ABCMdb

ABCC7 p.Ala462Cys

Predicted by SNAP2:	C: D (80%), D: D (95%), E: D (95%), F: D (95%), G: D (91%), H: D (95%), I: D (91%), K: D (95%), L: D (95%), M: D (91%), N: D (95%), P: D (95%), Q: D (95%), R: D (95%), S: N (61%), T: D (91%), V: D (91%), W: D (95%), Y: D (95%),
Predicted by PROVEAN:	C: N, D: D, E: N, F: D, G: N, H: D, I: D, K: N, L: D, M: D, N: N, P: D, Q: N, R: D, S: N, T: N, V: D, W: D, Y: D,

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Publications
[hide] Normal gating of CFTR requires ATP binding to both... Proc Natl Acad Sci U S A. 2005 Jan 11;102(2):455-60. Epub 2004 Dec 27. Berger AL, Ikuma M, Welsh MJ
Normal gating of CFTR requires ATP binding to both nucleotide-binding domains and hydrolysis at the second nucleotide-binding domain.
Proc Natl Acad Sci U S A. 2005 Jan 11;102(2):455-60. Epub 2004 Dec 27., 2005-01-11 [PMID:15623556]

Abstract [show]
ATP interacts with the two nucleotide-binding domains (NBDs) of CFTR to control gating. However, it is unclear whether gating involves ATP binding alone, or also involves hydrolysis at each NBD. We introduced phenylalanine residues into nonconserved positions of each NBD Walker A motif to sterically prevent ATP binding. These mutations blocked [alpha-(32)P]8-N(3)-ATP labeling of the mutated NBD and reduced channel opening rate without changing burst duration. Introducing cysteine residues at these positions and modifying with N-ethylmaleimide produced the same gating behavior. These results indicate that normal gating requires ATP binding to both NBDs, but ATP interaction with one NBD is sufficient to support some activity. We also studied mutations of the conserved Walker A lysine residues (K464A and K1250A) that prevent hydrolysis. By combining substitutions that block ATP binding with Walker A lysine mutations, we could differentiate the role of ATP binding vs. hydrolysis at each NBD. The K1250A mutation prolonged burst duration; however, blocking ATP binding prevented the long bursts. These data indicate that ATP binding to NBD2 allowed channel opening and that closing was delayed in the absence of hydrolysis. The corresponding NBD1 mutations showed relatively little effect of preventing ATP hydrolysis but a large inhibition of blocking ATP binding. These data suggest that ATP binding to NBD1 is required for normal activity but that hydrolysis has little effect. Our results suggest that both NBDs contribute to channel gating, NBD1 binds ATP but supports little hydrolysis, and ATP binding and hydrolysis at NBD2 are key for normal gating.

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No. Sentence Comment
156 A462F reduced Po, as did treating A462C channels with NEM. Login to comment
168 Fig. 5 shows that NEM treatment markedly reduced both A462C and A462C͞K464A current. Login to comment
179 (A) Examples of recordings from CFTR-A462F, and of A462C channels before and after NEM treatment. 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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No. Sentence Comment
82 'NBD1` composite site, with A462C and S1347C, S459C and V1379C, and S434C and D1336C At the NBD1 composite site, we first examined crosslinking between positions homologous to those tested successfully at the NBD2 composite site. 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
101 250 160 105 75 50 Anti-R-domainAnti-N-terminus kDa fsk BMOE BMH - - + - + + + - + + - +- - - - + -- + + 0ЊC23ЊC X-link CFTR 1-633 1 2 3 4 5 6 7 8 + + - 23ЊC - - + - + + + - + + - +- - - - + -- + + 0ЊC23ЊC fsk BMOE BMH X-link CFTR 634-1480 9 10 11 12 13 14 + + - 15 16 23ЊC 0ЊC23ЊC 0ЊC23ЊC 0ЊC23ЊC 0ЊC23ЊC fsk BMOE BMH - - + - + + + - + + - +- - - - + -- + + X-link CFTR 1-633 1 2 3 4 5 6 7 fsk BMOE BMH X-link CFTR 634-1480 8 9 10 11 12 13 14 - - + - + + + - + + - +- - - - + -- + + 250 160 105 75 50 kDa fsk BMOE BMH - - + - + + + - + + - +- - - - + -- + + X-link CFTR 1-633 1 2 3 4 5 6 7 fsk BMOE BMH X-link CFTR 634-1480 8 9 10 11 12 13 14 - - + - + + + - + + - +- - - - + -- + + kDa 250 150 100 75 50 A B C (1-633) A462C and (634-1480) 9CS+S1347C (1-633) S459C and (634-1480) 9CS+V1379C (1-633) S434C and (634-1480) 9CS+D1336C Figure 8 Crosslinking across the 'NBD1` composite site. Login to comment
104 (A) CFTR half channels (1-633) A462C (left panel) and (634-1480) 9CS þ S1347C (right panel). 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

[hide] Covalent modification of the nucleotide binding do... J Biol Chem. 1998 Nov 27;273(48):31873-9. Cotten JF, Welsh MJ
Covalent modification of the nucleotide binding domains of cystic fibrosis transmembrane conductance regulator.
J Biol Chem. 1998 Nov 27;273(48):31873-9., 1998-11-27 [PMID:9822656]

Abstract [show]
The cytosolic nucleotide binding domains of cystic fibrosis transmembrane conductance regulator (NBD1 and NBD2) mediate ATP-dependent opening and closing of the Cl- channel pore. To learn more about NBD structure and function, we introduced a cysteine residue into the Walker A motif or the LSGGQ motif of each NBD and examined modification by N-ethylmaleimide (NEM). Covalent modification of either Walker A motif partially inhibited cystic fibrosis transmembrane conductance regulator channel activity, decreasing the open state probability by prolonging the long closed duration. An increase in cytosolic ATP concentration slowed the rate of modification. The data suggest that both NBDs interact with ATP to influence channel opening and that inhibition by NEM modification was in part due to decreased ATP binding. When cysteine was placed in the NBD2 Walker A motif, it was modified more rapidly than when it was placed in NBD1, suggesting that the NBDs are not structurally or functionally identical. Modification of a cysteine inserted in the LSGGQ motif of either NBD1 or NBD2 also inhibited channel activity. The rate of modification was comparable with that of a thiol in free solution, suggesting that the LSGGQ motif resides in a surface-exposed position in both NBDs.

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Sentences [show]
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