scholarly journals Heterozygosity for the F508del Mutation in the Cystic Fibrosis Transmembrane Conductance Regulator Anion Channel Attenuates Influenza Severity

2013 ◽  
Vol 208 (5) ◽  
pp. 780-789 ◽  
Author(s):  
Famke Aeffner ◽  
Basant Abdulrahman ◽  
Judy M. Hickman-Davis ◽  
Paul M. Janssen ◽  
Amal Amer ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Anion Channel ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Cystic Fibrosis Transmembrane

scholarly journals Structural comparative modeling of multi-domain ΔF508 CFTR

2021 ◽  
Author(s):  
Eli Fritz McDonald ◽  
Hope Woods ◽  
Shannon Smith ◽  
Minsoo Kim ◽  
Clara T. Schoeder ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Rational Design ◽  
Anion Channel ◽  
Comparative Modeling ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Binding Domains ◽  
Δf508 Cftr ◽  
Domain Models ◽  
Cystic Fibrosis Transmembrane

Cystic Fibrosis (CF) is a common genetic disease caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR), an epithelial anion channel expressed in several vital organs. Absence of functional CFTR results in imbalanced osmotic equilibrium and subsequent mucus build up in the lungs - which increases the risk of infection and eventually causes death. CFTR is an ATP binding cassette (ABC) transporter composed of two transmembrane domains (TMDs), two nucleotide binding domains (NBDs), and an unstructured regulatory domain. The most prevalent patient mutation is the deletion of F508 (ΔF508), making ΔF508 CFTR the primary target for current FDA approved CF therapies. However, no experimental multi-domain ΔF508 CFTR structure has been determined and few studies have modeled ΔF508 using multi-domain WT CFTR structures. Here, we used cryo-EM density data and Rosetta comparative modeling (RosettaCM) to compare a ΔF508 model with published experimental data on CFTR NBD1 thermodynamics. We then apply this modeling method to generate multi-domain WT and ΔF508 CFTR structural models. These models demonstrate the destabilizing effects of ΔF508 on NBD1 and the NBD1/TMD interface in both the closed and open conformation of CFTR. Furthermore, we modeled ΔF508/R1070W and ΔF508 bound to a the CFTR corrector VX-809. Our models reveal the stabilizing effects of R1070W and VX-809 on multi-domain models of ΔF508 CFTR and pave the way for rational design of additional drugs that target ΔF508 CFTR for treatment of CF.

scholarly journals Molecular structure of the ATP-bound, phosphorylated human CFTR

2018 ◽  
Vol 115 (50) ◽  
pp. 12757-12762 ◽  
Author(s):  
Zhe Zhang ◽  
Fangyu Liu ◽  
Jue Chen
Keyword(s):  
Molecular Structure ◽  
Cystic Fibrosis ◽  
Conformational Changes ◽  
Anion Channel ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Common Features ◽  
Proper Functions ◽  
Cystic Fibrosis Transmembrane ◽  
Kinase A

The cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel important in maintaining proper functions of the lung, pancreas, and intestine. The activity of CFTR is regulated by ATP and protein kinase A-dependent phosphorylation. To understand the conformational changes elicited by phosphorylation and ATP binding, we present here the structure of phosphorylated, ATP-bound human CFTR, determined by cryoelectron microscopy to 3.2-Å resolution. This structure reveals the position of the R domain after phosphorylation. By comparing the structures of human CFTR and zebrafish CFTR determined under the same condition, we identified common features essential to channel gating. The differences in their structures indicate plasticity permitted in evolution to achieve the same function. Finally, the structure of CFTR provides a better understanding of why the G178R, R352Q, L927P, and G970R/D mutations would impede conformational changes of CFTR and lead to cystic fibrosis.

scholarly journals Rab11b Regulates the Apical Recycling of the Cystic Fibrosis Transmembrane Conductance Regulator in Polarized Intestinal Epithelial Cells

2009 ◽  
Vol 20 (8) ◽  
pp. 2337-2350 ◽  
Author(s):  
Mark R. Silvis ◽  
Carol A. Bertrand ◽  
Nadia Ameen ◽  
Franca Golin-Bisello ◽  
Michael B. Butterworth ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Epithelial Cells ◽  
Apical Membrane ◽  
Expression System ◽  
Anion Channel ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
T84 Cells ◽  
Anion Secretion ◽  
Cystic Fibrosis Transmembrane

The cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP/PKA-activated anion channel, undergoes efficient apical recycling in polarized epithelia. The regulatory mechanisms underlying CFTR recycling are understood poorly, yet this process is required for proper channel copy number at the apical membrane, and it is defective in the common CFTR mutant, ΔF508. Herein, we investigated the function of Rab11 isoforms in regulating CFTR trafficking in T84 cells, a colonic epithelial line that expresses CFTR endogenously. Western blotting of immunoisolated Rab11a or Rab11b vesicles revealed localization of endogenous CFTR within both compartments. CFTR function assays performed on T84 cells expressing the Rab11a or Rab11b GDP-locked S25N mutants demonstrated that only the Rab11b mutant inhibited 80% of the cAMP-activated halide efflux and that only the constitutively active Rab11b-Q70L increased the rate constant for stimulated halide efflux. Similarly, RNAi knockdown of Rab11b, but not Rab11a, reduced by 50% the CFTR-mediated anion conductance response. In polarized T84 monolayers, adenoviral expression of Rab11b-S25N resulted in a 70% inhibition of forskolin-stimulated transepithelial anion secretion and a 50% decrease in apical membrane CFTR as assessed by cell surface biotinylation. Biotin protection assays revealed a robust inhibition of CFTR recycling in polarized T84 cells expressing Rab11b-S25N, demonstrating the selective requirement for the Rab11b isoform. This is the first report detailing apical CFTR recycling in a native expression system and to demonstrate that Rab11b regulates apical recycling in polarized epithelial cells.

scholarly journals Cystic fibrosis transmembrane conductance regulator contributes to reacidification of alkalinized lysosomes in RPE cells

2012 ◽  
Vol 303 (2) ◽  
pp. C160-C169 ◽  
Author(s):  
Ji Liu ◽  
Wennan Lu ◽  
Sonia Guha ◽  
Gabriel C. Baltazar ◽  
Erin E. Coffey ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Anion Channel ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Rpe Cells ◽  
Lysosomal Ph ◽  
Baseline Levels ◽  
Interfering Rna ◽  
Cystic Fibrosis Transmembrane ◽  
Lysosomal Acidification

The role of the cystic fibrosis transmembrane conductance regulator (CFTR) in lysosomal acidification has been difficult to determine. We demonstrate here that CFTR contributes more to the reacidification of lysosomes from an elevated pH than to baseline pH maintenance. Lysosomal alkalinization is increasingly recognized as a factor in diseases of accumulation, and we previously showed that cAMP reacidified alkalinized lysosomes in retinal pigmented epithelial (RPE) cells. As the influx of anions to electrically balance proton accumulation may enhance lysosomal acidification, the contribution of the cAMP-activated anion channel CFTR to lysosomal reacidification was probed. The antagonist CFTRinh-172 had little effect on baseline levels of lysosomal pH in cultured human RPE cells but substantially reduced the reacidification of compromised lysosomes by cAMP. Likewise, CFTR activators had a bigger impact on cells whose lysosomes had been alkalinized. Knockdown of CFTR with small interfering RNA had a larger effect on alkalinized lysosomes than on baseline levels. Inhibition of CFTR in isolated lysosomes altered pH. While CFTR and Lamp1 were colocalized, treatment with cAMP did not increase targeting of CFTR to the lysosome. The inhibition of CFTR slowed lysosomal degradation of photoreceptor outer segments while activation of CFTR enhanced their clearance from compromised lysosomes. Activation of CFTR acidified RPE lysosomes from the ABCA4−/− mouse model of recessive Stargardt's disease, whose lysosomes are considerably alkalinized. In summary, CFTR contributes more to reducing lysosomal pH from alkalinized levels than to maintaining baseline pH. Treatment to activate CFTR may thus be of benefit in disorders of accumulation associated with lysosomal alkalinization.

scholarly journals Cystic fibrosis drug ivacaftor stimulates CFTR channels at picomolar concentrations

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
László Csanády ◽  
Beáta Töröcsik
Keyword(s):  
Cystic Fibrosis ◽  
Anion Channel ◽  
Aqueous Solubility ◽  
Transmembrane Conductance Regulator ◽  
Inherited Disease ◽  
Transmembrane Conductance ◽  
Molecular Mechanism Of Action ◽  
Intensive Investigation ◽  
Cystic Fibrosis Transmembrane

