ABCMdb

ABCC7 p.Ser1347Cys

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

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
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

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

24 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 &#c5;. Login to comment
135 The time constant of current decline on ATP withdrawal in the patch Targeting S1347C in LSHGH in the NBD2 tail, in the catalytically incompetent site, of CFTR channels opening and closing in ATP The position equivalent to S549 in the signature motif of the noncatalytic composite site is S1347, in sequence LSHGH in the NBD2 tail. 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
139 The residual current in MTSET+ -modified S1347C channels required ATP and declined on ATP withdrawal with a time course similar to that of the S1347C channels before modification (Fig. 5 E, red hatched bar). Login to comment
140 Thus, compared with unmodified S1347C channels, the presence of the MTSET+ adduct appears to stabilize closed-channel states without greatly affecting the open burst duration. Login to comment
141 S1347C CFTR channels opening and closing in ATP are also rapidly modified by MTSACE (1 mM in Fig. 5 B), causing current to decay with a time course closely similar to that seen on ATP washout (Fig. 5, B and C); the ratio of these time constants for ࣙ50 &#b5;M MTSACE averaged red bar). 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
159 The &#e07a;20% residual current of S1347C channels modified by MTSACE in ATP (see Fig. 6 B) declined on ATP removal with a similar time constant to that of unmodified S1347C channels (Fig. 5 E, green hatched bar). Login to comment
160 The MTSACE adduct therefore also appears to stabilize closed conformations of modified, relative to those of unmodified, S1347C channels, but to little influence the mechanism that times channel closing. Login to comment
161 Figure 6.ߓ S1347C CFTR channels are readily modified by MTS reagents when closed. Login to comment
162 (A) Immediately after &#e07a;60-s applications of 50 &#b5;M MTSET+ (red trace and bar), MTSACE (green trace and bar), or MTSES&#e032; (blue trace and bar) to closed S1347C channels in the absence of ATP, brief exposures to 3 mM ATP (black bars below record) assessed residual channel activity. Login to comment
164 (B) Amplitude of residual ATP-dependent current (Iresidual %), relative to ATP-activated current before modification, for S1347C channels modified, while closed (left, 0 ATP), by MTSET+ (red bar, 33 &#b1; 8%; n = 4 measurements in four patches), by MTSACE (green bar, 24 &#b1; 6%; n = 3 measurements in three patches), or by MTSES&#e032; (blue bar, 16 &#b1; 5%; n = 3 measurements in three patches), or while opening and closing (right, 3 mM ATP), by ࣙ50 &#b5;M MTSET+ (red bar, 42.4 &#b1; 4.5%, n = 8 measurements in four patches) or by ࣙ50 &#b5;M MTSACE (green bar, 19.5 &#b1; 2.0%, n = 9 measurements in four patches). Login to comment
165 Error bars represent mean &#b1; SEM. Figure 5.ߓ Similarly rapid decay of current in S1347C CFTR channels (containing a single target Cys in the dead composite site) upon ATP washout (w/o) or modification by MTS reagents. Login to comment
166 (A and B) S1347C CFTR channels were activated by 3 mM ATP (black bars below records) and modified by 1 mM MTSET+ or MTSACE (A and B, red and green bars below records, respectively). Login to comment
174 Experimental functional evidence indeed strongly suggests that ATP-induced tight dimerization, at least at CFTR`s catalytically active NBD2 composite S1347C CFTR channels after closed-state modification averaged 33 &#b1; 8% (n = 4) after MTSET+ , 24 &#b1; 6% (n = 3) after MTSACE, and 16 &#b1; 5% (n = 3) after MTSES&#e032; , of the control ATP-activated current amplitude before modification (Fig. 6 B, left; red, green, and blue bars). Login to comment
175 Those proportions were comparable to the average residual current amplitudes after modification of S1347C channels in the continued presence of ATP by MTSET+ , 42 &#b1; 5% (n = 8), and by MTSACE, 20 &#b1; 2% (n = 8) (Fig. 6 B, right; red and green bars). 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
208 When MTS-biotin-avidin complex was added to S1347C channels in the presence of ATP, or was added with ATP during channel activation (e.g., Fig. 9 B), the current was not diminished (Fig. 9, B and C). Login to comment
211 As noted for the smaller MTS reagents, the residual current of S1347C channels covalently modified with these larger adducts decayed rapidly once ATP was removed (e.g., Fig. 9, A and B); when they could be compared, the time courses matched (within a factor of 2) those observed before modification for MTS-biotin (n = 3), MTS-glucose (n = 3), and MTS-rhodamine (n = 2). Login to comment
216 Larger MTS reagents also modify S1347C channels opening and closing in ATP Target cysteine 1347, in the inactive composite site, was also readily modified by MTS-glucose, MTS-biotin, and MTS-rhodamine. Login to comment
217 To gauge the speed of modification, the reagents were applied to S1347C channels opening and closing in the presence of ATP (Fig. 9, A and B). Login to comment
