ABCC7 p.Cys491Ala
| ClinVar: |
c.1471T>C
,
p.Cys491Arg
?
, not provided
|
| CF databases: |
c.1471T>C
,
p.Cys491Arg
(CFTR1)
?
, This misense has been found in a CF patient of North African origin with [delta]F508 on the other CF chromosome. This mutation was found once out of 1460 CF chromosomes screened.
c.1472G>C , p.Cys491Ser (CFTR1) ? , |
| Predicted by SNAP2: | A: N (82%), D: D (85%), E: D (71%), F: D (71%), G: D (59%), H: D (85%), I: N (57%), K: D (71%), L: D (59%), M: D (59%), N: D (75%), P: D (75%), Q: D (63%), R: D (85%), S: N (66%), T: N (61%), V: N (87%), W: D (91%), Y: D (85%), |
| Predicted by PROVEAN: | A: 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, V: N, W: D, Y: N, |
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[hide] Modulation of cystic fibrosis transmembrane conduc... J Biol Chem. 2010 Dec 31;285(53):41591-6. Epub 2010 Oct 25. Melani R, Tomati V, Galietta LJ, Zegarra-Moran O
Modulation of cystic fibrosis transmembrane conductance regulator (CFTR) activity and genistein binding by cytosolic pH.
J Biol Chem. 2010 Dec 31;285(53):41591-6. Epub 2010 Oct 25., 2010-12-31 [PMID:20974851]
Abstract [show]
Potentiators are molecules that increase the activity of the cystic fibrosis transmembrane conductance regulator (CFTR). Some potentiators can also inhibit CFTR at higher concentrations. The activating binding site is thought to be located at the interface of the dimer formed by the two nucleotide-binding domains. We have hypothesized that if binding of potentiators involves titratable residues forming salt bridges, then modifications of cytosolic pH (pH(i)) would alter the binding affinity. Here, we analyzed the effect of pH(i) on CFTR activation and on the binding of genistein, a well known CFTR potentiator. We found that pH(i) does modify CFTR maximum current (I(m)) and half-activation concentration (K(d)): I(m) = 127.7, 185.5, and 231.8 muA/cm(2) and K(d) = 32.7, 56.6 and 71.9 mum at pH 6, 7.35, and 8, respectively. We also found that the genistein apparent dissociation constant for activation (K(a)) increased at alkaline pH(i), near cysteine pK (K(a) = 1.83, 1.81 and 4.99 mum at pH(i) 6, 7.35, and 8, respectively), suggesting the involvement of cysteines in the binding site. Mutations of cysteine residues predicted to be within (Cys-491) or outside (Cys-1344) the potentiator-binding site showed that Cys-491 is responsible for the sensitivity of potentiator binding to alkaline pH(i). Effects of pH(i) on inhibition by high genistein doses were also analyzed. Our results extend previous data about multiple effects of pH(i) on CFTR activity and demonstrate that binding of potentiators involves salt bridge formation with amino acids of nucleotide-binding domain 1.
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No. Sentence Comment
42 EXPERIMENTAL PROCEDURES Cell Culture-Fisher rat thyroid (FRT) cells stably transfected with WT-CFTR or with CFTR carrying the C491A or C1344A mutation were grown as described previously (23).
X
ABCC7 p.Cys491Ala 20974851:42:126
status: NEW73 pHi Modulates CFTR and Genistein-CFTR Interaction 41592 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285•NUMBER 53•DECEMBER 31, 2010 atUniversityofNorthCarolinaatChapelHill,onAugust8,www.jbc.orgDownloadedfrom pH 8 Has No Effect on the Potentiation of C491A-CFTR by Genistein-Among the amino acids predicted by molecular docking to make contacts with CFTR potentiators, we identified Cys-491 (12).
X
ABCC7 p.Cys491Ala 20974851:73:99
status: NEWX
ABCC7 p.Cys491Ala 20974851:73:260
status: NEW75 Both C491A and C1344A were produced on a wild-type CFTR background.
X
ABCC7 p.Cys491Ala 20974851:75:5
status: NEW77 Dose-response relationships to CPT-cAMP showed that the maximum current attainable from mutant C491A was 5-10-fold lower than that from WT-CFTR at all pHi values (Table 1).
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ABCC7 p.Cys491Ala 20974851:77:95
status: NEW82 The response to genistein of C491A-CFTR was bimodal, as in the WT channel, with dose-dependent current increase at low concentrations and inhibition when the genistein concentration was further raised (Fig. 2, B and C).
X
ABCC7 p.Cys491Ala 20974851:82:29
status: NEW87 Genistein Potentiation of C1344A-CFTR Maintains the Sensitivity to pH 8-The cysteine mutant outside the putative binding site for potentiators, C1344A, showed a CPT-cAMP maximum current even smaller than C491A, but the equilibrium constant (Kd) was very close to the equilibrium constants of C491A and WT-CFTR (Table 1).
