State-dependent modulation of CFTR gating by pyrophosphate

Abstract

Cystic fibrosis transmembrane conductance regulator (CFTR) is an adenosine triphosphate (ATP)-gated chloride channel. ATP-induced dimerization of CFTR's two nucleotide-binding domains (NBDs) has been shown to reflect the channel open state, whereas hydrolysis of ATP is associated with channel closure. Pyrophosphate (PPi), like nonhydrolytic ATP analogues, is known to lock open the CFTR channel for tens of seconds when applied with ATP. Here, we demonstrate that PPi by itself opens the CFTR channel in a Mg2+-dependent manner long after ATP is removed from the cytoplasmic side of excised membrane patches. However, the short-lived open state (τ ∼1.5 s) induced by MgPPi suggests that MgPPi alone does not support a stable NBD dimer configuration. Surprisingly, MgPPi elicits long-lasting opening events (τ ∼30 s) when administrated shortly after the closure of ATP-opened channels. These results indicate the presence of two different closed states (C1 and C2) upon channel closure and a state-dependent effect of MgPPi on CFTR gating. The relative amount of channels entering MgPPi-induced long-open bursts during the ATP washout phase decreases over time, indicating a time-dependent dissipation of the closed state (C2) that can be locked open by MgPPi. The stability of the C2 state is enhanced when the channel is initially opened by N6-phenylethyl-ATP, a high affinity ATP analogue, but attenuated by W401G mutation, which likely weakens ATP binding to NBD1, suggesting that an ATP molecule remains bound to the NBD1 site in the C2 state. Taking advantage of the slow opening rate of Y1219G-CFTR, we are able to identify a C2-equivalent state (C2*), which exists before the channel in the C1 state is opened by ATP. This closed state responds to MgPPi much more inefficiently than the C2 state. Finally, we show that MgAMP-PNP exerts its effects on CFTR gating via a similar mechanism as MgPPi. The structural and functional significance of our findings is discussed.

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

“…Furthermore, such prolongation of the opening burst became even more prominent at higher [ATP] (Fig. 3B), an observation not only corroborating the hypothesis that Vx-770 promotes the ATP-dependent reentry mechanism, but also distinguishing itself from the well-known increase of the open time by abolishing ATP hydrolysis (12,28,(30)(31)(32) We also measured the relaxation time constant of macroscopic current after removing different concentrations of ATP. In the presence of 200 nM Vx-770, these relaxation time constants are not significantly different over a wide range of [ATP] (Fig.…”

“…A fast solution exchange perfusion system (SF-77B; Warner Instruments) was used for recordings that require ligand exchange. The dead time of solution change is ∼30 ms (30).…”

“…Whereas the O2 state can bind ATP to initiate another round of ATP hydrolysis (i.e., O2 → O2ATP → O1 → O2 in Fig. 1A), it is expected that when the O2 state captures a nonhydrolyzable ATP analog such as PPi, the channel will sojourn to a locked-open state with a lifetime in tens of seconds (30,35,36). In a previous study, we have developed a protocol to quantify such effect of PPi (36).…”

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

“…Similar functional asymmetry of CFTR's two NBDs was observed when the Walker A lysine residues in NBD1 (K464) and 2 (K1250) were mutated (Powe et al 2002;Vergani et al 2003). The critical role of this p-electron stacking interaction between ATP and the binding pocket also explains why pyrophosphate (PPi), a ligand without the adenine ring for this stacking interaction, shows a very low apparent affinity as a channel opener (Tsai et al 2009). Lately, capitalizing on both structural information and bioinformatics, Szollosi et al (2010) were able to experimentally show an induced fit mechanism following ATP binding to the head subdomain of NBD2 during CFTR gating.…”

“…Using nonhydrolyzable ATP analogs (e.g., PPi or AMP-PNP) as ligands, Tsai et al (2009) showed that contrary to ATP-gated channels that show an open time of 400 ms (Zeltwanger et al 1999), the open time for PPi-opened channels is 30 s (inferred from deactivation rates) when this ligand is applied shortly after removal of ATP, but the open time becomes much shorter ( 1.5 s) if PPi is applied minutes after ATP washout (Tsai et al 2009). Thus, CFTR channels, once primed by ATP, respond to PPi differently depending on the washout time.…”