Atomic Structure of the Cystic Fibrosis Transmembrane Conductance Regulator.

The cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel evolved from the ATP-binding cassette (ABC) transporter family. In this study, we determined the structure of zebrafish CFTR in the absence of ATP by electron cryo-microscopy to 3.7 Å resolution. Human and zebrafish CFTR share 55% sequence identity, and 42 of the 46 cystic-fibrosis-causing missense mutational sites are identical. In CFTR, we observe a large anion conduction pathway lined by numerous positively charged residues. A single gate near the extracellular surface closes the channel. The regulatory domain, dephosphorylated, is located in the intracellular opening between the two nucleotide-binding domains (NBDs), preventing NBD dimerization and channel opening. The structure also reveals why many cystic-fibrosis-causing mutations would lead to defects either in folding, ion conduction, or gating and suggests new avenues for therapeutic intervention.

A combination therapy for cystic fibrosis.

The most prevalent form of cystic fibrosis arises from an amino acid deletion in the cystic fibrosis transmembrane conductance regulator, CFTR. A recently approved treatment for individuals homozygous for this mutation combines a chemical corrector, which helps CFTR fold, and a potentiator that increases CFTR channel activity.

Transgenic ferret models define pulmonary ionocyte diversity and function.

Speciation leads to adaptive changes in organ cellular physiology and creates challenges for studying rare cell-type functions that diverge between humans and mice. Rare cystic fibrosis transmembrane conductance regulator (CFTR)-rich pulmonary ionocytes exist throughout the cartilaginous airways of humans1,2, but limited presence and divergent biology in the proximal trachea of mice has prevented the use of traditional transgenic models to elucidate ionocyte functions in the airway. Here we describe the creation and use of conditional genetic ferret models to dissect pulmonary ionocyte biology and function by enabling ionocyte lineage tracing (FOXI1-CreERT2::ROSA-TG), ionocyte ablation (FOXI1-KO) and ionocyte-specific deletion of CFTR (FOXI1-CreERT2::CFTRL/L). By comparing these models with cystic fibrosis ferrets3,4, we demonstrate that ionocytes control airway surface liquid absorption, secretion, pH and mucus viscosity-leading to reduced airway surface liquid volume and impaired mucociliary clearance in cystic fibrosis, FOXI1-KO and FOXI1-CreERT2::CFTRL/L ferrets. These processes are regulated by CFTR-dependent ionocyte transport of Cl- and HCO3-. Single-cell transcriptomics and in vivo lineage tracing revealed three subtypes of pulmonary ionocytes and a FOXI1-lineage common rare cell progenitor for ionocytes, tuft cells and neuroendocrine cells during airway development. Thus, rare pulmonary ionocytes perform critical CFTR-dependent functions in the proximal airway that are hallmark features of cystic fibrosis airway disease. These studies provide a road map for using conditional genetics in the first non-rodent mammal to address gene function, cell biology and disease processes that have greater evolutionary conservation between humans and ferrets.

Requirements for efficient correction of ΔF508 CFTR revealed by analyses of evolved sequences.

Misfolding of ΔF508 cystic fibrosis (CF) transmembrane conductance regulator (CFTR) underlies pathology in most CF patients. F508 resides in the first nucleotide-binding domain (NBD1) of CFTR near a predicted interface with the fourth intracellular loop (ICL4). Efforts to identify small molecules that restore function by correcting the folding defect have revealed an apparent efficacy ceiling. To understand the mechanistic basis of this obstacle, positions statistically coupled to 508, in evolved sequences, were identified and assessed for their impact on both NBD1 and CFTR folding. The results indicate that both NBD1 folding and interaction with ICL4 are altered by the ΔF508 mutation and that correction of either individual process is only partially effective. By contrast, combination of mutations that counteract both defects restores ΔF508 maturation and function to wild-type levels. These results provide a mechanistic rationale for the limited efficacy of extant corrector compounds and suggest approaches for identifying compounds that correct both defective steps.

