Structural basis for the channel function of a degraded ABC transporter, CFTR (ABCC7)

Abstract

Cystic fibrosis transmembrane conductance regulator (CFTR) is a member of the ATP-binding cassette (ABC) transporter superfamily, but little is known about how this ion channel that harbors an uninterrupted ion permeation pathway evolves from a transporter that works by alternately exposing its substrate conduit to the two sides of the membrane. Here, we assessed reactivity of intracellularly applied thiol-specific probes with cysteine residues substituted into the 12th transmembrane segment (TM12) of CFTR. Our experimental data showing high reaction rates of substituted cysteines toward the probes, strong blocker protection of cysteines against reaction, and reaction-induced alterations in channel conductance support the idea that TM12 of CFTR contributes to the lining of the ion permeation pathway. Together with previous work, these findings raise the possibility that pore-lining elements of CFTR involve structural components resembling those that form the substrate translocation pathway of ABC transporters. In addition, comparison of reaction rates in the open and closed states of the CFTR channel leads us to propose that upon channel opening, the wide cytoplasmic vestibule tightens and the pore-lining TM12 rotates along its helical axis. This simple model for gating conformational changes in the inner pore domain of CFTR argues that the gating transition of CFTR and the transport cycle of ABC proteins share analogous conformational changes. Collectively, our data corroborate the popular hypothesis that degradation of the cytoplasmic-side gate turned an ABC transporter into the CFTR channel.

Molecular Dynamics Flexible Fitting Simulations Identify New Models of the Closed State of the Cystic Fibrosis Transmembrane Conductance Regulator Protein

“…Yet several residues on TM12 are known to be exposed to a more hydrophobic environment in closed channels, namely residues 1141, 1142, 1145, 1147 and 1150. 55 In accord with this observation, residues 1141, 1145 and 1150 have lower predicted averaged %SASA values in the MDFF simulations than in both initial models. Residue 1142 has a lower predicted %SASA only in the MDFF simulations initiated from the Dalton model, whereas residue 1147 has higher %SASA in MDFF simulations initiated from both models, but still lower than the cutoff value of 20%.…”

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

“…Kirk 2008;Jordan et al 2008). This proposition is supported by a very recent report providing strong evidence that the gate of CFTR is indeed located more to the extracellular part of the pore (Bai et al 2011;but cf. El Hiani and Linsdell 2010;Wang and Linsdell 2012).…”

“…TMs 5, 11, and 12 also have been argued to contribute to the pore based on mutagenesis and cysteine scanning results (Zhang et al 2000a,b;Fatehi and Linsdell 2009). Recent results from systematic cysteine scanning studies not only suggest that TM12 assumes an a-helical secondary structure, but also support the idea that TM12 is part of the pore-forming domain (Bai et al 2011;cf. Qian et al 2011).…”

“…First, the pattern of state-dependent accessibility for MTS reagents suggests that, during gating transitions, TM12 undergoes a rotational movement, which has long proposed to be part of the molecular motions for ABC transporters (Rosenberg et al 2001;Gutmann et al 2010). Second, using MTS reagents with different sizes, Bai et al (2011) showed that the opening of CFTR's gate is associated with a narrowing of the cytoplasmic entrance of the pore-a result that makes sense if CFTR's gating conformational changes involve movements that are analogous to the flip-flop motion seen in ABC transporters (cf. Wang and Linsdell 2012).…”

Two Salt Bridges Differentially Contribute to the Maintenance of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Channel Function

“…Based on functional data and homology models, CFTR has been predicted to contain five functional domains: two membranespanning domains (MSDs), each including six transmembrane (TM) helices; two nucleotide binding domains (NBD1 and NBD2); and a unique regulatory (R) domain, which carries multiple protein kinase A (PKA) consensus phosphorylation sites and is unique to CFTR in the ABC superfamily (1)(2)(3)(4)(5)(6). Functional studies from multiple groups have suggested that TM6 plays an essential role and TM12 contributes less to anion conduction and permeation properties in the CFTR channel pore (7)(8)(9)(10)(11).…”