Two Distinct Gating Cycles of CFTR Chloride Channels

Abstract

CFTR channels, at maximally effective [ATP], switch between open and closed states 1-2 s−1. Pyrophosphate (PPi), applied with ATP, slows down the gating cycle by locking the channel in a stable open state (τo ∼30 s). PPi alone, applied immediately following closing of ATP-opened channels, locks open the channel with the same open time constant. However, the open state induced by PPi long after ATP removal (> 2 min) assumes a lifetime of 1.5 s, indicating the presence of two different closed states with distinct responses to PPi. By altering the duration of ATP removal and measuring the response of closed channels to PPi, we estimated the lifetime of the closed state (C∗) that enters the lock-open state to be ∼30 s. Since the lifetime of the C∗ state can be modulated by N6-phenylethyl-ATP (P-ATP), a high-affinity ATP analog, or by mutations that lower the ATP binding affinity at NBD1, we propose that one ATP molecule remains tightly bound at NBD1 during this closed state. As the trapped ATP molecule should survive for numerous gating cycles before being replaced by a second ligand, we carried out single channel experiments where the perfusion solution containing 2 mM ATP (ropen = 3.33 ± 0.19 s−1; τopen = 235 ± 19.8 ms) was directly (dead time ∼40 ms) switched to one with 50 μM P-ATP. The Po of the channel increases in two steps. The channel opening rate was immediately increased (ropen = 4.88 ± 0.45 s−1) upon solution exchange, while the open time was not prolonged until ∼40s after the application of P-ATP (τopen = 386.7 ± 25.2 ms). This result indicates two gating cycles; one is sorely driven by fast ATP binding/hydrolysis in NBD2 while another involves slow dissociation of ATP in NBD1.