ABCMdb

ABCC7 p.Ser549Cys

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Publications

PMID: 16442101 [PubMed] Frelet A et al: "Insight in eukaryotic ABC transporter function by mutation analysis."

No. Sentence Comment

200 A cysteine instead of a serine in the LSGGQ motif in either NBD1 (S549C) or NBD2 (S1347C) inhibited CFTR channel activity [70]. 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
67 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
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
94 250 160 105 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 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
128 (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 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

PMID: 19114635 [PubMed] Wang X et al: "Mutations at the signature sequence of CFTR create a Cd(2+)-gated chloride channel."

No. Sentence Comment

22 The mutants G551C, L548C, and S549C, all in the signature sequence of CFTR`s NBD1, show robust response to Cd2+ . Login to comment
49 For the G551C and S549C mutants, because they are ATP dependent, we used the current in the presence of 1 mM ATP as control. Login to comment
50 The fold increase of the current in the presence of Cd2+ was normalized to the maximal fold increase for each mutant (100 μM for G551D, 10 μM G551D, and 5 μM for S549C). Login to comment
102 A representative S549C-CFTR current recording is shown in Fig. 6 A. Cd2+ elicited macroscopic current even at sub-micromolar [Cd2+ ]. Login to comment
107 However, for L548C, S549C, and G551C, the specificity of the ligand is altered so that Cd2+ becomes more effective at gating Cd2+ Is More Potent on G551C than on G551D We considered two possible mechanisms for the effect of Cd2+ on G551D-CFTR. Login to comment
132 The Binding Partner of Cd2+ Is Likely To Be a Cysteine Residue The micromolar affinity of Cd2+ in activating G551C or S549C mutants raises the possibility that some endogenous cysteine(s) or histidine(s) may participate in form- Figure 4. Login to comment
148 D I S C U S S I O N Here, we show that micromolar concentrations of Cd2+ can dramatically increase the activity of G551D-CFTR, a disease-associated mutant, as well as G551C-CFTR, L548C, and S549C-CFTR, in the absence of ATP. Login to comment
157 (A) Representative S549C-CFTR current recording in the presence of different [Cd2+ ]. Login to comment
159 (C) Dose-response relationship for S549C-CFTR fitted with the Hill equation (solid line); K1/2 = 2.4 ± 0.8 μM and n = 1.12 ± 0.39. Login to comment
216 First, the apparent affinities for G551C and S549C are at low micromolar range, supporting the idea that multiple cysteines are involved in coordinating Cd2+ . Login to comment
226 Our preliminary data (not depicted) indeed suggest that this may be the case because removing all six cysteines in NBD2 does not seem to affect the action of Cd2+ on S549C-CFTR. 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

