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Cysteine accessibility probes timing and extent of NBD separation along the dimer interface in gating CFTR channels.

Chaves LA, Gadsby DC - J. Gen. Physiol. (2015)

Bottom Line: These results suggest that the target cysteines can be modified only in closed channels; that after modification the attached MTS adduct interferes with ATP-mediated opening; and that modification in the presence of ATP occurs rapidly once channels close, before they can reopen.We conclude that, in every CFTR channel gating cycle, the NBD dimer interface separates simultaneously at both composite sites sufficiently to allow MTS reagents to access both signature-sequence serines.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 Å.

View Article: PubMed Central - HTML - PubMed

Affiliation: The Laboratory of Cardiac/Membrane Physiology, The Rockefeller University, New York, NY 10065.

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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. (A and B) S549C CFTR channels were activated by 3 mM ATP (black bars below records) and modified by 50 µM MTSET+ (A; red bars below record) or 5 and 50 µM MTSACE (B; green bars below record), and modification was reversed by DTT (A, 10 mM; B, 20 mM; black bars above record). Colored lines show single-exponential fits to current decay time courses on modification (τMTS) by MTSET+ (red) or by MTSACE (green), or on ATP removal (gray, τATPw/o) before modification, or on ATP removal after MTSACE modification (dotted green, τATPw/o(mod)); colored numbers give fit time constants; the time constants of Ca2+-dependent Cl− current decays in these patches were 0.3 s (A) and 0.2 s (B). (C) Average τATPw/o with corresponding average τMTS from the same patches (left: gray bar, w/o, 1.6 ± 0.2 s; red bar, ≥50 µM MTSET+, 1.4 ± 0.1 s; n = 6 measurements in five patches; right: gray bar, w/o, 3.5 ± 0.4 s; green bar, ≥50 µM MTSACE, 4.0 ± 1 s; n = 6 measurements in four patches); slower washout and modification time courses in the MTSACE experiments likely reflected slower superfusion influenced by patch geometry, but comparisons were always made within the same patch. (D) Averages of individual ratios of washout and modification time constants determined for each pair of measurements (from experiments of C; red open bar, τMTSET/τATPw/o, 0.9 ± 0.1; green open bar, τMTSACE/τATPw/o, 1.2 ± 0.3). (E) Average of washout time constant ratios, before and after MTSACE modification (green hatched bar, τATPw/o(mod)/τATPw/o, 0.9 ± 0.1; n = 5); absolute current amplitude of MTSACE-modified channels was 7–22 pA. Error bars represent mean ± SEM.
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fig3: 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. (A and B) S549C CFTR channels were activated by 3 mM ATP (black bars below records) and modified by 50 µM MTSET+ (A; red bars below record) or 5 and 50 µM MTSACE (B; green bars below record), and modification was reversed by DTT (A, 10 mM; B, 20 mM; black bars above record). Colored lines show single-exponential fits to current decay time courses on modification (τMTS) by MTSET+ (red) or by MTSACE (green), or on ATP removal (gray, τATPw/o) before modification, or on ATP removal after MTSACE modification (dotted green, τATPw/o(mod)); colored numbers give fit time constants; the time constants of Ca2+-dependent Cl− current decays in these patches were 0.3 s (A) and 0.2 s (B). (C) Average τATPw/o with corresponding average τMTS from the same patches (left: gray bar, w/o, 1.6 ± 0.2 s; red bar, ≥50 µM MTSET+, 1.4 ± 0.1 s; n = 6 measurements in five patches; right: gray bar, w/o, 3.5 ± 0.4 s; green bar, ≥50 µM MTSACE, 4.0 ± 1 s; n = 6 measurements in four patches); slower washout and modification time courses in the MTSACE experiments likely reflected slower superfusion influenced by patch geometry, but comparisons were always made within the same patch. (D) Averages of individual ratios of washout and modification time constants determined for each pair of measurements (from experiments of C; red open bar, τMTSET/τATPw/o, 0.9 ± 0.1; green open bar, τMTSACE/τATPw/o, 1.2 ± 0.3). (E) Average of washout time constant ratios, before and after MTSACE modification (green hatched bar, τATPw/o(mod)/τATPw/o, 0.9 ± 0.1; n = 5); absolute current amplitude of MTSACE-modified channels was 7–22 pA. Error bars represent mean ± SEM.

Mentions: S549 in the LSGGQ sequence of the NBD1 tail contributes to CFTR’s catalytically competent composite site and, by homology with nucleotide-bound NBD homodimer crystal structures, is expected to hydrogen bond to the γ phosphate of the ATP held by the NBD2 Walker A motif (Hopfner et al., 2000; Smith et al., 2002; Chen et al., 2003). To assess accessibility of introduced S549C, we applied the small hydrophilic, sulfhydryl-specific MTS reagent MTSET+ (50 µM; Fig. 3 A) to S549C-(C832S-C1458S) CFTR channels opening and closing in inside-out patches exposed to 3 mM MgATP (Fig. 3 A). Despite the maintained presence of ATP, the MTSET+ caused a rapid current decline (Fig. 3 A). The current decay reflects modification of the introduced cysteine because background (C832S-C1458S) CFTR channels, lacking the engineered target cysteine, are little affected (Fig. S2 A) by much higher concentrations of MTSET+ or of the similarly sized MTS reagents, negatively charged MTSES−, 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). Near abolition of CFTR current by MTSET+ while ATP was present indicates that CFTR channels with a covalently attached MTSET+ adduct at position 549 can essentially no longer be opened by ATP. This corroborates impaired activity of S549C CFTR after permanent attachment of similarly sized (Fig. 2), but neutral, NEM (Cotten and Welsh, 1998).


