ABCMdb

ABCC7 p.Ser1248Cys

None has been submitted yet.

Publications

PMID: 15623556 [PubMed] Berger AL et al: "Normal gating of CFTR requires ATP binding to both nucleotide-binding domains and hydrolysis at the second nucleotide-binding domain."

No. Sentence Comment

111 To directly compare these data with earlier results, we studied S1248C channels (22) and found that NEM altered gating in a similar way (Fig. 2 B and C). Login to comment
113 In addition, correspondence of the S1248F and NEM-modified S1248C data indicate that the gating effects of the S1248F substitution were not due to misfolding that might have occurred during channel biosynthesis. Login to comment
114 For WT and NEM-modified S1248C CFTR, increasing the ATP concentration from 1 mM to 10 mM produced only a small increase in current that was not affected by NEM modification (data not shown), indicating that the effects of NEM represented a block in ATP binding rather than a decreased affinity that could be overcome by increasing ATP concentration. Login to comment
128 (B) Recording from a membrane patch containing a small number of S1248C channels before and after treatment with 200 ␮M NEM. Login to comment
140 The finding that channels unable to bind nucleotide at NBD2 (S1248F and NEM-modified S1248C) had a normal burst duration suggested that the prolonged burst duration of K1250A (16-18, 20, 21) arose when ATP bound NBD2 but then did not undergo hydrolysis. Login to comment
141 To test this hypothesis, we combined the K1250A mutation with S1248C. Login to comment
221 Our results argue against this explanation because blocking NBD2 ATP binding (with either the S1248F mutation or NEM-modification of S1248C) did not prolong burst duration. Login to comment
233 However, it seems surprising that blocking ATP binding to NBD2 (with the S1248F mutation or the NEM-modified S1248C mutation) did not change the rate of channel closure compared to WT. Login to comment

PMID: 16246032 [PubMed] Vergani P et al: "Control of the CFTR channel's gates."

No. Sentence Comment

52 We first studied the functional consequence of thiol-specific cross-linking in CFTR channels containing cysteine residues introduced in the NBD1 tail (S549C) and in the NBD2 head (S1248C), in a background similar to that used for the biochemical Figure 2 Statistical coupling analysis detects co-evolution between two positions corresponding to CFTR`s Arg555 (putative hydrogen bond donor) and Thr1246 (putative hydrogen bond acceptor) (A) Side-chain distribution at acceptor position in total multiple sequence alignment (histogram on left) and in each of the two subsets of alignments obtained by 'fixing` the side chain at the donor site, either to an Arg (centre), or to a Lys residue (right). Login to comment

PMID: 17036051 [PubMed] Mense M et al: "In vivo phosphorylation of CFTR promotes formation of a nucleotide-binding domain heterodimer."