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
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
97 This corroborates impaired activity of S549C CFTR after permanent attachment of similarly sized (Fig. 2), but neutral, NEM (Cotten and Welsh, 1998). 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
104 Figure 3.ߓ Similarly rapid decay of current in S549C CFTR channels (containing a single target Cys in the active catalytic site) upon ATP washout (w/o) or modification by MTS reagents. Login to comment
105 (A and B) S549C CFTR channels were activated by 3 mM ATP (black bars below records) and modified by 50 &#b5;M MTSET+ (A; red bars below record) or 5 and 50 &#b5;M MTSACE (B; green bars below record), and modification was reversed by DTT (A, 10 mM; B, 20 mM; black bars above record). Login to comment
114 However, whereas that complete modification of a population of S549C channels by MTSET+ almost abolished channel current, steady-state modification of the same channels by MTSACE instead diminished ATP-activated current by only &#e07a;80% (see Fig. 4 B). Login to comment
115 The residual current depended on the presence of ATP and must have arisen from modified S549C CFTR channels containing an MTSACE adduct, rather than from a subpopulation of channels that avoided modification. Login to comment
118 If the residual current does flow through MTSACE-modified S549C CFTR channels, this implies that the timing of channel closure was little influenced by the modification (see Implications of functional observations on modified channels in Discussion). Login to comment
119 Modification of S549C in channels closed by removal of ATP To see whether S549C CFTR channels can be modified when they are closed, the MTS reagents were applied after removing ATP. 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
134 We next tested whether impairment of channel opening by the MTSET+ adduct simply reflected steric hindrance, independent of charge, by modifying S549C CFTR with neutral MTSACE (Fig. 3 B). Login to comment
136 As with S549C channels, application of MTSET+ (1 mM in the example in Fig. 5 A) to S1347C CFTR channels opening and closing in the presence of ATP caused rapid current decay, with a time constant (Fig. 5 C, red bar) comparable to that for current decline after ATP washout in the same patch (Fig. 5 C, left gray bar). Login to comment
138 However, unlike the near abolition of S549C CFTR current caused by MTSET+ , modification of S1347C channels by MTSET+ reduced ATP-activated current only by &#e07a;60% (see Fig. 6 B). Login to comment
142 This correlates well with the residual 4 &#b1; 1% (n = 10) of control current after modification of S549C CFTR by ࣙ50 &#b5;M MTSET+ applied in the presence of ATP (Fig. 4 B, right red bar; compare Fig. 3 A). Login to comment
144 This residual current amplitude agrees well with that after ࣙ50 &#b5;M MTSACE modification of S549C CFTR with ATP present (18 &#b1; 6%; n = 4; Fig. 4 B, right green bar; compare Fig. 3 B). Login to comment
146 Thus, closed S549C CFTR channels are readily modified by micromolar concentrations of MTSET+ , MTSACE, or MTSES&#e032; . Login to comment
147 The diminished open probability of modified S549C channels in ATP is smaller when the covalently attached adduct has a positive charge (MTSET+ ) than when it is neutral (MTSACE) or negatively charged (MTSES&#e032; ). Login to comment
148 Because unaltered decay rate of the residual current of MTSACE-modified S549C channels on ATP removal suggests little change in stability of the open state, this diminished open probability appears attributable (at least for the MTSACE adduct) to relative stabilization of the channel-closed state compared with unmodified S549C CFTR channels. Login to comment
149 Figure 4.ߓ S549C CFTR channels are readily modified by MTS reagents when closed. Login to comment
150 (A) Immediately after 60-s applications of 5 &#b5;M MTSET+ (red trace and bar), 100 &#b5;M MTSACE (green trace and bar), or 100 &#b5;M MTSES&#e032; (blue trace and bar) to closed S549C channels in the absence of ATP, brief exposures to 3 mM ATP (black bars below record) assessed residual channel activity; the time constant of Ca2+ - dependent Cl&#e032; current decay in this patch was 0.2 s. Login to comment
