ABCMdb

PMID: 25825169

Chaves LA, Gadsby DC
Cysteine accessibility probes timing and extent of NBD separation along the dimer interface in gating CFTR channels.
J Gen Physiol. 2015 Apr;145(4):261-83. doi: 10.1085/jgp.201411347., [PubMed]

Sentences

No. Mutations Sentence Comment

22 ABCC7 p.Lys1250Arg This interpretation was corroborated by the finding that, for either cysteine target, the addition of the hydrolysis-impairing mutation K1250R (catalytic site Walker A Lys) similarly slowed, by an order of magnitude, channel closing on ATP removal and the speed of modification by MTS reagent in ATP. Login to comment 24 ABCC7 p.Ser1347Cys 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 73 ABCC7 p.Cys1458Ser ABCC7 p.Cys832Ser ABCC7 p.Cys832Ser Fig. S2 shows tests of MTSET+ , MTSACE, and MTSES&#e032; effects on ATP-activated currents of background (C832S-C1458S) or C832S CFTR channels lacking any target cysteine. Login to comment 74 ABCC7 p.Cys1458Ser ABCC7 p.Cys832Ser ABCC7 p.Cys832Ser ABCC7 p.Ser549Cys 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 76 ABCC7 p.Cys1458Ser ABCC7 p.Cys832Ser Fig. S5 shows that MTS-glucose, MTS-rhodamine, and MTS-biotin do not affect background (C832S-C1458S) CFTR channels. Login to comment 79 ABCC7 p.Cys1458Ser ABCC7 p.Cys832Ser ABCC7 p.Ser549Cys 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 80 ABCC7 p.Lys1250Arg This template served for subsequent individual Ser- to-Cys mutations at positions 549, 605, and 1,347; Ala-to-Cys at 1,374 (Fig. S1); and the hydrolysis-impairing mutation K1250R, all introduced using QuikChange (Agilent Technologies). Login to comment 81 ABCC7 p.Cys832Ser ABCC7 p.Cys832Ser ABCC7 p.Ser549Cys 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 95 ABCC7 p.Cys1458Ser ABCC7 p.Cys832Ser MTS reagents (Toronto Research Chemicals) and avidin (Thermo Fisher Scientific) were (Fig. S2 A) by much higher concentrations of MTSET+ or of the similarly sized MTS reagents, negatively charged MTSES&#e032; , or neutral MTSACE; in these control (C832S-C1458S) CFTR channels, all eight native cysteines in the C-terminal half of CFTR have been replaced by serines, but all 10 native N-terminal cysteines remain (compare Mense et al., 2006). Login to comment 97 ABCC7 p.Ser549Cys 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 ABCC7 p.Cys1458Ser ABCC7 p.Cys832Ser ABCC7 p.Ser549Cys ABCC7 p.Ser549Cys 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 101 ABCC7 p.Cys1458Ser ABCC7 p.Cys832Ser The current decay reflects modification of the introduced cysteine because background (C832S-C1458S) CFTR channels, lacking the engineered target cysteine, are little affected Figure 2.ߓ Size comparison of MTS reagents, nucleotides, NEM, and MTS-biotin-avidin complex, shown as CPK-colored spheres, except for AMPPNP (yellow spheres; from TM287/288 structure; PDB accession no. 3QF4) and avidin tetramer (orange ribbon). Login to comment 104 ABCC7 p.Ser549Cys 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 ABCC7 p.Ser549Cys (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 ABCC7 p.Ser549Cys 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 ABCC7 p.Ser549Cys 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 ABCC7 p.Ser549Cys 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 ABCC7 p.Ser549Cys ABCC7 p.Ser549Cys 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 ABCC7 p.Cys832Ser ABCC7 p.Ser549Cys ABCC7 p.Ser549Cys 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 ABCC7 p.Cys1458Ser ABCC7 p.Cys832Ser ABCC7 p.Cys832Ser ABCC7 p.Ser549Cys ABCC7 p.Ser549Cys 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 133 ABCC7 p.Cys1458Ser ABCC7 p.Cys1458Ser ABCC7 p.Cys832Ser ABCC7 p.Cys832Ser ABCC7 p.Cys832Ser However, because (C832S) background CFTR channels were slowly modified by MTSES&#e032; and MTSACE (Fig. S2 B), unlike (C832S-C1458S) background CFTR channels, which were insensitive to these reagents (Fig. S2 A), (C832S-C1458S) channels were adopted as the background for cysteine targets for the rest of this work. Login to comment 134 ABCC7 p.Ser549Cys 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 135 ABCC7 p.Ser1347Cys 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 ABCC7 p.Ser549Cys ABCC7 p.Ser1347Cys 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 ABCC7 p.Ser549Cys ABCC7 