1250Ala details
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
X
ABCC7 p.Lys1250Ala 8741733:97:51
status: NEW
view ABCC7 p.Lys1250Ala details
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
X
ABCC7 p.Lys1250Ala 8741733:98:51
status: NEW
view ABCC7 p.Lys1250Ala details
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
X
ABCC7 p.Lys464Gln 8741733:99:33
status: NEW
view ABCC7 p.Lys464Gln details
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
X
ABCC7 p.Lys464Gln 8741733:100:33
status: NEW
view ABCC7 p.Lys464Gln details
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
X
ABCC7 p.Lys1250Ala 8741733:125:68
status: NEW
view ABCC7 p.Lys1250Ala details
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
X
ABCC7 p.Lys1250Ala 8741733:126:81
status: NEW
view ABCC7 p.Lys1250Ala details
(A) The plotted points (O) are means -+ SEM for 10 oocytes expressing the mutant K1250A. Login to comment
127 ABCC7 p.Lys1250Ala
X
ABCC7 p.Lys1250Ala 8741733:127:68
status: NEW
view ABCC7 p.Lys1250Ala details
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
X
ABCC7 p.Lys1250Ala 8741733:128:81
status: NEW
view ABCC7 p.Lys1250Ala details
(A) The plotted points (O) are means -+ SEM for 10 oocytes expressing the mutant K1250A. Login to comment
129 ABCC7 p.Lys1250Ala
X
ABCC7 p.Lys1250Ala 8741733:129:64
status: NEW
view ABCC7 p.Lys1250Ala details
ABCC7 p.Lys464Gln
X
ABCC7 p.Lys464Gln 8741733:129:108
status: NEW
view ABCC7 p.Lys464Gln details
(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
X
ABCC7 p.Lys1250Ala 8741733:131:64
status: NEW
view ABCC7 p.Lys1250Ala details
ABCC7 p.Lys464Gln
X
ABCC7 p.Lys464Gln 8741733:131:108
status: NEW
view ABCC7 p.Lys464Gln details
(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
X
ABCC7 p.Lys1250Ala 8741733:133:74
status: NEW
view ABCC7 p.Lys1250Ala details
ABCC7 p.Lys464Gln
X
ABCC7 p.Lys464Gln 8741733:133:85
status: NEW
view ABCC7 p.Lys464Gln details
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
X
ABCC7 p.Lys1250Ala 8741733:135:74
status: NEW
view ABCC7 p.Lys1250Ala details
ABCC7 p.Lys464Gln
X
ABCC7 p.Lys464Gln 8741733:135:85
status: NEW
view ABCC7 p.Lys464Gln details
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
X
ABCC7 p.Gly551Asp 8741733:150:650
status: NEW
view ABCC7 p.Gly551Asp details
ABCC7 p.Gly551Ser
X
ABCC7 p.Gly551Ser 8741733:150:568
status: NEW
view ABCC7 p.Gly551Ser details
ABCC7 p.Gly1349Asp
X
ABCC7 p.Gly1349Asp 8741733:150:907
status: NEW
view ABCC7 p.Gly1349Asp details
ABCC7 p.Gly1349Ser
X
ABCC7 p.Gly1349Ser 8741733:150:820
status: NEW
view ABCC7 p.Gly1349Ser details
ABCC7 p.Gly551Ala
X
ABCC7 p.Gly551Ala 8741733:150:486
status: NEW
view ABCC7 p.Gly551Ala details
ABCC7 p.Gly1349Ala
X
ABCC7 p.Gly1349Ala 8741733:150:732
status: NEW
view ABCC7 p.Gly1349Ala details
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
X
ABCC7 p.Gly551Asp 8741733:152:650
status: NEW
view ABCC7 p.Gly551Asp details
ABCC7 p.Gly551Ser
X
ABCC7 p.Gly551Ser 8741733:152:568