152 (B) Relative amplitude of residual ATP-dependent current (Iresidual %) of modified S549C channels. Login to comment
156 Modification of S1347C in channels closed by removal of ATP Closed S1347C channels in the absence of ATP, like closed S549C CFTR channels, were readily modified by MTS reagents. Login to comment
176 Delaying closure of S549C and S1347C channels delays their modification Evidently, engineered cysteines substituted for equivalent serines S549 in the catalytically active site and S1347 in the dead site are both readily accessible to small hydrophilic MTS reagents in closed CFTR channels. Login to comment
182 Accordingly, the current decay time constant on ATP withdrawal from S549C or S1347C CFTR channels bearing the K1250R mutation was slowed approximately 10-fold, to &#e07a;15 s (Fig. 7, A-C, gray fit curves and bars). Login to comment
183 Moreover, modification of both target cysteines, S549C by 50 &#b5;M MTSET+ (Fig. 7 A) and S1347C by 50 &#b5;M MTSACE (Fig. 7 B), was similarly slowed (Fig. 7, A-C). Login to comment
184 The ratios of the current decay time constants upon MTS modification in the presence of ATP (Fig. 7 C, red and green bars) to those upon ATP washout (Fig. 7 C, gray bars) therefore remained near unity, averaging 1.2 &#b1; 0.2 (n = 3) for MTSET+ action on S549C-K1250R, and 1.2 &#b1; 0.1 (n = 7) for MTSACE action on S1347C-K1250R (Fig. 7 D, red and green open bars). Login to comment
185 The matching time courses of current decline caused by ATP removal or to MTS modification of either target cysteine, S549C or S1347C, despite over an order of Figure 7.ߓ Hydrolysis-impairing mutation, K1250R, of the conserved Walker A lysine in the active composite site similarly slows current decay after ATP washout and upon MTS modification of both S549C and S1347C channels. Login to comment
186 (A and B) ATP-activated (3 mM, black bars below records) currents of S549C-K1250R (A) and S1347C-K1250R (B) CFTR channels with single-exponential fits to current decline upon ATP removal (gray, &#e074;ATP w/o) or modification (&#e074;MTS) by 50 &#b5;M MTSET+ (red) or MTSACE (green); 20 mM DTT (black bars above records) restored activation of currents by ATP; asterisks above the records mark brief activations of Ca2+ - dependent Cl&#e032; currents to monitor speed of solution exchange (0.3 s in A and 0.2 s in B). Login to comment
187 (C) Average &#e074;ATPw/o (gray bars) with corresponding average &#e074;MTS from the same patches (left, S549C-K1250R: gray bar, w/o, 17 &#b1; 3.8 s; red bar, MTSET+ , 20.9 &#b1; 7.6 s; n = 3 measurements in three patches; right, S1347C-K1250R: gray bar, w/o, 15.4 &#b1; 2.0 s; green bar, MTSACE, 18.6 &#b1; 3.5 s; n = 9 and 7 measurements, respectively, in three patches). Login to comment
188 (D) Averages of individual ratios of washout and modification time constants determined for each pair of measurements (from experiments of C; red open bar, S549C-K1250R, &#e074;MTSET/&#e074;ATPw/o, 1.2 &#b1; 0.2; green open bar, S1347C-K1250R, &#e074;MTSACE/&#e074;ATPw/o, 1.2 &#b1; 0.1). Login to comment
189 Error bars represent mean &#b1; SEM. also readily modified closed S549C CFTR (Fig. 8 A), diminishing ATP-activated current to &#e07a;5% of control (Fig. 8 C). Login to comment
191 The &#e07a;19 &#d7; &#e07a;7 &#d7; &#e07a;6-&#c5; (Fig. 2) reagent MTS-biotin (5 &#b5;M for 30 or 60 s; Fig. 8 B) similarly modified closed S549C CFTR channels, on average diminishing ATP-activated current to &#e07a;15% of control (Fig. 8 C), and the modification was promptly reversed by DTT (Fig. 8 B). Login to comment
199 Closed S549C channels are also readily modified by larger MTS reagents The substituted ethyl-MTS reagents used so far, MTSET+ , MTSACE, and MTSES&#e032; , all approximate cylinders &#e07a;12 &#c5; long and &#e07a;6 &#c5; in diameter (Fig. 2). Login to comment
201 We first examined S549C CFTR channels closed by withdrawal of ATP, as in Figs. 4 and 6. Login to comment
204 Larger still MTS-rhodamine (&#e07a;22 &#d7; &#e07a;14 &#d7; &#e07a;8 &#c5;; Fig. 2), applied for 60 s at 5 &#b5;M (Fig. 8 A) Figure 8.ߓ Larger MTS reagents also readily modify S549C CFTR channels when they are closed. Login to comment