p.Ser1347Cys 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 ABCC7 p.Ser1347Cys ABCC7 p.Ser1347Cys 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 ABCC7 p.Ser1347Cys 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 ABCC7 p.Ser1347Cys 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 142 ABCC7 p.Ser549Cys 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 ABCC7 p.Ser549Cys 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 ABCC7 p.Ser549Cys Thus, closed S549C CFTR channels are readily modified by micromolar concentrations of MTSET+ , MTSACE, or MTSES&#e032; . Login to comment 147 ABCC7 p.Ser549Cys 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 ABCC7 p.Ser549Cys ABCC7 p.Ser549Cys 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 ABCC7 p.Ser549Cys Figure 4.ߓ S549C CFTR channels are readily modified by MTS reagents when closed. Login to comment 150 ABCC7 p.Ser549Cys (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 ABCC7 p.Ser549Cys (B) Relative amplitude of residual ATP-dependent current (Iresidual %) of modified S549C channels. Login to comment 156 ABCC7 p.Ser549Cys ABCC7 p.Ser1347Cys ABCC7 p.Ser1347Cys 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 ABCC7 p.Ser1347Cys ABCC7 p.Ser1347Cys 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 ABCC7 p.Ser1347Cys 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 ABCC7 p.Ser1347Cys Figure 6.ߓ S1347C CFTR channels are readily modified by MTS reagents when closed. Login to comment 162 ABCC7 p.Ser1347Cys (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 ABCC7 p.Ser1347Cys (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 ABCC7 p.Ser1347Cys 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 ABCC7 p.Ser1347Cys (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 ABCC7 p.Ser1347Cys 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 ABCC7 p.Ser1347Cys 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 ABCC7 p.Ser549Cys ABCC7 p.Ser1347Cys 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 181 ABCC7 p.Lys1250Ala ABCC7 p.Lys1250Arg In CFTR channels mutated at NBD2 Walker A K1250, open burst durations are prolonged by about one (K1250R) or two (K1250A) orders of magnitude, in oocyte patches at room temperature (Vergani et al., 2003, 2005; Csan&#e1;dy et al., 2006). Login to comment 182 ABCC7 p.Lys1250Arg ABCC7 p.Ser549Cys ABCC7 p.Ser1347Cys 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 ABCC7 p.Ser549Cys ABCC7 p.Ser1347Cys 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 ABCC7 p.Lys1250Arg ABCC7 p.Lys1250Arg ABCC7 p.Ser549Cys ABCC7 p.Ser1347Cys 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 ABCC7 p.Lys1250Arg ABCC7 p.Ser549Cys ABCC7 p.Ser549Cys ABCC7 p.Ser1347Cys ABCC7 p.Ser1347Cys 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 ABCC7 p.Lys1250Arg ABCC7 p.Lys1250Arg ABCC7 p.Ser549Cys ABCC7 p.Ser1347Cys (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 ABCC7 p.Lys1250Arg ABCC7 p.Lys1250Arg ABCC7 p.Ser549Cys ABCC7 p.Ser1347Cys (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 ABCC7 p.Lys1250Arg ABCC7 p.Lys1250Arg ABCC7 p.Ser549Cys ABCC7 p.Ser1347Cys (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 ABCC7 p.Ser549Cys 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 ABCC7 p.Ser549Cys 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 192 ABCC7 p.Cys1458Ser ABCC7 p.Cys832Ser Control measurements with 50-&#b5;M applications of each of these larger MTS reagents confirmed that (like the smaller reagents; Fig. S2 A) they did not affect background (C832S-C1458S) CFTR channels with no added target cysteines (Fig. S5). Login to comment 199 ABCC7 p.Ser549Cys 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 ABCC7 p.Ser549Cys We first examined S549C CFTR channels closed by withdrawal of ATP, as in Figs. 4 and 6. Login to comment 204 ABCC7 p.Ser549Cys 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 ABCC7 p.Ser549Cys (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 ABCC7 p.Ser549Cys (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 208 ABCC7 p.Ser1347Cys 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 ABCC7 p.Ser1347Cys 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 214 ABCC7 p.Ser549Cys 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 216 ABCC7 p.Ser1347Cys 