Structural mechanisms of CFTR function and dysfunction

Single-channel behavior of human WT CFTR. (A) A representative continuous single-channel trace of human WT CFTR in an excised inside-out patch showing an incremental activation of the channel activity upon addition of PKA (25 IU) and ATP (2 mM). Currents were recorded at room temperature with symmetrical 154 mM [Cl⁻]. Membrane potential was held at −50 mV; upward deflections represent channel opening (signals were inverted purely for the purpose of presentation). Dashed lines on the bottom of each trace mark the zero-current level. Microscopic kinetic parameters including open time (Tb), interburst duration (Tib), and open probability (P_o) of each segment of the recording are presented above the trace. Of note, one can barely discern one single opening event in the first ∼20 s of the recording despite the presence of millimolar ATP, suggesting that before phosphorylation, the P_o is exceedingly low even at millimolar ATP. However, the status of R domain phosphorylation is not known; one cannot rule out the possibility that some serine/threonine in the R domain had been phosphorylated by cellular PKA before patch excision. (Thus, assuming that the CFTR channel upon patch excision is completely dephosphorylated could be erroneous.) In the second and third traces for an overall time of 6 min, the channel activity is relatively low with a closed time constant >1 s. A stable, high activity was not observed until ∼10 min after the addition of PKA and ATP. This result is consistent with the idea that phosphorylation-dependent activation of CFTR is more complex than a simple on–off switch. (B) ATP-dependent gating of a maximally phosphorylated human WT CFTR. Once the channel is fully activated, a P_o of ∼0.4 with a closed time constant <1 s was consistently observed in our laboratory (Zeltwanger et al., 1999; Zhou et al., 2005; Tsai et al., 2010; Jih and Hwang, 2013; Yeh et al., 2015). Because the degree of phosphorylation can alter the P_o of CFTR, the gating parameters reported in the literature are inevitably subject to the phosphorylation status of CFTR, which depends on the balance between kinase and phosphatase in the system. For example, when CFTR is expressed in X. laevis oocytes, the strong membrane-associated phosphatase activity will counter the action of exogenous PKA (Csanády et al., 2005); in some studies, CFTR currents in excised patches depend on the continuous presence of PKA as removal of PKA causes a sharp decrease of >50% of the currents in seconds (Weinreich et al., 1997; Csanády et al., 2000; Szellas and Nagel, 2003).

Interactions between the R domain and other components in CFTR. (A) The overall ribbon diagram of the front (left) and back (right) view of hCFTR (Liu et al., 2017). The EM density of the R domain is shown in red. (B) Bottom view of the structure revealing the interaction between the R domain and the NBDs. The side chains of the Walker A lysine of ATP binding site 1 (K464) and site 2 (K1250) are shown in sticks and labeled. (C) Side view of the structure demonstrating the proximity between the R domain and both the NBD1–ICL4 and NBD1–ICL1 interfaces. Individual TMs are labeled in number. (D) A graph highlighting the contacts between TMDs and the R domains. The dashed line marks the ion permeation pathway. M1140 in TM12 is labeled (see text).

Lateral view and top view of the TMDs in the cryo-EM structure of hCFTR. (A) Lateral view of the TMDs featuring a lateral entrance framed by TM4 (red) and TM6 (black), the surface view of the internal vestibule (gray), and a nonconductive region where close contacts among TMs obstruct the pore. Other 10 TMs are shown as ribbons in light purple. A yellow dot marks the end of the water-accessible space in the internal vestibule. Of note, the yellow dot is shifted away from the central axis of the pore. The functional implications of this structural feature are discussed in the text. (B) Top view of the TMDs. Color code is the same as used in A. Several residues in the external part of the TMDs (violet, R104; green, D110; gold, E116; brown, R117; salmon, R334; yellow, K335; and blue, E1126) were reported to affect the stability of the channel architecture or permeation properties. The distances between selected residues are shown in dashed lines, and the potential functional significance of these residues is discussed in the text.

