ABCMdb

ABCC7 p.Cys491Ala

None has been submitted yet.

Publications

PMID: 20974851 [PubMed] Melani R et al: "Modulation of cystic fibrosis transmembrane conductance regulator (CFTR) activity and genistein binding by cytosolic pH."

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). Login to comment
73 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). Login to comment
75 Both C491A and C1344A were produced on a wild-type CFTR background. Login to comment
77 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). Login to comment
82 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). Login to comment
87 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). Login to comment
100 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. Login to comment
102 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. Login to comment
105 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). Login to comment
106 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. Login to comment
119 Western blot of cysteine mutants and response of C491A-CFTR to genistein. Login to comment
120 A, Western blot showing CFTR protein expression in untransfected FRT cells (FRT-null) and in cells stably transfected with WT-, C491A-, and C1344A-CFTR. Login to comment
126 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. Login to comment
128 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). Login to comment
134 C, course of Ka measured on WT-, C491A-, and C1344A-CFTR at different pHi values. Login to comment
138 However, at pHi 8, the Ka for WT and C1344A-CFTR increased significantly (p Ͻ 0.05), whereas it remained unchanged for mutant C491A. Login to comment
164 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. Login to comment
165 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). Login to comment
174 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). Login to comment