ABCMdb

ABCC7 p.Ala554Glu

Predicted by SNAP2:	C: D (53%), D: D (80%), E: N (61%), F: D (85%), G: D (66%), H: D (80%), I: D (80%), K: D (85%), L: D (80%), M: D (75%), N: D (75%), P: D (85%), Q: N (66%), R: D (85%), S: D (63%), T: D (71%), V: D (75%), W: D (85%), Y: D (85%),
Predicted by PROVEAN:	C: D, D: D, E: N, F: D, G: D, H: D, I: D, K: D, L: D, M: D, N: D, P: D, Q: N, R: N, S: N, T: N, V: D, W: D, Y: D,

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Publications
[hide] Functional analysis of mutations in the putative b... J Biol Chem. 2007 Mar 23;282(12):9098-104. Epub 2007 Jan 23. Zegarra-Moran O, Monteverde M, Galietta LJ, Moran O
Functional analysis of mutations in the putative binding site for cystic fibrosis transmembrane conductance regulator potentiators. Interaction between activation and inhibition.
J Biol Chem. 2007 Mar 23;282(12):9098-104. Epub 2007 Jan 23., 2007-03-23 [PMID:17244607]

Abstract [show]
An increasing number of compounds able to potentiate the activity of mutants of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel have been identified by high throughput screening or by individual search of derivatives of known active compounds. Several lines of evidence suggest that most CFTR potentiators act through the same mechanism, probably by binding to the nucleotide binding domains to promote the activity of the protein and then, with lower affinity, to an inhibitory site. With the aim of identifying the activating binding site, we recently modeled the nucleotide binding domain dimer and predicted a common binding site for potentiators in its interface. To validate this model experimentally, we mutated some of the residues involved in the putative binding site, i.e. Arg(553), Ala(554), and Val(1293). The activity of CFTR potentiators was measured as apical membrane currents on polarized cells stably expressing wild type or mutated proteins. CFTR activity was elicited by application of a membrane-permeable cAMP analogue followed by increasing concentrations of potentiators. We found that all three mutants responded to cAMP, although the affinity of R553Q was higher than that of wild type CFTR. In R553Q and V1293G mutants, the dissociation constant of potentiators for the activating site was increased, whereas the dissociation constant for the inhibitory site was reduced. Our results show that the mutated residues are part of the activating binding site for potentiators, as suggested by the molecular model. In addition, these results suggest that the activating and inhibitory sites are not independent of each other.

