ABCMdb

PMID: 21486785

Jih KY, Li M, Hwang TC, Bompadre SG
The most common cystic fibrosis-associated mutation destabilizes the dimeric state of the nucleotide-binding domains of CFTR.
J Physiol. 2011 Jun 1;589(Pt 11):2719-31. Epub 2011 Apr 11., 2011-06-01 [PubMed]

Sentences

No. Mutations Sentence Comment

12 ABCC7 p.Phe494Asn ABCC7 p.Gln637Arg We found that both the PPi-induced locked-open time and the ATP/P-ATP ligand exchange time of F508-CFTR channels are dramatically shortened, suggesting that the F508 mutation destabilizes the full and partial NBD dimer states. We also tested if mutations that have been shown to improve trafficking of F508-CFTR, namely the solubilizing mutation F494N/Q637R and RI (deletion of the regulatory insertion), exert any effects on these newly identified functional defects associated with F508-CFTR. Login to comment 102 ABCC7 p.Glu1371Ser We introduced the E1371S mutation, which is known to demolish ATPase activity in ABC proteins including CFTR (Aleksandrov et al. 2000; Vergani et al. 2003; Bompadre et al. 2005), into WT and F508 backgrounds. Login to comment 103 ABCC7 p.Glu1371Ser ABCC7 p.Glu1371Ser For E1371S channels, the relaxation time constant of the current decay after ATP washout is ~110 s (Fig. 2A and C, Zhou et al. 2006), whereas that of F508/E1371S channels is only 32.45 ± 4.07 s (n = 4) (Fig. 2B and C). Login to comment 104 ABCC7 p.Glu1371Ser ABCC7 p.Glu1371Ser Although it is unclear why the F508 mutation shows less effect on the stability of NBD dimer under the E1371S background, the shortening of the locked-open time seen in F508/E1371S channels is consistent with the idea that the culprit is a destabilization of the NBD dimer rather than a lower affinity or efficacy of PPi. Login to comment 105 ABCC7 p.Trp401Phe ABCC7 p.His1348Gly Tight binding of nucleotides in NBD1 prolongs the channel locked-open time In a previous report (Tsai et al. 2010a), we demonstrated that the locked-open time of WT-CFTR induced by PPi is prolonged by replacing ATP with the high affinity ATP analogue N6 -phenylethyl-ATP (P-ATP), or by introducing 'gain-of-function` mutations to the ATP-binding site 1 (mutations which increase the Po of CFTR, such as W401F and H1348G) as the locked-open state reflects an NBD dimer with ATP-binding site 1 occupied by ATP and ATP-binding site 2 by PPi (Tsai et al. 2009). Login to comment 106 ABCC7 p.Trp401Phe ABCC7 p.His1348Gly In Fig. 3, we show that the gain-of-function mutations W401F and H1348G (Fig. 3A) and P-ATP (Fig. 3B) also prolong the locked-open time of F508-CFTR channels. Login to comment 107 ABCC7 p.Trp401Phe ABCC7 p.Trp401Phe ABCC7 p.His1348Gly Compared to F508-CFTR, the double mutant W401F/ F508-CFTR ( F508/DM)prolongedthelocked-opentimeby~2-fold, and the triple mutant W401F/H1348G/ F508-CFTR ( F508/TM) by ~4-fold. Login to comment 118 ABCC7 p.Glu1371Ser ABCC7 p.Glu1371Ser ABCC7 p.Glu1371Ser ABCC7 p.Glu1371Ser Comparison of the locked-open time of E1371S- and F508/E1371S-CFTR Representative traces of non-hydrolytic E1371S-CFTR (A) and F508/E1371S-CFTR (B) in the presence of 1 mM ATP. Login to comment 119 ABCC7 p.Glu1371Ser ABCC7 p.Glu1371Ser C, summary of the locked-open times for E1371S-CFTR (n = 15) and F508/E1371S-CFTR (n = 4) (P < 0.01). Login to comment 127 ABCC7 p.Trp401Phe ABCC7 p.Trp401Phe ABCC7 p.His1348Gly C, summary of PPi locked-open times for each construct ( F508/DM: W401F/ F508-CFTR, F508/TM: W401F/H1348G/ F508-CFTR). Login to comment 143 ABCC7 