No. Sentence Comment

66 'NBD2` composite site, with S549C and S1248C In contrast to these results with single introduced cysteines, BMOE (flexible spacer, reactive groups p8 A˚ apart) or BMH (flexible spacer length, 16 A˚ ) application to oocytes coexpressing CFTR half channels (1-633) S549C and (634-1480) 9CS þ S1248C, with both target cysteines in the NBD2 composite catalytic site (Figure 3), yielded a clear crosslinked product (Figure 6, arrows labeled X-link) not seen without crosslinking reagent (lanes 1 and 9). Login to comment
86 Crosslinking was weaker, but still evident, 250 150 100 75 kDa - - - + + - + - + - - - + + - + - + - - - + + - + - + - - - + + - + - + - - - + + - + - + - - - + + - + - + fsk Anti-R-domainAnti-N-terminus BMOE BMH Background S434C S459C A462C S549C S605C - - - + + - + - + - - - + + - + - + - - - + + - + - + - - - + + - + - + - - - + + - + - + S1248C D1336C S1347C A1374C V1379C 250 150 100 75 50 Figure 5 The absence of efficient crosslinking when no, or only one, engineered cysteine is present. Login to comment
95 Western blots identify the NH2-terminal half channel (1-633), S549C (left panel; lower arrow), the COOH-terminal half channel (634-1480) 9CS þ S1248C (right panel; core-glycosylated, B85-90-kDa, bands; fully glycosylated, lower arrow), and cross-linked product (both panels; arrows labeled X-link). Login to comment
120 Although no residual current was seen when 200 mM Cu(II)(o-phenanthroline)2 was added during withdrawal of ATP from split CFTR channels containing only one target cysteine, either S549C (Figure 10A and D) or S1248C (Figure 10B and D), in patches containing both half channels (1-633) S549C and (634-1480) 9CS þ S1248C, a substantial persistent current was observed (Figure 10C and D). Login to comment
123 The six include three crosslinks across the NBD1 composite site (between the NBD1 head, containing the Walker motifs, and the NBD2 tail, containing the ABC signature sequence: C462-C1347, C459-C1379, and C434- C1336), one crosslink between central regions of NBD1 and NBD2 (C605-C1374), one crosslink between the NBD1-tail 250 150 100 75 50 kDa fsk BMOE BMH - - + - + + + - + + - +- - - - + -- + + 0ЊC23ЊC - - + - + + + - + + - +- - - - + -- + + 0ЊC23ЊC fsk BMOE BMH X-link CFTR 1-633 X-link CFTR 634-1480 Anti-R-domainAnti-N-terminus 1 2 3 4 5 6 7 8 9 10 11 12 13 14 (1-633) S549C and (634-1480) 9CS+A1374CB 250 150 100 75 kDa - + + - + - - - + fsk BMOE BMH - + + - + - - - + - + + - + - - - + - + + - + - - - + S459C/S1248C S549C/D1336C S549C/V1379C S605C/D1336C 250 150 50 Anti-R-domainAnti-N-terminus - + + - + - - - + S434C/A1374C A Two engineered cysteine control experiments Figure 9 Tests of crosslinking between NBD1 and NBD2 using other combinations of the target cysteines. Login to comment
135 Nor did we find convincing evidence for 'homodimeric` interactions of NBD1 or of NBD2 (Figures 5-9): for example, we saw no efficient crosslinking between two NH2-terminal half chan- 0.00 0.04 0.08 0.12 0.16 0.20 S549C S1248C S549C/S1248C (1-633) and (634-1480) 9CS+S1248C(1-633) S549C and (634-1480) 9CS (1-633) S549C and (634-1480) 9CS+S1248C DTT ATP+PKA DTT DTTATP+PKA ATP+PKA ATP+PKA ATP+PKA ATP+PKADTT Cu(phen)2 Cu(phen)2 Cu(phen)2 DTT DTT 50 s 50 s 100 pA100 pA 50 s 100 pA Closure in bath solution Closure in Cu(phen)2 I0 I0/Imax Imax BA C D *** 5 5 7 7 9 10 Figure 10 Functional consequence of crosslinking S549C to S1248C. Login to comment
136 (A-C) Currents activated by 5 mM ATP and 300 nM PKA catalytic subunit in thousands of split CFTR channels in inside-out patches excised from oocytes expressing NH2-terminal (1-633), and COOH-terminal (634-1480) 9CS, half channels containing only one target cysteine, S549C (A) or S1248C (B), or both S549C and S1248C (C). Login to comment
139 For S549C/S1248C channels I0/Imax is significantly different (***Pp0.0005, Student`s t-test) after closure in the presence of Cu(II)(o-phenanthroline)2 compared to its absence (bath solution). Login to comment
159 Functional consequences of crosslinking The nucleotide-independent residual current induced by Cu(II)(o-phenanthroline)2 in the split channels comprising (1-633) S549C and (634-1480) 9CS þ S1248C (Figure 10C) provides direct evidence that artificial stabilization of the NBD1-NBD2 heterodimer tends to keep the affected channels open. Login to comment
160 Detailed characterization of that stabilized open state must await further analysis, but preliminary recordings from patches containing few Cu(II)(o-phenanthroline)2-modified channels show that the disulfide bond between S549C and S1248C results in a high channel open probability in the absence of ATP, with a persistent open state repeatedly interrupted by temporary closures. Login to comment
187 Primers for cysteine insertions S434C, S459C, A462C, S549C, S605C, S1248C, D1336C, S1347C, A1374C and V1379C are given 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

PMID: 9822656 [PubMed] Cotten JF et al: "Covalent modification of the nucleotide binding domains of cystic fibrosis transmembrane conductance regulator."

No. Sentence Comment

37 1 The abbreviations used are: CFTR, cystic fibrosis transmembrane conductance regulator; NEM, N-ethylmaleimide; PKA, catalytic subunit of cAMP-dependent protein kinase; ABC, ATP-binding cassette; NBD, nucleotide binding domain; NBD1-Cys, A462C/C832A; NBD2-Cys, C832A/S1248C; TB, mean burst duration; g, single channel conductance; ␶cs, slow, long closed time interval; ␶o, open time interval; ␶, time constant for rate of NEM modification; Iϱ, percentage of current remaining following complete NEM modification; TES, N-tris[hydroxymethyl]methyl-2-aminoethanesulfonic acid. Login to comment
73 NEM inhibits NBD1-Cys (CFTR-A462C/C832A) and NBD2-Cys(CFTR-S1248C/C832A) channel activity in an ATP-dependent manner. A, CFTR-C832A; B, CFTR-NBD1-Cys; C, CFTR-NBD2-Cys. 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

259 Evidence that S549 (NBD1 tail) does indeed closely approach Walker A residue S1248 (NBD2 head) across the dimer interface in open CFTR channels comes from the demonstration that a Cu2+ -phenanthroline- induced disulfide bond between cysteine pair S549C and S1248C keeps the channels in prolonged open burst states long after removal of ATP (Mense et al., 2006). Login to comment