Alternating Access to the Transmembrane Domain of the ATP-binding Cassette Protein Cystic Fibrosis Transmembrane Conductance Regulator (ABCC7)

Department of Physiology and Biophysics, Dalhousie University, Halifax, Nova Scotia B3H 4R2, Canada.
Journal of Biological Chemistry (Impact Factor: 4.57). 02/2012; 287(13):10156-65. DOI: 10.1074/jbc.M112.342972
Source: PubMed


The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is a member of the ATP-binding cassette (ABC) protein family, most members of which act as active transporters. Actively transporting ABC proteins are thought to alternate between "outwardly facing" and "inwardly facing" conformations of the transmembrane substrate pathway. In CFTR, it is assumed that the outwardly facing conformation corresponds to the channel open state, based on homology with other ABC proteins. We have used patch clamp recording to quantify the rate of access of cysteine-reactive probes to cysteines introduced into two different transmembrane regions of CFTR from both the intracellular and extracellular solutions. Two probes, the large [2-sulfonatoethyl]methanethiosulfonate (MTSES) molecule and permeant Au(CN)(2)(-) ions, were applied to either side of the membrane to modify cysteines substituted for Leu-102 (first transmembrane region) and Thr-338 (sixth transmembrane region). Channel opening and closing were altered by mutations in the nucleotide binding domains of the channel. We find that, for both MTSES and Au(CN)(2)(-), access to these two cysteines from the cytoplasmic side is faster in open channels, whereas access to these same sites from the extracellular side is faster in closed channels. These results are consistent with alternating access to the transmembrane regions, however with the open state facing inwardly and the closed state facing outwardly. Our findings therefore prompt revision of current CFTR structural and mechanistic models, as well as having broader implications for transport mechanisms in all ABC proteins. Our results also suggest possible locations of both functional and dysfunctional ("vestigial") gates within the CFTR permeation pathway.

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    • "Experimental data generally agree with the models of CFTR open states facing outwardly and a cytoplasmic vestigial gate [11] [18]. Cysteine-scanning studies indeed suggested the presence of a cytoplasmic gate and a scheme where the open state would face inwardly [19]. This last scheme is however hardly reconciled with the tight association of the NBDs in the open configuration. "
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    ABSTRACT: Background: CFTR is the only ABC transporter functioning as a chloride (Cl(-)) channel. We studied molecular determinants, which might distinguish CFTR from standard ABC transporters, and focused on the interface formed by the intracellular loops from the membrane spanning domains. Methods: Residues from ICL2 and ICL4 in close proximity were targeted, and their involvement in the functioning of CFTR was studied by whole cell patch clamp recording. Results: We identified 2 pairs of amino acids, at the extremity of the bundle formed by the four intracellular loops, whose mutation i) decreases the Cl(-) current of CFTR (couple E267-K1060) or ii) increases it with a change of the electrophysiological signature (couple S263-V1056). Conclusions: These results highlight the critical role of these ICL residues in the assembly of the different domains and/or in the Cl(-) permeation pathway of CFTR.
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    ABSTRACT: Multiple transmembrane (TM) segments line the pore of the cystic fibrosis transmembrane conductance regulator Cl(-) channel; however, the relative alignment of these TMs and their relative movements during channel gating are unknown. To gain three-dimensional structural information on the outer pore, we have used patch clamp recording to study the proximity of pairs of cysteine side chains introduced into TMs 6 and 11, using both disulfide cross-linking and Cd(2+) coordination. Following channel activation, disulfide bonds could apparently be formed between three cysteine pairs (of 15 studied): R334C/T1122C, R334C/G1127C, and T338C/S1118C. To examine the state dependence of cross-linking, we combined these cysteine mutations with a nucleotide-binding domain mutation (E1371Q) that stabilizes the channel open state. Investigation of the effects of the E1371Q mutation on disulfide bond formation and Cd(2+) coordination suggests that although R334C/T1122C and T338C/S1118C are closer together in the channel open state, R334C/G1127C are close together and can form disulfide bonds only when the channel is closed. These results provide important new information on the three-dimensional structure of the outer mouth of the cystic fibrosis transmembrane conductance regulator channel pore: TMs 6 and 11 are close enough together to form disulfide bonds in both open and closed channels. Moreover, the altered relative locations of residues in open and in closed channels that we infer allow us to propose that channel opening and closing may be associated with a relative translational movement of TMs 6 and 11, with TM6 moving "down" (toward the cytoplasm) during channel opening.
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    ABSTRACT: High-throughput screening has led to the identification of small-molecule blockers of the CFTR chloride channel, but the structural basis of blocker binding remains to be defined. We recently developed molecular models of the CFTR channel based on homology to the bacterial transporter, Sav1866, that could permit blocker binding to be analyzed in silico. The models accurately predicted the existence of a narrow region in the pore that is a likely candidate for the binding site of an open-channel pore blocker like GlyH-101, thought to act by entering the channel from the extracellular side. As a more stringent test of predictions of the CFTR pore model, we applied induced-fit, virtual ligand docking techniques to identify potential binding sites for GlyH-101 within the CFTR pore. The highest scoring, docked position was near two pore-lining residues, F337 and T338, and the rate of reaction of anionic thiol-directed reagents with cysteines substituted at either of these positions was slowed in the presence of the blocker, consistent with the predicted repulsive effect of the net negative charge on GlyH-101. When a bulky phenylalanine that forms part of the predicted binding pocket (F342) was replaced with alanine, the apparent affinity of the blocker increased by approximately 200 fold. A Molecular Mechanics-Generalized Born/Surface Area (MM-GB/SA) analysis of GlyH-101 binding predicted that substitution of F342 with alanine would substantially increase blocker affinity, primarily due to decreased intramolecular strain within the blocker-protein complex. This study suggests that GlyH-101 blocks the CFTR channel by binding within the pore bottleneck.
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