Structural identification of a selectivity filter in CFTR.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel that regulates transepithelial salt and fluid homeostasis. CFTR dysfunction leads to reduced chloride secretion into the mucosal lining of epithelial tissues, thereby causing the inherited disease cystic fibrosis. Although several structures of CFTR are available, our understanding of the ion-conduction pathway is incomplete. In particular, the route that connects the cytosolic vestibule with the extracellular space has not been clearly defined, and the structure of the open pore remains elusive. Furthermore, although many residues have been implicated in altering the selectivity of CFTR, the structure of the "selectivity filter" has yet to be determined. In this study, we identify a chloride-binding site at the extracellular ends of transmembrane helices 1, 6, and 8, where a dehydrated chloride is coordinated by residues G103, R334, F337, T338, and Y914. Alterations to this site, consistent with its function as a selectivity filter, affect ion selectivity, conductance, and open channel block. This selectivity filter is accessible from the cytosol through a large inner vestibule and opens to the extracellular solvent through a narrow portal. The identification of a chloride-binding site at the intra- and extracellular bridging point leads us to propose a complete conductance path that permits dehydrated chloride ions to traverse the lipid bilayer.

Cystic Fibrosis Transmembrane Conductance Regulator Attaches Tumor Suppressor PTEN to the Membrane and Promotes Anti Pseudomonas aeruginosa Immunity.

The tumor suppressor PTEN controls cell proliferation by regulating phosphatidylinositol-3-kinase (PI3K) activity, but the participation of PTEN in host defense against bacterial infection is less well understood. Anti-inflammatory PI3K-Akt signaling is suppressed in patients with cystic fibrosis (CF), a disease characterized by hyper-inflammatory responses to airway infection. We found that Ptenl-/- mice, which lack the NH2-amino terminal splice variant of PTEN, were unable to eradicate Pseudomonas aeruginosa from the airways and could not generate sufficient anti-inflammatory PI3K activity, similar to what is observed in CF. PTEN and the CF transmembrane conductance regulator (CFTR) interacted directly and this interaction was necessary to position PTEN at the membrane. CF patients under corrector-potentiator therapy, which enhances CFTR transport to the membrane, have increased PTEN amounts. These findings suggest that improved CFTR trafficking could enhance P. aeruginosa clearance from the CF airway by activating PTEN-mediated anti-bacterial responses and might represent a therapeutic strategy.

Global chemical effects of the microbiome include new bile-acid conjugations.

A mosaic of cross-phylum chemical interactions occurs between all metazoans and their microbiomes. A number of molecular families that are known to be produced by the microbiome have a marked effect on the balance between health and disease1-9. Considering the diversity of the human microbiome (which numbers over 40,000 operational taxonomic units10), the effect of the microbiome on the chemistry of an entire animal remains underexplored. Here we use mass spectrometry informatics and data visualization approaches11-13 to provide an assessment of the effects of the microbiome on the chemistry of an entire mammal by comparing metabolomics data from germ-free and specific-pathogen-free mice. We found that the microbiota affects the chemistry of all organs. This included the amino acid conjugations of host bile acids that were used to produce phenylalanocholic acid, tyrosocholic acid and leucocholic acid, which have not previously been characterized despite extensive research on bile-acid chemistry14. These bile-acid conjugates were also found in humans, and were enriched in patients with inflammatory bowel disease or cystic fibrosis. These compounds agonized the farnesoid X receptor in vitro, and mice gavaged with the compounds showed reduced expression of bile-acid synthesis genes in vivo. Further studies are required to confirm whether these compounds have a physiological role in the host, and whether they contribute to gut diseases that are associated with microbiome dysbiosis.

Pulmonary Vascular Dysfunctions in Cystic Fibrosis.

Cystic fibrosis (CF) is an inherited disorder caused by a deleterious mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Given that the CFTR protein is a chloride channel expressed on a variety of cells throughout the human body, mutations in this gene impact several organs, particularly the lungs. For this very reason, research regarding CF disease and CFTR function has historically focused on the lung airway epithelium. Nevertheless, it was discovered more than two decades ago that CFTR is also expressed and functional on endothelial cells. Despite the great strides that have been made in understanding the role of CFTR in the airway epithelium, the role of CFTR in the endothelium remains unclear. Considering that the airway epithelium and endothelium work in tandem to allow gas exchange, it becomes very crucial to understand how a defective CFTR protein can impact the pulmonary vasculature and overall lung function. Fortunately, more recent research has been dedicated to elucidating the role of CFTR in the endothelium. As a result, several vascular dysfunctions associated with CF disease have come to light. Here, we summarize the current knowledge on pulmonary vascular dysfunctions in CF and discuss applicable therapies.

Analysis of CF patient survival confirms STAT3 as a CF-modifying gene with changing impact over time.

