Allosteric inhibition of CFTR gating by CFTRinh-172 binding in the pore.

Loss-of-function mutations of the CFTR gene cause the life-shortening genetic disease cystic fibrosis (CF), whereas overactivity of CFTR may lead to secretory diarrhea and polycystic kidney disease. While effective drugs targeting the CFTR protein have been developed for the treatment of CF, little progress has been made for diseases caused by hyper-activated CFTR. Here, we solve the cryo-EM structure of CFTR in complex with CFTRinh-172 (Inh-172), a CFTR gating inhibitor with promising potency and efficacy. We find that Inh-172 binds inside the pore of CFTR, interacting with amino acid residues from transmembrane segments (TMs) 1, 6, 8, 9, and 12 through mostly hydrophobic interactions and a salt bridge. Substitution of these residues lowers the apparent affinity of Inh-172. The inhibitor-bound structure reveals re-orientations of the extracellular segment of TMs 1, 8, and 12, supporting an allosteric modulation mechanism involving post-binding conformational changes. This allosteric inhibitory mechanism readily explains our observations that pig CFTR, which preserves all the amino acid residues involved in Inh-172 binding, exhibits a much-reduced sensitivity to Inh-172 and that the apparent affinity of Inh-172 is altered by the CF drug ivacaftor (i.e., VX-770) which enhances CFTR's activity through binding to a site also comprising TM8.

Structure basis of CFTR folding, function and pharmacology.

The root cause of cystic fibrosis (CF), the most common life-shortening genetic disease in the Caucasian population, is the loss of function of the CFTR protein, which serves as a phosphorylation-activated, ATP-gated anion channel in numerous epithelia-lining tissues. In the past decade, high-throughput drug screening has made a significant stride in developing highly effective CFTR modulators for the treatment of CF. Meanwhile, structural-biology studies have succeeded in solving the high-resolution three-dimensional (3D) structure of CFTR in different conformations. Here, we provide a brief overview of some striking features of CFTR folding, function and pharmacology, in light of its specific structural features within the ABC-transporter superfamily. A particular focus is given to CFTR's first nucleotide-binding domain (NBD1), because folding of NBD1 constitutes a bottleneck in the CFTR protein biogenesis pathway, and ATP binding to this domain plays a unique role in the functional stability of CFTR. Unraveling the molecular basis of CFTR folding, function, and pharmacology would inspire the development of next-generation mutation-specific CFTR modulators.

CFTR Modulators: From Mechanism to Targeted Therapeutics.

People with cystic fibrosis (CF) suffer from a multi-organ disorder caused by loss-of-function variants in the gene encoding the epithelial anion channel cystic fibrosis transmembrane conductance regulator (CFTR). Tremendous progress has been made in both basic and clinical sciences over the past three decades since the identification of the CFTR gene. Over 90% of people with CF now have access to therapies targeting dysfunctional CFTR. This success was made possible by numerous studies in the field that incrementally paved the way for the development of small molecules known as CFTR modulators. The advent of CFTR modulators transformed this life-threatening illness into a treatable disease by directly binding to the CFTR protein and correcting defects induced by pathogenic variants. In this chapter, we trace the trajectory of structural and functional studies that brought CF therapies from bench to bedside, with an emphasis on mechanistic understanding of CFTR modulators.

Pharmacological Responses of the G542X-CFTR to CFTR Modulators.