status: NEW
view ABCC7 p.Gly551Ser details
ABCC7 p.Gly1349Asp
X
ABCC7 p.Gly1349Asp 8741733:152:907
status: NEW
view ABCC7 p.Gly1349Asp details
ABCC7 p.Gly1349Ser
X
ABCC7 p.Gly1349Ser 8741733:152:820
status: NEW
view ABCC7 p.Gly1349Ser details
ABCC7 p.Gly551Ala
X
ABCC7 p.Gly551Ala 8741733:152:486
status: NEW
view ABCC7 p.Gly551Ala details
ABCC7 p.Gly1349Ala
X
ABCC7 p.Gly1349Ala 8741733:152:732
status: NEW
view ABCC7 p.Gly1349Ala details
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
X
ABCC7 p.Gly551Ala 8741733:170:258
status: NEW
view ABCC7 p.Gly551Ala details
ABCC7 p.Gly1349Ala
X
ABCC7 p.Gly1349Ala 8741733:170:320
status: NEW
view ABCC7 p.Gly1349Ala details
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
X
ABCC7 p.Gly551Ala 8741733:172:258
status: NEW
view ABCC7 p.Gly551Ala details
ABCC7 p.Gly1349Ala
X
ABCC7 p.Gly1349Ala 8741733:172:320
status: NEW
view ABCC7 p.Gly1349Ala details
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
X
ABCC7 p.Gly551Asp 8741733:176:208
status: NEW
view ABCC7 p.Gly551Asp details
ABCC7 p.Gly551Ser
X
ABCC7 p.Gly551Ser 8741733:176:182
status: NEW
view ABCC7 p.Gly551Ser details
ABCC7 p.Gly551Ala
X
ABCC7 p.Gly551Ala 8741733:176:34
status: NEW
view ABCC7 p.Gly551Ala details
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
X
ABCC7 p.Gly1349Ala 8741733:177:67
status: NEW
view ABCC7 p.Gly1349Ala details
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
X
ABCC7 p.Gly551Asp 8741733:178:208
status: NEW
view ABCC7 p.Gly551Asp details
ABCC7 p.Gly551Ser
X
ABCC7 p.Gly551Ser 8741733:178:182
status: NEW
view ABCC7 p.Gly551Ser details
ABCC7 p.Gly1349Asp
X
ABCC7 p.Gly1349Asp 8741733:178:52
status: NEW
view ABCC7 p.Gly1349Asp details
ABCC7 p.Gly1349Asp
X
ABCC7 p.Gly1349Asp 8741733:178:159
status: NEW
view ABCC7 p.Gly1349Asp details
ABCC7 p.Gly1349Ser
X
ABCC7 p.Gly1349Ser 8741733:178:25
status: NEW
view ABCC7 p.Gly1349Ser details
ABCC7 p.Gly551Ala
X
ABCC7 p.Gly551Ala 8741733:178:34
status: NEW
view ABCC7 p.Gly551Ala details
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
X
ABCC7 p.Gly1349Ala 8741733:179:67
status: NEW
view ABCC7 p.Gly1349Ala details
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
X
ABCC7 p.Gly1349Asp 8741733:180:52
status: NEW
view ABCC7 p.Gly1349Asp details
ABCC7 p.Gly1349Asp
X
ABCC7 p.Gly1349Asp 8741733:180:159
status: NEW
view ABCC7 p.Gly1349Asp details
ABCC7 p.Gly1349Ser
X
ABCC7 p.Gly1349Ser 8741733:180:25
status: NEW
view ABCC7 p.Gly1349Ser details
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
X
ABCC7 p.Gly551Ser 8741733:194:32
status: NEW
view ABCC7 p.Gly551Ser details
ABCC7 p.Gly1349Asp
X
ABCC7 p.Gly1349Asp 8741733:194:65
status: NEW
view ABCC7 p.Gly1349Asp details
ABCC7 p.Gly1349Ser
X
ABCC7 p.Gly1349Ser 8741733:194:40
status: NEW
view ABCC7 p.Gly1349Ser details
""'"~"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
X
ABCC7 p.Lys1250Ala 8741733:195:219
status: NEW
view ABCC7 p.Lys1250Ala details
ABCC7 p.Asp1370Asn
