ABCC7 p.Val1293Gly
| CF databases: |
c.3877G>A
,
p.Val1293Ile
(CFTR1)
?
, The change was found in the asymptomatic husband of a CF carrier.
|
| Predicted by SNAP2: | A: N (72%), C: N (72%), D: D (80%), E: D (59%), F: N (66%), G: D (75%), H: D (75%), I: N (87%), K: D (66%), L: N (66%), M: N (78%), N: D (71%), P: D (59%), Q: D (66%), R: D (80%), S: N (53%), T: N (82%), W: D (75%), Y: D (75%), |
| Predicted by PROVEAN: | A: N, C: N, D: N, E: N, F: N, G: N, H: N, I: N, K: N, L: N, M: N, N: N, P: N, Q: N, R: N, S: N, T: N, W: D, Y: N, |
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[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
6 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.
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ABCC7 p.Val1293Gly 17244607:6:13
status: NEW44 We found that the stimulating effect of potentiators was reduced for R553Q and V1293G.
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ABCC7 p.Val1293Gly 17244607:44:79
status: NEW49 Mutant V1293G was introduced in a wild type CFTR construct contained in the expression vector pTracer-CMV (15) by a recombinant PCR method.
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ABCC7 p.Val1293Gly 17244607:49:7
status: NEW52 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.
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ABCC7 p.Val1293Gly 17244607:52:0
status: NEW53 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).
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ABCC7 p.Val1293Gly 17244607:53:51
status: NEW85 The apparent dissociation constant of CPTcAMP for mutants V1293G and A554E was not statistically different from that of the wild type protein (Fig. 2).
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ABCC7 p.Val1293Gly 17244607:85:58
status: NEW98 Continuous lines (WT, A554E, and V1293G) and broken lines (R553Q) indicate fitting of the data to Equation 1.
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ABCC7 p.Val1293Gly 17244607:98:33
status: NEW100 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.
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ABCC7 p.Val1293Gly 17244607:100:16
status: NEW102 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.
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ABCC7 p.Val1293Gly 17244607:102:112
status: NEW110 Mutant V1293G behavior was between A554E and R553Q.
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ABCC7 p.Val1293Gly 17244607:110:7
status: NEW113 In general, for these compounds Ka changed on mutants in the same direction as Ka for genistein; however, the effect was less marked, except for mutant V1293G, which tended to be more sensitive to Act-06 (see Table 2).
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ABCC7 p.Val1293Gly 17244607:113:152
status: NEW127 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.
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ABCC7 p.Val1293Gly 17244607:127:92
status: NEW130 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.
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ABCC7 p.Val1293Gly 17244607:130:190
status: NEWX
ABCC7 p.Val1293Gly 17244607:130:457
status: NEWX
ABCC7 p.Val1293Gly 17244607:130:718
status: NEW150 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.
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ABCC7 p.Val1293Gly 17244607:150:119
status: NEW159 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.
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ABCC7 p.Val1293Gly 17244607:159:155
status: NEW167 The CFTR proteins are indicated in red, yellow, green, and blue for R553Q, V1293G, A554E and WT, respectively.
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ABCC7 p.Val1293Gly 17244607:167:75
status: NEW176 Actually, we found that an increased Ka in R553Q and V1293G with respect to wild type CFTR is accompanied by a reduced Ki (Figs.
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ABCC7 p.Val1293Gly 17244607:176:53
status: NEW[hide] Identification of a NBD1-binding pharmacological c... Chem Biol. 2011 Feb 25;18(2):231-42. Sampson HM, Robert R, Liao J, Matthes E, Carlile GW, Hanrahan JW, Thomas DY
Identification of a NBD1-binding pharmacological chaperone that corrects the trafficking defect of F508del-CFTR.
Chem Biol. 2011 Feb 25;18(2):231-42., 2011-02-25 [PMID:21338920]
Abstract [show]
Most cases of cystic fibrosis (CF) are attributable to the F508del allele of CFTR, which causes the protein to be retained in the endoplasmic reticulum (ER) and subsequently degraded. One strategy for CF therapy is to identify corrector compounds that help traffic F508del-CFTR to the cell surface. Pharmacological chaperones, or correctors that bind specifically to F508del-CFTR and restore function, would be the most promising drug development candidates, but few pharmacological chaperones exist for F508del-CFTR. Using differential scanning fluorimetry (DSF), we have surveyed corrector compounds and identified one, RDR1, which binds directly to the first nucleotide binding domain (NBD1) of F508del-CFTR. We show that RDR1 treatment partially rescues F508del-CFTR function in both cells and in an F508del-CF mouse model. Thus, RDR1 is a pharmacological chaperone of F508del-CFTR and represents a novel scaffold for drug development.
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No. Sentence Comment
152 This hypothesis is consistent with the finding that the G551D and the V1293G mutations alter the affinity of these molecules for CFTR (Moran et al., 2005; Zegarra-Moran et al., 2007).
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ABCC7 p.Val1293Gly 21338920:152:70
status: NEW