ABCMdb

ABCC7 p.Cys1458Ser

None has been submitted yet.

Publications

PMID: 15272010 [PubMed] Chen EY et al: "The DeltaF508 mutation disrupts packing of the transmembrane segments of the cystic fibrosis transmembrane conductance regulator."

No. Sentence Comment

57 The construction of Cys-less CFTR (C76S/C126S/C225S/C276S/C343S/C491S/C524S/C590S/C592S/C657S/C832S/C866S/C1344S/C1355S/C1395S/C1400S/C1410S/C1458S) was performed using the following cDNA fragments. Login to comment
58 Point mutations C76/126S were generated in sequence in the PstI (bp 1) 3 XbaI (bp 573) fragment; point mutations C225S/C276S/C343S were generated in sequence in the XbaI (bp 573) 3 KpnI (bp 1370) fragment; point mutations C491S/C524S/C590S/C592S/C657S were generated in sequence in the KpnI (bp 1370) 3 ApaI (bp 2333) fragment; point mutations C832S/C866S were generated in sequence in the ApaI (bp 2333) 3 EcoRI (bp 3643) fragment; point mutations C1344S/C1355S/ C1395S/C1400S/C1410S/C1458S were generated in sequence in the EcoRI (bp 3643) 3 XhoI (bp 4560) fragment, the five insert fragments were then ligated and inserted into the PstI and XhoI sites of plasmid vector pMT21. Login to comment

PMID: 19754156 [PubMed] Alexander C et al: "Cystic fibrosis transmembrane conductance regulator: using differential reactivity toward channel-permeant and channel-impermeant thiol-reactive probes to test a molecular model for the pore."

No. Sentence Comment

42 The Cys-less CFTR construct (C76S, C126S, C225S, C276S, C343S, C491S, C524S, C590L, C592L, C657S, C832S, C866S, C1344S, C1355S, C1395S, C1400S, C1410S, C1458S) was a gift from Drs. Martin Mense and David Gadsby and was used in their pGEMHE vector previously described (13). Login to comment

PMID: 16766608 [PubMed] Serrano JR et al: "CFTR: Ligand exchange between a permeant anion ([Au(CN)2]-) and an engineered cysteine (T338C) blocks the pore."

No. Sentence Comment

23 MATERIALS AND METHODS Mutagenesis and in vitro transcription The Cys-less CFTR construct (C76S, C126S, C225S, C276S, C343S, C491S, C524S, C590L, C592L, C657S, C832S, C866S, C1344S, C1355S, C1395S, C1400S, C1410S, C1458S) was a gift from Drs. Martin Mense and Submitted December 28, 2005, and accepted for publication May 19, 2006. Login to comment

PMID: 25825169 [PubMed] Chaves LA et al: "Cysteine accessibility probes timing and extent of NBD separation along the dimer interface in gating CFTR channels."