Cystic fibrosis (CF) is a lethal hereditary disease caused by loss-of-function mutations of the chloride channel cystic fibrosis transmembrane conductance regulator (CFTR). With the development of small-molecule CFTR modulators, including correctors that facilitate protein folding and expression and potentiators that promote channel activity, about 90% of the CF patients are now receiving efficacious target therapies. G542X-CFTR, a premature termination codon (PTC) mutation, is the most common disease-associated mutation found in the remaining 10% of patients that await effective drugs to rectify the fundamental defects caused by PTC. In this study, we employed biophysical and biochemical techniques to characterize the pharmacological responses of the translational products of G542X-CFTR to a range of new CFTR modulators. Specifically, we identified two different proteins translated from the G542X-CFTR cDNA using western blotting: the C-terminus truncated protein that responds to the C1 corrector which binds to the N-terminal part of the protein and a full-length CFTR protein through the read-through process. Electrophysiological data suggest that the read-through protein, but not the C-terminus truncated one, is functional and responds well to CFTR potentiators despite a lower open probability compared to wild-type CFTR. As the expression of the read-through products can be increased synergistically with the read-through reagent G418 and C1 corrector, but not with combinations of different types of correctors, we concluded that an efficacious read-through reagent is a prerequisite for mitigating the deficits of G542X-CFTR. Moreover, the CFTR potentiators may help improve the effectiveness of future combinational therapy for patients carrying PTCs such as G542X.

Generation of human induced pluripotent stem cells from cystic fibrosis patient carrying nonsense mutation (p.S308X) in CFTR gene.

Cystic fibrosis (CF) is a genetic disease affects CFTR channel synthesis. While 90 percent of the CF patients now benefit from small molecule target therapies, this treatment has yet to extend to those bearing nonsense mutations. Studies of these rare mutations using cell lines with native pathological signatures of the disease may lead to breakthroughs in therapeutic development. Here, we report the generation of CF patient-derived induced pluripotent stem cells (iPSCs) carrying a nonsense mutation at position 308 (S308X). The pluripotency and genomic profile of the iPSC line was validated as a resource that can enable future research for CF.

Functional stability of CFTR depends on tight binding of ATP at its degenerate ATP-binding site.

Opening of the cystic fibrosis transmembrane conductance regulator (CFTR) channel is coupled to the motion of its two nucleotide-binding domains: they form a heterodimer sandwiching two functionally distinct ATP-binding sites (sites 1 and 2). While active ATP hydrolysis in site 2 triggers rapid channel closure, the functional role of stable ATP binding in the catalysis-incompetent (or degenerate) site 1, a feature conserved in many other ATP-binding cassette (ABC) transporter proteins, remains elusive. Here, we found that CFTR loses its prompt responsiveness to ATP after the channel is devoid of ATP for tens to hundreds of seconds. Mutants with weakened ATP binding in site 1 and the most prevalent disease-causing mutation, F508del, are more vulnerable to ATP depletion. In contrast, strengthening ligand binding in site 1 with N6 -(2-phenylethyl)-ATP, a high-affinity ATP analogue, or abolishing ATP hydrolysis in site 2 by the mutation D1370N, helps sustain a durable function of the otherwise unstable mutant channels. Thus, tight binding of ATP in the degenerate ATP-binding site is crucial to the functional stability of CFTR. Small molecules targeting site 1 may bear therapeutic potential to overcome the membrane instability of F508del-CFTR. KEY POINTS: During evolution, many ATP-binding cassette transporters - including the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel, whose dysfunction causes cystic fibrosis (CF) - lose the ability to hydrolyse ATP in one of the two ATP-binding sites. Here we show that tight ATP binding at this degenerate site in CFTR is central for maintaining the stable, robust function of normal CFTR. We also demonstrate that membrane instability of the most common CF-causing mutant, F508del-CFTR, can be rescued by strengthening ATP binding at CFTR's degenerate site. Our data thus explain an evolutionary puzzle and offer a potential therapeutic strategy for CF.

Organoids as a personalized medicine tool for ultra-rare mutations in cystic fibrosis: The case of S955P and 1717-2A>G.

