ABCMdb

PMID: 20435887

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 [PubMed]

Sentences

No. Mutations Sentence Comment

2 ABCC7 p.Gly622Asp ABCC7 p.His620Gln ABCC7 p.Gly628Arg ABCC7 p.Ser624Ala ABCC7 p.Ser623Ala ABCC7 p.Glu621Gly 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 ABCC7 p.Gly622Asp ABCC7 p.His620Gln ABCC7 p.Gly628Arg ABCC7 p.Ser624Ala ABCC7 p.Ser623Ala ABCC7 p.Glu621Gly 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 4 ABCC7 p.Gly622Asp ABCC7 p.Gly628Arg 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. Login to comment 5 ABCC7 p.Gly622Asp ABCC7 p.His620Gln ABCC7 p.Gly628Arg ABCC7 p.Ser624Ala ABCC7 p.Ser623Ala ABCC7 p.Glu621Gly 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 6 ABCC7 p.Gly622Asp ABCC7 p.Gly628Arg Finally, G622D and G628R were activated by the CFTR agonists genistein, RP-107, and isobutylmethylxanthine. Login to comment 33 ABCC7 p.Gly622Asp ABCC7 p.His620Gln ABCC7 p.Gly628Arg ABCC7 p.Ser624Ala ABCC7 p.Ser623Ala ABCC7 p.Glu621Gly 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 57 ABCC7 p.Gly628Arg C, focus on the Gly628 position, which was mutated in the three-dimensional model in an arginine residue (G628R), highlighting the steric clashes (orange lines), which would be associated with this mutation. Login to comment 87 ABCC7 p.Gly622Asp ABCC7 p.His620Gln ABCC7 p.Gly628Arg ABCC7 p.Ser624Ala ABCC7 p.Ser623Ala ABCC7 p.Glu621Gly 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 ABCC7 p.His620Gln ABCC7 p.Ser624Ala ABCC7 p.Ser623Ala ABCC7 p.Glu621Gly 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 91 ABCC7 p.Gly622Asp ABCC7 p.Gly628Arg However, no mature-glycosylated C-band form for G622D and G628R was detected (Fig. 2, lanes 6 and 7). Login to comment 102 ABCC7 p.Gly622Asp ABCC7 p.Gly628Arg Effect of the CFTR corrector miglustat on G622D and G628R. Login to comment 103 ABCC7 p.Gly622Asp ABCC7 p.Gly628Arg A, upper, Western blot showing wt-CFTR, G622D-, and G628R-CFTR expression with or without pretreatment with miglustat (100 ␮M) and detected with CFTR NBD2 C-terminal antibody. Login to comment 108 ABCC7 p.Ser624Ala ABCC7 p.Glu621Gly First, Cl- currents recorded for wt CFTR, E621G, and S624A CFTR mutants were not significantly different (mean current densities for each construct are reported in supplemental Table 2). Login to comment 109 ABCC7 p.His620Gln ABCC7 p.Ser623Ala 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 110 ABCC7 p.Gly622Asp ABCC7 p.Gly628Arg Third, the current densities for G622D and G628R, although not abolished, are both significantly reduced (p Ͻ 0.001) compared with wt (supplemental Table 2). Login to comment 115 ABCC7 p.Gly622Asp ABCC7 p.Gly628Arg Because we observed a pronounced reduction of the current density for the mutants G622D and G628R, we incubated transfected HEK293 and BHK-21 cells with this corrector and analyzed the corresponding CFTR activity. Login to comment 117 ABCC7 p.Gly622Asp ABCC7 p.Gly628Arg Similarly in BHK-21 cells the iodide efflux responses stimulated by Fsk and genistein was significantly increased for G622D and G628R after treatment with the corrector (Fig. 4B). Login to comment 118 ABCC7 p.Gly622Asp ABCC7 p.Gly628Arg This observation indicates that the diminution of cAMP-induced Cl- current is probably due to the diminution or to the absence of a mature form of G622D and G628R mutants. Login to comment 119 ABCC7 p.Gly622Asp ABCC7 p.Gly628Arg In support of this hypothesis, Western blot analysis of cells treated with miglustat shows enhanced mature C-band for G622D and G628R mutants (Fig. 4A). Login to comment 122 ABCC7 p.His620Gln ABCC7 p.His620Gln ABCC7 p.Ser623Ala ABCC7 p.Ser623Ala 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 ABCC7 p.His620Gln ABCC7 p.Ser623Ala 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 ABCC7 p.His620Gln ABCC7 p.Ser623Ala Our results indicated that H620Q and S623A channels could be activated more rapidly than the other CFTR mutant channels studied. Login to comment 130 ABCC7 p.His620Gln ABCC7 p.His620Gln ABCC7 p.Ser623Ala ABCC7 p.Ser623Ala 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 131 ABCC7 p.Glu621Gly ABCC7 p.Glu621Gly On the contrary, a significant increase of I/V slope (Fig. 7A) in presence of MPB-91 was observed for E621G CFTR channels compared with wt (I/V slope of wt, 0.390 Ϯ 0.014, n ϭ 6; E621G, 0.628 Ϯ 0.018, n ϭ 8). Login to comment 133 ABCC7 p.Gly622Asp ABCC7 p.Gly628Arg MPB-91 stimulated the Cl- current for each CFTR mutants studied except the glycine mutants G622D and G628R (Fig. 6 and supplemental Table 3). Login to comment 134 ABCC7 p.Gly622Asp ABCC7 p.Gly622Asp ABCC7 p.Gly628Arg ABCC7 p.Gly628Arg For them, comparison of the corresponding IV slope (Fig. 7A) did not reveal significant differences between basal condition (I/V slope of G622D, 0.032 Ϯ 0.001, n ϭ 7; G628R, 0.046 Ϯ 0.002, n