218 All three larger MTS reagents diminished S1347C CFTR current relatively rapidly, to a residual level &#e07a;15% of the control ATP-activated current before modification (Fig. 9 C). Login to comment
220 Using, instead, time courses of modification by 50 &#b5;M MTSACE as reference (e.g., Fig. 9 A), modification by MTS-biotin was 1.3-fold slower (n = 1), by MTS-glucose Figure 9.ߓ Larger MTS reagents relatively rapidly modify S1347C CFTR channels in the presence of ATP. Login to comment
221 (A and B) S1347C channels were activated by 3 mM ATP (black bars below records) and modified by 50 &#b5;M MTS-glucose (A and B; orange traces and bars), 50 &#b5;M MTSACE (A; green trace and bar), 50 &#b5;M MTS-biotin (A; dark yellow trace and bar), or 50 &#b5;M MTS-rhodamine (A; magenta trace and bar), but not by the MTS-biotin-avidin complex (B; 50 &#b5;M biotin plus 60 &#b5;M avidin, cyan trace and bar). Login to comment
224 (C) Amplitude of residual ATP-activated current (Iresidual %), as a fraction of ATP-dependent current before MTS treatment, for S1347C channels modified, while opening and closing (in 3 mM ATP), by MTS-biotin (dark yellow bar, 16 &#b1; 2%, n = 6 measurements in six patches), by MTS-glucose (orange bar, 14 &#b1; 3%, n = 6 measurements in three patches), by MTS-rhodamine (magenta bar, 18 &#b1; 6%, n = 6 measurements in five patches), and MTS-biotin-avidin complex (cyan bar, 119 &#b1; 2%, n = 3 measurements in two patches). 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
249 The inaccessibility of S1347C in open CFTR channels suggested by the present results argues that in the open-channel conformation, NBD1 and NBD2 are closely apposed also in the dead composite site, as they are in the active site (see below, Implications of functional observations on modified channels). 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
407 The fact that ATP-dependent current persisted after MTS modification means that CFTR channels continued to open and close, despite the presence of an &#e07a;8-&#c5; long, 6-&#c5; wide adduct covalently attached slowing of opening (C࢐O1; see above) and inferred severalfold speeding of nonhydrolytic closure (O1࢐C), could together explain the absence of measurable residual current of MTSACE-modified S1347C-K1250R channels. Login to comment
408 Assuming the K1250R mutation makes the rate k1 of the O1࢐O2 ATP hydrolysis step zero, the ATP washout time constant for unmodified S1347C-K1250R channels (Fig. 7 C) suggests that k&#e032;1 is &#e07a;0.06 s&#e032;1 , reflecting the considerable stability of the prehydrolytic NBD dimer. Login to comment
409 The ATP washout time constant for unmodified S1347C channels (Fig. 5 C) suggests that k1, which rate- limits hydrolytic closure, is &#e07a;1 s&#e032;1 (k2, the rate of post-hydrolytic dimer dissociation, O2࢐C, is &#e07a;11-fold faster than k1; Csan&#e1;dy et al., 2010). Login to comment
410 Because k1 is more than an order of magnitude larger than k&#e032;1, even the inferred severalfold increase of k&#e032;1 caused by the neutral adduct would be expected to little affect the hydrolysis-mediated closing rate of modified S1347C channels; and even larger destabilizing effects on k&#e032;1 might be masked by any tendency of the adducts to also delay ATP hydrolysis, i.e., to diminish k1. Login to comment
412 Finally, if the MTS adduct does destabilize the prehydrolytic dimer once S1347C-K1250R CFTR channels are modified, as the above analysis suggests, then our conclusion of strict state dependence of modification is further strengthened, particularly for nonhydrolytic channels. Login to comment
413 Otherwise, modification in the presence of ATP while S1347C-K1250R channels were open should have caused current to decay more rapidly than upon ATP washout before modification, contrary to observation (Figs. 7 and S4). Login to comment
418 Because no phosphate is released from the dead site, MTS access to S1347C requires dimer separation and hence channel closure, as for K1250R mutants. Login to comment
425 That observation is the apparent absence of residual current after MTSACE modification of S1374C-K1250R channels (Fig. 7 B) that are believed to close by nonhydrolytic dissociation of the NBD dimer (Vergani et al., 2005; Csan&#e1;dy et al., 2006, 2010), in contrast to the &#e07a;20% residual current after MTSACE observed (Figs. 5 B and 6 B) for S1347C channels that are closed by ATP hydrolysis. Login to comment
427 If the MTSACE adduct in the dead site influenced only channel opening, and exerted a similar approximate fivefold slowing effect on the opening of modified S1374C-K1250R channels as estimated above for modified S1347C channels, the residual current amplitude (percentage of control) of S1374C-K1250R ought to have been no smaller than that of S1347; in fact, it should be larger (ࣙ30%) because of their expected higher Po that results from slower closing. Login to comment
428 The lack of measurable (ࣙ5%) residual current in modified S1347C-K1250R channels, therefore, implies that the MTSACE adduct in the dead site exerted an additional effect, acceleration of nonhydrolytic closure; i.e., an increased rate k&#e032;1 of the step O1࢐C, the reversal of CFTR channel opening in the gating cycle C &#f083; O1࢐O2࢐C (Csan&#e1;dy et al., 2010). Login to comment