Current insights into the role of PKA phosphorylation in CFTR channel activity and the pharmacological rescue of cystic fibrosis disease-causing mutants

Cystic fibrosis transmembrane conductance regulator (CFTR) channel gating is predominantly regulated by protein kinase A (PKA)-dependent phosphorylation. In addition to regulating CFTR channel activity, PKA phosphorylation is also involved in enhancing CFTR trafficking and mediating conformational changes at the interdomain interfaces of the protein. The major cystic fibrosis (CF)-causing mutation is the deletion of phenylalanine at position 508 (F508del); it causes many defects that affect CFTR trafficking, stability, and gating at the cell surface. Due to the multiple roles of PKA phosphorylation, there is growing interest in targeting PKA-dependent signaling for rescuing the trafficking and functional defects of F508del-CFTR. This review will discuss the effects of PKA phosphorylation on wild-type CFTR, the consequences of CF mutations on PKA phosphorylation, and the development of therapies that target PKA-mediated signaling.     

Molecular models of the open and closed states of the whole human CFTR protein

Cystic fibrosis transmembrane conductance regulator (CFTR), involved in cystic fibrosis (CF), is a chloride channel belonging to the ATP-binding cassette (ABC) superfamily. Using the experimental structure of Sav1866 as template, we previously modeled the human CFTR structure, including membrane-spanning domains (MSD) and nucleotide-binding domains (NBD), in an outward-facing conformation (open channel state). Here, we constructed a model of the CFTR inward-facing conformation (closed channel) on the basis of the recent corrected structures of MsbA and compared the structural features of those two states of the channel. Interestingly, the MSD:NBD coupling interfaces including F508 (ΔF508 being the most common CF mutation) are mainly left unchanged. This prediction, completed by the modeling of the regulatory R domain, is supported by experimental data and provides a molecular basis for a better understanding of the functioning of CFTR, especially of the structural features that make CFTR the unique channel among the ABC transporters.     

Functional and pharmacological induced structural changes of the cystic fibrosis transmembrane conductance regulator in the membrane solved using SAXS

The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is a membrane-integral protein that belongs to the ATP-binding cassette superfamily. Mutations in the CFTR gene cause cystic fibrosis in which salt, water, and protein transports are defective in various tissues. To investigate the conformation of the CFTR in the membrane, we applied the small-angle x-ray scattering (SAXS) technique on microsomal membranes extracted from NIH/3T3 cells permanentely transfected with wild-type (WT) CFTR and with CFTR carrying the ΔF508 mutation. The electronic density profile of the membranes was calculated from the SAXS data, assuming the lipid bilayer electronic density to be composed by a series of Gaussian shells. The data indicate that membranes in the microsome vesicles, that contain mostly endoplasmic reticulum membranes, are oriented in the outside-out conformation. Phosphorylation does not change significantly the electronic density profile, while dephosphorylation produces a significant modification in the inner side of the profile. Thus, we conclude that the CFTR and its associated protein complex in microsomes are mostly phosphorylated. The electronic density profile of the ΔF508-CFTR microsomes is completely different from WT, suggesting a different assemblage of the proteins in the membranes. Low-temperature treatment of cells rescues the ΔF508-CFTR protein, resulting in a conformation that resembles the WT. Differently, treatment with the corrector VX-809 modifies the electronic profile of ΔF508-CFTR membrane, but does not recover completely the WT conformation. To our knowledge, this is the first report of a direct physical measurement of the structure of membranes containing CFTR in its native environment and in different functional and pharmacological conditions.     

A SAXS-based ensemble model of the native and phosphorylated regulatory domain of the CFTR

The cystic fibrosis transmembrane conductance regulator (CFTR), the defective protein in cystic fibrosis, is an anion channel activated by protein kinase A phosphorylation. The regulatory domain (RD) of CFTR has multiple phosphorylation sites, and is responsible for channel activation. This domain is intrinsically disordered, rendering the structural analysis a difficult task, as high-resolution techniques are barely applicable. In this work, we obtained a biophysical characterization of the native and phosphorylated RD in solution by employing complementary structural methods. The native RD has a gyration radius of 3.25 nm, and a maximum molecular dimension of 11.4 nm, larger than expected for a globular protein of the same molecular mass. Phosphorylation causes compaction of the structure, yielding a significant reduction of the gyration radius, to 2.92 nm, and on the maximum molecular dimension to 10.2 nm. Using an ensemble optimization method, we were able to generate a low-resolution, three-dimensional model of the native and the phosphorylated RD based on small-angle X-ray scattering data. We have obtained the first experiment-based model of the CFTR regulatory domain, which will be useful to understand the molecular mechanisms of normal and pathological CFTR functioning.     