BACKGROUND: For most of the >2000 CFTR gene variants reported, neither the associated disease liability nor the underlying basic defect are known, and yet these are essential for disease prognosis and CFTR-based therapeutics. Here we aimed to characterize two ultra-rare mutations - 1717-2A > G (c.1585-2A > G) and S955P (p.Ser955Pro) - as case studies for personalized medicine. METHODS: Patient-derived rectal biopsies and intestinal organoids from two individuals with each of these mutations and F508del (p.Phe508del) in the other allele were used to assess CFTR function, response to modulators and RNA splicing pattern. In parallel, we used cellular models to further characterize S955P independently of F508del and to assess its response to CFTR modulators. RESULTS: Results in both rectal biopsies and intestinal organoids from both patients evidence residual CFTR function. Further characterization shows that 1717-2A > G leads to alternative splicing generating <1% normal CFTR mRNA and that S955P affects CFTR gating. Finally, studies in organoids predict that both patients are responders to VX-770 alone and even more to VX-770 combined with VX-809 or VX-661, although to different levels. CONCLUSION: This study demonstrates the high potential of personalized medicine through theranostics to extend the label of approved drugs to patients with rare mutations.

CFTR: New insights into structure and function and implications for modulation by small molecules.

Structural biology and functional studies are a powerful combination to elucidate fundamental knowledge about the cystic fibrosis transmembrane conductance regulator (CFTR). Here, we discuss the latest findings, including how clinically-approved drugs restore function to mutant CFTR, leading to better clinical outcomes for people with cystic fibrosis (CF). Despite the prospect of regulatory approval of a CFTR-targeting therapy for most CF mutations, strenuous efforts are still needed to fully comprehend CFTR structure-and-function for the development of better drugs to enable people with CF to live full and active lives.

Biological Characterization of F508delCFTR Protein Processing by the CFTR Corrector ABBV-2222/GLPG2222.

Cystic fibrosis (CF) is the most common monogenic autosomal recessive disease in Caucasians caused by pathogenic mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene (CFTR). Significant small molecule therapeutic advances over the past two decades have been made to target the defective CFTR protein and enhance its function. To address the most prevalent defect of the defective CFTR protein (i.e., F508del mutation) in CF, two biomolecular activities are required, namely, correctors to increase the amount of properly folded F508delCFTR levels at the cell surface and potentiators to allow the effective opening, i.e., function of the F508delCFTR channel. Combined, these activities enhance chloride ion transport yielding improved hydration of the lung surface and subsequent restoration of mucociliary clearance. To enhance clinical benefits to CF patients, a complementary triple combination therapy consisting of two corrector molecules, type 1 (C1) and type 2, with additive mechanisms along with a potentiator are being investigated in the clinic for maximum restoration of mutated CFTR function. We report the identification and in vitro biologic characterization of ABBV-2222/GLPG2222 (4-[(2R,4R)-4-({[1-(2,2-difluoro-1,3-benzodioxol-5-yl)cyclopropyl]carbonyl}amino)-7-(difluoromethoxy)-3,4-dihydro-2H-chromen-2-yl]benzoic acid),-a novel, potent, and orally bioavailable C1 corrector developed by AbbVie-Galapagos and currently in clinical trials-which exhibits substantial improvements over the existing C1 correctors. This includes improvements in potency and drug-drug interaction (DDI) compared with 3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)benzoic acid (VX-809, Lumacaftor) and improvements in potency and efficacy compared with 1-(2,2-difluoro-1,3-benzodioxol-5-yl)-N-[1-[(2R)-2,3-dihydroxypropyl]-6-fluoro-2-(1-hydroxy-2-methylpropan-2-yl)indol-5-yl]cyclopropane-1-carboxamide (VX-661, Tezacaftor). ABBV-2222/GLPG2222 exhibits potent in vitro functional activity in primary patient cells harboring F508del/F508del CFTR with an EC50 value <10 nM. SIGNIFICANCE STATEMENT: To address the most prevalent defect of the defective CFTR protein (i.e., F508del mutation) in cystic fibrosis, AbbVie-Galapagos has developed ABBV-2222/GLPG2222, a novel, potent, and orally bioavailable C1 corrector of this protein. ABBV-2222/GLPG2222, which is currently in clinical trials, exhibits potent in vitro functional activity in primary patient cells harboring F508del/F508del CFTR and substantial improvements over the existing C1 correctors.

Positional effects of premature termination codons on the biochemical and biophysical properties of CFTR.

