ABCMdb

ABCC7 p.Lys464Gln

ClinVar:	c.1392G>T , p.Lys464Asn ? , not provided
Predicted by SNAP2:	A: D (95%), C: D (95%), D: D (95%), E: D (95%), F: D (95%), G: D (95%), H: D (95%), I: D (95%), L: D (95%), M: D (95%), N: D (91%), P: D (95%), Q: D (95%), R: D (95%), S: D (95%), T: D (95%), V: D (95%), W: D (95%), Y: D (95%),
Predicted by PROVEAN:	A: D, C: D, D: D, E: D, F: D, G: D, H: D, I: D, L: D, M: D, N: D, P: D, Q: D, R: D, S: D, T: D, V: D, W: D, Y: D,

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[hide] CFTR: the nucleotide binding folds regulate the ac... J Gen Physiol. 1996 Jan;107(1):103-19. 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., [PMID:8741733]

Abstract [show]
The functional roles of the two nucleotide binding folds, NBF1 and NBF2, in the activation of the cystic fibrosis transmembrane conductance regulator (CFTR) were investigated by measuring the rates of activation and deactivation of CFTR Cl- conductance in Xenopus oocytes. Activation of wild-type CFTR in response to application of forskolin and 3-isobutyl-1-methylxanthine (IBMX) was described by a single exponential. Deactivation after washout of the cocktail consisted of two phases: an initial slow phase, described by a latency, and an exponential decline. Rate analysis of CFTR variants bearing analogous mutations in NBF1 and NBF2 permitted us to characterize amino acid substitutions according to their effects on the accessibility and stability of the active state. Access to the active state was very sensitive to substitutions for the invariant glycine (G551) in NBF1, where mutations to alanine (A), serine (S), or aspartic acid (D) reduced the apparent on rate by more than tenfold. The analogous substitutions in NBF2 (G1349) also reduced the on rate, by twofold to 10-fold, but substantially destabilized the active state as well, as judged by increased deactivation rates. In the putative ATP-binding pocket of either NBF, substitution of alanine, glutamine (Q), or arginine (R) for the invariant lysine (K464 or K1250) reduced the on rate similarly, by two- to fourfold. In contrast, these analogous substitutions produced opposite effects on the deactivation rate. NBF1 mutations destabilized the active state, whereas the analogous substitutions in NBF2 stabilized the active state such that activation was prolonged compared with that seen with wild-type CFTR. Substitution of asparagine (N) for a highly conserved aspartic acid (D572) in the ATP-binding pocket of NBF1 dramatically slowed the on rate and destabilized the active state. 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. The relatively slow time courses for activation and deactivation suggest that slow processes modulate ATP-dependent gating.