X
ABCC7 p.Asp1370Asn 8741733:195:240
status: NEW
view ABCC7 p.Asp1370Asn details
ABCC7 p.Asp572Asn
X
ABCC7 p.Asp572Asn 8741733:195:126
status: NEW
view ABCC7 p.Asp572Asn details
ABCC7 p.Lys464Gln
X
ABCC7 p.Lys464Gln 8741733:195:11
status: NEW
view ABCC7 p.Lys464Gln details
ABCC7 p.Lys1250Cys
X
ABCC7 p.Lys1250Cys 8741733:195:226
status: NEW
view ABCC7 p.Lys1250Cys details
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
X
ABCC7 p.Gly551Ser 8741733:197:33
status: NEW
view ABCC7 p.Gly551Ser details
ABCC7 p.Gly1349Asp
X
ABCC7 p.Gly1349Asp 8741733:197:66
status: NEW
view ABCC7 p.Gly1349Asp details
ABCC7 p.Gly1349Ser
X
ABCC7 p.Gly1349Ser 8741733:197:41
status: NEW
view ABCC7 p.Gly1349Ser details
""'"~" 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
X
ABCC7 p.Lys1250Ala 8741733:198:221
status: NEW
view ABCC7 p.Lys1250Ala details
ABCC7 p.Asp1370Asn
X
ABCC7 p.Asp1370Asn 8741733:198:242
status: NEW
view ABCC7 p.Asp1370Asn details
ABCC7 p.Asp572Asn
X
ABCC7 p.Asp572Asn 8741733:198:128
status: NEW
view ABCC7 p.Asp572Asn details
ABCC7 p.Lys464Gln
X
ABCC7 p.Lys464Gln 8741733:198:11
status: NEW
view ABCC7 p.Lys464Gln details
ABCC7 p.Lys1250Cys
X
ABCC7 p.Lys1250Cys 8741733:198:228
status: NEW
view ABCC7 p.Lys1250Cys details
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
X
ABCC7 p.Asp1370Asn 8741733:201:127
status: NEW
view ABCC7 p.Asp1370Asn details
ABCC7 p.Asp572Asn
X
ABCC7 p.Asp572Asn 8741733:201:108
status: NEW
view ABCC7 p.Asp572Asn details
(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
X
ABCC7 p.Asp1370Asn 8741733:204:127
status: NEW
view ABCC7 p.Asp1370Asn details
ABCC7 p.Asp572Asn
X
ABCC7 p.Asp572Asn 8741733:204:108
status: NEW
view ABCC7 p.Asp572Asn details
(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
X
ABCC7 p.Gly1349Ala 8741733:218:46
status: NEW
view ABCC7 p.Gly1349Ala details
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
X
ABCC7 p.Gly1349Asp 8741733:219:141
status: NEW
view ABCC7 p.Gly1349Asp details
ABCC7 p.Gly1349Ser
X
ABCC7 p.Gly1349Ser 8741733:219:130
status: NEW
view ABCC7 p.Gly1349Ser details
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
X
ABCC7 p.Gly1349Ala 8741733:220:46
status: NEW
view ABCC7 p.Gly1349Ala details
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
X
ABCC7 p.Gly1349Asp 8741733:221:141
status: NEW
view ABCC7 p.Gly1349Asp details
ABCC7 p.Gly1349Ser
X
ABCC7 p.Gly1349Ser 8741733:221:130
status: NEW
view ABCC7 p.Gly1349Ser details
ABCC7 p.Gly551Ala
X
ABCC7 p.Gly551Ala 8741733:221:36
status: NEW
view ABCC7 p.Gly551Ala details
The most conservative substitution (G551A) did not appreciably alter the latency. Login to comment
222 ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 8741733:222:44
status: NEW
view ABCC7 p.Gly551Asp details
ABCC7 p.Gly551Ser
X
ABCC7 p.Gly551Ser 8741733:222:37
status: NEW
view ABCC7 p.Gly551Ser details
ABCC7 p.Gly1349Asp
X
ABCC7 p.Gly1349Asp 8741733:222:183
status: NEW
view ABCC7 p.Gly1349Asp details