KEY POINTS: Biochemical and biophysical characterizations of three nonsense mutations of cystic fibrosis transmembrane conductance regulator (CFTR) associated with a severe form of cystic fibrosis (CF) reveal the importance and heterogenous effects of the position of the premature termination codon (PTC) on the CFTR protein function. Electrophysiological studies of W1282X-CFTR, whose PTC is closer to the C-terminus of CFTR, suggest the presence of both C-terminus truncated CFTR proteins that are poorly functional and read-through, full-length products. For G542X- and E60X-CFTR, the only mechanism capable of generating functional proteins is the read-through, but the outcome of read-through products is highly variable depending on the interplay between the missense mutation caused by the read-through and the structural context of the protein. Pharmacological studies of these three PTCs with various CFTR modulators suggest position-dependent therapeutic strategies for these disease-inflicting mutations. ABSTRACT: About one-third of genetic diseases and cancers are caused by the introduction of premature termination codons (PTCs). In theory, the location of the PTC in a gene determines the alternative mechanisms of translation, including premature cessation or reinitiation of translation, and read-through, resulting in differential effects on protein integrity. In this study, we used CFTR as a model system to investigate the positional effect of the PTC because of its well-understood structure-function relationship and pathophysiology. The characterization of three PTC mutations, E60X-, G542X- and W1282X-CFTR revealed heterogenous effects of these PTCs on CFTR function. The W1282X mutation results in both C-terminus truncated and read-through proteins that are partially or fully functional. In contrast, only the read-through protein is functional with E60X- and G542X-CFTR, although abundant N-terminus truncated proteins due to reinitiation of translation were detected in E60X-CFTR. Single-channel studies of the read-through proteins of E60X- and G542X-CFTR demonstrated that both mutations have a single-channel amplitude similar to wild type (WT), and good responses to high-affinity ATP analogues, suggesting intact ion permeation pathways and nucleotide binding domains (NBDs), albeit with reduced open probability (Po ). The comparison of the Po of these mutations with the proposed missense mutations revealed potential identities of the read-through products. Importantly, a majority of the functional protein studied responds to CFTR modulators like GLPG1837 and Lumacaftor. These results not only expand current understanding of the molecular (patho)physiology of CFTR, but also infer therapeutic strategies for different PTC mutations at large.

Identifying the molecular target sites for CFTR potentiators GLPG1837 and VX-770.

The past two decades have witnessed major breakthroughs in developing compounds that target the chloride channel CFTR for the treatment of patients with cystic fibrosis. However, further improvement in affinity and efficacy for these CFTR modulators will require insights into the molecular interactions between CFTR modulators and their binding targets. In this study, we use in silico molecular docking to identify potential binding sites for GLPG1837, a CFTR potentiator that may share a common mechanism and binding site with VX-770, the FDA-approved drug for patients carrying mutations with gating defects. Among the five binding sites predicted by docking, the two top-scoring sites are located at the interface between CFTR's two transmembrane domains: site I consists of D924, N1138, and S1141, and site IIN includes F229, F236, Y304, F312, and F931. Using mutagenesis to probe the importance of these sites for GLPG187 binding, we find that disruption of predicted hydrogen-bonding interactions by mutation of D924 decreases apparent affinity, while hydrophobic amino acids substitutions at N1138 and introduction of positively charged amino acids at S1141 improve the apparent affinity for GLPG1837. Alanine substitutions at Y304, F312, and F931 (site IIN) decrease the affinity for GLPG1837, whereas alanine substitutions at F229 and F236 (also site IIN), or at residues in the other three lower-scoring sites, have little effect. In addition, current relaxation analysis to assess the apparent dissociation rate of VX-770 yields results consistent with the dose-response experiments for GLPG8137, with the dissociation rate of VX-770 accelerated by D924N, F236A, Y304A, and F312A, but decelerated by N1138L and S1141K mutations. Collectively, these data identify two potential binding sites for GLPG1837 and VX-770 in CFTR. We discuss the pros and cons of evidence for these two loci and the implications for future drug design.

Identification of GLPG/ABBV-2737, a Novel Class of Corrector, Which Exerts Functional Synergy With Other CFTR Modulators.

