Strict coupling between CFTR's catalytic cycle and gating of its Cl- ion pore revealed by distributions of open channel burst durations

Abstract

CFTR, the ABC protein defective in cystic fibrosis, functions as an anion channel. Once phosphorylated by protein kinase A, a CFTR channel is opened and closed by events at its two cytosolic nucleotide binding domains (NBDs). Formation of a head-to-tail NBD1/NBD2 heterodimer, by ATP binding in two interfacial composite sites between conserved Walker A and B motifs of one NBD and the ABC-specific signature sequence of the other, has been proposed to trigger channel opening. ATP hydrolysis at the only catalytically competent interfacial site is suggested to then destabilize the NBD dimer and prompt channel closure. But this gating mechanism, and how tightly CFTR channel opening and closing are coupled to its catalytic cycle, remains controversial. Here we determine the distributions of open burst durations of individual CFTR channels, and use maximum likelihood to evaluate fits to equilibrium and nonequilibrium mechanisms and estimate the rate constants that govern channel closure. We examine partially and fully phosphorylated wild-type CFTR channels, and two mutant CFTR channels, each bearing a deleterious mutation in one or other composite ATP binding site. We show that the wild-type CFTR channel gating cycle is essentially irreversible and tightly coupled to the ATPase cycle, and that this coupling is completely destroyed by the NBD2 Walker B mutation D1370N but only partially disrupted by the NBD1 Walker A mutation K464A.

Fig. 1.

Distributions of burst durations for WT and mutant CFTR channels. (A) Nonequilibrium cyclic full model (scheme 2) proposed to describe CFTR gating. The equilibrium closed-open model (scheme 1, encircled by blue dotted line) is a submodel of scheme 2. (B–D) Histograms of open burst durations for prephosphorylated WT (B), D1370N (C), and K464A (D) CFTR channels; 30-s segments of representative single-channel current recordings are shown above each panel. The lowest bins start at t_low = 6 ms. Blue dotted lines show ML fits to scheme 1. Red solid lines in B and D show ML fits to scheme 2; rate k₋₁ was fixed to zero in B, but left free in D. In C, red solid line shows the ML fit to a double-exponential distribution. Obtained rate estimates, as well as time constants and fractional amplitudes of the resulting exponential components, are printed in red. ATP was 2 mM for WT and D1370N, but 5 mM for K464A.

Fig. 2.

Slow nonhydrolytic closing rate and its acceleration by the K464A mutation. (A and B) Macroscopic currents of prephosphorylated K1250A (A) and K464A/K1250A (B) CFTR channels were activated by application of 10 mM ATP. Time courses of current decay upon ATP removal were fitted by single exponentials (colored lines). (C) Mean (±SEM) closing rates estimated as the inverses of the current relaxation time constants (τ_relax), for K1250A (blue) and K464A/K1250A (red).

Fig. 3.

Phosphorylation slows both sequential steps involved in CFTR channel closure. (A) Channel activity of WT CFTR rapidly declines upon PKA removal, but then remains steady for a prolonged period, thereby defining gating patterns for “fully” (in the presence of PKA) and “partially” phosphorylated CFTR (in just ATP, following PKA withdrawal). (B) Histogram of open burst durations for fully phosphorylated WT CFTR channels, and 30-s segment of single-channel current recording. Lowest bin starts at t_low = 6 ms. Blue dotted line is a ML fit to scheme 1. Red solid line is a ML fit to scheme 2 with rate k₋₁ fixed to zero.

Fig. 4.

Average gating parameters, gating mechanisms, and microscopic transition rate estimates for WT and mutant CFTR channels. (A–D) Open probabilities (A), mean burst (B), and interburst (C) durations obtained from multichannel fits, and calculated channel cycle times (D) for fully (navy blue) and partially (royal blue) phosphorylated WT, and partially phosphorylated D1370N (green) and K464A (red) CFTR. [ATP] was 2 mM for WT and D1370N, but 5 mM for K464A. Error bars represent SEM. (E) ML estimates of rates k₁ (Left), k₂ (Center), and k₋₁ (Right) for fully (navy blue) and partially (royal blue) phosphorylated WT, and partially phosphorylated D1370N (green) and K464A (red) CFTR channels. Error bars represent 0.5-unit log-likelihood intervals. ^(a)k₋₁ for partially phosphorylated WT is modeled by the closing rate of partially phosphorylated K1250R. (F) Cartoon of gating mechanisms. NBD1, green; NBD2, blue; TMDs, cyan; ATP, yellow; ADP, red. Probabilities for exiting state O₁ (Top Right) in either of two possible directions are printed in color for partially phosphorylated WT (blue), K464A (red), and D1370N (green). Note subtle shape change in TMDs between O₁ and O₂ (see text).