ATP-independent CFTR channel gating and allosteric modulation by phosphorylation

Abstract

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) channel, an ATP binding cassette (ABC) transporter. CFTR gating is linked to ATP binding and dimerization of its two nucleotide binding domains (NBDs). Channel activation also requires phosphorylation of the R domain by poorly understood mechanisms. Unlike conventional ligand-gated channels, CFTR is an ATPase for which ligand (ATP) release typically involves nucleotide hydrolysis. The extent to which CFTR gating conforms to classic allosteric schemes of ligand activation is unclear. Here, we describe point mutations in the CFTR cytosolic loops that markedly increase ATP-independent (constitutive) channel activity. This finding is consistent with an allosteric gating mechanism in which ligand shifts the equilibrium between inactive and active states but is not essential for channel opening. Constitutive mutations mapped to the putative symmetry axis of CFTR based on the crystal structures of related ABC transporters, a common theme for activating mutations in ligand-gated channels. Furthermore, the ATP sensitivity of channel activation was strongly enhanced by these constitutive mutations, as predicted for an allosteric mechanism (reciprocity between protein activation and ligand occupancy). Introducing constitutive mutations into CFTR channels that cannot open in response to ATP (i.e., the G551D CF mutant and an NBD2-deletion mutant) substantially rescued their activities. Importantly, constitutive mutants that opened without ATP or NBD2 still required R domain phosphorylation for optimal activity. Our results confirm that (i) CFTR gating exhibits features of protein allostery that are shared with conventional ligandgated channels and (ii) the R domain modulates CFTR activity independent of ATP-induced NBD dimerization.ATP binding cassette transporter | cystic fibrosis | ligand | constitutive | mutant C ystic fibrosis transmembrane conductance regulator (CFTR) is a member of the ATP binding cassette (ABC) transporter superfamily, although it is the only known ion channel in this transporter family (1). Like other ABC transporters, CFTR uses ATP binding to its two nucleotide binding domains (NBDs) to drive conformational rearrangements of its transmembrane domains (2, 3). CFTR channel opening is linked to ATP binding to each of two sites at the interface of an NBD1-NBD2 dimer (2, 3). Subsequent hydrolysis, typically at site 2 (primarily composed of sequences from NBD2), promotes channel closure by clearing ligand from this site (4, 5). The coupling between ATP binding and pore opening is presumably mediated by the cytosolic loops that physically link the NBDs to the transmembrane domains (6, 7).Because CFTR is an enzyme that normally hydrolyzes its ligand as part of the channel gating cycle, the extent to which its properties are similar to those of more conventional ligand-gated channels is an interesting issue. Ligand-gated channels such as acetylcholine receptors obey the principles of protein alloster...

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

“…GOF mutations for conventional ligand-gated channels typically locate along the axis that links the ligand-binding site to the pore. The GOF mutations/modifications that have been discovered for CFTR locate near the NBD dimer interface (Szollosi et al 2010), along the cytosolic loops that connect the NBDs to the TMDs (Wang et al 2010) and within a TM that has been argued to line the pore (Bai et al 2010). Thus, the CFTR gating mechanism appears to share features with the gating of conventional ligand-gated channels; notably, unliganded openings and GOF mutations along the axis that links the ligand-binding domains to the pore.…”

“…This ATP-independent gating is readily seen with mutants with dysfunctional site 2 (e.g., the G551D mutation) (Bompadre et al 2007), or interestingly constructs that lack the entire NBD2 (Cui et al 2007;Wang et al 2007). Furthermore, gain-of-function (GOF) mutations that enhance ATP-free CFTR gating and increase the otherwise low activities of constructs that are insensitive to ATP (G551D and NBD-deletion mutants) have been produced (Szollosi et al 2010;Wang et al 2010). ATP-independent gating also can be strongly enhanced by chemically modifying cysteines placed at specific locations in TM 6 (Bai et al 2010).…”

“…As noted earlier CFTR truncation mutants that lack NBD2 show detectable channel activity (Cui et al 2007). Wang et al (2007Wang et al ( , 2010 observed that the channel activity of an NBD2-deletion mutant was strongly dependent on PKA phosphorylation but not ATP binding, an effect that required the R domain. Similarly, G551D -CFTR activity is dependent on PKA phosphorylation but not ATP binding (Bompadre et al 2007;Wang et al 2010).…”

An Electrostatic Interaction at the Tetrahelix Bundle Promotes Phosphorylation-dependent Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Channel Opening

“…Site 1 is catalytically inactive but binds ATP tightly; thus, most cycles of channel opening and closing are temporally linked to ATP binding and subsequent hydrolysis at site 2 (6 -9). R domain phosphorylation controls channel gating by unknown mechanisms that may include regulating both NBD dimerization (10) and the conformations or flexibilities of the TMs (11,12).…”

Regulation of Activation and Processing of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) by a Complex Electrostatic Interaction between the Regulatory Domain and Cytoplasmic Loop 3

“…The pore formed by TMD1 and TMD2 is gated by binding/hydrolysis of ATP at the interface of NBD1 and NBD2 and regulated by R domain phosphorylation/dephosphorylation (11,12). Although CLs have been found to play a central role in channel gating (6,7,9,10,13,14), it is largely unknown how CLs couple ATP actions at NBDs to gating rearrangements of TMDs and how R domain phosphorylation by cAMP-dependent PKA mediates the coupling.…”