Ligand binding to a remote site thermodynamically corrects the F508del mutation in the human cystic fibrosis transmembrane conductance regulator.

Many disease-causing mutations impair protein stability. Here, we explore a thermodynamic strategy to correct the disease-causing F508del mutation in the human cystic fibrosis transmembrane conductance regulator (hCFTR). F508del destabilizes nucleotide-binding domain 1 (hNBD1) in hCFTR relative to an aggregation-prone intermediate. We developed a fluorescence self-quenching assay for compounds that prevent aggregation of hNBD1 by stabilizing its native conformation. Unexpectedly, we found that dTTP and nucleotide analogs with exocyclic methyl groups bind to hNBD1 more strongly than ATP and preserve electrophysiological function of full-length F508del-hCFTR channels at temperatures up to 37 °C. Furthermore, nucleotides that increase open-channel probability, which reflects stabilization of an interdomain interface to hNBD1, thermally protect full-length F508del-hCFTR even when they do not stabilize isolated hNBD1. Therefore, stabilization of hNBD1 itself or of one of its interdomain interfaces by a small molecule indirectly offsets the destabilizing effect of the F508del mutation on full-length hCFTR. These results indicate that high-affinity binding of a small molecule to a remote site can correct a disease-causing mutation. We propose that the strategies described here should be applicable to identifying small molecules to help manage other human diseases caused by mutations that destabilize native protein conformation.

R-Domain Phosphorylation by Protein Kinase A Stimulates Dissociation of Unhydrolyzed ATP from the First Nucleotide-Binding Site of the Cystic Fibrosis Transmembrane Conductance Regulator.

The cystic fibrosis transmembrane conductance regulator (CFTR) is an asymmetric ATP-binding cassette transporter in which ATP hydrolysis occurs only at the second of the two composite nucleotide-binding sites whereas there are noncanonical substitutions of key catalytic residues in the first site. Therefore, in widely accepted models of CFTR function, ATP is depicted as remaining bound at the first site while it is hydrolyzed at the second site. However, the long lifetime of ATP at nucleotide-binding domain 1 (NBD1) had been measured under conditions where the channel had not been activated by phosphorylation. Here we show that phosphorylation by protein kinase A (PKA), obligatory for channel activation, strongly accelerates dissociation of the unhydrolyzed ATP from NBD1 of both full-length and NBD2-deleted CFTR. This stimulation of nucleotide release results from phosphorylation of the CFTR regulatory domain (residues 634-835) (R-domain). Mimicking phosphorylation by mutating the eight phosphorylation sites in the R-domain (8SE) has the same robust effect on accelerating the dissociation of ATP from NBD1. These findings provide new insight into relationships between R-domain phosphorylation and ATP binding and hydrolysis, the two main CFTR regulatory pathways.

Cryo-EM Visualization of an Active High Open Probability CFTR Anion Channel.

The cystic fibrosis transmembrane conductance regulator (CFTR) anion channel, crucial to epithelial salt and water homeostasis, and defective due to mutations in its gene in patients with cystic fibrosis, is a unique member of the large family of ATP-binding cassette transport proteins. Regulation of CFTR channel activity is stringently controlled by phosphorylation and nucleotide binding. Structural changes that underlie transitions between active and inactive functional states are not yet fully understood. Indeed the first 3D structures of dephosphorylated, ATP-free, and phosphorylated ATP-bound states were only recently reported. Here we have determined the structure of inactive and active states of a thermally stabilized CFTR, the latter with a very high channel open probability, confirmed after reconstitution into proteoliposomes. These structures, obtained at nominal resolution of 4.3 and 6.6 Å, reveal a unique repositioning of the transmembrane helices and regulatory domain density that provide insights into the structural transition between active and inactive functional states of CFTR. Moreover, we observe an extracellular vestibule that may provide anion access to the pore due to the conformation of transmembrane helices 7 and 8 that differs from the previous orthologue CFTR structures. In conclusion, our work contributes detailed structural information on an active, open state of the CFTR anion channel.

