ABCMdb

ABCC7 p.Cys832Ser

ClinVar:	c.2496C>A , p.Cys832* ? , not provided
Predicted by SNAP2:	A: N (82%), D: N (57%), E: N (61%), F: N (72%), G: N (66%), H: N (78%), I: N (72%), K: N (66%), L: N (72%), M: N (66%), N: N (78%), P: N (53%), Q: N (78%), R: N (66%), S: N (78%), T: N (78%), V: N (87%), W: N (66%), Y: N (72%),
Predicted by PROVEAN:	A: N, D: D, E: N, F: N, G: N, H: N, I: N, K: N, L: N, M: N, N: D, P: D, Q: N, R: N, S: N, T: N, V: N, W: N, Y: N,

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[hide] The DeltaF508 mutation disrupts packing of the tra... J Biol Chem. 2004 Sep 17;279(38):39620-7. Epub 2004 Jul 21. Chen EY, Bartlett MC, Loo TW, Clarke DM
The DeltaF508 mutation disrupts packing of the transmembrane segments of the cystic fibrosis transmembrane conductance regulator.
J Biol Chem. 2004 Sep 17;279(38):39620-7. Epub 2004 Jul 21., 2004-09-17 [PMID:15272010]

Abstract [show]
The most common mutation in cystic fibrosis (deletion of Phe-508 in the first nucleotide binding domain (DeltaF508)) in the cystic fibrosis transmembrane conductance regulator (CFTR) causes retention of the mutant protein in the endoplasmic reticulum. We previously showed that the DeltaF508 mutation causes the CFTR protein to be retained in the endoplasmic reticulum in an inactive and structurally altered state. Proper packing of the transmembrane (TM) segments is critical for function because the TM segments form the chloride channel. Here we tested whether the DeltaF508 mutation altered packing of the TM segments by disulfide cross-linking analysis between TM6 and TM12 in wild-type and DeltaF508 CFTRs. These TM segments were selected because TM6 appears to line the chloride channel, and cross-linking between these TM segments has been observed in the CFTR sister protein, the multidrug resistance P-glycoprotein. We first mapped potential contact points in wild-type CFTR by cysteine mutagenesis and thiol cross-linking analysis. Disulfide cross-linking was detected in CFTR mutants M348C(TM6)/T1142C(TM12), T351C(TM6)/T1142C(TM12), and W356C(TM6)/W1145C(TM12) in a wild-type background. The disulfide cross-linking occurs intramolecularly and was reducible by dithiothreitol. Introduction of the DeltaF508 mutation into these cysteine mutants, however, abolished cross-linking. The results suggest that the DeltaF508 mutation alters interactions between the TM domains. Therefore, a potential target to correct folding defects in the DeltaF508 mutant of CFTR is to identify compounds that promote correct folding of the TM domains.

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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

[hide] In vivo phosphorylation of CFTR promotes formation... EMBO J. 2006 Oct 18;25(20):4728-39. Epub 2006 Oct 12. Mense M, Vergani P, White DM, Altberg G, Nairn AC, Gadsby DC
In vivo phosphorylation of CFTR promotes formation of a nucleotide-binding domain heterodimer.
EMBO J. 2006 Oct 18;25(20):4728-39. Epub 2006 Oct 12., 2006-10-18 [PMID:17036051]

Abstract [show]
The human ATP-binding cassette (ABC) protein CFTR (cystic fibrosis transmembrane conductance regulator) is a chloride channel, whose dysfunction causes cystic fibrosis. To gain structural insight into the dynamic interaction between CFTR's nucleotide-binding domains (NBDs) proposed to underlie channel gating, we introduced target cysteines into the NBDs, expressed the channels in Xenopus oocytes, and used in vivo sulfhydryl-specific crosslinking to directly examine the cysteines' proximity. We tested five cysteine pairs, each comprising one introduced cysteine in the NH(2)-terminal NBD1 and another in the COOH-terminal NBD2. Identification of crosslinked product was facilitated by co-expression of NH(2)-terminal and COOH-terminal CFTR half channels each containing one NBD. The COOH-terminal half channel lacked all native cysteines. None of CFTR's 18 native cysteines was found essential for wild type-like, phosphorylation- and ATP-dependent, channel gating. The observed crosslinks demonstrate that NBD1 and NBD2 interact in a head-to-tail configuration analogous to that in homodimeric crystal structures of nucleotide-bound prokaryotic NBDs. CFTR phosphorylation by PKA strongly promoted both crosslinking and opening of the split channels, firmly linking head-to-tail NBD1-NBD2 association to channel opening.