INTRODUCTION AND AIM: The signal transducer and activator of transcription 3 (STAT3) has been identified as one of the cystic fibrosis (CF) modifying genes. In this study, we aimed to assess the association between STAT3 genotype and CF patient survival over several decades and to investigate the effect of STAT3 inhibition on epithelial CFTR expression. METHODS: We analyzed the informative genetic marker STAT3Sat for its association with survival in 174 p.Phe508del-CFTR homozygous CF patients treated at the CF center in Hannover spanning birth cohorts from >3 decades (1959-1994). Furthermore, we treated two epithelial cell lines with STAT3 inhibitors and monitored changes of CFTR protein expression by western blot. RESULTS: Only for p.Phe508del-CFTR homozygous patients born prior to 1975, survival was significantly influenced by STAT3sat genotype (P = 0.023). The expression levels of STAT3 and CFTR positively correlated in epithelial cell lines (P = 0.01). CONCLUSIONS: Our results in different birth cohorts identified a time-dependent impact of STAT3 genotype on CF patients' survival and found that improved symptomatic treatment of later-born CF patients obviates STAT3's modifying influence. Consistent with our previous results, STAT3-specific inhibition resulted in increased CFTR expression in the epithelial cell line 16HBE14o-. Thus, care should be taken when CF-modifying genes are studied in cross-sectional cohorts as the impact of modifying genes might not be invariant in the light of changing therapeutic regimens.

Cystic Fibrosis Modulator Therapies.

Cystic fibrosis (CF) is an inherited multisystemic disease that can cause progressive bronchiectasis, pancreatic endocrine and exocrine insufficiency, distal intestinal obstruction syndrome, liver dysfunction, and other disorders. Traditional therapies focused on the treatment or prevention of damage to each organ system with incremental modalities such as nebulized medications for the lungs, insulin for diabetes, and supplementation with pancreatic enzymes. However, the advent of highly effective modulator therapies that target specific cystic fibrosis transmembrane conductance regulator protein malformations resulting from individual genetic mutations has transformed the lives and prognosis for persons with CF.

Pleiotropic modifiers of age-related diabetes and neonatal intestinal obstruction in cystic fibrosis.

Individuals with cystic fibrosis (CF) develop complications of the gastrointestinal tract influenced by genetic variants outside of CFTR. Cystic fibrosis-related diabetes (CFRD) is a distinct form of diabetes with a variable age of onset that occurs frequently in individuals with CF, while meconium ileus (MI) is a severe neonatal intestinal obstruction affecting ∼20% of newborns with CF. CFRD and MI are slightly correlated traits with previous evidence of overlap in their genetic architectures. To better understand the genetic commonality between CFRD and MI, we used whole-genome-sequencing data from the CF Genome Project to perform genome-wide association. These analyses revealed variants at 11 loci (6 not previously identified) that associated with MI and at 12 loci (5 not previously identified) that associated with CFRD. Of these, variants at SLC26A9, CEBPB, and PRSS1 associated with both traits; variants at SLC26A9 and CEBPB increased risk for both traits, while variants at PRSS1, the higher-risk alleles for CFRD, conferred lower risk for MI. Furthermore, common and rare variants within the SLC26A9 locus associated with MI only or CFRD only. As expected, different loci modify risk of CFRD and MI; however, a subset exhibit pleiotropic effects indicating etiologic and mechanistic overlap between these two otherwise distinct complications of CF.

A spectrum of recessiveness among Mendelian disease variants in UK Biobank.

Recent work has found increasing evidence of mitigated, incompletely penetrant phenotypes in heterozygous carriers of recessive Mendelian disease variants. We leveraged whole-exome imputation within the full UK Biobank cohort (n ∼ 500K) to extend such analyses to 3,475 rare variants curated from ClinVar and OMIM. Testing these variants for association with 58 quantitative traits yielded 102 significant associations involving variants previously implicated in 34 different diseases. Notable examples included a POR missense variant implicated in Antley-Bixler syndrome that associated with a 1.76 (SE 0.27) cm increase in height and an ABCA3 missense variant implicated in interstitial lung disease that associated with reduced FEV1/FVC ratio. Association analyses with 1,134 disease traits yielded five additional variant-disease associations. We also observed contrasting levels of recessiveness between two more-common, classical Mendelian diseases. Carriers of cystic fibrosis variants exhibited increased risk of several mitigated disease phenotypes, whereas carriers of spinal muscular atrophy alleles showed no evidence of altered phenotypes. Incomplete penetrance of cystic fibrosis carrier phenotypes did not appear to be mediated by common allelic variation on the functional haplotype. Our results show that many disease-associated recessive variants can produce mitigated phenotypes in heterozygous carriers and motivate further work exploring penetrance mechanisms.