Stabilization of a nucleotide-binding domain of the cystic fibrosis transmembrane conductance regulator yields insight into disease-causing mutations.

Characterization of the second nucleotide-binding domain (NBD2) of the cystic fibrosis transmembrane conductance regulator (CFTR) has lagged behind research into the NBD1 domain, in part because NBD1 contains the F508del mutation, which is the dominant cause of cystic fibrosis. Research on NBD2 has also been hampered by the overall instability of the domain and the difficulty of producing reagents. Nonetheless, multiple disease-causing mutations reside in NBD2, and the domain is critical for CFTR function, because channel gating involves NBD1/NBD2 dimerization, and NBD2 contains the catalytically active ATPase site in CFTR. Recognizing the paucity of structural and biophysical data on NBD2, here we have defined a bioinformatics-based method for manually identifying stabilizing substitutions in NBD2, and we used an iterative process of screening single substitutions against thermal melting points to both produce minimally mutated stable constructs and individually characterize mutations. We present a range of stable constructs with minimal mutations to help inform further research on NBD2. We have used this stabilized background to study the effects of NBD2 mutations identified in cystic fibrosis (CF) patients, demonstrating that mutants such as N1303K and G1349D are characterized by lower stability, as shown previously for some NBD1 mutations, suggesting a potential role for NBD2 instability in the pathology of CF.

Structural stability of purified human CFTR is systematically improved by mutations in nucleotide binding domain 1.

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is an ABC transporter containing two transmembrane domains forming a chloride ion channel, and two nucleotide binding domains (NBD1 and NBD2). CFTR has presented a formidable challenge to obtain monodisperse, biophysically stable protein. Here we report a comprehensive study comparing effects of single and multiple NBD1 mutations on stability of both the NBD1 domain alone and on purified full length human CFTR. Single mutations S492P, A534P, I539T acted additively, and when combined with M470V, S495P, and R555K cumulatively yielded an NBD1 with highly improved structural stability. Strategic combinations of these mutations strongly stabilized the domain to attain a calorimetric Tm > 70 °C. Replica exchange molecular dynamics simulations on the most stable 6SS-NBD1 variant implicated fluctuations, electrostatic interactions and side chain packing as potential contributors to improved stability. Progressive stabilization of NBD1 directly correlated with enhanced structural stability of full-length CFTR protein. Thermal unfolding of the stabilized CFTR mutants, monitored by changes in intrinsic fluorescence, demonstrated that Tm could be shifted as high as 67.4 °C in 6SS-CFTR, more than 20 °C higher than wild-type. H1402S, an NBD2 mutation, conferred CFTR with additional thermal stability, possibly by stabilizing an NBD-dimerized conformation. CFTR variants with NBD1-stabilizing mutations were expressed at the cell surface in mammalian cells, exhibited ATPase and channel activity, and retained these functions to higher temperatures. The capability to produce enzymatically active CFTR with improved structural stability amenable to biophysical and structural studies will advance mechanistic investigations and future cystic fibrosis drug development.

Transmembrane helical interactions in the CFTR channel pore.

Mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene affect CFTR protein biogenesis or its function as a chloride channel, resulting in dysregulation of epithelial fluid transport in the lung, pancreas and other organs in cystic fibrosis (CF). Development of pharmaceutical strategies to treat CF requires understanding of the mechanisms underlying channel function. However, incomplete 3D structural information on the unique ABC ion channel, CFTR, hinders elucidation of its functional mechanism and correction of cystic fibrosis causing mutants. Several CFTR homology models have been developed using bacterial ABC transporters as templates but these have low sequence similarity to CFTR and are not ion channels. Here, we refine an earlier model in an outward (OWF) and develop an inward (IWF) facing model employing an integrated experimental-molecular dynamics simulation (200 ns) approach. Our IWF structure agrees well with a recently solved cryo-EM structure of a CFTR IWF state. We utilize cysteine cross-linking to verify positions and orientations of residues within trans-membrane helices (TMHs) of the OWF conformation and to reconstruct a physiologically relevant pore structure. Comparison of pore profiles of the two conformations reveal a radius sufficient to permit passage of hydrated Cl- ions in the OWF but not the IWF model. To identify structural determinants that distinguish the two conformations and possible rearrangements of TMHs within them responsible for channel gating, we perform cross-linking by bifunctional reagents of multiple predicted pairs of cysteines in TMH 6 and 12 and 6 and 9. To determine whether the effects of cross-linking on gating observed are the result of switching of the channel from open to close state, we also treat the same residue pairs with monofunctional reagents in separate experiments. Both types of reagents prevent ion currents indicating that pore blockage is primarily responsible.