p.Phe494Asn ABCC7 p.Gln637Arg We introduced into F508-CFTR the 'solubilizing mutations` F494N/Q637R (Pissarra et al. 2008) and the regulatory insertion deletion ( RI, deletion of residues 404-435) (Aleksandrov et al. 2010) to test whether they have any effects on the F508-CFTR gating defects described above. Login to comment 145 ABCC7 p.Phe494Asn ABCC7 p.Gln637Arg As seen in Figs 5A and 6A, in either case, the current relaxation upon removal of ATP and PPi was significantly slower compared with that for WT-CFTR (F494N/Q637R-CFTR: τ = 86.14 ± 12.61s, n = 6; RI-CFTR: τ = 75.33 ± 14.36 s, n = 7). Login to comment 147 ABCC7 p.Phe494Asn ABCC7 p.Gln637Arg For ligand exchange experiments, these mutations also significantly prolong the second phase of current changes upon switching the ligand from ATP to P-ATP (F494N/Q637R-CFTR: τ = 76.41 ± 12.31 s, n = 6; RI-CFTR: τ = 81.78 ± 6.66 s, n = 7) (Figs 5A, 6A and 7). Login to comment 149 ABCC7 p.Phe494Asn ABCC7 p.Phe494Asn ABCC7 p.Gln637Arg ABCC7 p.Gln637Arg We found that although the locked-open time (F494N/Q637R/ F508-CFTR: τ = 5.95 ± 0.36 s, n = 8; RI/ F508-CFTR: τ =5.52 ± 0.45 s, n = 11) and ligand exchange time (F494N/Q637R/ F508-CFTR: τ=8.44 ± 1.3s, n=6; RI/ Figure 5. Login to comment 150 ABCC7 p.Phe494Asn ABCC7 p.Phe494Asn ABCC7 p.Phe494Asn ABCC7 p.Gln637Arg ABCC7 p.Gln637Arg ABCC7 p.Gln637Arg Effects of 'solubilizing mutations`, F494N/Q637R, on WTand F508-CFTR channels A, representative current traces of F494N/Q637R-CFTR channels locked opened by 1 mM ATP and 2 mM PPi (left) and ATP/P-ATP ligand exchange (right). Login to comment 151 ABCC7 p.Phe494Asn ABCC7 p.Gln637Arg B, representative current traces of F494N/Q637R/ F508-CFTR channels locked opened by 1 mM ATP and 2 mM PPi (left) and ATP/P-ATP ligand exchange (right). Login to comment 157 ABCC7 p.Phe494Asn ABCC7 p.Gln637Arg F508-CFTR: τ = 8.95 ± 1.75 s, n = 4) of F494N/ Q637R/ F508 and F508/ RI channels are prolonged (Figs 5B, 6B and 7), they are still much shorter than those of WT channels. Login to comment 159 ABCC7 p.Phe494Asn ABCC7 p.Gln637Arg Besides prolonging the PPi locked-open time and the ligand exchange time, F494N/Q637R/ F508 and F508/ RI have been previously shown to improve the function of F508-CFTR (Pissarra et al. 2008; Aleksandrov et al. 2010). Login to comment 161 ABCC7 p.Phe494Asn ABCC7 p.Gln637Arg To our surprise, we found that P-dATP still increases the current dramatically for both compound mutants: 6.96 ± 0.17-fold increase for RI/ F508-CFTR (n = 5), and 12.36 ± 1.21-fold increase for F494N/Q637R-CFTR (n = 8) (Fig. 8). Login to comment 165 ABCC7 p.Phe494Asn ABCC7 p.Gln637Arg sol: solubilizing mutation, F494N/Q637R. Login to comment 170 ABCC7 p.Phe494Asn ABCC7 p.Gln637Arg Since P-dATP does not alter single channel conductance (Miki et al. 2010), the increase in the macroscopic current induced by P-dATP in F494N/Q637R/ F508 and F508/ RI (12-and 7-fold, respectively) suggests that the Po of these mutant channels is still lower than that of WT channels when ATP is used as the ligand. Login to comment 191 ABCC7 p.Phe494Asn ABCC7 p.Phe494Asn ABCC7 p.Gln637Arg ABCC7 p.Gln637Arg Effect of P-dATP on F494N/Q637R/ F508-CFTR and RI/ F508-CFTR Representative current traces of F494N/Q637R/ F508-CFTR (A) and RI/ F508-CFTR (B) in the presence of 50 μM P-dATP. Login to comment 192 ABCC7 p.Phe494Asn ABCC7 p.Gln637Arg C, current increase induced by P-dATP for F494N/Q637R/ F508-CFTR (n = 8) and RI/ F508-CFTR (n = 5). Login to comment