The gating of the CFTR channel

Cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel expressed in the apical membrane of epithelia. Mutations in the CFTR gene are the cause of cystsic fibrosis. CFTR is the only ABC-protein that constitutes an ion channel pore forming subunit. CFTR gating is regulated in complex manner as phosphorylation is mandatory for channel activity and gating is directly regulated by binding of ATP to specific intracellular sites on the CFTR protein. This review covers our current understanding on the gating mechanism in CFTR and illustrates the relevance of alteration of these mechanisms in the onset of cystic fibrosis.     

Molecular modelling and molecular dynamics of CFTR

The cystic fibrosis transmembrane conductance regulator (CFTR) protein is a member of the ATP-binding cassette (ABC) transporter superfamily that functions as an ATP-gated channel. Considerable progress has been made over the last years in the understanding of the molecular basis of the CFTR functions, as well as dysfunctions causing the common genetic disease cystic fibrosis (CF). This review provides a global overview of the theoretical studies that have been performed so far, especially molecular modelling and molecular dynamics (MD) simulations. A special emphasis is placed on the CFTR-specific evolution of an ABC transporter framework towards a channel function, as well as on the understanding of the effects of disease-causing mutations and their specific modulation. This in silico work should help structure-based drug discovery and design, with a view to develop CFTR-specific pharmacotherapeutic approaches for the treatment of CF in the context of precision medicine.     

Nucleotide binding domains of human CFTR: a structural classification of critical residues and disease-causing mutations.

Defective function of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) causes CF, the most frequent lethal inherited disease among the Caucasian population. The structure of this chloride ion channel includes two nucleotide-binding domains (NBDs), whose ATPase activity controls channel gating. Recently, the experimental structures of mouse and human CFTR NBD1 and our model of the human CFTR NBD1/NBD2 heterodimer have provided new insights into specific structural features of the CFTR NBD dimer. In the present work, we provide a structural classification of CF-causing mutations which may complement the existing functional classification. Our analysis also identified amino acid residues which may play a critical role in interdomain interaction and are located at the NBD1-NBD2 interface or on the surface of the dimer. In particular, a cluster of aromatic amino acids, which includes F508 and straddles the two NBDs, might be directly involved in the interaction of the NBD1/NBD2 heterodimer with the channel-forming membrane-spanning domains.Received 24 May 2005; received after revision 13 June 2005; accepted 18 June 2005     

New paradigms of CFTR chloride channel regulation

The Cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel controls salt and water transport across epithelial tissues. Alterations in the activity of this ion channel lead to two major human diseases: cystic fibrosis (low CFTR activity) and secretory diarrhea (excessive CFTR activity). The goal of this article is to review recent developments in our understanding of two aspects of CFTR biology: (i) interactions between CFTR domains (intramolecular interactions) that control the gating of this epithelial chloride channel and (ii) interactions between CFTR and other proteins (intermolecular interactions) that couple the activity of this ion channel to additional cellular processes in epithelial cells (e.g. membrane traffic). Clarifying the nature of these interactions may lead to the development of novel strategies for treating diseases that involve the CFTR chloride channel. Received 12 October 1999; accepted 31 December 1999     

From the endoplasmic reticulum to the plasma membrane: mechanisms of CFTR folding and trafficking