ABCC7 p.Gly1349Ser
X
ABCC7 p.Gly1349Ser 8741733:222:175
status: NEW
view ABCC7 p.Gly1349Ser details
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
X
ABCC7 p.Gly551Asp 8741733:223:111
status: NEW
view ABCC7 p.Gly551Asp details
ABCC7 p.Gly551Ser
X
ABCC7 p.Gly551Ser 8741733:223:101
status: NEW
view ABCC7 p.Gly551Ser details
ABCC7 p.Gly551Ala
X
ABCC7 p.Gly551Ala 8741733:223:36
status: NEW
view ABCC7 p.Gly551Ala details
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
X
ABCC7 p.Gly551Asp 8741733:224:44
status: NEW
view ABCC7 p.Gly551Asp details
ABCC7 p.Gly551Ser
X
ABCC7 p.Gly551Ser 8741733:224:37
status: NEW
view ABCC7 p.Gly551Ser details
ABCC7 p.Gly1349Asp
X
ABCC7 p.Gly1349Asp 8741733:224:183
status: NEW
view ABCC7 p.Gly1349Asp details
ABCC7 p.Gly1349Ser
X
ABCC7 p.Gly1349Ser 8741733:224:175
status: NEW
view ABCC7 p.Gly1349Ser details
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
X
ABCC7 p.Gly551Asp 8741733:225:111
status: NEW
view ABCC7 p.Gly551Asp details
ABCC7 p.Gly551Ser
X
ABCC7 p.Gly551Ser 8741733:225:101
status: NEW
view ABCC7 p.Gly551Ser details
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
X
ABCC7 p.Lys464Ala 8741733:229:44
status: NEW
view ABCC7 p.Lys464Ala details
ABCC7 p.Lys464Gln
X
ABCC7 p.Lys464Gln 8741733:229:34
status: NEW
view ABCC7 p.Lys464Gln details
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
X
ABCC7 p.Lys464Ala 8741733:231:44
status: NEW
view ABCC7 p.Lys464Ala details
ABCC7 p.Lys464Gln
X
ABCC7 p.Lys464Gln 8741733:231:34
status: NEW
view ABCC7 p.Lys464Gln details
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
X
ABCC7 p.Gly551Asp 8741733:236:138
status: NEW
view ABCC7 p.Gly551Asp details
ABCC7 p.Gly1349Asp
X
ABCC7 p.Gly1349Asp 8741733:236:146
status: NEW
view ABCC7 p.Gly1349Asp details
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
X
ABCC7 p.Lys464Gln 8741733:237:225
status: NEW
view ABCC7 p.Lys464Gln details
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
X
ABCC7 p.Gly551Asp 8741733:238:137
status: NEW
view ABCC7 p.Gly551Asp details
ABCC7 p.Gly1349Asp
X
ABCC7 p.Gly1349Asp 8741733:238:145
status: NEW
view ABCC7 p.Gly1349Asp details
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
X
ABCC7 p.Lys464Gln 8741733:239:227
status: NEW
view ABCC7 p.Lys464Gln details
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
X
ABCC7 p.Asp1370Asn 8741733:247:32
status: NEW
view ABCC7 p.Asp1370Asn details
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
X
ABCC7 p.Asp1370Asn 8741733:249:32
status: NEW
view ABCC7 p.Asp1370Asn details
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
X
ABCC7 p.Asp572Asn 8741733:254:32
status: NEW
view ABCC7 p.Asp572Asn details
ABCC7 p.Gly551Ala
X
ABCC7 p.Gly551Ala 8741733:254:196
status: NEW
view ABCC7 p.Gly551Ala details
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
X
ABCC7 p.Asp1370Asn 8741733:256:49
status: NEW
view ABCC7 p.Asp1370Asn details
ABCC7 p.Asp572Asn
X
ABCC7 p.Asp572Asn 8741733:256:32
status: NEW