205 (A and B) ATP-activated current (3 mM, black bars below record) was strongly diminished after modification of closed S549C CFTR channels by ࣘ60-s exposures to larger MTS reagents, 20 &#b5;M MTS-glucose (A; orange bar), 5 &#b5;M MTS-rhodamine (A; magenta bar), 5 &#b5;M MTS-biotin (B; dark yellow bar), and MTS-biotin-avidin complex (B; 5 &#b5;M biotin plus 5 &#b5;M avidin, cyan bar), all in the absence of ATP; 10 mM DTT (black bars above records) released adducts after each modification; the time constants of Ca2+ -dependent Cl&#e032; current decays in these patches were 0.5 s (A) and 0.2 s (B). Login to comment
206 (C) Amplitude of residual ATP-activated current (Iresidual %), relative to ATP-activated current before modification, for S549C channels modified, while closed (in 0 ATP), by MTS-biotin (dark yellow bar, 15 &#b1; 3%, n = 7 measurements), by MTS-glucose (orange bar, 7 &#b1; 1%, n = 3 measurements), by MTS-rhodamine (magenta bar, 5 &#b1; 2%, n = 5 measurements), or by MTS-biotin-avidin complex (cyan bar, 90 &#b1; 8%, n = 5 measurements). Login to comment
214 We conclude, therefore, that the MTS-biotin-avidin complex failed to modify S549C in the catalytically competent site in closed CFTR channels because separation of the NBD1 tail from the NBD2 head was insufficient to allow the complex to reach the target cysteine. Login to comment
227 Unlike the readily reversible modification of S549C or S1347C targets, however, the MTSET+ adduct was only poorly, if at all, released from MTSET+ -modified S605C channels by up to 85-s exposures to 10 mM DTT. Login to comment
233 Timing, and gating-state dependence, of modification Ready reversal of MTS modification of target cysteines S549C and S1347C by DTT reduction of the mixed disulfide bonds, to release the adducts deposited during the reaction with MTS reagents, allowed the CFTR channels in each patch to serve as their own controls. Login to 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
282 In a single patch of S549C channels subjected to repeated exposures to three concentrations of MTSET+ in ATP, the mean current decay time constant in response to near saturating, 1 mM, MTSET+ was 1.1 &#b1; 0.1 s (n = 4), to 50 &#b5;M MTSET+ was 1.9 &#b1; 0.1 s (n = 8), and to 5 &#b5;M MTSET+ was 4.0 &#b1; 0.2 s (n = 6), whereas a further approximately ninefold slowing was observed in other patches on lowering [MTSET+ ] from 5 to 0.5 &#b5;M. Login to comment
285 From the approximately linear dependence of reaction rate on reagent concentration at low micromolar levels, and the 4-s modification time constant at 5 &#b5;M, the second-order reaction rate constant for MTSET+ modification of S549C is estimated to be 5 &#d7; 104 M&#e032;1 s&#e032;1 . 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
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
390 The residual current after MTSACE modification of S549C was &#e07a;18%, and of S1347C was &#e07a;20%, of control current, indicating that the neutral MTS adduct, whether in the active or dead composite site, made channel opening about fivefold less probable (assuming control Po of &#e07a;0.1 for both S549C and S1347C; compare Csan&#e1;dy et al., 2000; Vergani et al., 2003; Fig. S1 in Mense et al., 2006). Login to comment
392 In contrast, the residual current after MTSET+ modification varied with position along the dimer interface, and was &#e07a;4% of control with the adduct at S549C (Fig. 4), 10-20% at A1374C (Fig. 11), &#e07a;20% at S605C (Fig. 10), and &#e07a;40% at S1347C (Fig. 6). Login to comment
420 One interpretation of the unaltered timing of S549C channel closure after MTSACE modification is that ATP hydrolysis at the active site still controls closing, even though the modified channels contain a 6-&#c5;-thick neutral adduct extending 8 &#c5; from the position of the signature-sequence serine that, when present, contacts the &#e067; phosphate of the hydrolyzed ATP. Login to comment
470 Effects of pyrophosphate and nucleotide analogs suggest a role for ATP hydrolysis in cystic any MTS modification of open S549C channels after phosphate release would be limited to <10% of the open time, i.e., <1% of the gating cycle for Po of &#e07a;0.1. Login to comment