ABCMdb

PMID: 17036051

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 [PubMed]

Sentences

No. Mutations Sentence Comment

25 ABCC7 p.Cys592Ser ABCC7 p.Cys590Ser Functional expression was markedly diminished when C590 and C592 were both mutated to serine, regardless of whether the other 16 cysteines remained (data not shown) or had all been replaced by serines (Figure 2B, 16CS þ C590S/ C592S). Login to comment 26 ABCC7 p.Cys590Leu ABCC7 p.Cys592Val ABCC7 p.Cys590Val ABCC7 p.Cys592Leu Nor could C590 and C592 be replaced by alanine, threonine or phenylalanine (Figure 2B), but function was similar to that of the 16CS background when they were replaced by leucines (16CS þ C590L/C592L; Figure 2A and B) or valines (16CS þ C590V/C592V; Figure 2B). Login to comment 29 ABCC7 p.Cys592Ser ABCC7 p.Cys592Val ABCC7 p.Cys592Val ABCC7 p.Cys592Val ABCC7 p.Cys590Val ABCC7 p.Cys592Leu ABCC7 p.Cys592Leu ABCC7 p.Cys592Thr ABCC7 p.Cys592Ala ABCC7 p.Cys592Phe B Resting Stimulated 16CS+C590V/C592V 16CS+C590L/C592L 16CS+C590F/C592F 16CS+C590T/C592T 16CS+C590S/C592S 16CS+C590/C592 16CS+C590A/C592A A 200 s 5 µA WT CFTR Cys-free CFTR 16CS+C590L/C592L 0 25 50 75 100 125 150 175 WT CFTR Whole-oocyte conductance (µS) 16CS+C590V/C592V 16CS+C590/C592 C (2.5 ng cRNA) (20 ng cRNA) 250 160 105 75 kD cRNA (ng) 0.25 20 2.5 20 WT CFTR 16CS+ C590V/C592V 1 2 3 4 Mature Core D Uninjected oocyte 40 µM forskolin 40 µM forskolin 40 µM forskolin Washout Washout Washout Figure 2 Expression and function of cysteine-deficient CFTR channels in Xenopus oocytes. Login to comment 30 ABCC7 p.Cys590Leu ABCC7 p.Cys592Leu (A) Two-microelectrode voltage-clamp current recordings from uninjected oocyte and oocytes expressing WT CFTR (2.5 ng cRNA) or HA-tagged Cys-free CFTR 16CS þ C590L/C592L (20 ng cRNA); vertical current deflections monitor conductance, which was transiently increased by brief exposure to 40 mM forskolin (between arrows). Login to comment 33 ABCC7 p.Cys590Val (C) Conductances from oocytes injected with 2.5 ng cRNA, and measured 1 day later for WT (153717 mS, n ¼ 3), or 3 days later for HA-tagged 16CS mutants with C590/C592 (42710 mS, n ¼ 6) or C590V/C592 V (5173 mS, n ¼ 6). Login to comment 35 ABCC7 p.Cys592Val ABCC7 p.Cys590Val (D) WT CFTR and Cys-free CFTR (16CS þ C590V/C592V) were immunoprecipitated from membranes of oocytes injected with cRNA amounts indicated, and subjected to SDS-PAGE and Western blot analysis; arrows mark core-glycosylated and mature fully glycosylated CFTR. Login to comment 36 ABCC7 p.Cys592Val ABCC7 p.Cys590Val independently, Cys-free (16CS þ C590V/C592V) CFTR channels, like WT, required phosphorylation by PKA before they could be opened by ATP, closed upon ATP removal, and were activated half-maximally by B50 mM [ATP] (Supplementary Figure S1); their single-channel conductance was very slightly larger than that of WT CFTR channels. Login to comment 41 ABCC7 p.Phe508Ala Introduction of target cysteines for crosslinking studies On the basis of crystal structures of nucleotide-bound prokaryotic NBD homodimers and of monomeric NBD1 F508A from human CFTR, we made a homology model of the anticipated CFTR NBD1-NBD2 complex (Figure 3; see Materials and methods). Login to comment 55 ABCC7 p.Phe508Ala NNBD1 NBD2 A462 S605 S1347 S459 S434 D1336 V1379 A1374 S549 S1248 Figure 3 Homology model of a head-to-tail CFTR NBD1-NBD2 heterodimer, based on crystal structures of human CFTR NBD1 F508A and of ATPor AMPPNP-bound NBDs of other ABC proteins (Materials and methods). Login to comment 66 ABCC7 p.Ser1248Cys ABCC7 p.Ser1248Cys ABCC7 p.Ser549Cys ABCC7 p.Ser549Cys '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 67 ABCC7 p.Ser549Cys ABCC7 p.Ser434Cys The product band detected with antibody against the NH2-terminal half channel was of identical molecular mass to that identified with antibody against the COOH-terminal half channel, strongly sug- 250 150 100 75 434 459 462 549 605 1248 1336 1347 1374 1379 kDa A 5 µA 300 s (1-633) S549C and (634-1480) 9CS+S1248C Washout Washout Washout (1-633) and (634-1480) 9CS+D1336C (1-633) S434C and (634-1480) 9CS B C 549 549 no C no C no C 1248 1248 462 462 1336 1336 1347 1347 605 605 459 459 434 434 1374 1379 1379 0 20 40 60 80 100 0 2 4 6 8 Whole-oocyteconductance/expressionlevel Normalized expression level Resting conductance Stimulated conductance 1374 40 µM forskolin 40 µM forskolin 40 µM forskolin Figure 4 Expression (A, B) and function (A, C) of split CFTR channels containing introduced cysteines. Login to comment 78 ABCC7 p.Ser605Cys ABCC7 p.Ala1374Cys Central region, with S605C and A1374C Similar results were obtained with the pair of cysteines introduced at positions 605 and 1374, predicted to lie near the center of the proposed NBD1-NBD2 interface. Login to comment 82 ABCC7 p.Ala462Cys ABCC7 p.Ser434Cys ABCC7 p.Ser459Cys ABCC7 p.Val1379Cys ABCC7 p.Asp1336Cys ABCC7 p.Ser1347Cys '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 ABCC7 p.Ser1248Cys ABCC7 p.Ala462Cys ABCC7 p.Ser549Cys ABCC7 p.Ser434Cys ABCC7 p.Ser605Cys ABCC7 p.Ala1374Cys ABCC7 p.Ser459Cys ABCC7 p.Val1379Cys ABCC7 p.Asp1336Cys ABCC7 p.Ser1347Cys 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 94 ABCC7 p.Ser549Cys 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 (1-633) S549C and (634-1480) 9CS+S1248C Figure 6 Crosslinking across the 'NBD2` composite catalytic site, between position 1248 in NBD2 Walker A and position 549 in NBD1 LSGGQ. Login to comment 95 ABCC7 p.Ser1248Cys ABCC7 p.Ser549Cys 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 98 ABCC7 p.Ser605Cys 250 160 105 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) S605C and (634-1480) 9CS+A1374C Figure 7 Crosslinking between central region residues, 605 of NBD1 and 1374 of NBD2. Login to comment 99 ABCC7 p.Ser605Cys Western blots show CFTR half channels (1-633) S605C (left panel; lower arrow), (634-1480) 9CS þA1374C (right panel; core-glycosylated, B85-90 kDa, bands; fully glycosylated, lower arrow), and crosslinked product (both panels; arrows labeled X-link). Login to comment 101 ABCC7 p.Ala462Cys ABCC7 p.Ser434Cys ABCC7 p.Ser459Cys 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 ABCC7 p.Ala462Cys ABCC7 p.Ser1347Cys (A) CFTR half channels (1-633) A462C (left panel) and (634-1480) 9CS þ S1347C (right panel). Login to comment 106 ABCC7 