view ABCC7 p.Asp572Asn details
ABCC7 p.Gly551Ala
X
ABCC7 p.Gly551Ala 8741733:256:196
status: NEW
view ABCC7 p.Gly551Ala details
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
X
ABCC7 p.Asp1370Asn 8741733:258:49
status: NEW
view ABCC7 p.Asp1370Asn details
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
X
ABCC7 p.Lys1250Arg 8741733:260:191
status: NEW
view ABCC7 p.Lys1250Arg details
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
X
ABCC7 p.Asp1370Asn 8741733:261:46
status: NEW
view ABCC7 p.Asp1370Asn details
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
X
ABCC7 p.Lys1250Arg 8741733:262:191
status: NEW
view ABCC7 p.Lys1250Arg details
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
X
ABCC7 p.Asp1370Asn 8741733:263:46
status: NEW
view ABCC7 p.Asp1370Asn details
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
X
ABCC7 p.Gly551Asp 8741733:270:290
status: NEW
view ABCC7 p.Gly551Asp details
ABCC7 p.Gly551Ser
X
ABCC7 p.Gly551Ser 8741733:270:280
status: NEW
view ABCC7 p.Gly551Ser details
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
X
ABCC7 p.Gly551Asp 8741733:272:290
status: NEW
view ABCC7 p.Gly551Asp details
ABCC7 p.Gly551Ser
X
ABCC7 p.Gly551Ser 8741733:272:280
status: NEW
view ABCC7 p.Gly551Ser details
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
X
ABCC7 p.Gly551Asp 8741733:275:43
status: NEW
view ABCC7 p.Gly551Asp details
ABCC7 p.Gly551Ser
X
ABCC7 p.Gly551Ser 8741733:275:33
status: NEW
view ABCC7 p.Gly551Ser details
For mutants of the NBF1 glycine (G551S and G551D), the values of *kon. Login to comment
277 ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 8741733:277:43
status: NEW
view ABCC7 p.Gly551Asp details
ABCC7 p.Gly551Ser
X
ABCC7 p.Gly551Ser 8741733:277:33
status: NEW
view ABCC7 p.Gly551Ser details
ABCC7 p.Asp1370Asn
X
ABCC7 p.Asp1370Asn 8741733:277:35
status: NEW
view ABCC7 p.Asp1370Asn details
ABCC7 p.Lys1250Arg
X
ABCC7 p.Lys1250Arg 8741733:277:24
status: NEW
view ABCC7 p.Lys1250Arg details
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
X
ABCC7 p.Asp1370Asn 8741733:279:35
status: NEW
view ABCC7 p.Asp1370Asn details
ABCC7 p.Lys1250Arg
X
ABCC7 p.Lys1250Arg 8741733:279:24
status: NEW
view ABCC7 p.Lys1250Arg details
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
X
ABCC7 p.Lys1250Ala 8741733:281:1008
status: NEW
view ABCC7 p.Lys1250Ala details
ABCC7 p.Lys464Ala
X
ABCC7 p.Lys464Ala 8741733:281:495
status: NEW
view ABCC7 p.Lys464Ala details
ABCC7 p.Asp1370Asn
X
ABCC7 p.Asp1370Asn 8741733:281:1122
status: NEW
view ABCC7 p.Asp1370Asn details
ABCC7 p.Asp572Asn
X
ABCC7 p.Asp572Asn 8741733:281:635
status: NEW
view ABCC7 p.Asp572Asn details
ABCC7 p.Lys1250Arg
X
ABCC7 p.Lys1250Arg 8741733:281:749
status: NEW
view ABCC7 p.Lys1250Arg details
ABCC7 p.Lys1250Gln
X
ABCC7 p.Lys1250Gln 8741733:281:883
status: NEW
view ABCC7 p.Lys1250Gln details
ABCC7 p.Lys464Arg
X
ABCC7 p.Lys464Arg 8741733:281:234
status: NEW
view ABCC7 p.Lys464Arg details