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No. Sentence Comment
19 In contrast, mutations in the putative ATP-binding pockets of the two NBFs produced opposite results, a reduction in sensitivity for mutations in NBF1 (K464Q, D572N) and an increase in sensitivity for the analogous mutations in NBF2 (K12500~ D1370N). Login to comment
60 Representative time courses are shown for wild type CFTR and two variants, K464Q and K1250Q. Login to comment
65 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 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 (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 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 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 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
99 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
129 (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 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
195 a~ODiap- 9 K464Q a I ' ' ' ' I ' ' ' ' I ' ' 0 10 20 ~ O -0 0, 9 -0~176176176o ....... 9.... -o*- -o*- 9 i~ e'~176176176176 9 D572N o i , , , , i , , , , i , , , , I , , , , i , , , , i , , , , 0 I0 20 30 40 50 minutes K1250A K1250C I i 30 D1370N 6O FIGURE4. Login to comment
229 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
237 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
281 + kott) (10-3 min-l kon kofr latency *k~m CFTR (mM) n (10-3min-]) mM-1) (10-3min 1) (10-3min-l) n (min) (10 3min i) n wt 0.65 • 0.08 26 664 • 51 118 • 9 558 • 45 76-+ 6 20 6.0 • 0.3 88 • 6 16 K464R 2.6 • 0.1": 4 153 + 20**+ 20 • 3*** 101 • 13''` 52 • 7*: 5 1.3 • 0.2*++ 174 • 14"** 7 K464Q 3.3 • 0.5"* 5 331 • 56*** 40 -+ 7* 199 • 34* 132 • 22*'` 5 1.9 • 0.3"I 142 -+ 19''` 5 K464A 4.6 • 0.7** 6 289 • 49* 30 • 5** 151 • 26*** 139 • 24*: 7 1.1 • 0.1"** 133 • 14"** 8 D572N 9.3 + 0.02*: 6 106 • 7*: 7-+0.5*: 37-+3*** 69 • 5+* 4 0.9 • 0.2*** 245 • 32*: 3 K1250R 0.17 • 0.07*: 5 239 •33*** 46 -+ 6"+* 231 • 32*: 8 • 1": 10 10.4 • 0.8"~ 100 • 7** 6 K1250Q 0.12 • 0.04*** 5 150 • 18''` 29 • 4* 146 -+ 18" 4 + 0.4"I 5 22.3 • 2.4*: 30 •5": 5 K1250A 0.07 + 0.02*: 10 218 • 18" 43 • 4*'` 215 • 18": 3 -+0.3*~* 5 15.6-+ 1.0"** 43 -+5** 5 D1370N 0.16 + 0.04*'` 7 449 - 79*: 87 • 15: 435 +76** 14 - 2*: 5 16.3-4-1.2"" 69-+ 6** 5 The symbols (*) and ('`) indicate significant differences from wild-type CFTR and the analogous mutant, respectively (P < 0.05). Login to comment
61 Representative time courses are shown for wild type CFTR and two variants, K464Q and K1250Q. Login to comment
66 In contrast, the decline in gel for mutants such as K464Q was rapid, showing only a slight increase after IBMX withdrawal. Login to comment
77 (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
80 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
100 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
131 (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
135 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
198 a~ODiap- 9 K464Q a I ' ' ' ' I ' ' ' ' I ' ' 0 10 20 ~ O -0 0, 9 -0 ~176176176 o ....... 9.... -o*- -o*- 9 i~ e'~176176176176 9 D572N o i , , , , i , , , , i , , , , I , , , , i , , , , i , , , , 0 I0 20 30 40 50 minutes K1250A K1250C I i 30 D1370N 6O FIGURE4. Login to comment
231 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
239 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
283 + kott) (10-3 min-l kon kofr latency *k~m CFTR (mM) n (10-3 min-]) mM-1) (10-3 min 1) (10-3min-l) n (min) (10 3min i) n wt 0.65 ߦ 0.08 26 664 ߦ 51 118 ߦ 9 558 ߦ 45 76 -+ 6 20 6.0 ߦ 0.3 88 ߦ 6 16 K464R 2.6 ߦ 0.1": 4 153 + 20**+ 20 ߦ 3*** 101 ߦ 13''` 52 ߦ 7*: 5 1.3 ߦ 0.2*++ 174 ߦ 14"** 7 K464Q 3.3 ߦ 0.5"* 5 331 ߦ 56*** 40 -+ 7* 199 ߦ 34* 132 ߦ 22*'` 5 1.9 ߦ 0.3"I 142 -+ 19''` 5 K464A 4.6 ߦ 0.7** 6 289 ߦ 49* 30 ߦ 5** 151 ߦ 26*** 139 ߦ 24*: 7 1.1 ߦ 0.1"** 133 ߦ 14"** 8 D572N 9.3 + 0.02*: 6 106 ߦ 7*: 7 -+0.5*: 37 -+3*** 69 ߦ 5+* 4 0.9 ߦ 0.2*** 245 ߦ 32*: 3 K1250R 0.17 ߦ 0.07*: 5 239 ߦ 33*** 46 -+ 6"+* 231 ߦ 32*: 8 ߦ 1": 10 10.4 ߦ 0.8"~ 100 ߦ 7** 6 K1250Q 0.12 ߦ 0.04*** 5 150 ߦ 18''` 29 ߦ 4* 146 -+ 18" 4 + 0.4"I 5 22.3 ߦ 2.4*: 30 ߦ 5": 5 K1250A 0.07 + 0.02*: 10 218 ߦ 18" 43 ߦ 4*'` 215 ߦ 18": 3 -+0.3*~* 5 15.6 -+ 1.0"** 43 -+5** 5 D1370N 0.16 + 0.04*'` 7 449 - 79*: 87 ߦ 15: 435 + 76** 14 - 2*: 5 16.3 -4-1.2"" 69 -+ 6** 5 The symbols (*) and ('`) indicate significant differences from wild-type CFTR and the analogous mutant, respectively (P < 0.05). Login to comment