ϭ 5) and in presence of 50 ␮M MPB-91 (I/V slope of G622D, 0.037 Ϯ 0.001, n ϭ 7; G628R, 0.053 Ϯ 0.003, n ϭ 5) indicating the absence of a response of the two mutated channels to that agent. Login to comment 136 ABCC7 p.Gly622Asp ABCC7 p.Gly628Arg Results in Fig. 8 show a potentiation of the cAMP-dependent Cl- current by MPB-91 for wt channels but neither for G622D nor G628R FIGURE 5. Login to comment 138 ABCC7 p.Ser623Ala 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 141 ABCC7 p.Gly622Asp ABCC7 p.Gly628Arg We also tested analogues of MPB-91 (12), but again for the mutant G622D or G628R, the Cl-channel function of CFTR was not stimulated by MPB-95 and MPB-97 (Fig. 9A). Login to comment 143 ABCC7 p.Gly622Asp ABCC7 p.Gly622Asp ABCC7 p.Gly628Arg ABCC7 p.Gly628Arg Importantly, all of these CFTR activators stimulated the Cl-channel activity of G622D and G628R CFTR, suggesting the relative specificity of the effect observed in the presence of the benzoquinolizinium drugs (Fig. 9B). Login to comment 146 ABCC7 p.Gly622Asp ABCC7 p.Gly628Arg Using Western blot analysis, we now observed a strong decrease of the mature-glycosylated form for the two mutants G622D and G628R. Login to comment 158 ABCC7 p.Gly622Asp ABCC7 p.Gly628Arg Consistently, the defect in the maturation process induced by the G622D and G628R mutations, which causes the retention of the mutated CFTR protein, can be clearly explained by the key role of these glycine residues at the structure level. Login to comment 163 ABCC7 p.His620Gln ABCC7 p.Ser623Ala In particular, the cAMP-activated Cl- current densities elicited by H620Q and S623A channels were increased compared with wt. Login to comment 165 ABCC7 p.His620Gln For H620Q mutant, this increase could be explained by the reported increased of open probability (15). Login to comment 169 ABCC7 p.His620Gln The H620Q mutation might thus disturb the contacts existing between the beta hairpin and the P-loop of NBD1 and thus might result in a perturbation of the channel gating through an effect on the ATP binding and/or hydrolysis in the noncanonical site. Login to comment 173 ABCC7 p.Ser624Ala ABCC7 p.Glu621Gly Indeed, our recordings of Cl currents supported by CFTR mutants E621G and S624A were not different from the non-mutated channels stimulated either by Fsk or by MPB agents. Login to comment 176 ABCC7 p.Ser623Ala 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 ABCC7 p.His620Gln ABCC7 p.Ser624Ala ABCC7 p.Ser623Ala ABCC7 p.Glu621Gly In addition, MPB-91 was also able to activate H620Q, E621G, S623A, and S624A mutants. Login to comment 181 ABCC7 p.Glu621Gly The Cl- current densities elicited by these mutants, except for E621G, were similar to wt. Login to comment 182 ABCC7 p.His620Gln ABCC7 p.Ser623Ala Contrary to the activation by Fsk, no difference of kinetic parameters was detected with the two mutants H620Q and S623A. Login to comment 184 ABCC7 p.Gly622Asp ABCC7 p.Gly628Arg In contrast, for the two mutants G622D and G628R, no activation was recorded in the presence of MPB-91 or other MPB compounds. Login to comment 187 ABCC7 p.Gly622Asp ABCC7 p.Gly628Arg However, this is not the case since xanthine (isobutylmethylxanthine), RP-107, or iso- flavonoide (genistein) successfully stimulated the channel activity of G622D and G628R CFTR channels. Login to comment 188 ABCC7 p.Gly622Asp ABCC7 p.Gly628Arg Therefore, one could hypothesize that the mechanism of activation of CFTR by MPB was itself affected by the mutations G622D and G628R and that the binding site of MPB might be located, on the folded protein, in the vicinity of the last beta hairpin of CFTR NBD1. Login to comment 189 ABCC7 p.Gly622Asp ABCC7 p.Gly628Arg However, the perturbation induced by the G622D and G628R mutations on the overall NBD1 structure might be felt at long range and thus influence potential binding sites, which may be distant at the three-dimensional level. Login to comment 190 ABCC7 p.Glu621Gly For the CFTR E621G mutant, Cl- current activated by MPB-91 was increased compared with wt. Login to comment 193 ABCC7 p.Gly622Asp A sliding of the two NBDs has been predicted to occur when CFTR evolves toward a closed form of the channel (inward-facing conformation) (29) in which, FIGURE8.KineticofactivationofwholecellCl- currentsforwt,G622D,andG628RCFTR.A,representative time course of whole cell Cl- current densities at 40 mV recorded in presence of 1 ␮M Fsk and 1 ␮M ϩ50 ␮M MPB-91. Login to comment 196 ABCC7 p.Gly622Asp ABCC7 p.Gly628Arg Iodide efflux of G622D and G628R CFTR-expressing cells in the presence of different MPB compounds or different activators. Login to comment 202 ABCC7 p.Glu621Gly Thus, one might hypothesize that the E621G mutation may reinforce the effect of MPB-91 by acting on critical features of the NBD interface, for example by contributing to reinforce it. Login to comment