A flexible summary statistics-based colocalization method with application to the mucin cystic fibrosis lung disease modifier locus.

Mucus obstruction is a central feature in the cystic fibrosis (CF) airways. A genome-wide association study (GWAS) of lung disease by the CF Gene Modifier Consortium (CFGMC) identified a significant locus containing two mucin genes, MUC20 and MUC4. Expression quantitative trait locus (eQTL) analysis using human nasal epithelia (HNE) from 94 CF-affected Canadians in the CFGMC demonstrated MUC4 eQTLs that mirrored the lung association pattern in the region, suggesting that MUC4 expression may mediate CF lung disease. Complications arose, however, with colocalization testing using existing methods: the locus is complex and the associated SNPs span a 0.2 Mb region with high linkage disequilibrium (LD) and evidence of allelic heterogeneity. We previously developed the Simple Sum (SS), a powerful colocalization test in regions with allelic heterogeneity, but SS assumed eQTLs to be present to achieve type I error control. Here we propose a two-stage SS (SS2) colocalization test that avoids a priori eQTL assumptions, accounts for multiple hypothesis testing and the composite null hypothesis, and enables meta-analysis. We compare SS2 to published approaches through simulation and demonstrate type I error control for all settings with the greatest power in the presence of high LD and allelic heterogeneity. Applying SS2 to the MUC20/MUC4 CF lung disease locus with eQTLs from CF HNE revealed significant colocalization with MUC4 (p = 1.31 × 10-5) rather than with MUC20. The SS2 is a powerful method to inform the responsible gene(s) at a locus and guide future functional studies. SS2 has been implemented in the application LocusFocus.

Small-molecule ion channels increase host defences in cystic fibrosis airway epithelia.

Loss-of-function mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) compromise epithelial HCO3- and Cl- secretion, reduce airway surface liquid pH, and impair respiratory host defences in people with cystic fibrosis1-3. Here we report that apical addition of amphotericin B, a small molecule that forms unselective ion channels, restored HCO3- secretion and increased airway surface liquid pH in cultured airway epithelia from people with cystic fibrosis. These effects required the basolateral Na+, K+-ATPase, indicating that apical amphotericin B channels functionally interfaced with this driver of anion secretion. Amphotericin B also restored airway surface liquid pH, viscosity, and antibacterial activity in primary cultures of airway epithelia from people with cystic fibrosis caused by different mutations, including ones that do not yield CFTR, and increased airway surface liquid pH in CFTR-null pigs in vivo. Thus, unselective small-molecule ion channels can restore host defences in cystic fibrosis airway epithelia via a mechanism that is independent of CFTR and is therefore independent of genotype.

Targeting ubiquitination machinery in cystic fibrosis: Where do we stand?

Cystic Fibrosis (CF) is a genetic disease caused by mutations in CFTR gene expressing the anion selective channel CFTR located at the plasma membrane of different epithelial cells. The most commonly investigated variant causing CF is F508del. This mutation leads to structural defects in the CFTR protein, which are recognized by the endoplasmic reticulum (ER) quality control system. As a result, the protein is retained in the ER and degraded via the ubiquitin-proteasome pathway. Although blocking ubiquitination to stabilize the CFTR protein has long been considered a potential pharmacological approach in CF, progress in this area has been relatively slow. Currently, no compounds targeting this pathway have entered clinical trials for CF. On the other hand, the emergence of Orkambi initially, and notably the subsequent introduction of Trikafta/Kaftrio, have demonstrated the effectiveness of molecular chaperone-based therapies for patients carrying the F508del variant and even showed efficacy against other variants. These treatments directly target the CFTR variant protein without interfering with cell signaling pathways. This review discusses the limits and potential future of targeting protein ubiquitination in CF.

Exon-skipping antisense oligonucleotides for cystic fibrosis therapy.