[hide] Sequence homologies between nucleotide binding reg... FEBS Lett. 1995 Jun 12;366(2-3):87-91. Manavalan P, Dearborn DG, McPherson JM, Smith AE
Sequence homologies between nucleotide binding regions of CFTR and G-proteins suggest structural and functional similarities.
FEBS Lett. 1995 Jun 12;366(2-3):87-91., [PMID:7540563]

Abstract [show]
Sequence homology between the alpha-subunits of G-proteins and other GTP-binding proteins and certain regions within the nucleotide binding domains (NBDs) of cystic fibrosis transmembrane conductance regulator (CFTR) indicates that these protein structures may be similar. A sequence alignment of the NBDs of CFTR and NBDs from other membrane transporters, forms the basis of a structural model. This model predicts that one of the conserved sequences GGQR, within which a number of CF mutations occur, forms part of the nucleotide binding pocket and serves as an ON/OFF conformational switch as observed in GTP binding proteins. Furthermore, based on subtle sequence differences between the first and second NBDs of CFTR and from structure-activity data, we suggest that the nucleotide binding site environments of the two NBDs are different.

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No. Sentence Comment
171 The NBDI mutations (K464Q, D572N) showed a decrease in sensitivity to IBMX activation while the equivalent NBD2 mutations (KI250Q, DI370N) produced an increase in IBMX sensitivity, These results together with our sequence analysis data suggest that the nature of nuclcotide binding and the subsequent conforma- tior~:,lchanges may differ for the two domains. Login to comment
170 The NBDI mutations (K464Q, D572N) showed a decrease in sensitivity to IBMX activation while the equivalent NBD2 mutations (KI250Q, DI370N) produced an increase in IBMX sensitivity, These results together with our sequence analysis data suggest that the nature of nuclcotide binding and the subsequent conforma- tior~:,lchanges may differ for the two domains. Login to comment

[hide] Functional roles of the nucleotide-binding folds i... Proc Natl Acad Sci U S A. 1993 Nov 1;90(21):9963-7. Smit LS, Wilkinson DJ, Mansoura MK, Collins FS, Dawson DC
Functional roles of the nucleotide-binding folds in the activation of the cystic fibrosis transmembrane conductance regulator.
Proc Natl Acad Sci U S A. 1993 Nov 1;90(21):9963-7., [PMID:7694298]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR), a member of the traffic ATPase superfamily, possesses two putative nucleotide-binding folds (NBFs). The NBFs are sufficiently similar that sequence alignment of highly conserved regions can be used to identify analogous residues in the two domains. To determine whether this structural homology is paralleled in function, we compared the activation of chloride conductance by forskolin and 3-isobutyl-1-methylxanthine in Xenopus oocytes expressing CFTRs bearing mutations in NBF1 or NBF2. Mutation of a conserved glycine in the putative linker domain in either NBF produced virtually identical changes in the sensitivity of chloride conductance to activating conditions, and mutation of this site in both NBFs produced additive effects, suggesting that in the two NBFs this region plays a similar and critical role in the activation process. In contrast, amino acid substitutions in the Walker A and B motifs, thought to form an integral part of the nucleotide-binding pockets, produced strikingly different effects in NBF1 and NBF2. Substitutions for the conserved lysine (Walker A) or aspartate (Walker B) in NBF1 resulted in a marked decrease in sensitivity to activation, whereas the same changes in NBF2 produced an increase in sensitivity. These results are consistent with a model for the activation of CFTR in which both NBF1 and NBF2 are required for normal function but in which either the nature or the exact consequences of nucleotide binding differ for the two domains.

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No. Sentence Comment
92 The dose- 100- g 80-E C3) ° 60- V ai) X 40- E co 20 NBF1 O O G551S (9) v-v G551D (6) 0 Owt (12) T 6o NBF1 + NBF2 O--O G551S + G1349S (7) *--* G551 D + G 1 349D (5) Oz0 6 0 / T/ * , * /1 ° T 0 r / / / 3 _ -- ........ 0.02 0.05 0.2 0.5 1 2 IBMX, mM O-O wt (12) V-V K464Q ( 4) 1OOT 9 80E 0I-) I00160- -0 . Login to comment