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.ATPase cycle | maximum likelihood | nonequilibrium | phosphorylation | Walker motifsA TP-binding cassette (ABC) proteins bind and hydrolyze ATP, usually to power transport of substrates across membranes. Their common architecture comprises two transmembrane domains (TMDs) and two cytoplasmic nucleotide binding domains (NBDs) containing conserved sequences for interacting with ATP. Once ATP binds to the conserved Walker A and B motifs of each NBD, they dimerize in head-to-tail fashion, burying two ATP molecules in composite interfacial sites (1, 2). Cyclic formation and disruption of the dimer requires hydrolysis of at least one of the ATPs per cycle (3-6).The ion channel CFTR contains, in addition to canonical ABC protein domains (TMD1, NBD1, TMD2, NBD2), a unique regulatory (R) domain (7) with multiple cAMP-dependent protein kinase (PKA) targets that must be phosphorylated for ATP to activate bursts of channel openings reviewed in ref. 8). But the mechanism of CFTR channel gating remains controversial. Early observations that the gating pattern violates microscopic reversibility suggested that the process underlying bursting operates far from thermodynamic equilibrium (9-11). Building on this, with functional evidence from mutants and nucleotide homologs (12-15) and with structural and biochemical data from prokaryotic NBDs (1-4), a CFTR channel open burst was proposed to be initiated by ATP-mediated formation of a stable NBD1-NBD2 heterodimer, and terminated by dimer dissociation after ATP hydrolysis (16). The ATP hydrolysis was posited to occur at the NBD2 composite site (between NBD2 Walke...

The CFTR Ion Channel: Gating, Regulation, and Anion Permeation

“…By scrutinizing their single-channel data, they were able to discern two conductance levels and an uneven distributions of transitions between these two open channel levels, indicating a violation of microscopic reversibility and hence a necessity of an input of free energy-in this case, from ATP hydrolysis (Gunderson and Kopito 1995). This idea of an infusion of free energy in driving (Csanády et al 2010). These investigators showed a paucity of shortlived events that results in an unusual double exponential distributions with a negative component, indicative of a violation of microscopic reversibility.…”

Thermodynamics of Statistical Inference by Cells

“…Extrapolating this result suggests that there may be fundamental thermodynamic bounds on statistical inference. The trade-off between accuracy and energy is general and may be relevant for other signal transduction systems, such as gene regulation [30], light-activated proteins [31] or ligand-gated ion channels [32]. We note that following the tradition of Berg and Purcell, in this paper we only considered estimating a concentration after a long time T .…”

Vx-770 potentiates CFTR function by promoting decoupling between the gating cycle and ATP hydrolysis cycle

“…1A) for CFTR gating offers a unique mechanism for prolonging the open time of WT-CFTR besides delaying hydrolysis and/or NBD dimer separation implicated in other gating models (25,26). As shown in Fig.…”

“…Notably, there are brief closings buried within each opening burst known as flickers. However, because these flickering closings were traditionally considered ATP-independent events (12,(26)(27)(28), a computer program had been developed by Csanady et al (29) to isolate the ATP-dependent opening bursts for single-channel kinetic analysis.…”