The deletion of phenylalanine at position 508 (F508del) in cystic fibrosis transmembrane conductance regulator (CFTR) causes a severe defect in folding and trafficking of the chloride channel resulting in its absence at the plasma membrane of epithelial cells leading to cystic fibrosis. Progress in the understanding of the disease increased over the past decades and led to the awareness that combinations of mechanistically different CFTR modulators are required to obtain meaningful clinical benefit. Today, there remains an unmet need for identification and development of more effective CFTR modulator combinations to improve existing therapies for patients carrying the F508del mutation. Here, we describe the identification of a novel F508del corrector using functional assays. We provide experimental evidence that the clinical candidate GLPG/ABBV-2737 represents a novel class of corrector exerting activity both on its own and in combination with VX809 or GLPG/ABBV-2222.

Structural mechanisms for defective CFTR gating caused by the Q1412X mutation, a severe Class VI pathogenic mutation in cystic fibrosis.

KEY POINTS: Electrophysiological characterization of Q1412X-CFTR, a C-terminal truncation mutation of cystic fibrosis transmembrane conductance regulator (CFTR) associated with the severe form of cystic fibrosis (CF), reveals a gating defect that has not been reported previously. Mechanistic investigations of the gating deficit in Q1412X-CFTR suggest that the reduced open probability in Q1412X-CFTR is the result of a disruption of the function of the second ATP binding site (or site 2) in the nucleotide binding domains (NBDs). Detailed comparisons of several mutations with different degrees of truncation in the C-terminal region of NBD2 reveal the importance of the last two beta-strands in NBD2 for maintaining proper gating functions. The results of the present study also show that the application of clinically-approved drugs (VX-770 and VX-809) can greatly enhance the function of Q1412X, providing in vitro evidence for a therapeutic strategy employing both reagents for patients bearing Q1412X or similar truncation mutations. ABSTRACT: Cystic fibrosis (CF) is caused by loss-of-function mutations of cystic fibrosis transmembrane conductance regulator (CFTR), a phosphorylation-activated but ATP-gated chloride channel. Based on the molecular mechanism of CF pathogenesis, disease-associated mutations are categorized into six classes. Among them, Class VI, whose members include some of the C-terminal truncation mutations such as Q1412X, is defined as decreased membrane expression because of a faster turnover rate. In the present study, we characterized the functional properties of Q1412X-CFTR, a severe-form premature stop codon mutation. We confirmed previous findings of a ∼90% decrease in membrane expression but found a ∼95% reduction in the open probability (Po ). Detailed kinetic studies support the idea that the gating defect is the result of a dysfunctional ATP-binding site 2 in the nucleotide binding domains (NBDs). Because the Q1412X mutation results in a deletion of the last two beta-strands in NBD2 and the whole C-terminal region, we further characterized truncation mutations with different degrees of deletion in this segment. Mutations that completely or partially remove the C-terminus of CFTR at the same time as keeping an intact NBD2 (i.e. D1425X and S1455X) assume gating function almost identical to that of wild-type channels. However, the deletion of the last beta-strand in the NBD2 (i.e. N1419X) causes gating dysfunction that is milder than that of Q1412X. Thus, normal CFTR gating requires structural integrity of NBD2. Moreover, our observation that clinically-approved VX-809 (Lumacaftor, Vertex Pharmaceuticals, Boston, MA, USA) and VX-770 (Ivacaftor, Vertex Pharmaceuticals, Boston, MA, USA) significantly enhance the overall function of Q1412X-CFTR provides the conceptual basis for the treatment of patients carrying this mutation.

Characterization of Δ(G970-T1122)-CFTR, the most frequent CFTR mutant identified in Japanese cystic fibrosis patients.

