ABCMdb

PMID: 20974851

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 [PubMed]

Sentences

No. Mutations Sentence Comment

23 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp First, CFTR carrying NBD mutations, such as G551D and G1349D, exhibit a lower affinity for several potentiators, indicating that mutations are close to the binding site (21-23). Login to comment 42 ABCC7 p.Cys1344Ala ABCC7 p.Cys491Ala 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 ABCC7 p.Cys491Ala ABCC7 p.Cys491Ala 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 ABCC7 p.Cys1344Ala ABCC7 p.Cys491Ala Both C491A and C1344A were produced on a wild-type CFTR background. Login to comment 77 ABCC7 p.Cys491Ala 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 ABCC7 p.Cys491Ala 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 ABCC7 p.Cys1344Ala ABCC7 p.Cys1344Ala ABCC7 p.Cys491Ala ABCC7 p.Cys491Ala 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 88 ABCC7 p.Cys1344Ala Similar to the other two proteins, also C1344A-CFTR showed a higher apparent affinity (lower Kd) for CPT-cAMP at pH 6. Login to comment 100 ABCC7 p.Cys1344Ala ABCC7 p.Cys1344Ala ABCC7 p.Cys491Ala ABCC7 p.Cys491Ala 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 ABCC7 p.Cys1344Ala ABCC7 p.Cys1344Ala ABCC7 p.Cys1344Ala ABCC7 p.Cys491Ala ABCC7 p.Cys491Ala ABCC7 p.Cys491Ala 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 104 ABCC7 p.Cys1344Ala The Ka was found to be higher than that of the WT-CFTR, but as in the WT channel, the Ka of C1344A-CFTR increased when pHi was set at a value of 8. Login to comment 105 ABCC7 p.Cys1344Ala ABCC7 p.Cys491Ala 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 ABCC7 p.Cys491Ala 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 111 ABCC7 p.Gly551Asp A recent report on phase II clinical trials using potentiator VX-770 in patients carrying at least one G551D allele (severe gating mutation) indicated that short-term treatment (2-4 weeks) improved nasal potential difference and lung function (clinicaltrials.gov/ct2/results?intrϭ%22VX- 770%22). Login to comment 119 ABCC7 p.Cys491Ala Western blot of cysteine mutants and response of C491A-CFTR to genistein. Login to comment 120 ABCC7 p.Cys1344Ala ABCC7 p.Cys491Ala 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 ABCC7 p.Cys491Ala 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 ABCC7 p.Cys491Ala 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 131 ABCC7 p.Cys1344Ala Dose-response relationship of C1344A-CFTR to genistein. Login to comment 132 ABCC7 p.Cys1344Ala A, representative traces showing the response of mutant C1344A-CFTR to increasing doses of genistein (indicated by arrows). Login to comment 133 ABCC7 p.Cys1344Ala Dashed lines under the curves indicate the zero current level. B, normalized dose-response relationships in experiments on mutant C1344A at pH 6 (n ϭ 4), 7.35 (n ϭ 4), and 8 (n ϭ 4). Login to comment 134 ABCC7 p.Cys1344Ala ABCC7 p.Cys491Ala C, course of Ka measured on WT-, C491A-, and C1344A-CFTR at different pHi values. Login to comment 138 ABCC7 p.Cys1344Ala ABCC7 p.Cys491Ala 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 ABCC7 p.Cys491Ala 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 ABCC7 p.Cys1344Ala ABCC7 p.Cys491Ala 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 ABCC7 p.Cys1344Ala ABCC7 p.Cys491Ala 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 175 ABCC7 p.Cys1344Ala This effect was more marked at pH 6 and for C1344A-CFTR (Ki ϭ 24 ␮M). Login to comment