X
ABCC7 p.Cys491Ala 20974851:87:204
status: NEWX
ABCC7 p.Cys491Ala 20974851:87:292
status: NEW100 Parameter pH 6.0 7.35 8.0 Im (A⅐cm-2 ) WT 127.7 Ϯ 15.8 (10)a 185.5 Ϯ 17.3 (23) 231.8 Ϯ 27.4 (14) C491A 13.9 Ϯ 3.7 (9)a 36.1 Ϯ 4.5 (8) 29.9 Ϯ 2.2 (8) C1344A 2.2 Ϯ 0.5 (5)a 6.2 Ϯ 1.7 (9) 11.7 Ϯ 2.2 (8) Kd (M) WT 32.7 Ϯ 6 (10)a 56.6 Ϯ 5.9 (23) 71.9 Ϯ 13.3 (14) C491A 10.3 Ϯ 1.2 (9)a 56.7 Ϯ 6.2 (8) 67.7 Ϯ 8.6 (9) C1344A 11.2 Ϯ 2 (5)a 63.4 Ϯ 9.4 (9) 104.3 Ϯ 12.7 (8) a p Ͻ 0.05 compared with the same parameter on the same protein at pH 7.35.
X
ABCC7 p.Cys491Ala 20974851:100:130
status: NEWX
ABCC7 p.Cys491Ala 20974851:100:353
status: NEW102 Parameter pH 6 7.35 8.0 fA WT 1.24 Ϯ 0.22 (12) 1.87 Ϯ 0.34 (14) 4.36 Ϯ 0.83 (16)a C491A 2.57 Ϯ 0.58 (11) 3.29 Ϯ 0.53 (13) 3.85 Ϯ 0.54 (11) C1344A 1.84 Ϯ 0.64 (4) 4.5 Ϯ 1.2 (9) 5.23 Ϯ 0.89 (13) Ka (M) WT 1.83 Ϯ 0.43 (12) 1.81 Ϯ 0.37 (14) 4.99 Ϯ 0.89 (16)a C491A 17.2 Ϯ 4 (11) 17.8 Ϯ 5.44 (13) 16.4 Ϯ 3.29 (11) C1344A 8.2 Ϯ 1.27 (4) 10.1 Ϯ 2.17 (9) 20 Ϯ 3.53 (13)a Ki (M) WT 250.9 Ϯ 29.5 (12) 368 Ϯ 50.9 (14) 237.9 Ϯ 44.95 (16) C491A 78.5 Ϯ 21.2 (11) 108.5 Ϯ 24.3 (13) 394.9 Ϯ 70.4 (12)a C1344A 23.8 Ϯ 2.48 (4)a 73.9 Ϯ 12.7 (9) 398.75 Ϯ 87.1 (13)a a p Ͻ 0.05 compared with the same parameter at pH 7.35 on the same protein.
X
ABCC7 p.Cys491Ala 20974851:102:100
status: NEWX
ABCC7 p.Cys491Ala 20974851:102:335
status: NEWX
ABCC7 p.Cys491Ala 20974851:102:568
status: NEW105 This indicates that, in contrast to what was found with C491A, the C1344A mutation did not modify the sensitivity of genistein binding to the activating site at alkaline pHi (Fig. 3C).
X
ABCC7 p.Cys491Ala 20974851:105:56
status: NEW106 As with C491A, the Ki values at pHi 6 and 7.35 were significantly smaller than for WT-CFTR, whereas at pH 8, the Ki did not change.
X
ABCC7 p.Cys491Ala 20974851:106:8
status: NEW119 Western blot of cysteine mutants and response of C491A-CFTR to genistein.
X
ABCC7 p.Cys491Ala 20974851:119:49
status: NEW120 A, Western blot showing CFTR protein expression in untransfected FRT cells (FRT-null) and in cells stably transfected with WT-, C491A-, and C1344A-CFTR.
X
ABCC7 p.Cys491Ala 20974851:120:128
status: NEW126 B, representative traces showing the response of mutant C491A-CFTR to the application of genistein (indicated by arrows) after activating CFTR with 20 M CPT-cAMP.
X
ABCC7 p.Cys491Ala 20974851:126:56
status: NEW128 C, normalized dose-response relationships in experiments on mutant C491A at pH 6 (n ϭ 11), 7.35 (n ϭ 13), and 8 (n ϭ 12).
X
ABCC7 p.Cys491Ala 20974851:128:67
status: NEW134 C, course of Ka measured on WT-, C491A-, and C1344A-CFTR at different pHi values.
X
ABCC7 p.Cys491Ala 20974851:134:33
status: NEW138 However, at pHi 8, the Ka for WT and C1344A-CFTR increased significantly (p Ͻ 0.05), whereas it remained unchanged for mutant C491A.
X
ABCC7 p.Cys491Ala 20974851:138:132
status: NEW164 Consequently, we next mutated Cys-491 to alanine. As a control, we mutated to alanine also Cys-1344, a NBD2 cysteine situated outside the putative binding site for potentiators.
X
ABCC7 p.Cys491Ala 20974851:164:30
status: NEW165 Analysis of epithelia stably expressing these mutant CFTR proteins showed that, whereas C1344A-CFTR behaved as WT-CFTR, exhibiting reduced affinity for genistein at pH 8, the C491A mutation kept the same affinity for genistein at the three pHi values (Fig. 3C and Table 2).
X
ABCC7 p.Cys491Ala 20974851:165:175
status: NEW174 Substitution of either Cys-491 or Cys-1344 with alanine resulted in an increased affinity for the inhibitory site at pHi 6 and 7.35 (Table 2).
X
ABCC7 p.Cys491Ala 20974851:174:23
status: NEW