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181 Primers for the mutations C76S, C276S and C832S are listed in Table I. Login to comment
199 For recording macroscopic currents of split CFTR channels in excised patches (Figure 10), oocytes were Table I Forward primers for site-directed mutagenesis PCR C76S 50 -GCCCTTCGGCGATcgTTTTTCTGGAG-30 C276S 50 -CTGTTAAGGCCTACTcCTGGGAAGAAGC-30 C832S 50 -CGAAGAAGACCTTAAGGAGTcCTTTTTTGATGATATGGAGAGC-30 EagI site 50 -GGTAAAATTAAGCACAGcGGccGAATTTCATTCTGTTCTC-30 HA epitope 50 -CGGGCCGCCATGtAcccatAcGACGttccgGAttAcgcaAGGTCGCCTCTGG-30 CFTR 16CS C590A/C592A 50 -GGAGATCTTCGAGAGCgCTGTCgCTAAACTGATGGC-30 CFTR 16CS C590F/C592F 50 -GGAGATCTTCGAGAGCTtTGTCTtTAAACTGATGGC-30 CFTR 16CS C590L/C592L 50 -GGAGATCTTCGAGAGCctTGTCctTAAACTGATGGC-30 CFTR 16CS C590T/C592T 50 -GGAGATCTTCGAGAGCaCTGTCaCTAAACTGATGGC-30 CFTR 16CS C590V/C592V 50 -GGAGATCTTCGAGAGCgtcGTCgtTAAACTGATGGC-30 S434C 50 -CCTCTTCTTCAGTAATTTCTgtCTaCTTGGTACTCCTGTC-30 S459C 50 -GTTGGCGGTTGCTGGATgCACTGGAGCAGGCAAG-3 A462C 50 -GCTGGATCCACTGGGtgcGGCAAGACTTCACTTC-30 L549C 50 -GGTGGAATCACACtatGcGGAGGTCAACGAGCACG-30 S605C 50 -GGATTTTGGTCACaTgTAAAATGGAAC-30 S1248C 50 -CCTCTTGGGAAGAACCGGtTgtGGGAAGAGTAC-30 D1336C 50 -GTTTCCTGGGAAGCTTtgCTTTGTCCTTGTGG-30 L1346C 50 -GGATGGGGGCTCTGTCTgtAGTCATGGCCACAAGC-30 A1374C 50 -GATGAACCAAGCtgTCATTTAGATCC-30 V1379C 50 -GCTCATTTAGATCCgtgcACATACCAAATAATTCG-30 The underlined bases are the codons for the introduced serines, cysteines or other residues; lowercase letters mark base changes from the original sequence, including those for introducing diagnostic restriction endonuclease sites. Login to comment

[hide] Cystic fibrosis transmembrane conductance regulato... Biochemistry. 2009 Oct 27;48(42):10078-88. Alexander C, Ivetac A, Liu X, Norimatsu Y, Serrano JR, Landstrom A, Sansom M, Dawson DC
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.
Biochemistry. 2009 Oct 27;48(42):10078-88., 2009-10-27 [PMID:19754156]

Abstract [show]
The sixth transmembrane segment (TM6) of the CFTR chloride channel has been intensively investigated. The effects of amino acid substitutions and chemical modification of engineered cysteines (cysteine scanning) on channel properties strongly suggest that TM6 is a key component of the anion-conducting pore, but previous cysteine-scanning studies of TM6 have produced conflicting results. Our aim was to resolve these conflicts by combining a screening strategy based on multiple, thiol-directed probes with molecular modeling of the pore. CFTR constructs were screened for reactivity toward both channel-permeant and channel-impermeant thiol-directed reagents, and patterns of reactivity in TM6 were mapped onto two new, molecular models of the CFTR pore: one based on homology modeling using Sav1866 as the template and a second derived from the first by molecular dynamics simulation. Comparison of the pattern of cysteine reactivity with model predictions suggests that nonreactive sites are those where the TM6 side chains are occluded by other TMs. Reactive sites, in contrast, are generally situated such that the respective amino acid side chains either project into the predicted pore or lie within a predicted extracellular loop. Sites where engineered cysteines react with both channel-permeant and channel-impermeant probes occupy the outermost extent of TM6 or the predicted TM5-6 loop. Sites where cysteine reactivity is limited to channel-permeant probes occupy more cytoplasmic locations. The results provide an initial validation of two, new molecular models for CFTR and suggest that molecular dynamics simulation will be a useful tool for unraveling the structural basis of anion conduction by CFTR.

