ABCMdb

PMID: 10893239

Berger AL, Welsh MJ
Differences between cystic fibrosis transmembrane conductance regulator and HisP in the interaction with the adenine ring of ATP.
J Biol Chem. 2000 Sep 22;275(38):29407-12., 2000-09-22 [PubMed]

Sentences

No. Mutations Sentence Comment

8 ABCC7 p.Phe430Cys ABCC7 p.Phe429Cys ABCC7 p.Phe433Cys ABCC7 p.Phe1232Cys The mutant channels CFTR-F429C, F430C, F433C, and F1232C showed no difference from wild-type CFTR, indicating that either the residues were not accessible to modification, or cysteine modification did not affect function. Login to comment 9 ABCC7 p.Tyr1219Cys ABCC7 p.Phe446Cys Although modification inactivated CFTR-Y1219C more rapidly than wild-type CFTR, and inactivation of CFTR-F446C was nucleotide-dependent; failure of these mutations to alter gating suggested that Tyr1219 and Phe446 were not important for nucleotide binding. Login to comment 77 ABCC7 p.Phe430Cys ABCC7 p.Phe429Cys ABCC7 p.Phe433Cys ABCC7 p.Phe1232Cys ABCC7 p.Tyr1219Cys ABCC7 p.Phe446Cys When expressed in HeLa cells, CFTR-F429C, F430C, F433C, F446C, Y1219C, and F1232C all generated Cl-channel activity in excised, inside-out patches of membrane. Login to comment 98 ABCC7 p.Phe1232Cys ABCC7 p.Tyr1219Cys CFTR-Y1219C was more rapidly inactivated than wild-type, and CFTR-F1232C showed a tendency for more rapid inactivation (0.05 Ͻ p Ͻ 0.10). Login to comment 114 ABCC7 p.Phe430Cys ABCC7 p.Phe429Cys ABCC7 p.Phe433Cys ABCC7 p.Phe1232Cys A summary of these data is shown in Fig. 4B. Wild-type, CFTR-F429C, F430C, F433C, and F1232C all exhibited similar levels of activity after treatment with 200 ␮M MTSET in the absence of ATP. Login to comment 116 ABCC7 p.Tyr1219Cys In these experiments of modification without ATP, MTSET inhibited CFTR-Y1219C to a greater extent than wild-type CFTR and the other NBD mutants (Fig. 4, A and B). Login to comment 117 ABCC7 p.Tyr1219Cys However, because MTSET also inactivated CFTR-Y1219C faster than the other channels in the presence of 1 mM ATP (Table II), we expected a greater inhibition during the timed application of MTSET in the absence of ATP. Login to comment 118 ABCC7 p.Tyr1219Cys In the presence of ATP (Fig. 3), a 60-s treatment with MTSET reduced CFTR-Y1219C current to 47 Ϯ 7% (n ϭ 8) of the initial value. Login to comment 120 ABCC7 p.Tyr1219Cys When we subtract the measured channel rundown of 37 Ϯ 16% for CFTR-Y1219C (because of the time for washing and the absence of ATP), we expected a current that was 30% of the basal value. Login to comment 126 ABCC7 p.Phe430Cys ABCC7 p.Phe429Cys ABCC7 p.Phe433Cys ABCC7 p.Phe1232Cys ABCC7 p.Tyr1219Cys ABCC7 p.Phe446Cys Construct Po WT 0.29 Ϯ 0.04 F429C 0.21 Ϯ 0.03 F430C 0.30 Ϯ 0.03 F433C 0.21 Ϯ 0.03 F446C 0.34 Ϯ 0.06 Y1219C 0.26 Ϯ 0.07 F1232C 0.35 Ϯ 0.02 FIG. 2. Login to comment 142 ABCC7 p.Phe430Cys ABCC7 p.Phe429Cys ABCC7 p.Phe433Cys ABCC7 p.Phe1232Cys ABCC7 p.Tyr1219Cys ABCC7 p.Tyr1219Cys ABCC7 p.Phe446Cys CFTR variant k M -1 s-1 Wild-type 23.1 Ϯ 6.4 F429C 27.0 Ϯ 16.7 F430C 34.6 Ϯ 22.8 F433C 19.0 Ϯ 5.5 F446C 36.1 Ϯ 15.8 Y1219C 75.5 Ϯ 20.1* F1232C 68.5 Ϯ 19.6 not interfere with the ability of MTSET to inactivate CFTR-Y1219C. Login to comment 143 ABCC7 p.Tyr1219Cys To confirm more directly that the rate at which MTSET inactivated CFTR-Y1219C was independent of ATP, we examined the rate of inhibition in the presence of 25 ␮M ATP. Login to comment 146 ABCC7 p.Tyr1219Cys If ATP interferes with MTSET modification and inactivation of CFTR-Y1219C, we expect to see a faster inactivation rate constant. Login to comment 148 ABCC7 p.Tyr1219Cys ABCC7 p.Tyr1219Cys The rate of inactivation for CFTR-Y1219C in 25 ␮M ATP (140 Ϯ 81 M -1 s-1 , n ϭ 4) was ϳ4 times the rate of inactivation for wild-type CFTR in 25 ␮M ATP (38 Ϯ 3 M -1 s-1 , n ϭ 3), similar to the ratio of CFTR-Y1219C inactivation to wild-type CFTR inactivation in 1 mM ATP. Login to comment 150 ABCC7 p.Tyr1219Cys These results indicate that ATP did not alter MTSET modification of CFTR-Y1219C. Login to comment 151 ABCC7 p.Phe446Cys In contrast to the other channels, CFTR-F446C showed a more striking inhibition by MTSET in the absence than in the presence of ATP (compare Fig. 4, A and B, with Fig. 3) that was not explained by the rate of inhibition observed in the presence of ATP (Table II). Login to comment 155 ABCC7 p.Phe446Cys Reaction of CFTR-F446C with MTSET for 60 s in the presence of 1 mM ADP preserved almost all CFTR activity (Fig. 6). Login to comment 168 ABCC7 p.Tyr1219Cys Even though CFTR-Y1219C showed a FIG. 4. Login to comment 179 ABCC7 p.Tyr1219Cys MTSET inactivation of wild-type CFTR and CFTR-Y1219C-CFTR in 25 ␮M ATP. Login to comment 191 ABCC7 p.Phe446Cys CFTR-F446C was the only channel in which nucleotide altered the inactivation rate. Login to comment