ABCMdb

ABCC7 p.Ser623Ala

Predicted by SNAP2:	A: N (87%), C: N (93%), D: N (82%), E: N (93%), F: N (61%), G: N (87%), H: N (93%), I: N (72%), K: N (87%), L: N (66%), M: N (72%), N: N (87%), P: N (66%), Q: N (87%), R: N (78%), T: N (93%), V: N (82%), W: D (71%), Y: D (59%),
Predicted by PROVEAN:	A: N, C: D, D: N, E: N, F: D, G: N, H: N, I: D, K: N, L: N, M: N, N: N, P: N, Q: N, R: N, T: N, V: N, W: D, Y: N,

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Publications
[hide] C terminus of nucleotide binding domain 1 contains... J Biol Chem. 2010 Jul 16;285(29):22132-40. Epub 2010 Apr 30. Billet A, Melin P, Jollivet M, Mornon JP, Callebaut I, Becq F
C terminus of nucleotide binding domain 1 contains critical features for cystic fibrosis transmembrane conductance regulator trafficking and activation.
J Biol Chem. 2010 Jul 16;285(29):22132-40. Epub 2010 Apr 30., 2010-07-16 [PMID:20435887]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl(-) channel physiologically important in fluid-transporting epithelia and pathologically relevant in several human diseases. Here, we show that mutations in the C terminus of the first nucleotide binding domain comprising the latest beta strands (beta(c)5 and beta(c)6) influence the trafficking, channel activity, and pharmacology of CFTR. We mutated CFTR amino acids located in the beta(c)5-beta(c)6 hairpin, within the beta(c)5 strand (H620Q), within the beta-turn linking the two beta strands (E621G, G622D), as well as within (S623A, S624A) and at the extremity (G628R) of the beta(c)6 strand. Functional analysis reveals that the current density was largely reduced for G622D and G628R channels compared with wt CFTR, similar for E621G and S624A, but increased for H620Q and S623A. For G622D and G628R, the abnormal activity is likely due to a defective maturation process, as assessed by the augmented activity and mature C-band observed in the presence of the trafficking corrector miglustat. In addition, in presence of the CFTR activator benzo[c]quinolizinium, the CFTR current density compared with that of wt CFTR was abolished for G622D and G628R channels, but similar for H620Q, S623A, and S624A or slightly increased for E621G. Finally, G622D and G628R were activated by the CFTR agonists genistein, RP-107, and isobutylmethylxanthine. Our results identify the C terminus of the CFTR first nucleotide binding domain as an important molecular site for the trafficking of CFTR protein, for the control of CFTR channel gating, and for the pharmacological effect of a dual activity agent.

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No. Sentence Comment
2 We mutated CFTR amino acids located in the betac5-betac6 hairpin, within the betac5 strand (H620Q), within the beta-turn linking the two beta strands (E621G, G622D), as well as within (S623A, S624A) and at the extremity (G628R) of the betac6 strand. Login to comment
3 Functional analysis reveals that the current density was largely reduced for G622D and G628R channels compared with wt CFTR, similar for E621G and S624A, but increased for H620Q and S623A. Login to comment
5 In addition, in presence of the CFTR activator benzo[c]quinolizinium, the CFTR current density compared with that of wt CFTR was abolished for G622D and G628R channels, but similar for H620Q, S623A, and S624A or slightly increased for E621G. Login to comment
33 We have mutated these two glycine residues in aspartic acid (G622D) and arginine (G628R) and considered other mutants in their neighborhood (H620Q, E621G, S623A, and S624A). Login to comment
87 RESULTS Expression of the NBD1 C-terminal CFTR Mutants-We have introduced EGFP-tagged CFTR proteins into HEK293 cells, wt CFTR and six CFTR mutants, i.e. H620Q, E621G, G622D, S623A, S624A, and G628R. Login to comment
90 For the expression of CFTR mutants H620Q, E621G, S623A, and S624A, the profiles of core-glycosylated and mature-glycosylated forms were similar to that of the wt protein. Login to comment
109 Second, with the two mutations introduced on both sides of the beta turn, H620Q and S623A, we recorded an increased (p Ͻ 0.001) Cl- current (supplemental Table 2) compared with wt CFTR. Login to comment
122 Concerning the activation kinetics, only the two CFTR mutants with an increased Cl-transport activity (H620Q and S623A) also present a faster time course of activation (Fig. 5B). Login to comment
124 T50 of the two mutated channels (T50 ϭ 124.45 Ϯ 8.8 s for H620Q, n ϭ 8 and T50 ϭ 114.63 Ϯ 8.8 s for S623A, n ϭ 8) were significantly different compared with wt (T50 ϭ 164.85 Ϯ 10.2 s, n ϭ 8). Login to comment
125 Our results indicated that H620Q and S623A channels could be activated more rapidly than the other CFTR mutant channels studied. Login to comment
130 Interestingly, although the level of current for H620Q and S623A was greater than the response of wt channels (Fig. 3), both mutated CFTRs were activated by MPB-91 to a level similar to that of wt CFTR (Fig. 6) (at 40 mV, current densities were: H620Q, 27.86 Ϯ 4.2 pA/pF, n ϭ 5; S623A, 30.83 Ϯ 3.4 pA/pF, n ϭ 6). Login to comment
138 Dotted line corresponds to the wt level. B, upper, representatives time course of wt and S623A mutant in the presence of 10 ␮M Fsk. Login to comment
163 In particular, the cAMP-activated Cl- current densities elicited by H620Q and S623A channels were increased compared with wt. Login to comment
176 Dotted line corresponds to the wt level. B, upper, representatives time course of wt and S623A mutant in presence of 50 ␮M MPB-91. Login to comment
180 In addition, MPB-91 was also able to activate H620Q, E621G, S623A, and S624A mutants. Login to comment
182 Contrary to the activation by Fsk, no difference of kinetic parameters was detected with the two mutants H620Q and S623A. Login to comment