CFTR biogenesis starts with its co-translational insertion into the membrane of endoplasmic reticulum and folding of the cytosolic domains, towards the acquisition of a fully folded compact native structure. Efficiency of this process is assessed by the ER quality control system that allows the exit of folded proteins but targets unfolded/misfolded CFTR to degradation. If allowed to leave the ER, CFTR is modified at the Golgi and reaches the post-Golgi compartments to be delivered to the plasma membrane where it functions as a cAMP- and phosphorylation-regulated chloride/bicarbonate channel. CFTR residence at the membrane is a balance of membrane delivery, endocytosis, and recycling. Several adaptors, motor, and scaffold proteins contribute to the regulation of CFTR stability and are involved in continuously assessing its structure through peripheral quality control systems. Regulation of CFTR biogenesis and traffic (and its dysregulation by mutations, such as the most common F508del) determine its overall activity and thus contribute to the fine modulation of chloride secretion and hydration of epithelial surfaces. This review covers old and recent knowledge on CFTR folding and trafficking from its synthesis to the regulation of its stability at the plasma membrane and highlights how several of these steps can be modulated to promote the rescue of mutant CFTR.     

Cystic fibrosis transmembrane conductance regulator—emerging regulator of cancer

Mutations of cystic fibrosis transmembrane conductance regulator (CFTR) cause cystic fibrosis, the most common life-limiting recessive genetic disease among Caucasians. CFTR mutations have also been linked to increased risk of various cancers but remained controversial for a long time. Recent studies have begun to reveal that CFTR is not merely an ion channel but also an important regulator of cancer development and progression with multiple signaling pathways identified. In this review, we will first present clinical findings showing the correlation of genetic mutations or aberrant expression of CFTR with cancer incidence in multiple cancers. We will then focus on the roles of CFTR in fundamental cellular processes including transformation, survival, proliferation, migration, invasion and epithelial–mesenchymal transition in cancer cells, highlighting the signaling pathways involved. Finally, the association of CFTR expression levels with patient prognosis, and the potential of CFTR as a cancer prognosis indicator in human malignancies will be discussed.     

A “SYDE” effect of hierarchical phosphorylation: possible relevance to the cystic fibrosis basic defect

The motif “SYDE”, incorporating the protein kinase CK2 consensus sequence (S-x-x-E) has been found to be phosphorylated at both its serine and tyrosine residues in several proteins. Of special interest is the case of cystic fibrosis Transmembrane-conductance Regulator (CFTR), where this motif is close to the residue (F508), whose deletion is the by far commonest cause of cystic fibrosis. Intriguingly, however, CFTR S511 cannot be phosphorylated by CK2 to any appreciable extent. Using a number of peptide substrates encompassing the CFTR “SYDE” site we have recently shown that: (1) failure of CK2 to phosphorylate the S⁵¹¹YDE motif is due to the presence of Y512; (2) CK2 readily phosphorylates S511 if Y512 is replaced by a phospho-tyrosine; (3) the Src family protein tyrosine kinase Lyn phosphorylates Y512 in a manner that is enhanced by the deletion of F508. These data, in conjunction with the recent observation that by inhibiting CK2 the degradation of F508delCFTR is reduced, lead us to hypothesize that the hierarchical phosphorylation of the motif SYDE by the concerted action of protein tyrosine kinases and CK2 is one of the mechanisms that cooperate to the premature degradation of F508delCFTR.     

Architecture and functional properties of the CFTR channel pore

The main function of the cystic fibrosis transmembrane conductance regulator (CFTR) is as an ion channel for the movement of small anions across epithelial cell membranes. As an ion channel, CFTR must form a continuous pathway across the cell membrane—referred to as the channel pore—for the rapid electrodiffusional movement of ions. This review summarizes our current understanding of the architecture of the channel pore, as defined by electrophysiological analysis and molecular modeling studies. This includes consideration of the characteristic functional properties of the pore, definition of the overall shape of the entire extent of the pore, and discussion of how the molecular structure of distinct regions of the pore might control different facets of pore function. Comparisons are drawn with closely related proteins that are not ion channels, and also with structurally unrelated proteins with anion channel function. A simple model of pore function is also described.     