The devastating inherited disease cystic fibrosis (CF) is caused by mutations of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) anion channel. The recent approval of the CFTR potentiator drug ivacaftor (Vx-770) for the treatment of CF patients has marked the advent of causative CF therapy. Currently, thousands of patients are being treated with the drug, and its molecular mechanism of action is under intensive investigation. Here we determine the solubility profile and true stimulatory potency of Vx-770 towards wild-type (WT) and mutant human CFTR channels in cell-free patches of membrane. We find that its aqueous solubility is ~200 fold lower (~60 nanomolar), whereas the potency of its stimulatory effect is >100 fold higher, than reported, and is unexpectedly fully reversible. Strong, but greatly delayed, channel activation by picomolar Vx-770 identifies multiple sequential slow steps in the activation pathway. These findings provide solid guidelines for the design of in vitro studies using Vx-770.

scholarly journals A Topological Switch Enables Misfolding of the Cystic Fibrosis Transmembrane Conductance Regulator

2020 ◽  
Author(s):  
Daniel Scholl ◽  
Maud Sigoillot ◽  
Marie Overtus ◽  
Rafael Colomer Martinez ◽  
Chloé Martens ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Single Molecule ◽  
Genetic Disorder ◽  
Resonance Energy ◽  
Anion Channel ◽  
Atp Binding ◽  
Transmembrane Conductance Regulator ◽  
Wild Type ◽  
Transmembrane Conductance ◽  
Cystic Fibrosis Transmembrane

AbstractCystic Fibrosis (CF) is a common lethal genetic disorder caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel. Misfolding and degradation of CFTR are the hallmarks of the predominant mutation, F508del, located in the first nucleotide binding domain (NBD1). While the mutation is known to affect the thermal stability of NBD1 and assembly of CFTR domains, the molecular events that lead to misfolding of F508del-CFTR remain elusive. Here, we demonstrate that NBD1 of CFTR can adopt an alternative conformation that departs from the canonical NBD fold previously observed for CFTR and other ATP-binding cassette (ABC) transporter proteins. Crystallography studies reveal that this conformation involves a topological reorganization of the β-subdomain of NBD1. This alternative state is adopted by wild-type CFTR in cells and enhances channel activity. Single-molecule fluorescence resonance energy transfer microscopy shows that the equilibrium between the conformations is regulated by ATP binding. Under destabilizing conditions, however, this conformational flexibility enables unfolding of the β-subdomain. Our data indicate that in wild-type CFTR switching to this topologically-swapped conformation of NBD1 regulates channel function, but, in the presence of the F508del mutation, it allows domain misfolding and subsequent protein degradation. Our work provides a framework to design conformation-specific therapeutics to prevent noxious transitions.

A Topological Switch in the Cystic Fibrosis Transmembrane Conductance Regulator Modulates Channel Activity and Sensitivity to Disease-Causing Mutation

2020 ◽  
Author(s):  
Daniel Scholl ◽  
Maud Sigoillot ◽  
Marie Overtus ◽  
Rafael Colomer ◽  
Chloé Martens ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Single Molecule ◽  
Genetic Disorder ◽  
Anion Channel ◽  
Atp Binding ◽  
Transmembrane Conductance Regulator ◽  
Channel Activity ◽  
Wild Type ◽  
Transmembrane Conductance ◽  
Cystic Fibrosis Transmembrane

Abstract The cystic fibrosis transmembrane conductance regulator (CFTR) anion channel is essential to maintain fluid homeostasis in key organs such as the lungs or the digestive systems. Functional impairment of CFTR due to mutation in the cftr gene lead to Cystic Fibrosis (CF) the most common lethal genetic disorder. Here we observe that the first nucleotide-binding domain (NBD1) of CFTR can spontaneously adopt an alternative conformation that departs from the canonical NBD fold previously observed for CFTR and other ATP-binding cassette (ABC) transporter proteins. Crystallography studies reveal that this conformation involves a topological reorganization of the β-subdomain of NBD1. This alternative state is adopted by wild-type CFTR in cells, where it leads to enhanced channel activity. Single-molecule fluorescence resonance energy transfer microscopy shows that the equilibrium between the conformations is regulated by ATP binding. However, under destabilizing conditions, such as a the prominent disease-causing mutation F508el , this conformational flexibility enables unfolding of the β-subdomain. Our data indicate that in wild-type CFTR switching to this topologically-swapped conformation of NBD1 regulates channel function, but, in the presence of the F508del mutation, it allows domain misfolding and subsequent protein degradation. Our work provides a framework to design conformation-specific therapeutics to prevent noxious transitions.

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