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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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S1347C CFTR channels are readily modified by MTS reagents when closed. (A) Immediately after ∼60-s applications of 50 µM MTSET+ (red trace and bar), MTSACE (green trace and bar), or MTSES− (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. Exposures to 20 mM DTT (black bars above record) restored ATP-activated current by releasing adducts after each modification; the asterisk above the record marks brief activation of Ca2+-dependent Cl− current to monitor solution exchange speed (time constant = 0.3 s for this patch). (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 ± 8%; n = 4 measurements in four patches), by MTSACE (green bar, 24 ± 6%; n = 3 measurements in three patches), or by MTSES− (blue bar, 16 ± 5%; n = 3 measurements in three patches), or while opening and closing (right, 3 mM ATP), by ≥50 µM MTSET+ (red bar, 42.4 ± 4.5%, n = 8 measurements in four patches) or by ≥50 µM MTSACE (green bar, 19.5 ± 2.0%, n = 9 measurements in four patches). Error bars represent mean ± SEM.
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fig6: S1347C CFTR channels are readily modified by MTS reagents when closed. (A) Immediately after ∼60-s applications of 50 µM MTSET+ (red trace and bar), MTSACE (green trace and bar), or MTSES− (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. Exposures to 20 mM DTT (black bars above record) restored ATP-activated current by releasing adducts after each modification; the asterisk above the record marks brief activation of Ca2+-dependent Cl− current to monitor solution exchange speed (time constant = 0.3 s for this patch). (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 ± 8%; n = 4 measurements in four patches), by MTSACE (green bar, 24 ± 6%; n = 3 measurements in three patches), or by MTSES− (blue bar, 16 ± 5%; n = 3 measurements in three patches), or while opening and closing (right, 3 mM ATP), by ≥50 µM MTSET+ (red bar, 42.4 ± 4.5%, n = 8 measurements in four patches) or by ≥50 µM MTSACE (green bar, 19.5 ± 2.0%, n = 9 measurements in four patches). Error bars represent mean ± SEM.

Mentions: The position equivalent to S549 in the signature motif of the noncatalytic composite site is S1347, in sequence LSHGH in the NBD2 tail. 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). The ratio of the time constants of current decay caused by ≥50 µM MTSET+ and by ATP withdrawal averaged 1.0 ± 0.1 (n = 8; Fig. 5 D, red open bar). However, unlike the near abolition of S549C CFTR current caused by MTSET+, modification of S1347C channels by MTSET+ reduced ATP-activated current only by ∼60% (see Fig. 6 B). 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). 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.


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)

S1347C CFTR channels are readily modified by MTS reagents when closed. (A) Immediately after ∼60-s applications of 50 µM MTSET+ (red trace and bar), MTSACE (green trace and bar), or MTSES− (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. Exposures to 20 mM DTT (black bars above record) restored ATP-activated current by releasing adducts after each modification; the asterisk above the record marks brief activation of Ca2+-dependent Cl− current to monitor solution exchange speed (time constant = 0.3 s for this patch). (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 ± 8%; n = 4 measurements in four patches), by MTSACE (green bar, 24 ± 6%; n = 3 measurements in three patches), or by MTSES− (blue bar, 16 ± 5%; n = 3 measurements in three patches), or while opening and closing (right, 3 mM ATP), by ≥50 µM MTSET+ (red bar, 42.4 ± 4.5%, n = 8 measurements in four patches) or by ≥50 µM MTSACE (green bar, 19.5 ± 2.0%, n = 9 measurements in four patches). Error bars represent mean ± SEM.
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fig6: S1347C CFTR channels are readily modified by MTS reagents when closed. (A) Immediately after ∼60-s applications of 50 µM MTSET+ (red trace and bar), MTSACE (green trace and bar), or MTSES− (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. Exposures to 20 mM DTT (black bars above record) restored ATP-activated current by releasing adducts after each modification; the asterisk above the record marks brief activation of Ca2+-dependent Cl− current to monitor solution exchange speed (time constant = 0.3 s for this patch). (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 ± 8%; n = 4 measurements in four patches), by MTSACE (green bar, 24 ± 6%; n = 3 measurements in three patches), or by MTSES− (blue bar, 16 ± 5%; n = 3 measurements in three patches), or while opening and closing (right, 3 mM ATP), by ≥50 µM MTSET+ (red bar, 42.4 ± 4.5%, n = 8 measurements in four patches) or by ≥50 µM MTSACE (green bar, 19.5 ± 2.0%, n = 9 measurements in four patches). Error bars represent mean ± SEM.
Mentions: The position equivalent to S549 in the signature motif of the noncatalytic composite site is S1347, in sequence LSHGH in the NBD2 tail. 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). The ratio of the time constants of current decay caused by ≥50 µM MTSET+ and by ATP withdrawal averaged 1.0 ± 0.1 (n = 8; Fig. 5 D, red open bar). However, unlike the near abolition of S549C CFTR current caused by MTSET+, modification of S1347C channels by MTSET+ reduced ATP-activated current only by ∼60% (see Fig. 6 B). 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). 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.

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