Regulation of the cystic fibrosis transmembrane conductance regulator anion channel by tyrosine phosphorylation.

The cystic fibrosis transmembrane conductance regulator (CFTR) channel is activated by PKA phosphorylation of a regulatory domain that interacts dynamically with multiple CFTR domains and with other proteins. The large number of consensus sequences for phosphorylation by PKA has naturally focused most attention on regulation by this kinase. We report here that human CFTR is also phosphorylated by the tyrosine kinases p60c-Src (proto-oncogene tyrosine-protein kinase) and the proline-rich tyrosine kinase 2 (Pyk2), and they can also cause robust activation of quiescent CFTR channels. In excised patch-clamp experiments, CFTR activity during exposure to Src or Pyk2 reached ∼80% of that stimulated by PKA. Exposure to PKA after Src or Pyk2 caused a further increase to the level induced by PKA alone, implying a common limiting step. Channels became spontaneously active when v-Src or the catalytic domain of Pyk2 was coexpressed with CFTR and were further stimulated by the tyrosine phosphatase inhibitor dephostatin. Exogenous Src also activated 15SA-CFTR, a variant that lacks 15 potential PKA sites and has little response to PKA. PKA-independent activation by tyrosine phosphorylation has implications for the mechanism of regulation by the R domain and for the physiologic functions of CFTR.

Thermal stability of purified and reconstituted CFTR in a locked open channel conformation.

CFTR is unique among ABC transporters as the only one functioning as an ion channel and from a human health perspective because mutations in its gene cause cystic fibrosis. Although considerable advances have been made towards understanding CFTR's mechanism of action and the impact of mutations, the lack of a high-resolution 3D structure has hindered progress. The large multi-domain membrane glycoprotein is normally present at low copy number and when over expressed at high levels it aggregates strongly, limiting the production of stable mono-disperse preparations. While the reasons for the strong self-association are not fully understood, its relatively low thermal stability seems likely to be one. The major CF causing mutation, ΔF508, renders the protein very thermally unstable and therefore a great deal of attention has been paid to this property of CFTR. Multiple second site mutations of CFTR in NBD1 where F508 normally resides and small molecule binders of the domain increase the thermal stability of the mutant. These manipulations also stabilize the wild-type protein. Here we have applied ΔF508-stabilizing changes and other modifications to generate wild-type constructs that express at much higher levels in scaled-up suspension cultures of mammalian cells. After purification and reconstitution into liposomes these proteins are active in a locked-open conformation at temperatures as high as 50 °C and remain monodisperse at 4 °C in detergent or lipid for at least a week. The availability of adequate amounts of these and related stable active preparations of homogeneous CFTR will enable stalled structural and ligand binding studies to proceed.

Targeted proteomic quantitation of the absolute expression and turnover of cystic fibrosis transmembrane conductance regulator in the apical plasma membrane.