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause cystic fibrosis (CF), and the CFTR-W1282X nonsense mutation causes a severe form of CF. Although Trikafta and other CFTR-modulation therapies benefit most CF patients, targeted therapy for patients with the W1282X mutation is lacking. The CFTR-W1282X protein has residual activity but is expressed at a very low level due to nonsense-mediated messenger RNA (mRNA) decay (NMD). NMD-suppression therapy and read-through therapy are actively being researched for CFTR nonsense mutants. NMD suppression could increase the mutant CFTR mRNA, and read-through therapies may increase the levels of full-length CFTR protein. However, these approaches have limitations and potential side effects: because the NMD machinery also regulates the expression of many normal mRNAs, broad inhibition of the pathway is not desirable, and read-through drugs are inefficient partly because the mutant mRNA template is subject to NMD. To bypass these issues, we pursued an exon-skipping antisense oligonucleotide (ASO) strategy to achieve gene-specific NMD evasion. A cocktail of two splice-site-targeting ASOs induced the expression of CFTR mRNA without the premature-termination-codon-containing exon 23 (CFTR-Δex23), which is an in-frame exon. Treatment of human bronchial epithelial cells with this cocktail of ASOs that target the splice sites flanking exon 23 results in efficient skipping of exon 23 and an increase in CFTR-Δex23 protein. The splice-switching ASO cocktail increases the CFTR-mediated chloride current in human bronchial epithelial cells. Our results set the stage for developing an allele-specific therapy for CF caused by the W1282X mutation.

Cellular and molecular architecture of submucosal glands in wild-type and cystic fibrosis pigs.

Submucosal glands (SMGs) protect lungs but can also contribute to disease. For example, in cystic fibrosis (CF), SMGs produce abnormal mucus that disrupts mucociliary transport. CF is an ion transport disease, yet knowledge of the ion transporters expressed by SMG acini, which produce mucus, and SMG ducts that carry it to the airway lumen is limited. Therefore, we isolated SMGs from newborn pigs and used single-cell messenger RNA sequencing, immunohistochemistry, and in situ hybridization to identify cell types, gene expression, and spatial distribution. Cell types and transcript levels were the same in non-CF and CF SMGs, suggesting that loss of epithelial anion secretion rather than an intrinsic cell defect causes CF mucus abnormalities. Gene signatures of acinar mucous and acinar serous cells revealed specialized functions in producing mucins and antimicrobials, respectively. However, surprisingly, these two cell types expressed the same ion transporters and neurohumoral receptors, suggesting the importance of balancing mucin and liquid secretion to produce optimal mucus properties. SMG duct cell transcripts suggest that they secrete HCO3- and Cl-, and thus have some similarity to pancreatic ducts that are also defective in CF. These and additional findings suggest the functions of the SMG acinus and duct and provide a baseline for understanding how environmental and genetic challenges impact their contribution to lung disease.

Open reading frame correction using splice-switching antisense oligonucleotides for the treatment of cystic fibrosis.

CFTR gene mutations that result in the introduction of premature termination codons (PTCs) are common in cystic fibrosis (CF). This mutation type causes a severe form of the disease, likely because of low CFTR messenger RNA (mRNA) expression as a result of nonsense-mediated mRNA decay, as well as the production of a nonfunctional, truncated CFTR protein. Current therapeutics for CF, which target residual protein function, are less effective in patients with these types of mutations due in part to low CFTR protein levels. Splice-switching antisense oligonucleotides (ASOs), designed to induce skipping of exons in order to restore the mRNA open reading frame, have shown therapeutic promise preclinically and clinically for a number of diseases. We hypothesized that ASO-mediated skipping of CFTR exon 23 would recover CFTR activity associated with terminating mutations in the exon, including CFTR p.W1282X, the fifth most common mutation in CF. Here, we show that CFTR lacking the amino acids encoding exon 23 is partially functional and responsive to corrector and modulator drugs currently in clinical use. ASO-induced exon 23 skipping rescued CFTR expression and chloride current in primary human bronchial epithelial cells isolated from a homozygote CFTR-W1282X patient. These results support the use of ASOs in treating CF patients with CFTR class I mutations in exon 23 that result in unstable CFTR mRNA and truncations of the CFTR protein.

Fecal dysbiosis and inflammation in intestinal-specific Cftr knockout mice on regimens preventing intestinal obstruction.