ABCC7 p.Lys464Gln
X
ABCC7 p.Lys464Gln 8741733:281:368
status: NEW
view ABCC7 p.Lys464Gln details
+ 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
X
ABCC7 p.Lys1250Ala 8741733:283:976
status: NEW
view ABCC7 p.Lys1250Ala details
ABCC7 p.Lys464Ala
X
ABCC7 p.Lys464Ala 8741733:283:481
status: NEW
view ABCC7 p.Lys464Ala details
ABCC7 p.Asp1370Asn
X
ABCC7 p.Asp1370Asn 8741733:283:1088
status: NEW
view ABCC7 p.Asp1370Asn details
ABCC7 p.Asp572Asn
X
ABCC7 p.Asp572Asn 8741733:283:614
status: NEW
view ABCC7 p.Asp572Asn details
ABCC7 p.Lys1250Arg
X
ABCC7 p.Lys1250Arg 8741733:283:726
status: NEW
view ABCC7 p.Lys1250Arg details
ABCC7 p.Lys1250Gln
X
ABCC7 p.Lys1250Gln 8741733:283:855
status: NEW
view ABCC7 p.Lys1250Gln details
ABCC7 p.Lys464Arg
X
ABCC7 p.Lys464Arg 8741733:283:231
status: NEW
view ABCC7 p.Lys464Arg details
ABCC7 p.Lys464Gln
X
ABCC7 p.Lys464Gln 8741733:283:359
status: NEW
view ABCC7 p.Lys464Gln details
+ 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
X
ABCC7 p.Asp1370Asn 8741733:321:75
status: NEW
view ABCC7 p.Asp1370Asn details
Of particular interest in this regard was the effect of the NBF2 mutation, D1370N. Login to comment
323 ABCC7 p.Asp1370Asn
X
ABCC7 p.Asp1370Asn 8741733:323:75
status: NEW
view ABCC7 p.Asp1370Asn details
Of particular interest in this regard was the effect of the NBF2 mutation, D1370N. Login to comment
339 ABCC7 p.Asp1370Asn
X
ABCC7 p.Asp1370Asn 8741733:339:210
status: NEW
view ABCC7 p.Asp1370Asn details
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
X
ABCC7 p.Gly551Asp 8741733:340:163
status: NEW
view ABCC7 p.Gly551Asp details
ABCC7 p.Gly551Ser
X
ABCC7 p.Gly551Ser 8741733:340:156
status: NEW
view ABCC7 p.Gly551Ser details
ABCC7 p.Gly1349Asp
X
ABCC7 p.Gly1349Asp 8741733:340:174
status: NEW
view ABCC7 p.Gly1349Asp details
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
X
ABCC7 p.Asp1370Asn 8741733:341:210
status: NEW
view ABCC7 p.Asp1370Asn details
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
X
ABCC7 p.Gly551Asp 8741733:342:163
status: NEW
view ABCC7 p.Gly551Asp details
ABCC7 p.Gly551Ser
X
ABCC7 p.Gly551Ser 8741733:342:156
status: NEW
view ABCC7 p.Gly551Ser details
ABCC7 p.Gly1349Asp
X
ABCC7 p.Gly1349Asp 8741733:342:174
status: NEW
view ABCC7 p.Gly1349Asp details
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
X
ABCC7 p.Gly551Asp 8741733:345:78
status: NEW
view ABCC7 p.Gly551Asp details
ABCC7 p.Gly1349Asp
X
ABCC7 p.Gly1349Asp 8741733:345:88
status: NEW
view ABCC7 p.Gly1349Asp details
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
X
ABCC7 p.Gly551Ser 8741733:346:89
status: NEW
view ABCC7 p.Gly551Ser details
ABCC7 p.Gly1349Asp
X
ABCC7 p.Gly1349Asp 8741733:346:99
status: NEW
view ABCC7 p.Gly1349Asp details
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
X
ABCC7 p.Gly551Asp 8741733:347:78
status: NEW
view ABCC7 p.Gly551Asp details
ABCC7 p.Gly1349Asp
X
ABCC7 p.Gly1349Asp 8741733:347:88
status: NEW
view ABCC7 p.Gly1349Asp details