Proteomic interaction profiling reveals KIFC1 as a factor involved in early targeting of F508del-CFTR to degradation

Misfolded F508del-CFTR, the main molecular cause of the recessive disorder cystic fibrosis, is recognized by the endoplasmic reticulum (ER) quality control (ERQC) resulting in its retention and early degradation. The ERQC mechanisms rely mainly on molecular chaperones and on sorting motifs, whose presence and exposure determine CFTR retention or exit through the secretory pathway. Arginine-framed tripeptides (AFTs) are ER retention motifs shown to modulate CFTR retention. However, the interactions and regulatory pathways involved in this process are still largely unknown. Here, we used proteomic interaction profiling and global bioinformatic analysis to identify factors that interact differentially with F508del-CFTR and F508del-CFTR without AFTs (F508del-4RK-CFTR) as putative regulators of this specific ERQC checkpoint. Using LC–MS/MS, we identified kinesin family member C1 (KIFC1) as a stronger interactor with F508del-CFTR versus F508del-4RK-CFTR. We further validated this interaction showing that decreasing KIFC1 levels or activity stabilizes the immature form of F508del-CFTR by reducing its degradation. We conclude that the current approach is able to identify novel putative therapeutic targets that can be ultimately used to the benefit of CF patients.     

CFTR pharmacology

CFTR protein is an ion channel regulated by cAMP-dependent phosphorylation and expressed in many types of epithelial cells. CFTR-mediated chloride and bicarbonate secretion play an important role in the respiratory and gastrointestinal systems. Pharmacological modulators of CFTR represent promising drugs for a variety of diseases. In particular, correctors and potentiators may restore the activity of CFTR in cystic fibrosis patients. Potentiators are also potentially useful to improve mucociliary clearance in patients with chronic obstructive pulmonary disease. On the other hand, CFTR inhibitors may be useful to block fluid and electrolyte loss in secretory diarrhea and slow down the progression of polycystic kidney disease.     

Cystic fibrosis: a clinical view

Cystic fibrosis (CF), a monogenic disease caused by mutations in the CFTR gene on chromosome 7, is complex and greatly variable in clinical expression. Airways, pancreas, male genital system, intestine, liver, bone, and kidney are involved. The lack of CFTR or its impaired function causes fat malabsorption and chronic pulmonary infections leading to bronchiectasis and progressive lung damage. Previously considered lethal in infancy and childhood, CF has now attained median survivals of 50 years of age, mainly thanks to the early diagnosis through neonatal screening, recognition of mild forms, and an aggressive therapeutic attitude. Classical treatment includes pancreatic enzyme replacement, respiratory physiotherapy, mucolitics, and aggressive antibiotic therapy. A significant proportion of patients with severe symptoms still requires lung or, less frequently, liver transplantation. The great number of mutations and their diverse effects on the CFTR protein account only partially for CF clinical variability, and modifier genes have a role in modulating the clinical expression of the disease. Despite the increasing understanding of CFTR functioning, several aspects of CF need still to be clarified, e.g., the worse outcome in females, the risk of malignancies, the pathophysiology, and best treatment of comorbidities, such as CF-related diabetes or CF-related bone disorder. Research is focusing on new drugs restoring CFTR function, some already available and with good clinical impact, others showing promising preliminary results that need to be confirmed in phase III clinical trials.     

Atomic model of human cystic fibrosis transmembrane conductance regulator: Membrane-spanning domains and coupling interfaces

We describe herein an atomic model of the outward-facing three-dimensional structure of the membrane-spanning domains (MSDs) and nucleotide-binding domains (NBDs) of human cystic fibrosis transmembrane conductance regulator (CFTR), based on the experimental structure of the bacterial transporter Sav1866. This model, which is in agreement with previous experimental data, highlights the role of some residues located in the transmembrane passages and directly involved in substrate translocation and of some residues within the intracellular loops (ICL1-ICL4) making MSD/NBD contacts. In particular, our model reveals that D173 ICL1 and N965 ICL3 likely interact with the bound nucleotide and that an intricate H-bond network (involving especially the ICL4 R1070 and the main chain of NBD1 F508) may stabilize the interface between MSD2 and the NBD1F508 region. These observations allow new insights into the ATP-binding sites asymmetry and into the molecular consequences of the F508 deletion, which is the most common cystic fibrosis mutation.     