Deficient chloride transport through cystic fibrosis (CF) transmembrane conductance regulator (CFTR) causes lethal complications in CF patients. CF is the most common autosomal recessive genetic disease, which is caused by mutations in the CFTR gene; thus, CFTR mutants can serve as primary targets for drugs to modulate and rescue the ion channel's function. The first step of drug modulation is to increase the expression of CFTR in the apical plasma membrane (PM); thus, accurate measurement of CFTR in the PM is desired. This work reports a tandem enrichment strategy to prepare PM CFTR and uses a stable isotope labeled CFTR sample as the quantitation reference to measure the absolute amount of apical PM expression of CFTR in CFBE 41o- cells. It was found that CFBE 41o- cells expressing wild-type CFTR (wtCFTR), when cultured on plates, had 2.9 ng of the protein in the apical PM per million cells; this represented 10% of the total CFTR found in the cells. When these cells were polarized on filters, the apical PM expression of CFTR increased to 14%. Turnover of CFTR in the apical PM of baby hamster kidney cells overexpressing wtCFTR (BHK-wtCFTR) was also quantified by targeted proteomics based on multiple reaction monitoring mass spectrometry; wtCFTR had a half-life of 29.0 ± 2.5 h in the apical PM. This represents the first direct measurement of CFTR turnover using stable isotopes. The absolute quantitation and turnover measurements of CFTR in the apical PM can significantly facilitate understanding the disease mechanism of CF and thus the development of new disease-modifying drugs. Absolute CFTR quantitation allows for direct result comparisons among analyses, analysts, and laboratories and will greatly amplify the overall outcome of CF research and therapy.

Correctors of ΔF508 CFTR restore global conformational maturation without thermally stabilizing the mutant protein.

Most cystic fibrosis is caused by the deletion of a single amino acid (F508) from CFTR and the resulting misfolding and destabilization of the protein. Compounds identified by high-throughput screening to improve ΔF508 CFTR maturation have already entered clinical trials, and it is important to understand their mechanisms of action to further improve their efficacy. Here, we showed that several of these compounds, including the investigational drug VX-809, caused a much greater increase (5- to 10-fold) in maturation at 27 than at 37°C (<2-fold), and the mature product remained short-lived (T(1/2)∼4.5 h) and thermally unstable, even though its overall conformational state was similar to wild type, as judged by resistance to proteolysis and interdomain cross-linking. Consistent with its inability to restore thermodynamic stability, VX-809 stimulated maturation 2-5-fold beyond that caused by several different stabilizing modifications of NBD1 and the NBD1/CL4 interface. The compound also promoted maturation of several disease-associated processing mutants on the CL4 side of this interface. Although these effects may reflect an interaction of VX-809 with this interface, an interpretation supported by computational docking, it also rescued maturation of mutants in other cytoplasmic loops, either by allosteric effects or via additional sites of action. In addition to revealing the capabilities and some of the limitations of this important investigational drug, these findings clearly demonstrate that ΔF508 CFTR can be completely assembled and evade cellular quality control systems, while remaining thermodynamically unstable. He, L., Kota, P., Aleksandrov, A. A., Cui, L., Jensen, T., Dokholyan, N. V., Riordan, J. R. Correctors of ΔF508 CFTR restore global conformational maturation without thermally stabilizing the mutant protein.

Cigarette smoke exposure induces CFTR internalization and insolubility, leading to airway surface liquid dehydration.

Cigarette smoke (CS) exposure induces mucus obstruction and the development of chronic bronchitis (CB). While many of these responses are determined genetically, little is known about the effects CS can exert on pulmonary epithelia at the protein level. We, therefore, tested the hypothesis that CS exerts direct effects on the CFTR protein, which could impair airway hydration, leading to the mucus stasis characteristic of both cystic fibrosis and CB. In vivo and in vitro studies demonstrated that CS rapidly decreased CFTR activity, leading to airway surface liquid (ASL) volume depletion (i.e., dehydration). Further studies revealed that CS induced internalization of CFTR. Surprisingly, CS-internalized CFTR did not colocalize with lysosomal proteins. Instead, the bulk of CFTR shifted to a detergent-resistant fraction within the cell and colocalized with the intermediate filament vimentin, suggesting that CS induced CFTR movement into an aggresome-like, perinuclear compartment. To test whether airway dehydration could be reversed, we used hypertonic saline (HS) as an osmolyte to rehydrate ASL. HS restored ASL height in CS-exposed, dehydrated airway cultures. Similarly, inhaled HS restored mucus transport and increased clearance in patients with CB. Thus, we propose that CS exposure rapidly impairs CFTR function by internalizing CFTR, leading to ASL dehydration, which promotes mucus stasis and a failure of mucus clearance, leaving smokers at risk for developing CB. Furthermore, our data suggest that strategies to rehydrate airway surfaces may provide a novel form of therapy for patients with CB.