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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

[hide] CFTR: Ligand exchange between a permeant anion ([A... Biophys J. 2006 Sep 1;91(5):1737-48. Epub 2006 Jun 9. Serrano JR, Liu X, Borg ER, Alexander CS, Shaw CF 3rd, Dawson DC
CFTR: Ligand exchange between a permeant anion ([Au(CN)2]-) and an engineered cysteine (T338C) blocks the pore.
Biophys J. 2006 Sep 1;91(5):1737-48. Epub 2006 Jun 9., [PMID:16766608]

Abstract [show]
Previous attempts to identify residues that line the pore of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel have utilized cysteine-substituted channels in conjunction with impermeant, thiol-reactive reagents like MTSET+ and MTSES-. We report here that the permeant, pseudohalide anion [Au(CN)2]- can also react with a cysteine engineered into the pore of the CFTR channel. Exposure of Xenopus oocytes expressing the T338C CFTR channel to as little as 100 nM [Au(CN)2]- produced a profound reduction in conductance that was not reversed by washing but was reversed by exposing the oocytes to a competing thiol like DTT (dithiothreitol) and 2-ME (2-mercaptoethanol). In detached, inside out patches single-channel currents were abolished by [Au(CN)2]- and activity was not restored by washing [Au(CN)2]- from the bath. Both single-channel and macroscopic currents were restored, however, by exposing [Au(CN)2]- -blocked channels to excess [CN]-. The results are consistent with the hypothesis that [Au(CN)2]- can participate in a ligand exchange reaction with the cysteine thiolate at 338 such that the mixed-ligand complex, with a charge of -1, blocks the anion conduction pathway.

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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

[hide] Cysteine accessibility probes timing and extent of... J Gen Physiol. 2015 Apr;145(4):261-83. doi: 10.1085/jgp.201411347. Chaves LA, Gadsby DC
Cysteine accessibility probes timing and extent of NBD separation along the dimer interface in gating CFTR channels.
J Gen Physiol. 2015 Apr;145(4):261-83. doi: 10.1085/jgp.201411347., [PMID:25825169]

Abstract [show]
Cystic fibrosis transmembrane conductance regulator (CFTR) channel opening and closing are driven by cycles of adenosine triphosphate (ATP) binding-induced formation and hydrolysis-triggered disruption of a heterodimer of its cytoplasmic nucleotide-binding domains (NBDs). Although both composite sites enclosed within the heterodimer interface contain ATP in an open CFTR channel, ATP hydrolysis in the sole catalytically competent site causes channel closure. Opening of the NBD interface at that site then allows ADP-ATP exchange. But how frequently, and how far, the NBD surfaces separate at the other, inactive composite site remains unclear. We assessed separation at each composite site by monitoring access of nucleotide-sized hydrophilic, thiol-specific methanothiosulfonate (MTS) reagents to interfacial target cysteines introduced into either LSGGQ-like ATP-binding cassette signature sequence (replacing equivalent conserved serines: S549 and S1347). Covalent MTS-dependent modification of either cysteine while channels were kept closed by the absence of ATP impaired subsequent opening upon ATP readdition. Modification while channels were opening and closing in the presence of ATP caused macroscopic CFTR current to decline at the same speed as when the unmodified channels shut upon sudden ATP withdrawal. These results suggest that the target cysteines can be modified only in closed channels; that after modification the attached MTS adduct interferes with ATP-mediated opening; and that modification in the presence of ATP occurs rapidly once channels close, before they can reopen. This interpretation was corroborated by the finding that, for either cysteine target, the addition of the hydrolysis-impairing mutation K1250R (catalytic site Walker A Lys) similarly slowed, by an order of magnitude, channel closing on ATP removal and the speed of modification by MTS reagent in ATP. We conclude that, in every CFTR channel gating cycle, the NBD dimer interface separates simultaneously at both composite sites sufficiently to allow MTS reagents to access both signature-sequence serines. Relatively rapid modification of S1347C channels by larger reagents-MTS-glucose, MTS-biotin, and MTS-rhodamine-demonstrates that, at the noncatalytic composite site, this separation must exceed 8 A.

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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
81 Point mutations in the pGEMHE-wild-type template (Chan et al., 2000) yielded C832S and S549C-C832S constructs (Figs. S2 B and S3). 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
131 To confirm that this rapid MTSET+ modification of S549C CFTR channels did not depend on the eight C-terminal cysteine-to-serine mutations, we tested modification of S549C CFTR that otherwise differed from wild-type by only the single mutation C832S needed to render CFTR unresponsive to NEM (Cotten and Welsh, 1997). 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
302 Further evidence that the isosteric replacement of eight native cysteines little affected the results is the fact that our findings with the S549C target mutation in NBD1 (Fig. 3 A) were reproduced in an almost native background CFTR (C832S) lacking only 1 of the 18 cysteines (Fig. S3); likewise, NEM was found to rapidly modify both S549C and S1347C targets introduced into that same C832S CFTR background (Cotten and Welsh, 1998). Login to comment
310 They studied S549C-C832S CFTR (compare Fig. S3) and S1347C-C832S CFTR channels, at 35&#b0;C, in patches excised from HeLa cells, and found modification of S1347C "slightly slower" than that of S549C, but nevertheless concluded that the signature sequence lies in a "solvent-exposed position" and "at the protein surface" (Cotten and Welsh, 1998). 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