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 Mg ²⁺-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 (C ₁ and C ₂) 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 (C ₂) that can be locked open by MgPPi. The stability of the C ₂ state is enhanced when the channel is initially opened by N ⁶-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 C ₂ state. Taking advantage of the slow opening rate of Y1219G-CFTR, we are able to identify a C ₂-equivalent state (C ₂*), which exists before the channel in the C ₁ state is opened by ATP. This closed state responds to MgPPi much more inefficiently than the C ₂ 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.