The cystic fibrosis transmembrane conductance regulator (CFTR): three-dimensional structure and localization of a channel gate.

Cystic fibrosis affects about 1 in 2500 live births and involves loss of transmembrane chloride flux due to a lack of a membrane protein channel termed the cystic fibrosis transmembrane conductance regulator (CFTR). We have studied CFTR structure by electron crystallography. The data were compared with existing structures of other ATP-binding cassette transporters. The protein was crystallized in the outward facing state and resembled the well characterized Sav1866 transporter. We identified regions in the CFTR map, not accounted for by Sav1866, which were potential locations for the regulatory region as well as the channel gate. In this analysis, we were aided by the fact that the unit cell was composed of two molecules not related by crystallographic symmetry. We also identified regions in the fitted Sav1866 model that were missing from the map, hence regions that were either disordered in CFTR or differently organized compared with Sav1866. Apart from the N and C termini, this indicated that in CFTR, the cytoplasmic end of transmembrane helix 5/11 and its associated loop could be partly disordered (or alternatively located).

Domain location within the cystic fibrosis transmembrane conductance regulator protein investigated by electron microscopy and gold labelling.

The domain organisation of the cystic fibrosis transmembrane conductance regulator (CFTR) protein was studied using electron microscopy of detergent-solubilised dimeric complexes. Ni-NTA nanogold labelling data suggest that in the nonphosphorylated, nucleotide-free state, the C-terminus is intimately associated with the cytoplasmic ATP-binding regions, whilst part of the regulatory domain occupies a position close to the cytoplasmic surface of the lipid membrane. Removal of the entire second nucleotide binding domain (NBD2) results in a deficit in the CFTR structure that is consistent with the size and shape of a single NBD. The data suggest that NBD2 lies closer to the C2 symmetry axis than the first nucleotide binding domain (NBD1) and that NBD2 from one CFTR monomer also contacts NBD1 from the opposing one. These data suggest that current homology models for CFTR based on other ATP-binding cassette proteins appear to be reasonable, at least to low resolution. We also find that Ni-NTA nanogold labelling of an internal hexa-Histidine sequence is a valuable approach to locate individual protein domains.

Reduced histone deacetylase 7 activity restores function to misfolded CFTR in cystic fibrosis.

Chemical modulation of histone deacetylase (HDAC) activity by HDAC inhibitors (HDACi) is an increasingly important approach for modifying the etiology of human disease. Loss-of-function diseases arise as a consequence of protein misfolding and degradation, which lead to system failures. The DeltaF508 mutation in cystic fibrosis transmembrane conductance regulator (CFTR) results in the absence of the cell surface chloride channel and a loss of airway hydration, leading to the premature lung failure and reduced lifespan responsible for cystic fibrosis. We now show that the HDACi suberoylanilide hydroxamic acid (SAHA) restores surface channel activity in human primary airway epithelia to levels that are 28% of those of wild-type CFTR. Biological silencing of all known class I and II HDACs reveals that HDAC7 plays a central role in restoration of DeltaF508 function. We suggest that the tunable capacity of HDACs can be manipulated by chemical biology to counter the onset of cystic fibrosis and other human misfolding disorders.

Restoration of domain folding and interdomain assembly by second-site suppressors of the DeltaF508 mutation in CFTR.

