Cysteine accessibility probes timing and extent of NBD separation along the dimer interface in gating CFTR channels.
Bottom Line: Modification while channels were opening and closing in the presence of ATP caused macroscopic CFTR current to decline at the same speed as when the unmodified channels shut upon sudden ATP withdrawal.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
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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).
Affiliation: The Laboratory of Cardiac/Membrane Physiology, The Rockefeller University, New York, NY 10065.