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
X
ABCC7 p.Gly551Ser 8741733:348:89
status: NEW
view ABCC7 p.Gly551Ser details
ABCC7 p.Gly1349Asp
X
ABCC7 p.Gly1349Asp 8741733:348:99
status: NEW
view ABCC7 p.Gly1349Asp details
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
X
ABCC7 p.Gly551Ala 8741733:355:52
status: NEW
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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
X
ABCC7 p.Gly551Ala 8741733:357:52
status: NEW
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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
X
ABCC7 p.Gly551Asp 8741733:362:118
status: NEW
view ABCC7 p.Gly551Asp details
ABCC7 p.Gly1349Asp
X
ABCC7 p.Gly1349Asp 8741733:362:136
status: NEW
view ABCC7 p.Gly1349Asp details
ABCC7 p.Asp572Asn
X
ABCC7 p.Asp572Asn 8741733:362:125
status: NEW
view ABCC7 p.Asp572Asn details
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
X
ABCC7 p.Gly551Asp 8741733:364:118
status: NEW
view ABCC7 p.Gly551Asp details
ABCC7 p.Gly1349Asp
X
ABCC7 p.Gly1349Asp 8741733:364:136
status: NEW
view ABCC7 p.Gly1349Asp details
ABCC7 p.Asp572Asn
X
ABCC7 p.Asp572Asn 8741733:364:125
status: NEW
view ABCC7 p.Asp572Asn details
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
X
ABCC7 p.Lys1250Ala 8741733:371:143
status: NEW
view ABCC7 p.Lys1250Ala details
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
X
ABCC7 p.Lys1250Ala 8741733:373:143
status: NEW
view ABCC7 p.Lys1250Ala details
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
X
ABCC7 p.Lys1250Ala 8741733:382:26
status: NEW
view ABCC7 p.Lys1250Ala details
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
X
ABCC7 p.Lys464Ala 8741733:383:24
status: NEW
view ABCC7 p.Lys464Ala details
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
X
ABCC7 p.Lys1250Ala 8741733:384:26
status: NEW
view ABCC7 p.Lys1250Ala details
ABCC7 p.Lys1250Ala
X
ABCC7 p.Lys1250Ala 8741733:384:33
status: NEW
view ABCC7 p.Lys1250Ala details
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
X
ABCC7 p.Lys464Ala 8741733:385:24
status: NEW
view ABCC7 p.Lys464Ala details
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
X
ABCC7 p.Lys1250Ala 8741733:386:33
status: NEW
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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
X
ABCC7 p.Gly551Asp 8741733:406:125
status: NEW
view ABCC7 p.Gly551Asp details
ABCC7 p.Gly551Ser
X
ABCC7 p.Gly551Ser 8741733:406:107
status: NEW
view ABCC7 p.Gly551Ser details
ABCC7 p.Gly1349Asp
X
ABCC7 p.Gly1349Asp 8741733:406:132
status: NEW
view ABCC7 p.Gly1349Asp details
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
X
ABCC7 p.Gly551Asp 8741733:408:125
status: NEW
view ABCC7 p.Gly551Asp details
ABCC7 p.Gly551Ser
X
ABCC7 p.Gly551Ser 8741733:408:107
status: NEW
view ABCC7 p.Gly551Ser details
ABCC7 p.Gly1349Asp
X
ABCC7 p.Gly1349Asp 8741733:408:132
status: NEW
view ABCC7 p.Gly1349Asp details
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