Role of CFTR in epithelial physiology

Salt and fluid absorption and secretion are two processes that are fundamental to epithelial function and whole body fluid homeostasis, and as such are tightly regulated in epithelial tissues. The CFTR anion channel plays a major role in regulating both secretion and absorption in a diverse range of epithelial tissues, including the airways, the GI and reproductive tracts, sweat and salivary glands. It is not surprising then that defects in CFTR function are linked to disease, including life-threatening secretory diarrhoeas, such as cholera, as well as the inherited disease, cystic fibrosis (CF), one of the most common life-limiting genetic diseases in Caucasian populations. More recently, CFTR dysfunction has also been implicated in the pathogenesis of acute pancreatitis, chronic obstructive pulmonary disease (COPD), and the hyper-responsiveness in asthma, underscoring its fundamental role in whole body health and disease. CFTR regulates many mechanisms in epithelial physiology, such as maintaining epithelial surface hydration and regulating luminal pH. Indeed, recent studies have identified luminal pH as an important arbiter of epithelial barrier function and innate defence, particularly in the airways and GI tract. In this chapter, we will illustrate the different operational roles of CFTR in epithelial function by describing its characteristics in three different tissues: the airways, the pancreas, and the sweat gland.     

Nucleotide-binding domains of human cystic fibrosis transmembrane conductance regulator: detailed sequence analysis and three-dimensional modeling of the heterodimer

The cystic fibrosis transmembrane conductance regulator (CFTR) protein is encoded by the gene that is defective in cystic fibrosis, the most common lethal inherited disease among the Caucasian population. CFTR belongs to the ABC transporter superfamily, whose members form macromolecular architectures composed of two membrane-spanning domains and two nucleotide-binding domains (NBDs). The experimental structures of NBDs from several ABC transporters have recently been solved, opening new avenues for understanding the structure/function relationships and the consequences of some disease-causing mutations of CFTR. Based on a detailed sequence/structure analysis, we propose here a three-dimensional model of the human CFTR NBD heterodimer. This model, which is in agreement with recent experimental data, highlights the specific features of the CFTR asymmetric active sites located at the interface between the two NBDs. Moreover, additional CFTR-specific features can be identified at the subunit interface, which may play critical roles in active site interdependence and are uncommon in other NBD dimers.Received 16 October 2003; received after revision 16 November 2003; accepted 21 November 2003     

Conformational change of the extracellular parts of the CFTR protein during channel gating

Cystic fibrosis can be treated by potentiators, drugs that interact directly with the cystic fibrosis transmembrane conductance regulator (CFTR) Cl^? channel to increase its open probability. These substances likely target key conformational changes occurring during channel opening and closing, however, the molecular bases of these conformational changes, and their susceptibility to manipulation are poorly understood. We have used patch clamp recording to identify changes in the three-dimensional organization of the extracellularly accessible parts of the CFTR protein during channel opening and closing. State-dependent formation of both disulfide bonds and Cd²⁺ bridges occurred for pairs of cysteine side-chains introduced into the extreme extracellular ends of transmembrane helices (TMs) 1, 6, and 12. Between each of these three TMs, we found that both disulfide bonds and metal bridges formed preferentially or exclusively in the closed state and that these inter-TM cross-links stabilized the closed state. These results indicate that the extracellular ends of these TMs are close together when the channel is closed and that they separate from each other when the channel opens. These findings identify for the first time key conformational changes in the extracellular parts of the CFTR protein that can potentially be manipulated to control channel activity.     

Combining theoretical and experimental data to decipher CFTR 3D structures and functions

Cryo-electron microscopy (cryo-EM) has recently provided invaluable experimental data about the full-length cystic fibrosis transmembrane conductance regulator (CFTR) 3D structure. However, this experimental information deals with inactive states of the channel, either in an apo, quiescent conformation, in which nucleotide-binding domains (NBDs) are widely separated or in an ATP-bound, yet closed conformation. Here, we show that 3D structure models of the open and closed forms of the channel, now further supported by metadynamics simulations and by comparison with the cryo-EM data, could be used to gain some insights into critical features of the conformational transition toward active CFTR forms. These critical elements lie within membrane-spanning domains but also within NBD1 and the N-terminal extension, in which conformational plasticity is predicted to occur to help the interaction with filamin, one of the CFTR cellular partners.