Nonintegral stoichiometry in CFTR gating revealed by a pore-lining mutation

Abstract

Cystic fibrosis transmembrane conductance regulator (CFTR) is a unique member of the ATP-binding cassette (ABC) protein superfamily. Unlike most other ABC proteins that function as active transporters, CFTR is an ATP-gated chloride channel. The opening of CFTR’s gate is associated with ATP-induced dimerization of its two nucleotide-binding domains (NBD1 and NBD2), whereas gate closure is facilitated by ATP hydrolysis-triggered partial separation of the NBDs. This generally held theme of CFTR gating—a strict coupling between the ATP hydrolysis cycle and the gating cycle—is put to the test by our recent finding of a short-lived, post-hydrolytic state that can bind ATP and reenter the ATP-induced original open state. We accidentally found a mutant CFTR channel that exhibits two distinct open conductance states, the smaller O1 state and the larger O2 state. In the presence of ATP, the transition between the two states follows a preferred O1→O2 order, a telltale sign of a violation of microscopic reversibility, hence demanding an external energy input likely from ATP hydrolysis, as such preferred gating transition was abolished in a hydrolysis-deficient mutant. Interestingly, we also observed a considerable amount of opening events that contain more than one O1→O2 transition, indicating that more than one ATP molecule may be hydrolyzed within an opening burst. We thus conclude a nonintegral stoichiometry between the gating cycle and ATP consumption. Our results lead to a six-state gating model conforming to the classical allosteric mechanism: both NBDs and transmembrane domains hold a certain degree of autonomy, whereas the conformational change in one domain will facilitate the conformational change in the other domain.

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

“…*Data for R352C-CFTR and R352C/W401F-CFTR in the absence of Vx-770 were taken from ref. 24. gate in the absence of ATP, C2 ↔ O2) primarily affected by Vx-770 are supposed to take place in CFTR's TMDs (24). The observation that Vx-770 increases the opening rate of ATP-gated WT-CFTR (Fig.…”

“…Table 1 summarizes the number of events with different characteristic gating patterns with or without Vx-770. As shown in our recent report (24), most of the opening bursts in R352C-CFTR contain one O1 → O2 transition; only ∼10% of the openings are categorized as events with more than one O1 → O2 transition. However, the percentage of this latter event was nearly doubled in the presence of 200 nM Vx-770, indicating that Vx-770 increases the frequency of reentry.…”

“…Notably, the reentry pathway was first suggested by a report using PPi as a tool to fish out a unique posthydrolytic state (36) and later established by scrutinizing the R352C mutant channel that exhibits hydrolysis-dependent open channel conductance: unequal single-channel current amplitudes between pre-and posthydrolytic states (24). Direct evidence for the existence of reentry events in WT-CFTR was lacking because most previous studies failed to observe Table 1) and the mean open time is prolonged (Fig.…”

“…Lately, we discovered that W401F, a conserved mutation in NBD1, increases the reentry frequency without significantly altering the O2 → C2 transition (24). Although how the W401F mutation modifies the function of NBDs is unclear, this finding does raise the possibility that manipulating NBD function can complement the action of Vx-770.…”

“…Perhaps the most puzzling observation is that Vx-770 not only prolongs the open time of WT-CFTR, which is controlled by ATP hydrolysis; it also increases the activity of G551D-CFTR, a mutation that likely prevents ATPinduced NBD dimerization from happening due to its unique position in the signature sequence (14) and for the same reason eliminates ATP hydrolysis (22,23). This apparent paradox is no longer indecipherable as unique insights into CFTR's gating mechanism have emerged (24).…”