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No. Sentence Comment
50 For mutations A554E and R553Q, CFTR cDNA was subcloned on pcDNA3.1. Login to comment
52 V1293G clones were selected and maintained in 800 ␮g/ml Zeocin, and A554E and R553Q constructs were selected and maintained in 1 mg/ml G418. Login to comment
53 Cell Cultures-FRT cells expressing wild type (WT), V1293G, A554E, or R553Q CFTR were cultured on 60-mm Petri dishes with Coon`s modified F12 containing 5% fetal bovine serum, 2 mM L-glutamine, 50 units/ml penicillin, and 50 ␮g/ml streptomycin and selection antibiotics, as described previously (15). Login to comment
85 The apparent dissociation constant of CPTcAMP for mutants V1293G and A554E was not statistically different from that of the wild type protein (Fig. 2). Login to comment
98 Continuous lines (WT, A554E, and V1293G) and broken lines (R553Q) indicate fitting of the data to Equation 1. Login to comment
100 Wild type A554E V1293G R553Q G551D n ϭ 5 n ϭ 4 n ϭ 4 n ϭ 6 n ϭ 7 Imax (␮A/cm2 ) 282.2 Ϯ 13.5 66.9 Ϯ 16.4a 70.2 Ϯ 14a 93.8 Ϯ 19a 10.1 Ϯ 2.8a Kd (␮M) 54.2 Ϯ 11.5 40.5 Ϯ 5.9 48.4 Ϯ 13.5 21.5 Ϯ 4.5a 74.2 Ϯ 12.1 I(20)/I(max) 0.3 Ϯ 0.04 0.34 Ϯ 0.03 0.32 Ϯ 0.05 0.51 Ϯ 0.05a 0.23 Ϯ 0.03 a p Ͻ 0.05. Login to comment
102 It is interesting to note that 20 ␮M CPTcAMP produced a similar current fraction (ϳ0.3) on mutants V1293G and A554E but half of the maximum current (ϳ0.5) on R553Q (see I(20)/ I(max) in Table 1), in agreement with the lower Kd of CPT-cAMP found on this mutant. Login to comment
107 The response of mutant A554E to genistein, with almost no change on Ka or Ki, was similar to the response of wild type CFTR. Login to comment
110 Mutant V1293G behavior was between A554E and R553Q. Login to comment
127 C-E, comparison of normalized and averaged genistein dose-response relationships of mutants V1293G,A554E,andR553Q(seesymbolkeys)toWT(dashedlines).Eachsymbol is the mean of 4-6 experiments, and vertical bars show S.E. Login to comment
130 Compounds Protein n fA Ka Ki ␮M ␮M Genistein Wild type 5 1.33 Ϯ 0.41 3.08 Ϯ 0.74 562.4 Ϯ 111.1 A554E 6 2.75 Ϯ 0.62 4.58 Ϯ 0.51 408.6 Ϯ 84.8 V1293G 6 4.87 Ϯ 1.91 12 Ϯ 3.9a 270.5 Ϯ 36.4a R553Q 6 1.87 Ϯ 0.12 22.28 Ϯ 5.2a 119.9 Ϯ 34.9a UCCF029 Wild type 4 0.41 Ϯ 0.04 0.021 Ϯ 0.002 1036 Ϯ 523 A554E 4 1.56 Ϯ 0.45a 0.036 Ϯ 0.009 1519 Ϯ 651 V1293G 5 3.19 Ϯ 1.09 0.042 Ϯ 0.008 843 Ϯ 126 R553Q 5 1.08 Ϯ 0.17a 0.154 Ϯ 0.029a 547 Ϯ 99 Act-06 Wild type 5 0.8 Ϯ 0.17 0.69 Ϯ 0.33 439.6 Ϯ 108 A554E 4 1.67 Ϯ 0.42 0.74 Ϯ 0.25 319.1 Ϯ 42.4 V1293G 4 1.25 Ϯ 0.16 0.35 Ϯ 0.09 383.8 Ϯ 28.9 R553Q 6 1.77 Ϯ 0.47a 2.11 Ϯ 0.73 179.1 Ϯ 73.2a a Student`s t test indicated that these values were statistically different from those on WT CFTR with p Ͻ 0.05. Login to comment
150 Mutations of NBDs, like the CF mutation G551D (see Table 1 and Ref. 15) or the other mutations studied here, A554E and V1293G, do not seem to change significantly the sensitivity of the protein to CPTcAMP. Login to comment
159 Conversion of the Ala554 residue to glutamic acid had little effect on the capability of potentiators to favor the conductive state, whereas conversion of Val1293 to glycine increased the Ka for genistein by 4-fold. Login to comment
167 The CFTR proteins are indicated in red, yellow, green, and blue for R553Q, V1293G, A554E and WT, respectively. Login to comment
169 Mutations in Binding Site for CFTR Potentiators 9102 potentiators (see Fig. 1; the distance between these amino acids and potentiators is less than 3.6 Å, whereas in A554E this distance is larger), and therefore a modification of any of them is predicted to produce a strong effect on the activation dissociation constant. Login to comment
170 The A554E mutant is different. Login to comment

[hide] Identification of 12 novel mutations in the CFTR g... Hum Mol Genet. 1993 Jan;2(1):51-4. Audrezet MP, Mercier B, Guillermit H, Quere I, Verlingue C, Rault G, Ferec C
Identification of 12 novel mutations in the CFTR gene.
Hum Mol Genet. 1993 Jan;2(1):51-4., [PMID:7683952]

Abstract [show]
Over 200 mutations, besides the deletion delta F508, have been identified in the CFTR gene and are known to cause CF. In order to characterize the molecular defects of non delta F508 CF chromosomes of various French origin, we have combined the techniques of denaturing gradient gel electrophoresis (DGGE) and direct sequencing to screen for mutations in the whole coding sequence of the CFTR gene corresponding to the 27 exons and their exon-intron boundaries. This approach enabled us to identify 12 novel mutations which are described here. We have systematically tested a large number of other nucleotide changes distributed in the 27 exons, each of them was clearly detected. These data support the notion that the DGGE conditions we have defined for screening coding sequence of the CFTR gene allows the identification of most of, if not all, the CFTR gene mutations.

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Sentences [show]
No. Sentence Comment
47 A554E The nucleotide at position 1733 was changed from a C to T leading to a glutamic acid for an alanine. Login to comment