Separation and Accessibility of the Nucleotide-Binding Domain (NBD) Heterodimerization Interface When a CFTR Channel Closes

CFTR channel gating is driven by cyclic formation and dissociation of a head-to-tail NBD1-NBD2 heterodimer enclosing two composite ATP sites, one catalytically active and one dead. NBD separation at the active site is believed to follow ATP hydrolysis there that triggers channel closure. But whether the NBDs also separate at the dead site each cycle remains unclear. To address this we determined, in patches from Xenopus oocytes, gating-state-dependent accessibility to MTS reagents of interfacial target cysteines in CFTR channels with one (C832S), or eight (C832-1458S), native Cys replaced by Ser. Target S549C (S in NBD1 LSGGQ) in the catalytically-active site was rapidly modified, diminishing CFTR current (τMTS ≤ 2 s), by micromolar MTSACE, MTSET+, or MTSES- applied during channel opening and closing in 3 mM ATP. Similarly rapid current diminution attended modification, in 3 mM ATP, of the equivalent target in the catalytically-dead site, S1347C (S in NBD2 LSHGH). For both S549C and S1347C, the current decay time course upon MTS modification in ATP (τMTS) matched that of channel closure on ATP washout (τwashout), implying that modification occurred as soon as channels closed. Supporting that interpretation, τMTS and τwashout were both slowed ∼10-fold in S1347C channels bearing the hydrolysis-impairing mutation K1250R, suggesting S1347C cannot be modified in open CFTR channels. Hence, in open CFTR channels, a tight heterodimer interface in the catalytically-dead site prevents MTS access to S1347C, but NBD1-NBD2 separation allows access when channels close. Rapid modification of S549C and S1347C in 3 mM ATP by larger reagents, MTS-glucose (∼14A x 9A x 9A), MTS-biotin (∼15A x 11A x 8A), and MTS-rhodamine (∼17A x 16A x 11A), suggests that active and dead sites simultaneously separate by ≥11A each gating cycle. [DK51767].