Deletion of PHE508 (DeltaF508) from the first nucleotide-binding domain (NBD1) of CFTR, which causes most cystic fibrosis, disrupts the folding and assembly of the protein. Although the folding pathways and yield of isolated NBD1 are altered, its global structure is not, and details of the changes in the rest of the protein remain unclear. To gain further insight into how the whole mutant protein is altered, we have determined the influence of known second-site suppressor mutations in NBD1 on the conformation of this domain and key interfaces between domains. We found that the suppressors restored maturation of only those processing mutations located in NBD1, but not in other domains, including those in the C-terminal cytoplasmic loop of the second membrane-spanning domain, which forms an interface with the NBD1 surface. Nevertheless, the suppressors promoted the formation of this interface and others in the absence of F508. The suppressors restored maturation in a DeltaF508 construct from which NBD2 was absent but to a lesser extent than in the full-length, indicating that DeltaF508 disrupts interactions involving NBD2, as well as other domains. Rescue of DeltaF508-CFTR by suppressors required the biosynthesis of the entire full-length protein in continuity, as it did not occur when N- and C-terminal "halves" were coexpressed. Simultaneous with these interdomain perturbations, DeltaF508 resulted in suppressor reversed alterations in accessibility of residues both in the F508-containing NBD1 surface loop and in the Q loop within the domain core. Thus, in the context of the full-length protein, DeltaF508 mutation causes detectable changes in NBD1 conformation, as well as interdomain interactions.

Phenylalanine-508 mediates a cytoplasmic-membrane domain contact in the CFTR 3D structure crucial to assembly and channel function.

Deletion of phenylalanine-508 (Phe-508) from the N-terminal nucleotide-binding domain (NBD1) of the cystic fibrosis transmembrane conductance regulator (CFTR), a member of the ATP-binding cassette (ABC) transporter family, disrupts both its folding and function and causes most cystic fibrosis. Most mutant nascent chains do not pass quality control in the ER, and those that do remain thermally unstable, only partially functional, and are rapidly endocytosed and degraded. Although the lack of the Phe-508 peptide backbone diminishes the NBD1 folding yield, the absence of the aromatic side chain is primarily responsible for defective CFTR assembly and channel gating. However, the site of interdomain contact by the side chain is unknown as is the high-resolution 3D structure of the complete protein. Here we present a 3D structure of CFTR, constructed by molecular modeling and supported biochemically, in which Phe-508 mediates a tertiary interaction between the surface of NBD1 and a cytoplasmic loop (CL4) in the C-terminal membrane-spanning domain (MSD2). This crucial cytoplasmic membrane interface, which is dynamically involved in regulation of channel gating, explains the known sensitivity of CFTR assembly to many disease-associated mutations in CL4 as well as NBD1 and provides a sharply focused target for small molecules to treat CF. In addition to identifying a key intramolecular site to be repaired therapeutically, our findings advance understanding of CFTR structure and function and provide a platform for focused biochemical studies of other features of this unique ABC ion channel.

Chemical and biological folding contribute to temperature-sensitive DeltaF508 CFTR trafficking.

Proteostasis (Balch WE, Morimoto RI, Dillin A, Kelly JW. Adapting proteostasis for disease intervention. Science 2008;319:916-919) refers to the biology that maintains the proteome in health and disease. Proteostasis is challenged by the most common mutant in cystic fibrosis, DeltaF508, a chloride channel [the cystic fibrosis transmembrane conductance regulator (CFTR)] that exhibits a temperature-sensitive phenotype for coupling to the coatomer complex II (COPII) transport machine for exit from the endoplasmic reticulum. Whether rescue of export of DeltaF508 CFTR at reduced temperature simply reflects energetic stabilization of the chemical fold defined by its primary sequence or requires a unique proteostasis environment is unknown. We now show that reduced temperature (30 degrees C) export of DeltaF508 does not occur in some cell types, despite efficient export of wild-type CFTR. We find that DeltaF508 export requires a local biological folding environment that is sensitive to heat/stress-inducible factors found in some cell types, suggesting that the energetic stabilization by reduced temperature is necessary, but not sufficient, for export of DeltaF508. Thus, the cell may require a proteostasis environment that is in part distinct from the wild-type pathway to restore DeltaF508 coupling to COPII. These results are discussed in the context of the energetics of the protein fold and the potential application of small molecules to achieve a proteostasis environment favoring export of a functional form of DeltaF508.