p.Ser459Cys ABCC7 p.Val1379Cys (B) CFTR half channels (1-633) S459C (left panel) and (634-1480) 9CSþ V1379C (right panel). Login to comment 108 ABCC7 p.Ser434Cys ABCC7 p.Asp1336Cys (C) CFTR half channels (1-633) S434C (left panel) and (634-1480) 9CSþ D1336C (right panel), as well as crosslinked product (arrow labeled X-link, both panels). Login to comment 120 ABCC7 p.Ser1248Cys ABCC7 p.Ser1248Cys ABCC7 p.Ser549Cys ABCC7 p.Ser549Cys 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 ABCC7 p.Ser1248Cys ABCC7 p.Ser549Cys ABCC7 p.Ser549Cys ABCC7 p.Ser549Cys ABCC7 p.Ser434Cys ABCC7 p.Ser605Cys ABCC7 p.Ala1374Cys ABCC7 p.Ser459Cys ABCC7 p.Val1379Cys ABCC7 p.Asp1336Cys ABCC7 p.Asp1336Cys 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 128 ABCC7 p.Ser549Cys (B) Western blots show CFTR half channels (1-633) S549C (left panel, lower arrow) and (634-1480) 9CS þA1374C (right panel, core-glycosylated, B85-90-kDa bands; fully glycosylated, lower arrow), as well as crosslinked product (both panels, arrows labeled X-link). Login to comment 135 ABCC7 p.Ser1248Cys ABCC7 p.Ser1248Cys ABCC7 p.Ser1248Cys ABCC7 p.Ser549Cys ABCC7 p.Ser549Cys ABCC7 p.Ser549Cys ABCC7 p.Ser549Cys ABCC7 p.Ser549Cys 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 ABCC7 p.Ser1248Cys ABCC7 p.Ser1248Cys ABCC7 p.Ser549Cys ABCC7 p.Ser549Cys (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 ABCC7 p.Ser1248Cys ABCC7 p.Ser549Cys 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 ABCC7 p.Ser1248Cys ABCC7 p.Ser549Cys 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 ABCC7 p.Ser1248Cys ABCC7 p.Ser549Cys 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 180 ABCC7 p.Cys225Ser ABCC7 p.Cys866Ser ABCC7 p.Cys343Ser ABCC7 p.Cys128Ser CFTR with substitutions C128S, C225S, C343S and C866S was provided by Dr DC Dawson (OHSU, Portland, OR, USA). Login to comment 181 ABCC7 p.Cys276Ser ABCC7 p.Cys832Ser ABCC7 p.Cys76Ser Primers for the mutations C76S, C276S and C832S are listed in Table I. Login to comment 187 ABCC7 p.Ser1248Cys ABCC7 p.Ala462Cys ABCC7 p.Ser549Cys ABCC7 p.Ser434Cys ABCC7 p.Ser605Cys ABCC7 p.Ala1374Cys ABCC7 p.Ser459Cys ABCC7 p.Val1379Cys ABCC7 p.Asp1336Cys ABCC7 p.Ser1347Cys Primers for cysteine insertions S434C, S459C, A462C, S549C, S605C, S1248C, D1336C, S1347C, A1374C and V1379C are given in Table I. Login to comment 199 ABCC7 p.Cys276Ser ABCC7 p.Cys832Ser ABCC7 p.Cys76Ser ABCC7 p.Ser1248Cys ABCC7 p.Ala462Cys ABCC7 p.Cys590Leu ABCC7 p.Cys592Val ABCC7 p.Cys590Val ABCC7 p.Cys592Leu ABCC7 p.Cys592Thr ABCC7 p.Cys592Ala ABCC7 p.Cys592Phe ABCC7 p.Ser434Cys ABCC7 p.Ser605Cys ABCC7 p.Ala1374Cys ABCC7 p.Ser459Cys ABCC7 p.Val1379Cys ABCC7 p.Asp1336Cys ABCC7 p.Cys590Phe ABCC7 p.Cys590Thr ABCC7 p.Cys590Ala ABCC7 p.Leu1346Cys 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 220 ABCC7 p.Phe508Ala Structural alignments were created including only ATP- (or AMPPNP-) bound NBD structures (HisP, PDB ID 1B0U (Hung et al, 1998); MJ0796, 1L2T (Smith et al, 2002); MalK, 1Q12 (Chen et al, 2003); GlcV, 1OXV (Verdon et al, 2003) and HlyB, 1XEF (Zaitseva et al, 2005), as well as human CFTR NBD1 F508A, 1XMI (Lewis et al, 2005). Login to comment