Cysteine accessibility probes timing and extent of NBD separation along the dimer interface in gating CFTR channels.

Chaves LA, Gadsby DC - J. Gen. Physiol. (2015)

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. (A and B) S549C CFTR channels were activated by 3 mM ATP (black bars below records) and modified by 50 µM MTSET+ (A; red bars below record) or 5 and 50 µM MTSACE (B; green bars below record), and modification was reversed by DTT (A, 10 mM; B, 20 mM; black bars above record). Colored lines show single-exponential fits to current decay time courses on modification (τMTS) by MTSET+ (red) or by MTSACE (green), or on ATP removal (gray, τATPw/o) before modification, or on ATP removal after MTSACE modification (dotted green, τATPw/o(mod)); colored numbers give fit time constants; the time constants of Ca2+-dependent Cl− current decays in these patches were 0.3 s (A) and 0.2 s (B). (C) Average τATPw/o with corresponding average τMTS from the same patches (left: gray bar, w/o, 1.6 ± 0.2 s; red bar, ≥50 µM MTSET+, 1.4 ± 0.1 s; n = 6 measurements in five patches; right: gray bar, w/o, 3.5 ± 0.4 s; green bar, ≥50 µM MTSACE, 4.0 ± 1 s; n = 6 measurements in four patches); slower washout and modification time courses in the MTSACE experiments likely reflected slower superfusion influenced by patch geometry, but comparisons were always made within the same patch. (D) Averages of individual ratios of washout and modification time constants determined for each pair of measurements (from experiments of C; red open bar, τMTSET/τATPw/o, 0.9 ± 0.1; green open bar, τMTSACE/τATPw/o, 1.2 ± 0.3). (E) Average of washout time constant ratios, before and after MTSACE modification (green hatched bar, τATPw/o(mod)/τATPw/o, 0.9 ± 0.1; n = 5); absolute current amplitude of MTSACE-modified channels was 7–22 pA. Error bars represent mean ± SEM.
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fig3: 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. (A and B) S549C CFTR channels were activated by 3 mM ATP (black bars below records) and modified by 50 µM MTSET+ (A; red bars below record) or 5 and 50 µM MTSACE (B; green bars below record), and modification was reversed by DTT (A, 10 mM; B, 20 mM; black bars above record). Colored lines show single-exponential fits to current decay time courses on modification (τMTS) by MTSET+ (red) or by MTSACE (green), or on ATP removal (gray, τATPw/o) before modification, or on ATP removal after MTSACE modification (dotted green, τATPw/o(mod)); colored numbers give fit time constants; the time constants of Ca2+-dependent Cl− current decays in these patches were 0.3 s (A) and 0.2 s (B). (C) Average τATPw/o with corresponding average τMTS from the same patches (left: gray bar, w/o, 1.6 ± 0.2 s; red bar, ≥50 µM MTSET+, 1.4 ± 0.1 s; n = 6 measurements in five patches; right: gray bar, w/o, 3.5 ± 0.4 s; green bar, ≥50 µM MTSACE, 4.0 ± 1 s; n = 6 measurements in four patches); slower washout and modification time courses in the MTSACE experiments likely reflected slower superfusion influenced by patch geometry, but comparisons were always made within the same patch. (D) Averages of individual ratios of washout and modification time constants determined for each pair of measurements (from experiments of C; red open bar, τMTSET/τATPw/o, 0.9 ± 0.1; green open bar, τMTSACE/τATPw/o, 1.2 ± 0.3). (E) Average of washout time constant ratios, before and after MTSACE modification (green hatched bar, τATPw/o(mod)/τATPw/o, 0.9 ± 0.1; n = 5); absolute current amplitude of MTSACE-modified channels was 7–22 pA. Error bars represent mean ± SEM.
Mentions: S549 in the LSGGQ sequence of the NBD1 tail contributes to CFTR’s catalytically competent composite site and, by homology with nucleotide-bound NBD homodimer crystal structures, is expected to hydrogen bond to the γ phosphate of the ATP held by the NBD2 Walker A motif (Hopfner et al., 2000; Smith et al., 2002; Chen et al., 2003). To assess accessibility of introduced S549C, we applied the small hydrophilic, sulfhydryl-specific MTS reagent MTSET+ (50 µM; Fig. 3 A) to S549C-(C832S-C1458S) CFTR channels opening and closing in inside-out patches exposed to 3 mM MgATP (Fig. 3 A). Despite the maintained presence of ATP, the MTSET+ caused a rapid current decline (Fig. 3 A). The current decay reflects modification of the introduced cysteine because background (C832S-C1458S) CFTR channels, lacking the engineered target cysteine, are little affected (Fig. S2 A) by much higher concentrations of MTSET+ or of the similarly sized MTS reagents, negatively charged MTSES−, 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). Near abolition of CFTR current by MTSET+ while ATP was present indicates that CFTR channels with a covalently attached MTSET+ adduct at position 549 can essentially no longer be opened by ATP. This corroborates impaired activity of S549C CFTR after permanent attachment of similarly sized (Fig. 2), but neutral, NEM (Cotten and Welsh, 1998).

Bottom Line: These results suggest that the target cysteines can be modified only in closed channels; that after modification the attached MTS adduct interferes with ATP-mediated opening; and that modification in the presence of ATP occurs rapidly once channels close, before they can reopen.We conclude that, in every CFTR channel gating cycle, the NBD dimer interface separates simultaneously at both composite sites sufficiently to allow MTS reagents to access both signature-sequence serines.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 Å.

View Article: PubMed Central - HTML - PubMed

Affiliation: The Laboratory of Cardiac/Membrane Physiology, The Rockefeller University, New York, NY 10065.

Show MeSH
Related in: MedlinePlus