Role of individual R domain phosphorylation sites in CFTR regulation by protein kinase A.

The cystic fibrosis transmembrane conductance regulator (CFTR) plays a critical role in transcellular ion transport and when defective, results in the genetic disease cystic fibrosis. CFTR is novel in the ATP-binding cassette superfamily as an ion channel that is enabled by a unique unstructured regulatory domain. This R domain contains multiple protein kinase A sites, which when phosphorylated allow channel gating. Most of the sites have been indicated to stimulate channel activity, while two of them have been suggested to be inhibitory. It is unknown whether individual sites act coordinately or distinctly. To address this issue, we raised monoclonal antibodies recognizing the unphosphorylated, but not the phosphorylated states of four functionally relevant sites (700, 737, 768, and 813). This enabled simultaneous monitoring of their phosphorylation and dephosphorylation and revealed that both processes occurred rapidly at the first three sites, but more slowly at the fourth. The parallel phosphorylation rates of the stimulatory 700 and the putative inhibitory 737 and 768 sites prompted us to reexamine the role of the latter two. With serines 737 and 768 reintroduced individually into a PKA insensitive variant, in which serines at 15 sites had been replaced by alanines, a level of channel activation by PKA was restored, showing that these sites can mediate stimulation. Thus, we have provided new tools to study the CFTR regulation by phosphorylation and found that sites proposed to inhibit channel activity can also participate in stimulation.

Modulation of endocytic trafficking and apical stability of CFTR in primary human airway epithelial cultures.

CFTR is a highly regulated apical chloride channel of epithelial cells that is mutated in cystic fibrosis (CF). In this study, we characterized the apical stability and intracellular trafficking of wild-type and mutant CFTR in its native environment, i.e., highly differentiated primary human airway epithelial (HAE) cultures. We labeled the apical pool of CFTR and subsequently visualized the protein in intracellular compartments. CFTR moved from the apical surface to endosomes and then efficiently recycled back to the surface. CFTR endocytosis occurred more slowly in polarized than in nonpolarized HAE cells or in a polarized epithelial cell line. The most common mutation in CF, DeltaF508 CFTR, was rescued from endoplasmic reticulum retention by low-temperature incubation but transited from the apical membrane to endocytic compartments more rapidly and recycled less efficiently than wild-type CFTR. Incubation with small-molecule correctors resulted in DeltaF508 CFTR at the apical membrane but did not restore apical stability. To stabilize the mutant protein at the apical membrane, we found that the dynamin inhibitor Dynasore and the cholesterol-extracting agent cyclodextrin dramatically reduced internalization of DeltaF508, whereas the proteasomal inhibitor MG-132 completely blocked endocytosis of DeltaF508. On examination of intrinsic properties of CFTR that may affect its apical stability, we found that N-linked oligosaccharides were not necessary for transport to the apical membrane but were required for efficient apical recycling and, therefore, influenced the turnover of surface CFTR. Thus apical stability of CFTR in its native environment is affected by properties of the protein and modulation of endocytic trafficking.

Architecture of the cystic fibrosis transmembrane conductance regulator protein and structural changes associated with phosphorylation and nucleotide binding.

We describe biochemical and structural studies of the isolated cystic fibrosis transmembrane conductance regulator (CFTR) protein. Using electron cryomicroscopy, low resolution three-dimensional structures have been obtained for the non-phosphorylated protein in the absence of nucleotide and for the phosphorylated protein with ATP. In the latter state, the cytosolic nucleotide-binding domains move closer together, forming a more compact packing arrangement. Associated with this is a reorganization within the cylindrical transmembrane domains, consistent with a shift from an inward-facing to outward-facing configuration. A region of density in the non-phosphorylated protein that extends from the bottom of the cytosolic regions up to the transmembrane domains is hypothesised to represent the unique regulatory region of CFTR. These data offer insights into the architecture of this ATP-binding cassette protein, and shed light on the global motions associated with nucleotide binding and priming of the chloride channel via phosphorylation of the regulatory region.