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 ABCC7 p.Ser1347Cys 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 ABCC7 p.Ser1347Cys 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 ABCC7 p.Ser1347Cys 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 ABCC7 p.Ser1347Cys (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 ABCC7 p.Ser1347Cys (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 226 ABCC7 p.Ser605Cys The &#e07a;20% residual current of MTSET+ -modified S605C channels (Fig. 10 D) declined with a comparable time course to that of the unmodified channels on ATP withdrawal (Fig. 10, A and E). Login to comment 227 ABCC7 p.Ser549Cys ABCC7 p.Ser605Cys ABCC7 p.Ser1347Cys 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 228 ABCC7 p.Ala1374Cys A1374C CFTR channels, with the target in the NBD2 D-loop, were also readily modified by MTSET+ applied in the absence (Fig. 11 A) or presence of ATP (Fig. 11 B), in either case leaving a residual current 10-20% of control (Fig. 11 C). Login to comment 230 ABCC7 p.Ser605Cys ABCC7 p.Ala1374Cys In contrast to the irreversible modification of S605C channels, the MTSET+ adduct could be released from modified A1374C channels by treatment with DTT (Fig. 11, A and B). Login to comment 233 ABCC7 p.Ser549Cys ABCC7 p.Ser1347Cys 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 239 ABCC7 p.Ser605Cys The current of S605C CFTR channels opening and closing in the presence of ATP declined rapidly on modification by 1 mM MTSET+ (Fig. 10 A). Login to comment 240 ABCC7 p.Ser605Cys The time course was similar to that of the current decay when the Figure 10.ߓ Similarly rapid decay of current in S605C CFTR channels (containing a mid-interface target Cys-in the NBD1 H loop-between the two composite sites) upon ATP washout (w/o) or modification by MTSET+ . Login to comment 241 ABCC7 p.Cys1458Ser ABCC7 p.Cys832Ser ABCC7 p.Ser605Cys (A) S605C-(C832S-C1458S) CFTR channels were activated by 3 mM ATP (black bars below records) and modified by 1 mM MTSET+ (red bar). Login to comment 244 ABCC7 p.Ser605Cys (B) Amplitude of residual current (Iresidual %), relative to ATP-activated current before modification, for S605C channels modified in 3 mM ATP by 1 mM MTSET+ (red bar, 21 &#b1; 4%, n = 4 measurements in four patches). Login to comment 249 ABCC7 p.Ser1347Cys 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 253 ABCC7 p.Ser605Cys Moreover, relatively rapid modification, on the time scale of the gating cycle, of interfacial target cysteines S605C and removal argue that, once closed, each channel was modified before it could reopen. Login to comment 259 ABCC7 p.Ser1248Cys ABCC7 p.Ser549Cys 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 260 ABCC7 p.Ala1374Cys In addition, mutant cycle analysis indicates that NBD2 Walker A T1246 hydrogen bonds to NBD1 R555, Figure 11.ߓ Rapid decay of current in A1374C CFTR channels (containing a mid-interface target Cys-in the NBD2 D loop-between the two composite sites) upon ATP washout (w/o) or modification by MTSET+ . Login to comment 261 ABCC7 p.Cys1458Ser ABCC7 p.Cys832Ser ABCC7 p.Ala1374Cys (A and B) A1374C-(C832S-C1458S) CFTR channels were modified by 1 mM MTSET+ (red trace bar) either while closed in the absence of ATP (A), as assessed by subsequent diminished ATP-activated current (3 mM, black bars below records), or in the presence of ATP (B). Login to comment 264 ABCC7 p.Ala1374Cys (C) Amplitude of residual current (Iresidual %), relative to ATP-activated current before modification, for A1374C channels modified, while closed (left, 0 ATP), by 5 &#b5;M-1 mM MTSET+ (red bar, 9 &#b1; 2%; n = 8 measurements in six patches), or while opening and closing (right, 3 mM ATP), by 10 &#b5;M-1 mM MTSET+ (red bar, 17 &#b1; 6%, n = 3 measurements in two patches). Login to comment 280 ABCC7 p.Ala1374Cys Analysis of tryptic digests (Aleksandrov et al., 2002, 2008) or of split A1374C, located between the two composite sites, implies separation all along the dimer interface each cycle. Login to comment 282 ABCC7 p.Ser549Cys 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 ABCC7 p.Ser549Cys 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 ABCC7 p.Cys1458Ser ABCC7 p.Cys832Ser ABCC7 p.Ser549Cys ABCC7 p.Cys592Val ABCC7 p.Cys590Val 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 300 ABCC7 