Chronic intestinal inflammation is a poorly understood manifestation of cystic fibrosis (CF), which may be refractory to ion channel CF transmembrane conductance regulator (CFTR) modulator therapy. People with CF exhibit intestinal dysbiosis, which has the potential for stimulating intestinal and systemic inflammation. CFTR is expressed in organ epithelia, leukocytes, and other tissues. Here, we investigate the contribution of intestinal epithelium-specific loss of Cftr [iCftr knockout (KO)] to dysbiosis and inflammation in mice treated with either of two antiobstructive dietary regimens necessary to maintain CF mouse models [polyethylene glycol (PEG) laxative or a liquid diet (LiqD)]. Feces collected from iCftr KO mice and their wild-type (WT) sex-matched littermates were used to measure fecal calprotectin to evaluate inflammation and to perform 16S rRNA sequencing to characterize the gut microbiome. Fecal calprotectin was elevated in iCftr KO relative to WT mice that consumed either PEG or LiqD. PEG iCftr KO mice did not show a change in α diversity versus WT mice but demonstrated a significant difference in microbial composition (ß diversity) with included increases in the phylum Proteobacteria, the family Peptostreptococcaceae, four genera of Clostridia including C. innocuum, and the mucolytic genus Akkermansia. Fecal microbiome analysis of LiqD-fed iCftr KO mice showed both decreased α diversity and differences in microbial composition with increases in the Proteobacteria family Enterobacteriaceae, Firmicutes families Clostridiaceae and Peptostreptococcaceae, and enrichment of Clostridium perfringens, C. innocuum, C. difficile, mucolytic Ruminococcus gnavus, and reduction of Akkermansia. It was concluded that epithelium-specific loss of Cftr is a major driver of CF intestinal dysbiosis and inflammation with significant similarities to previous studies of pan Cftr KO mice.NEW & NOTEWORTHY Chronic intestinal inflammation is a manifestation of cystic fibrosis (CF), a disease caused by loss of the anion channel CF transmembrane conductance regulator (CFTR) that is expressed in many tissues. This study shows that intestinal epithelial cell-specific loss of CFTR [inducible Cftr knockout (KO)] in mice is sufficient to induce intestinal dysbiosis and inflammation. Experiments were performed on mice consuming two dietary regimens routinely used to prevent obstruction in CF mice.

Two rare variants that affect the same amino acid in CFTR have distinct responses to ivacaftor.

Some residues in the cystic fibrosis transmembrane conductance regulator (CFTR) channel are the site of more than one CFTR variant that cause cystic fibrosis. Here, we investigated the function of S1159F and S1159P, two variants associated with different clinical phenotypes, which affect the same pore-lining residue in transmembrane segment 12 that are both strongly potentiated by ivacaftor when expressed in CFBE41o- bronchial epithelial cells. To study the single-channel behaviour of CFTR, we applied the patch-clamp technique to Chinese hamster ovary cells heterologously expressing CFTR variants incubated at 27°C to enhance channel residence at the plasma membrane. S1159F- and S1159P-CFTR formed Cl- channels activated by cAMP-dependent phosphorylation and gated by ATP that exhibited thermostability at 37°C. Both variants modestly reduced the single-channel conductance of CFTR. By severely attenuating channel gating, S1159F- and S1159P-CFTR reduced the open probability (Po ) of wild-type CFTR by ≥75% at ATP (1 mM); S1159F-CFTR caused the greater decrease in Po consistent with its more severe clinical phenotype. Ivacaftor (10-100 nM) doubled the Po of both CFTR variants without restoring Po values to wild-type levels, but concomitantly, ivacaftor decreased current flow through open channels. For S1159F-CFTR, the reduction of current flow was marked at high (supersaturated) ivacaftor concentrations (0.5-1 µM) and voltage-independent, identifying an additional detrimental action of elevated ivacaftor concentrations. In conclusion, S1159F and S1159P are gating variants, which also affect CFTR processing and conduction, but not stability, necessitating the use of combinations of CFTR modulators to optimally restore their channel activity. KEY POINTS: Dysfunction of the ion channel cystic fibrosis transmembrane conductance regulator (CFTR) causes the genetic disease cystic fibrosis (CF). This study investigated two rare pathogenic CFTR variants, S1159F and S1159P, which affect the same amino acid in CFTR, to understand the molecular basis of disease and response to the CFTR-targeted therapy ivacaftor. Both rare variants diminished CFTR function by modestly reducing current flow through the channel and severely inhibiting ATP-dependent channel gating with S1159F exerting the stronger adverse effect, which correlates with its association with more severe disease. Ivacaftor potentiated channel gating by both rare variants without restoring their activity to wild-type levels, but concurrently reduced current flow through open channels, particularly those of S1159F-CFTR. Our data demonstrate that S1159F and S1159P cause CFTR dysfunction by multiple mechanisms that require combinations of CFTR-targeted therapies to fully restore channel function.