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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.

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

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Larger MTS reagents also readily modify S549C CFTR channels when they are closed. (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 µM MTS-glucose (A; orange bar), 5 µM MTS-rhodamine (A; magenta bar), 5 µM MTS-biotin (B; dark yellow bar), and MTS-biotin–avidin complex (B; 5 µM biotin plus 5 µM 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− current decays in these patches were 0.5 s (A) and 0.2 s (B). (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 ± 3%, n = 7 measurements), by MTS-glucose (orange bar, 7 ± 1%, n = 3 measurements), by MTS-rhodamine (magenta bar, 5 ± 2%, n = 5 measurements), or by MTS-biotin–avidin complex (cyan bar, 90 ± 8%, n = 5 measurements). Error bars represent mean ± SEM.
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fig8: Larger MTS reagents also readily modify S549C CFTR channels when they are closed. (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 µM MTS-glucose (A; orange bar), 5 µM MTS-rhodamine (A; magenta bar), 5 µM MTS-biotin (B; dark yellow bar), and MTS-biotin–avidin complex (B; 5 µM biotin plus 5 µM 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− current decays in these patches were 0.5 s (A) and 0.2 s (B). (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 ± 3%, n = 7 measurements), by MTS-glucose (orange bar, 7 ± 1%, n = 3 measurements), by MTS-rhodamine (magenta bar, 5 ± 2%, n = 5 measurements), or by MTS-biotin–avidin complex (cyan bar, 90 ± 8%, n = 5 measurements). Error bars represent mean ± SEM.

Mentions: The substituted ethyl-MTS reagents used so far, MTSET+, MTSACE, and MTSES−, all approximate cylinders ∼12 Å long and ∼6 Å in diameter (Fig. 2). For comparison with the various NBD separations observed in ABC exporter crystal structures, we tested whether larger MTS reagents could also access the NBD1–NBD2 interface in functioning CFTR. We first examined S549C CFTR channels closed by withdrawal of ATP, as in Figs. 4 and 6. Indeed, exposure to 20 µM MTS-glucose for ∼60 s diminished subsequent current activation by ATP (Fig. 8 A) to ∼7% of the control amplitude (Fig. 8 C), demonstrating that the ∼16 × ∼8 × ∼7–Å reagent (Fig. 2) could readily reach the target cysteine. The deposited adduct was partially removed by a 30-s application of DTT, and completely removed by further DTT treatment, as judged by restoration of ATP-activated current amplitude (Fig. 8 A). Larger still MTS-rhodamine (∼22 × ∼14 × ∼8 Å; Fig. 2), applied for 60 s at 5 µM (Fig. 8 A) also readily modified closed S549C CFTR (Fig. 8 A), diminishing ATP-activated current to ∼5% of control (Fig. 8 C). The MTS-rhodamine adduct appeared to be more slowly removed by DTT than the MTS-glucose adduct (Fig. 8 A).


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)

Larger MTS reagents also readily modify S549C CFTR channels when they are closed. (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 µM MTS-glucose (A; orange bar), 5 µM MTS-rhodamine (A; magenta bar), 5 µM MTS-biotin (B; dark yellow bar), and MTS-biotin–avidin complex (B; 5 µM biotin plus 5 µM 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− current decays in these patches were 0.5 s (A) and 0.2 s (B). (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 ± 3%, n = 7 measurements), by MTS-glucose (orange bar, 7 ± 1%, n = 3 measurements), by MTS-rhodamine (magenta bar, 5 ± 2%, n = 5 measurements), or by MTS-biotin–avidin complex (cyan bar, 90 ± 8%, n = 5 measurements). Error bars represent mean ± SEM.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4380215&req=5

fig8: Larger MTS reagents also readily modify S549C CFTR channels when they are closed. (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 µM MTS-glucose (A; orange bar), 5 µM MTS-rhodamine (A; magenta bar), 5 µM MTS-biotin (B; dark yellow bar), and MTS-biotin–avidin complex (B; 5 µM biotin plus 5 µM 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− current decays in these patches were 0.5 s (A) and 0.2 s (B). (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 ± 3%, n = 7 measurements), by MTS-glucose (orange bar, 7 ± 1%, n = 3 measurements), by MTS-rhodamine (magenta bar, 5 ± 2%, n = 5 measurements), or by MTS-biotin–avidin complex (cyan bar, 90 ± 8%, n = 5 measurements). Error bars represent mean ± SEM.
Mentions: The substituted ethyl-MTS reagents used so far, MTSET+, MTSACE, and MTSES−, all approximate cylinders ∼12 Å long and ∼6 Å in diameter (Fig. 2). For comparison with the various NBD separations observed in ABC exporter crystal structures, we tested whether larger MTS reagents could also access the NBD1–NBD2 interface in functioning CFTR. We first examined S549C CFTR channels closed by withdrawal of ATP, as in Figs. 4 and 6. Indeed, exposure to 20 µM MTS-glucose for ∼60 s diminished subsequent current activation by ATP (Fig. 8 A) to ∼7% of the control amplitude (Fig. 8 C), demonstrating that the ∼16 × ∼8 × ∼7–Å reagent (Fig. 2) could readily reach the target cysteine. The deposited adduct was partially removed by a 30-s application of DTT, and completely removed by further DTT treatment, as judged by restoration of ATP-activated current amplitude (Fig. 8 A). Larger still MTS-rhodamine (∼22 × ∼14 × ∼8 Å; Fig. 2), applied for 60 s at 5 µM (Fig. 8 A) also readily modified closed S549C CFTR (Fig. 8 A), diminishing ATP-activated current to ∼5% of control (Fig. 8 C). The MTS-rhodamine adduct appeared to be more slowly removed by DTT than the MTS-glucose adduct (Fig. 8 A).

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