p.Cys1458Ser ABCC7 p.Cys1458Ser ABCC7 p.Cys832Ser ABCC7 p.Cys832Ser The cysteine-depleted (C832S-C1458S) CFTR used as the background construct here lacks only eight native cysteines (via eight conservative Cys-Ser substitutions, from C832S to C1458S) and is the full-length (concatenated) version of the split CFTR (coexpressed halves) into which cysteine target pairs were introduced for cross-linking studies to define CFTR`s NBD1-NBD2 interface (Mense et al., 2006). Login to comment 302 ABCC7 p.Cys832Ser ABCC7 p.Cys832Ser ABCC7 p.Ser549Cys ABCC7 p.Ser549Cys ABCC7 p.Ser1347Cys 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 ABCC7 p.Cys832Ser ABCC7 p.Cys832Ser ABCC7 p.Ser549Cys ABCC7 p.Ser549Cys ABCC7 p.Ser1347Cys ABCC7 p.Ser1347Cys 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 328 ABCC7 p.Ser1347Gly Even in CFTR channels, the nonconservative mutation S1347G in the NBD2 signature sequence diminished open burst duration, and open probability, by only about one third (Tsai et al., 2010). Login to comment 329 ABCC7 p.Cys1458Ser ABCC7 p.Cys832Ser ABCC7 p.Ser549Cys ABCC7 p.Ser1347Cys 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 ABCC7 p.Cys1458Ser ABCC7 p.Cys832Ser ABCC7 p.Lys1250Arg ABCC7 p.Ser549Cys ABCC7 p.Ser1347Cys 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 ABCC7 p.Cys1458Ser ABCC7 p.Cys1458Ser ABCC7 p.Cys832Ser ABCC7 p.Cys832Ser ABCC7 p.Lys1250Arg ABCC7 p.Lys1250Arg ABCC7 p.Lys1250Arg ABCC7 p.Ser549Cys ABCC7 p.Ser1347Cys 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 346 ABCC7 p.Ser605Cys However, cysteines introduced at either location in CFTR are rapidly modified by MTSET+ (Figs. S6 and S7), and so both must be easily accessible; the reason for the impaired reversal of S605C modification by DTT is unclear but perhaps reflects electrostatic interaction with phosphates of the bound nucleotide. Login to comment 390 ABCC7 p.Ser549Cys ABCC7 p.Ser549Cys ABCC7 p.Ser1347Cys ABCC7 p.Ser1347Cys 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 ABCC7 p.Ser549Cys ABCC7 p.Ser605Cys ABCC7 p.Ala1374Cys ABCC7 p.Ser1347Cys 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 398 ABCC7 p.Lys1250Arg Closure of these cysteine-depleted channels containing a target cysteine is slowed at least an order of magnitude after the addition of the K1250R mutation (Figs. 7 and S4), arguing that closure of the unmodified channels, like that of wild-type CFTR, is normally rate-limited by hydrolysis of of hydrolysis appears sensitive to perturbations in and around the dead site, including mutation of the NBD1 Walker A motif (Powe et al., 2002; Vergani et al., 2003; Csan&#e1;dy et al., 2010, 2013) or NBD2 signature sequence (Tsai et al., 2010; Csan&#e1;dy et al., 2013), or replacement of the ATP bound there with an unnatural nucleotide (Tsai et al., 2010; Csan&#e1;dy et al., 2013). Login to comment 407 ABCC7 p.Lys1250Arg ABCC7 p.Ser1347Cys 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 ABCC7 p.Lys1250Arg ABCC7 p.Lys1250Arg ABCC7 p.Ser1347Cys 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 ABCC7 p.Ser1347Cys 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 ABCC7 p.Ser1347Cys 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 ABCC7 p.Lys1250Arg ABCC7 p.Ser1347Cys 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 ABCC7 p.Lys1250Arg ABCC7 p.Ser1347Cys 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 ABCC7 p.Lys1250Arg ABCC7 p.Ser1347Cys 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 420 ABCC7 p.Ser549Cys 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 425 ABCC7 p.Lys1250Arg ABCC7 p.Ser1347Cys 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 426 ABCC7 p.Lys1250Arg ABCC7 p.Lys1250Arg Opening rate of K1250R CFTR channels is maximal at 3 mM ATP (half-maximal [ATP] is &#e07a;150 &#b5;M; Szollosi et al., 2010) and comparable to that of wild-type CFTR (within a factor of 2 given the 10-fold longer open bursts, and Po of &#e07a;0.5, for K1250R; Vergani et al., 2005; Csan&#e1;dy et al., 2006, 2010; Szollosi et al., 2010). Login to comment 427 ABCC7 p.Lys1250Arg ABCC7 p.Lys1250Arg ABCC7 p.Ser1347Cys 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 ABCC7 p.Lys1250Arg ABCC7 p.Ser1347Cys 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 470 ABCC7 p.Ser549Cys 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