A massive deletion over three exons 16-17b in the CFTR gene was identified in Japanese CF patients with the highest frequency (about 70% of Japanese CF patients definitely diagnosed). This pathogenic mutation results in a deletion of 153 amino acids from glycine at position 970 (G970) to threonine at 1122 (T1122) in the CFTR protein without a frameshift. We name it Δ(G970-T1122)-CFTR. In the present study, we characterized the Δ(G970-T1122)-CFTR expressed in CHO cells using immunoblots and a super resolution microscopy. Δ(G970-T1122)-CFTR proteins were synthesized and core-glycosylated but not complex-glycosylated. This observation suggests that the Δ(G970-T1122) mutation can be categorized into the class II mutation like ΔF508. However, VX-809 a CFTR corrector that can help maturation of ΔF508, had no effect on Δ(G970-T1122). Interestingly C-terminal FLAG tag seems to partially rescue the trafficking defect of Δ(G970-T1122)-CFTR; however the rescued Δ(G970-T1122)-CFTR proteins do not assume channel function. Japanese, and perhaps people in other Asian nations, carry a class II mutation Δ(G970-T1122) with a higher frequency than previously appreciated. Further study of the Δ(G970-T1122)-CFTR is essential for understanding CF and CFTR-related diseases particularly in Asian countries.

Identification and Characterization of Novel CFTR Potentiators.

There is still a high unmet need for the treatment of most patients with cystic fibrosis (CF). The identification and development of new Cystic Fibrosis Transmembrane conductance Regulator (CFTR) modulators is necessary to achieve higher clinical benefit in patients. In this report we describe the characterization of novel potentiators. From a small screening campaign on F508del CFTR, hits were developed leading to the identification of pre-clinical candidates GLPG1837 and GLPG2451, each derived from a distinct chemical series. Both drug candidates enhance WT CFTR activity as well as low temperature or corrector rescued F508del CFTR, and are able to improve channel activity on a series of Class III, IV CFTR mutants. The observed activities in YFP halide assays translated well to primary cells derived from CF lungs when measured using Trans-epithelial clamp circuit (TECC). Both potentiators improve F508del CFTR channel opening in a similar manner, increasing the open time and reducing the closed time of the channel. When evaluating the potentiators in a chronic setting on corrected F508del CFTR, no reduction of channel activity in presence of potentiator was observed. The current work identifies and characterizes novel CFTR potentiators GLPG1837 and GLPG2451, which may offer new therapeutic options for CF patients.

Physiological and pharmacological characterization of the N1303K mutant CFTR.

BACKGROUND: N1303K, one of the common, severe disease-causing mutations in the CFTR gene, causes both defective biogenesis and gating abnormalities of the CFTR protein. The goals of the present study are to quantitatively assess the gating defects associated with the N1303K mutation and its pharmacological response to CFTR modulators including potentiators VX-770 and GLPG1837 and correctors VX-809, and VX-661. METHODS: Gating behavior and pharmacological responses to CFTR potentiators were assessed using patch-clamp technique in the excised, inside-out mode. We also examined the effects of GLPG1837, VX-770, VX-809 and VX-661 on N1303K-CFTR surface expression using Western blot analysis. RESULTS: Like wild-type (WT) CFTR, N1303K-CFTR channels were activated by protein kinase A-dependent phosphorylation, but the open probability (Po) of phosphorylated N1303K-CFTR was extremely low (~0.03 vs ~0.45 in WT channels). N1303K mutants showed abnormal responses to ATP analogs or mutations that disrupt ATP hydrolysis and/or dimerization of CFTR's two nucleotide-binding domains (NBDs). However, the Po of N1303K-CFTR was dramatically increased by GLPG1837 (~17-fold) and VX-770 (~8-fold). VX-809 or VX-661 enhanced N1303K-CFTR maturation by 2-3 fold, and co-treatment with GLPG1837 or VX-770 did not show any negative drug-drug interaction. CONCLUSION: N1303K has a severe gating defect, reduced ATP-dependence and aberrant response to ATP analogs. These results suggest a defective function of the NBDs in N1303K-CFTR. An improvement of channel function by GLPG1837 or VX-770 and an increase of Band C protein by VX-809 or VX-661 support a therapeutic strategy of combining CFTR potentiator and corrector for patients carrying the N1303K mutation.

