Cysteine accessibility probes timing and extent of NBD separation along the dimer interface in gating CFTR channels.
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.
Affiliation: The Laboratory of Cardiac/Membrane Physiology, The Rockefeller University, New York, NY 10065.Show MeSH
Related in: MedlinePlus
License 1 - License 2
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.
Affiliation: The Laboratory of Cardiac/Membrane Physiology, The Rockefeller University, New York, NY 10065.