Comparison of the architecture of TMDs between hCFTR and P-glycoprotein. (A) Top view of the TMDs in the cryo-EM structure of hCFTR (Protein Data Bank [PDB] accession no. 5UAK). Arrows indicate the cytoplasmic gaps of the internal vestibule between TM4 and TM6 and between TM10 and TM12. Dark blue, TMD1; cyan, TMD2. (B) Symmetrical architecture of the TMDs in P-glycoprotein (PDB accession no. 4KSB). The color code is the same as in A. Although CFTR evolves from ABC transporters and serves as an anion channel, the basic architecture of the TMDs in CFTR follows the pseudo-symmetrical mode found in ABC exporters. However, some modifications of this basic architecture are needed for CFTR to work as a channel (an issue discussed throughout this article).

Distortion of CFTR’s TMD2 breaks twofold symmetry of the TMDs. (A) Lateral view of part of CFTR’s TMDs featuring asymmetrical arrangements between TM1 (blue) and TM7 (purple) and between TM2 (violet) and TM8 (red). The internal vestibule is shown in a gray surface view. The loop-like structure in a small segment of TM8 (G921–L935 in hCFTR) is distinct from the typical helical structure of TM2, and the impingement of this loop-like segment of TM8 toward the central vertical axis likely pushes TM7 away from the permeation pathway so that TM7, contrary to TM1, is not pore lining. The dashed box is enlarged in B. (B) E92 (black) and K95 (dark blue) in TM1 form an intra-helical salt bridge. (C) Top view of the TM pairs in A shows clearly that TM7 is located at the periphery of the protein with little contact with the internal vestibule (in gray surface view).

Narrow region of the internal vestibule largely contributed by TM1, TM6, and TM12. (A) Top view of the narrow region of the internal vestibule lined by TM1, TM6, and TM12. The external end of the internal vestibule is indicated with a yellow dot as in Fig. 4. Marine, TM1; deep purple, TM6; and cyan, TM12. Arrow indicates the lateral entrance between TM4 and TM6. (B) Lateral view of the alignment of TM1, TM6, and TM12 contributing to the narrow region. Residues are shown as sticks and labeled in the same color as the TMs. Of note, the yellow dot is in close proximity to S341 in TM6 and L102 in TM1, both of which define the internal limits of the narrowest region of the pore from SCAM studies.

Conserved sequences and motifs in CFTR’s NBDs. Left: a cartoon depicting the relative positions of the conserved motifs in NBD1 and NBD2 with the characteristic motifs highlighted in different colors. The color code is also used for the cartoon on the right showing the head and tail subdomains of NBDs with two ATP molecules sandwiched in the dimer interface.

Conformational changes of NBDs in CFTR. (A) Widely separated NBDs in unphosphorylated CFTR. Top left: Superimposed cryo-EM structures for hCFTR (blue; Protein Data Bank [PDB] accession no. 5UAK) and zCFTR (yellow; PDB accession no. 5UAR). Bottom: Top view of NBDs. All conserved motifs are labeled as ABC, ABC signature sequence; Q, Q loop; H, H loop; A, Walker A; B, Walker B; and D, D loop. Right: magnified views of those labeled i–vi in the top view. (B) Phosphorylated E1372Q-zCFTR (PDB accession no. 5W81). The catalytic glutamate E1372 (E1371 in hCFTR) was mutated to glutamine to abolish ATP hydrolysis in site 2. Top left: cryo-EM structure of zCFTR (PDB accession no. 5W81; TMD1 in blue; TMD2 in cyan; NBD1 in gray; NBD2 in green). Bottom: Top view of NBD. Right: i and ii are magnified images showing the interactions between conserved motifs and ATP in dimeric NBDs.

Two gating models for CFTR. (A) Strict coupling scheme: The strict coupling model adopted from Liu et al. (2017) dictates that the gating cycle is strictly coupled to the ATP hydrolysis cycle. The opening of the channel is initiated by the formation of NBD dimer, and terminated by dimer disruption triggered by ATP hydrolysis. Of note, although in this model the NBDs are completely separated in the closed channel conformation (state C), a different thesis was proposed in Csanády et al. (2010) where the NBD dimer is not completely separated in the closed state. (B) Energetic coupling mechanism proposed in several previous studies (Jih and Hwang, 2012; Lin et al., 2014, 2016). This model follows the classical allosteric modulation principle that the conformational change in one domain facilitates the conformational change in the other domain. Note subtle shape change in TMDs between O₁ and O₂ (see text). Pi, inorganic phosphate.