No. Sentence Comment

73 Fig. S2 shows tests of MTSET+ , MTSACE, and MTSES&#e032; effects on ATP-activated currents of background (C832S-C1458S) or C832S CFTR channels lacking any target cysteine. Login to comment
74 Fig. S3 shows the action of MTSET+ on the S549C target in the C832S background (missing only a single native cysteine) for comparison with the (C832S-C1458S) background. Login to comment
76 Fig. S5 shows that MTS-glucose, MTS-rhodamine, and MTS-biotin do not affect background (C832S-C1458S) CFTR channels. Login to comment
79 R E S U L T S Targeting S549C in the NBD1 tail, in the catalytically competent site, of CFTR channels opening and closing in ATP S549 in the LSGGQ sequence of the NBD1 tail contributes to CFTR`s catalytically competent composite site M A T E R I A L S A N D M E T H O D S Molecular biology The full-length human pGEMHE-CFTR (C832S-C1458S) construct, in which all eight C-terminal cysteines (C832, C866, C1344, C1355, C1395, C1400, C1410, and C1458 out of CFTR`s 18 native cysteines) were replaced by serine, was generated by de novo PCR gene synthesis combined with site-directed mutagenesis (Mense et al., 2006). Login to comment
95 MTS reagents (Toronto Research Chemicals) and avidin (Thermo Fisher Scientific) were (Fig. S2 A) by much higher concentrations of MTSET+ or of the similarly sized MTS reagents, negatively charged MTSES&#e032; , or neutral MTSACE; in these control (C832S-C1458S) CFTR channels, all eight native cysteines in the C-terminal half of CFTR have been replaced by serines, but all 10 native N-terminal cysteines remain (compare Mense et al., 2006). Login to comment
99 To assess accessibility of introduced S549C, we applied the small hydrophilic, sulfhydryl-specific MTS reagent MTSET+ (50 &#b5;M; Fig. 3 A) to S549C- (C832S-C1458S) CFTR channels opening and closing in inside-out patches exposed to 3 mM MgATP (Fig. 3 A). Login to comment
101 The current decay reflects modification of the introduced cysteine because background (C832S-C1458S) CFTR channels, lacking the engineered target cysteine, are little affected Figure 2.ߓ Size comparison of MTS reagents, nucleotides, NEM, and MTS-biotin-avidin complex, shown as CPK-colored spheres, except for AMPPNP (yellow spheres; from TM287/288 structure; PDB accession no. 3QF4) and avidin tetramer (orange ribbon). Login to comment
132 The current decline on modification of S549C-C832S CFTR by MTSET+ in 3 mM ATP (Fig. S3, red fit lines) was as rapid as that of S549C-(C832S-C1458S) CFTR (Fig. 3, A and C), and it similarly matched the time course of current decay on ATP washout (Fig. S3, gray fit line). Login to comment
133 However, because (C832S) background CFTR channels were slowly modified by MTSES&#e032; and MTSACE (Fig. S2 B), unlike (C832S-C1458S) background CFTR channels, which were insensitive to these reagents (Fig. S2 A), (C832S-C1458S) channels were adopted as the background for cysteine targets for the rest of this work. Login to comment
192 Control measurements with 50-&#b5;M applications of each of these larger MTS reagents confirmed that (like the smaller reagents; Fig. S2 A) they did not affect background (C832S-C1458S) CFTR channels with no added target cysteines (Fig. S5). Login to comment
241 (A) S605C-(C832S-C1458S) CFTR channels were activated by 3 mM ATP (black bars below records) and modified by 1 mM MTSET+ (red bar). Login to comment
261 (A and B) A1374C-(C832S-C1458S) CFTR channels were modified by 1 mM MTSET+ (red trace bar) either while closed in the absence of ATP (A), as assessed by subsequent diminished ATP-activated current (3 mM, black bars below records), or in the presence of ATP (B). Login to comment
287 As Po of Cys-free (16CS + C590V/C592V) CFTR was approximately twofold larger (Mense et al., 2006) than that of wild-type CFTR, which is &#e07a;0.1 under these conditions (Csan&#e1;dy et al., 2000; Vergani et al., 2003), if Po of these S549C- (C832S-C1458S) CFTR channels lies between these values, then our second-order rate constant estimate should be increased by up to 25%. Login to comment
300 The cysteine-depleted (C832S-C1458S) CFTR used as the background construct here lacks only eight native cysteines (via eight conservative Cys-Ser substitutions, from C832S to C1458S) and is the full-length (concatenated) version of the split CFTR (coexpressed halves) into which cysteine target pairs were introduced for cross-linking studies to define CFTR`s NBD1-NBD2 interface (Mense et al., 2006). Login to comment
329 Consistent with these small effects, the mean time constant of current decay on ATP withdrawal, a measure of open burst duration, averaged 1.0 &#b1; 0.1 s (n = 19) for our background (C832S-C1458S) CFTR channels (e.g., Fig. S2 A), and was unaltered after insertion of the dead-site signature-sequence target cysteine, S1347C (1.0 &#b1; 0.1 s; n = 22), but was somewhat slowed by the corresponding cysteine in the live site, S549C (2.2 &#b1; 0.2 s; n = 24). Login to comment
331 That closure of (C832S-C1458S) channels containing S1347C or S549C target cysteines is indeed rate-limited by ATP hydrolysis (like wild type) is confirmed by the order of magnitude slowing of closure caused by the addition of the hydrolysis-impairing K1250R mutation (Figs. 7 and S4). Login to comment
332 Moreover, the &#e07a;15-s time constants for nonhydrolytic closure of those S1347C-K1250R-(C832S-C1458S) and S549C-K1250R-(C832S-C1458S) channels upon ATP washout are no shorter than those, 6-9 s, of K1250R CFTR channels bearing no other mutation (Vergani et al., 2005; Csan&#e1;dy et al., 2006; Szollosi et al., 2010, 2011). Login to comment