Structural mechanisms of CFTR function and dysfunction.

Cystic fibrosis (CF) transmembrane conductance regulator (CFTR) chloride channel plays a critical role in regulating transepithelial movement of water and electrolyte in exocrine tissues. Malfunction of the channel because of mutations of the cftr gene results in CF, the most prevalent lethal genetic disease among Caucasians. Recently, the publication of atomic structures of CFTR in two distinct conformations provides, for the first time, a clear overview of the protein. However, given the highly dynamic nature of the interactions among CFTR's various domains, better understanding of the functional significance of these structures requires an integration of these new structural insights with previously established biochemical/biophysical studies, which is the goal of this review.

Cystic fibrosis research topics featured at the 14th ECFS Basic Science Conference: Chairman's summary.

In recent years, tremendous progress has been made in the development of novel drugs targeting the basic defect in patients with cystic fibrosis (CF). This breakthrough is based on a solid foundation of knowledge on CFTR's function in health and how mutations in CFTR cause CF multi-organ disease. This knowledge has been collected and continuously expanded by an active and persistent CF research community and has paved the way for precision medicine for CF. Since 2004, the European Cystic Fibrosis Society (ECFS) has held an annual Basic Science Conference that has evolved as an international forum for interdisciplinary discussion of hot topics and unsolved questions related to CF research. This Special Issue reviews CF research topics featured at the 14th ECFS Basic Science Conference and provides an up-to-date overview of recent progress in our understanding of CFTR structure and function, disease mechanisms implicated in airway mucus plugging, inflammation and abnormal host-pathogen interactions, and advancements with enhanced cell and animal model systems and breakthrough therapies directed at mutant CFTR or alternative targets. In addition, this Special Issue also identifies a number of fundamental questions and hurdles that still have to be overcome to realize the full potential of precision medicine and develop transformative therapies for all patients with CF.

Functional characterization reveals that zebrafish CFTR prefers to occupy closed channel conformations.

Cystic fibrosis transmembrane conductance regulator (CFTR), the culprit behind the genetic disease cystic fibrosis (CF), is a phosphorylation-activated, but ATP-gated anion channel. Studies of human CFTR over the past two decades have provided an in-depth understanding of how CFTR works as an ion channel despite its structural resemblance to ABC transporters. Recently-solved cryo-EM structures of unphosphorylated human and zebrafish CFTR (hCFTR and zCFTR), as well as phosphorylated ATP-bound zebrafish and human CFTR offer an unprecedented opportunity to understand CFTR's function at a molecular level. Interestingly, despite millions of years of phylogenetic distance between human and zebrafish, the structures of zCFTR and hCFTR exhibit remarkable similarities. In the current study, we characterized biophysical and pharmacological properties of zCFTR with the patch-clamp technique, and showed surprisingly very different functional properties between these two orthologs. First, while hCFTR has a single-channel conductance of 8.4 pS with a linear I-V curve, zCFTR shows an inwardly-rectified I-V relationship with a single-channel conductance of ~3.5 pS. Second, single-channel gating behaviors of phosphorylated zCFTR are very different from those of hCFTR, featuring a very low open probability Po (0.03 ± 0.02, vs. ~0.50 for hCFTR) with exceedingly long closed events and brief openings. In addition, unlike hCFTR where each open burst is clearly defined with rare short-lived flickery closures, the open bursts of zCFTR are not easily resolved. Third, although abolishing ATP hydrolysis by replacing the catalytic glutamate with glutamine (i.e., E1372Q) drastically prolongs the open bursts defined by the macroscopic relaxation analysis in zCFTR, the Po within a "locked-open" burst of E1372Q-zCFTR is only ~ 0.35 (vs. Po > 0.94 in E1371Q-hCFTR). Collectively, our data not only provide a reasonable explanation for the unexpected closed-state structure of phosphorylated E1372Q-zCFTR with a canonical ATP-bound dimer of the nucleotide binding domains (NBDs), but also implicate significant structural and functional differences between these two evolutionarily distant orthologs.