Accessibility of the NBD dimer interface. (A) Structural comparison between Sav1866 (left) and CFTR (right). The structures are presented in space-filling model. As a prototypical ABC exporter in an outward-facing conformation, Sav1866 (Protein Data Bank [PDB] accession no. 2ONJ) shows a complete coalesce of the transmembrane helices at the junction between its TMDs (green and blue) and NBDs (lime and cyan). In contrast, a clear gap representing the lateral entrance to the pore is seen in the structure of zCFTR with dimerized NBDs (PDB accession no. 5W81). Yellow marks the bound ATP, and target residues S1348 (equivalent to S1347 in hCFTR) and S548 (S549 in hCFTR) as space-filling spheres in purple. The equivalent residues in Sav1866 are completely buried in the NBD dimer. In contrast, although S548 in site 2 is buried in zCFTR, S1348 is exposed. This analysis suggests that MTSET (labeled as space-filling sphere), even much larger reagents such as MTS-rhodamine, may be able to access the S1348C through the lateral entrance, which is unique to CFTR, an ion channel, not a transporter. (B) Solvent-accessible surface in the cryo-EM structure of zCFTR (PDB accession no. 5W81). The solvent-accessible regions were assessed by molecular visualization software PyMOL. The residues were labeled with numbers that indicate relative accessibilities. (The range of 0–1 is used to rank the accessibility: 0, hardly accessible; 1, highly accessible.)

Molecular interactions at TMD–NBD interfaces. (A) CFTR topology showing its 12 TMs (1–12), two NBDs (gray, NBD1; green, NBD2), the R domain, and interfaces between ICL1 and ICL4). Note the two ball-and-socket joints are between ICL2–NBD2 and ICL4–NBD1. Labeled residues next to each ICL are mutations that cause a severe form of CF. (B) (i) Locations of the four ICLs in the cryo-EM structures of CFTR (Protein Data Bank [PDB] accession nos. 5UAR and 5W81). Left: unphosphorylated zCFTR (PDB accession no. 5UAR). Right: phosphorylated ATP-bound zCFTR (PDB accession no. 5W81). The color codes of ICLs are the same as those in A (blue, ICL1; cyan, ICL2; red, ICL3; and orange, ICL4). (ii) Close-up front view of the ICL–NBD interfaces. The surface views of NBD are shown. (iii) Cartoon depicting the dynamic change of hydrogen bond network upon dimerization. The relative position of each segment is based on the structures of interfaces shown in ii; only TM4, 6, 10, and 12 are shown. Dashed lines mark the regions connecting TMs and their corresponding ICLs. The relatively constant number (shown in the yellow symbols at each interface) of hydrogen bonds before and after NBD dimerization suggests that the ball-in-the-socket interfaces (ICL2–NBD2 and ICL4–NBD1) move in sync with NBD. In contrast, the loose interfaces (ICL1–NBD1 and ICL3–NBD2) lag behind because of many weakened interactions (see text). (C) Close-up view of the interfaces between ICL1, ICL4, and NBD1. Left: Unphosphorylated zCFTR (PDB accession no. 5UAR). Right: Phosphorylated ATP-bound zCFTR (PDB accession no. 5W81). (D) Similar presentation as C except ICL2, ICL4, and NBD2 interfaces are shown.

Locations of pathogenic mutations covered in the current article. A lateral view of hCFTR structure showing the positions of all disease-associated mutations discussed. Pink, residues in NBDs; blue, residues at NBD–TMD interfaces; red, residues that line the pore; and black, non–pore-lining positions.

Chemical structures of representative CFTR modulators. (A) CFTR corrector. (B and C) Pore blocker. (D and E) Gating inhibitor. (F–I) CFTR potentiators.